Lipid Mediators and Their Metabolism in the Brain

Akhlaq A. Farooqui

Lipid Mediators and Their Metabolism in the Brain Akhlaq A. Farooqui Department of Molecular and Cellular Biochemistry The Ohio State University Columbus, OH 43210 USA [email protected]

ISBN 978-1-4419-9939-9 e-ISBN 978-1-4419-9940-5 DOI 10.1007/978-1-4419-9940-5 Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011934260

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Springer is part of Springer Science+Business Media (www.springer.com) Dedicated to my teachers for their passion to teach and stimulate the desire to learn and integrate knowledge. Akhlaq A. Farooqui

Preface

Neural membranes are highly dynamic and interactive structures composed of glycerophospholipids, sphingolipids, cholesterol, and transmembrane and peripheral proteins of various shapes, molecular masses, and functions. The binding between phospholipids and proteins is necessary for vertical positioning and tight integration of proteins into the membrane. Phospholipids and sphingolipids contribute to the lipid bilayer asymmetry, whereas cholesterol and sphingolipids form lipid rafts, which act as platforms for molecular sorting, trafficking, and signal transduction processes. Lipid mediators are chemical messengers that are released in response to cell stimulation or injury from membrane phospholipids, sphingolipid, and cholesterol. Lipid mediators play important roles in internal and external communication and modulate cellular responses such as the growth arrest, differentiation, adhesion, and migration. These processes are modulated by eicosanoids (prostaglandins, leukotrienes, thromboxanes, and lipoxins) and docosanoids (resolvins, protectins, neuroprotectins, and maresins), which are generated by the action of phospholipases A2, cyclooxygenases, and lipoxygenases on arachidonic and docosahexaenoic acids (ARA and DHA), respectively. The non-enzymic lipid mediators of ARA and DHA metabolism include isoprostanes, neuroprostanes, isoketals, neuroketals, isofurans, 4-hydroxynonenal, and 4-hydroxyhexanal. Action of sphingomyelinases on sphingomyelin generates ceramide, a metabolite closely associated with apoptotic cell death. Further degradation of ceramide generates sphingosine, which in its phosphorylated form induces cell proliferation, and thus produces the balance between cell death and cell survival. In the brain, cholesterol is hydroxylated into oxycholesterols or hydroxycholesterols (24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 22-hydroxycholesterol) by cytochrome P450- dependent oxygenases. Conversion of cholesterol to hydroxycholesterols is a major mechanism for the elimination of cholesterol from the brain. Collective evidence suggests that under normal conditions low levels of lipid mediators are needed for signal transduction, gene expression, and neural cell proliferation and differentiation, resulting in neural cell survival but high levels of enzymic and non-enzymic lipid mediators may cause neurodegeneration through the induction of oxidative stress, neuroinflammation, and apoptosis. Thus, neural membranes are not only

vii viii Preface simple inert barrier, but a Pandora’s box of lipid mediators; many of which have powerful neurochemical effects on cell growth, proliferation, differentiation, survival, and apoptosis. Levels of lipid mediators in neural and non-neural tissues are partly regulated by diet. The high intake of food enriched in ARA (vegetable oils) elevates levels of eicosanoids and upregulates the expression of proinflammatory cytokines. ARA and its metabolites have prothrombotic, proaggregatory, and proinflammatory properties. In contrast, diet enriched in DHA (fish and fish oil) generates docosanoids, which not only downregulate proinflammatory cytokines but also have antiinflammatory, antithrombotic, antiarrhythmic, hypolipidemic, and vasodilatory effects. At present, the threshold concentrations of lipid mediators that promote and facilitate neural cell injury and death are not known. In neurological disorders, cell death not only depends upon elevated levels of lipid mediators, but also on cross-talk (interplay) among glycerophospholipid-, glycosphingolipid-, and cholesterol-derived lipid mediators. Studies on lipid-derived mediators fall in a fast- paced research area related to neurodegeneration and provide opportunities for target-based therapeutic intervention using inhibitors of lipid mediator synthesizing enzymes. The goal of this monograph is to present readers with cutting edge and comprehensive information on lipid mediators in a manner that is useful not only to students and teachers, but also to researchers and physicians. This monograph has 11 chapters. Chapters 1 and 2 describe metabolism, roles, and involvement of eicosanoids and docosanoids in neurological disorders. Chapters 3 and 4 describe cutting edge information on the synthesis, degradation, roles, and association of lyso-glycerophospholipids and platelet activating factor in neurological disorders. Chapter 5 describes metabolism and roles of cannabinoids in brain and their relationship with neurological disorders. Chapters 6 and 7 are devoted to the metabolism of nonenzymic lipid mediators of arachidonic acid metabolism (4-hydroxynonenal and isoprostanes) and docosahexaenoic acid metabolism (4-hydoxyhexanal and neuroprostanes). Chapters 8 and 9 describe metabolism, roles, and involvement of ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively, in neurological disorders. Chapter 10 describes metabolism, role, and association of cholesterol and hydroxycholesterols with neurological disorders. Finally, Chap. 11 provides readers and researchers with perspective that will be important for future research work on bioactive lipid mediators. My writing style and demonstrated ability to present complicated material on lipid mediators makes this monograph particularly accessible to neuroscience graduate students, teachers, and fellow researchers. It can be used as supplement text for a range of neuroscience courses. Clinicians and pharmacologists will find this book useful for understanding molecular aspects of lipid mediators in neurodegeneration in acute neural trauma (stroke, spinal cord trauma, and head injury) and neurodegenerative diseases (Alzheimer disease, Parkinson disease, and Huntington disease). To the best of my knowledge no one has written a monograph on the role of lipid mediators in the brain. This monograph will be the first to provide a comprehensive description of glycerophospholipid, sphingolipid, and cholesterol-derived mediators, their interactions with each others in normal brain and in brain tissue from neurological disorders. I also hope that this Preface ix monograph will provide senior researchers some guidance for overcoming problems on lipid mediator research that they are encountering in their laboratories. One of the hallmark of this monograph is the presentation of a unifying concept of lipid mediator-mediated signal transduction processes associated with excitotoxicity, oxidative stress, and neuroinflammation. The presentation of this monograph is based on uniformity and logical progression of subject from one topic to another with an extensive bibliography. For the sake of explanation, simplicity, and uniformity a large number of figures and line diagrams of signal transduction pathways with chemical structures of lipid mediators are also presented. It is hoped that my attempt to integrate and consolidate the knowledge of lipid mediators and signal transduction processes in normal and diseased brain will provide the basis of more dramatic advances and developments on the determination, characterization, and roles of glycerophospholipid-, sphingolipid-, and cholesterol-derived lipid mediators in neurological disorders.

Columbus, OH Akhlaq A. Farooqui

Acknowledgments

I thank my wife, Tahira, for critical reading of the manuscript, providing colored figures, and useful discussion during the writing of this monograph. This monograph would not have been possible without her unrelenting support, constructive criti- cisms, and constant encouragement. I also thank Dr. Wei-Yi Ong, Department of Anatomy, National University of Singapore, Singapore, for many years of collabora- tion on phospholipases A2, lipid mediators, and their roles in neurodegeneration. Finally, I also express my gratitude to Ann H. Avouris and Melissa Higgs of Springer, New York, for their cooperation, rapid responses to my queries, and professional manuscript handing. It has been a pleasure working with them for many years.

About My Teachers

Late Professor Bimal Kumar Bachhawat was my Ph.D. advisor and Chief of Neurochemistry Laboratory, Christian Medical College, Vellore, India. Later he became Director of Indian Institute of Chemical Biology, Calcutta, Professor Head, Department of Biochemistry, and Dean Faculty of Interdisciplinary and Applied Sciences, University of Delhi South Campus, New Delhi. He was elected to all of the scientific academies in India (such as Fellow of National Academy of Sciences, Indian Academy of Sciences and Indian National Science Academy). He received numerous national awards and honors, including the Shanti Swarup Bhatnagar award (1962), the Golden Jubilee Medal (1976) in India. He was elected President of Federation of Asian and Oceanian Biochemistry (1983–1985), Indian Society of Biological Chemists (1970–1972 and 1990–1994); and National Organizing Committee, International Union of Biochemistry and Molecular Biology (1994). Late Professor Bachhawat was a leader, organizer, and teacher, who nurtured a generation of biochemists not only in India, but around the world. He was a man of many admirable qualities, known for his keen interest in others with great sense of humor. Late Professor Bachhawat and his colleagues discovered that metachromatic leukodystrophy is a glycolipid storage disease that is caused by the deficiency of arylsulfatase A. This pathbreaking study set the stage for the elucidation of enzymic defects in other glycolipid storage disorders, including Gaucher’s disease and Tay–Sachs disease. He pioneered the development of carbohydrate-bearing lipo- somes for the site-specific delivery of drugs and enzymes to diseased organs. He was also involved in the development of liposomal formulations for treating systemic fungal infections.

Professor Abdul Majid Siddiqi taught me biochemistry during my M.S. classes. He is a retired Professor and Chairman of Biochemistry Department, and first Dean of the Faculty of Life Sciences at Aligarh Muslim University (AMU), Aligarh. During a long career at AMU, Professor Siddiqi taught Biochemistry to thousands of B.Sc. and M.Sc. students and mentored 39 M.Phil. and 40 Ph.D. students, who now hold important academic positions in India and elsewhere. His research at AMU showed for the first time the utility of inhibitors of cholesterol biosynthesis,

xiii xiv About My Teachers such as 3-hydroxy-3-methylglutaric acid (HMG) for treating hyperlipidemia. For this work he was awarded US Patent 36,29,44,9 titled “Process of Combating Hypercholesterolemia” in 1971, the global rights for which were taken by M/S Calbiochem-Behring Corporation, USA. Professor Siddiqi is an active member of the Society of Biological Chemists (India) and an active participant in various national and international groups on Biochemical Education.

Professor V.P. Jaiswal, Retired Principal, Government Postgraduate College, Department of Higher Education, Uttar Pradesh, India, has a devoted academic, professional, and administrative career. After achieving a high merit at postgraduate level in Zoology from University of Allahabad (1956), Professor V.P. Jaiswal was appointed lecturer in Zoology at St. Andrews College Gorakhpur (Gorakhpur University, Gorakhpur), Govt. Raza Degree College Rampur (U.P.), where he taught me zoology and put me to the path of higher studies in biological sciences. He was promoted to Professor and Head of Zoology Department at Birla Government College, Srinagar, Garhwal, Government College, Pithoragarh, Gopeshwar (chamoli), Government P.G. College, Rampur, and Government P.G. College Kotdawara, Garhwal, where he was appointed as a Principal. In the year 1990 he retired as Principal, P.G. College, Hamirpur, (Bundelkhand University) where in his tenure he was a member of Executive Council Bundelkhand University, Jhansi (U.P.), India. At present he is an active member of Bombay Natural History Society, Bombay, India.

Professor Mahdi Hasan is a well-known neuroscientist, neurotoxicologist and a neurogerontologist, who has inspired and mentored me in studies on neurological disorders. He has been teaching human anatomy to medical students for over 52 years and some 10,000 of his former students are globally active and productive. His publications include 135 research papers, 6 books and 14 book reviews. He is a recipient of India’s most prestigious research awards, Dr. B.C. Roy National Award for eminent teacher, Dr. Bachhawat Life Time Achievement Award of Indian Academy of Neurosciences. He is a Fellow of Alexander von Humboldt Foundation, International College of Surgeons, National Academy of Medical Sciences (India) & Indian National Science Academy (INSA). Currently, he is an Emeritus Professor and INSA Honorary Scientist at King George Medical University, Lucknow (India). About the Author

Akhlaq A. Farooqui is a leader in the field of signal transduction, brain phospholipases

A2, bioactive ether lipid metabolism, polyunsaturated fatty acid metabolism, glycerophospholipid-, sphingolipid-, and cholesterol-derived lipid mediators, and glutamate-induced neurotoxicity. Akhlaq A. Farooqui has discovered the stimulation of plasmalogen-selective phospholipase A2 (PlsEtn-PLA2) and diacyl- and monoacylglycerol lipases in brains from patients with Alzheimer disease. Stimulation of

PlsEtn-PLA2 produces plasmalogen deficiency and increases levels of eicosanoids that may be related to the loss of synapses in brains of patients with Alzheimer disease. Akhlaq A. Farooqui has published cutting edge research on the generation and identification of glycerophospholipid-, sphingolipid-, and cholesterol-derived lipid mediators in kainic acid-mediated neurotoxicity by lipidomics. Akhlaq A. Farooqui has authored six monographs: Glycerophospholipids in Brain: Phospholipase A2 in Neurological Disorders (2007); Neurochemical Aspects of Excitotoxicity (2008); Metabolism and Functions of Bioactive Ether Lipids in Brain (2008); and Hot Topics in Neural Membrane Lipidology (2009); Beneficial Effects of Fish Oil in Human Brain (2009); and Neurochemical Aspects of Neurotraumatic and Neurodegenerative Diseases (2010). All monographs are published by Springer, New York. In addition, Akhlaq A. Farooqui has edited four books (Biogenic Amines: Pharmacological, Neurochemical and Molecular Aspects in the CNS (2010) Nova Science Publisher, Hauppauge, N.Y, Molecular Aspects of Neurodegeneration and Neuroprotection, Bentham Science Publishers Ltd (2011); Phytotherapeutics and Human Health: Molecular and pharmacological Aspects, Nova Science Publisher, Hauppauge, N.Y In Press (2011) and Molecular Aspects of Oxidative Stress on Cell Signaling in Vertebrates and Invertebrates, Wiley Blackwell Publishing Company, New York in press, 2011).

Contents

1 Metabolism and Roles of Eicosanoids in Brain...... 1 1.1 Introduction...... 1 1.2 Multiplicity of Cyclooxygenases, Lipoxygenases, and Epoxygenases in the Brain...... 4 1.2.1 Cyclooxygenases (COXs)...... 6 1.2.2 Lipoxygenases (LOXs)...... 11 1.2.3 Cytochrome P450 Epoxygenases (EPOXs)...... 13 1.3 Eicosanoids and Their Receptors in Brain...... 15 1.4 Interplay Among COX, LOX, and EPOX-derived Products,

and Relationship to Upstream PLA2 Isoforms...... 22 1.5 Roles of Eicosanoids in the Brain...... 25 1.5.1 Eicosanoids in Neuroinflammation...... 25 1.5.2 Eicosanoids in Neurodegeneration...... 27 1.5.3 Eicosanoids in Nociception (Pain State)...... 28 1.5.4 Eicosanoids in Synaptic Plasticity...... 28 1.6 Eicosanoids in Neurotraumatic Diseases...... 29 1.6.1 Eicosanoids in Ischemic Injury...... 30 1.6.2 Eicosanoids in Spinal Cord Trauma...... 31 1.6.3 Eicosanoids in Traumatic Brain Injury...... 31 1.6.4 Eicosanoids in Epilepsy...... 32 1.7 Eicosanoids in Neurodegenerative Diseases...... 32 1.7.1 COX, LOX, EPOX, and Eicosanoids in AD...... 33 1.7.2 COX, LOX, EPOX, and Eicosanoids in PD...... 35 1.7.3 COX, LOX, EPOX, and Eicosanoids in Amyotrophic Lateral Sclerosis...... 35 1.7.4 COX, LOX, EPOX, and Eicosanoids in Creutzfeldt-Jakob Disease (CJD)...... 36 1.8 Conclusions...... 37 References...... 37

xvii xviii Contents

2 Recent Development on the Neurochemistry of Docosanoids...... 49 2.1 Introduction...... 49 2.2 Importance of n-3 Polyunsaturated Fatty Acids in the Brain...... 50 2.3 EPA-Derived Lipid Mediators in the Brain...... 52 2.4 DHA-Derived Lipid Mediators in the Brain...... 58 2.4.1 17S D Series Resolvins...... 60 2.4.2 Protectins and Neuroprotectin...... 60 2.5 Conclusion...... 68 References...... 68 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids in Neurological Disorders...... 73 3.1 Introduction...... 73 3.2 Deacylation/Reacylation Cycle (Land’s Cycle) and Its Importance...... 74 3.2.1 Acyl-CoA Synthetases in the Brain...... 75 3.2.2 Acyl-CoA: Lyso-phospholipid Acyltransferase in the Brain...... 77

3.2.3 Phospholipases A2 (PLA2) in the Brain...... 78 3.2.4 Long-Chain Acyl-CoA Hydrolase or Thioesterases in the Brain...... 80 3.2.5 CoA-independent Reacylation in the Brain...... 81 3.3 Effects of Lysophospholipids on Neural Membrane Metabolism...... 82 3.3.1 Lysophosphatidylcholine (Lyso-PtdCho)...... 82 3.3.2 Lyso-phosphatidylethanolamine (Lyso-PtdEtn)...... 85 3.3.3 Lyso-phosphatidylserine (Lyso-PtdSer)...... 86 3.3.4 Lyso-phosphatidylinositol (Lyso-PtdIns)...... 87

3.3.5 Lyso-cardiolipin (Lyso-Ptd2Gro)...... 88 3.3.6 Lyso-ethanolamine and Choline Plasmalogens (Lyso-PlsEtn and Lyso-PlsCho)...... 89 3.3.7 Lyso-phosphatidic Acid (Lyso-PtdH)...... 90 3.4 Lyso-phospholipids in Neurotraumatic and Neurodegenerative Diseases...... 93 3.4.1 Lyso-phospholipids in Ischemic Injury...... 94 3.4.2 Lyso-phospholipids in Alzheimer Disease...... 95 3.4.3 Lyso-phospholipids in MS and EAE...... 96 3.4.4 Lyso-phospholipids in NCL...... 97 3.5 Conclusion...... 97 References...... 97 Contents xix

4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement in Neurological Disorders...... 107 4.1 Introduction...... 107 4.2 PAF Biosynthesis in the Brain...... 108 4.2.1 De Novo Synthesis of PAF...... 109 4.2.2 Remodeling Pathway for PAF Synthesis...... 111 4.2.3 Oxidative Fragmentation Pathway for PAF Synthesis...... 114 4.3 Catabolism of PAF in the Brain...... 114 4.4 PAF Receptors in the Brain...... 116 4.5 PAF in Neurological and Visceral Disorders...... 118 4.5.1 PAF in Neurological Disorders...... 118 4.5.2 PAF in Visceral Disorders...... 123 4.5.3 PAF in Kainic Acid Neurotoxicity...... 124 4.6 Molecular Mechanism Associated with PAF-Mediated Neural Injury...... 124 4.7 Conclusion...... 125 References...... 126 5 Cannabinoids in the Brain: Their Metabolism, Roles, and Involvement in Neurological Disorders...... 133 5.1 Introduction...... 133 5.2 Cannabinoid Receptor-Mediated Signaling in the Brain...... 136 5.3 Occurrence and Synthesis of Endocannabinoids in the Brain...... 137 5.3.1 Metabolism of 2-Arachidonylglycerol in the Brain...... 139 5.3.2 Metabolism of Anandamide in the Brain...... 142 5.4 Interplay Among Cannabinoid, Glutamate, and Dopamine Receptors in the Basal Ganglia...... 143 5.5 Endocannabinoids and Neurological Disorders...... 144 5.5.1 Endocannabinoids in Neurotraumatic Diseases...... 145 5.5.2 Endocannabinoids in Neurodegenerative Disorders...... 147 5.6 Use of FAAH and MGL Inhibitors for Inflammatory Pain...... 149 5.7 Conclusion...... 150 References...... 151 6 Neurochemical Aspects of 4-Hydroxynonenal...... 159 6.1 Introduction...... 159 6.2 Synthesis of 4-HNE in the Brain...... 160 6.3 Metabolism of 4-HNE in the Brain...... 161 6.4 Effect of 4-HNE on Enzyme Activities...... 163 6.4.1 Modulation of Kinases by 4-HNE...... 164 6.4.2 Modulation of Caspases by 4-HNE...... 166 6.4.3 Modulation of Glutathione S-Transferases by 4-HNE...... 167 xx Contents

6.4.4 Modulation of ATPases by 4-HNE...... 169 6.4.5 Modulation of Other Enzymes and Cell Cycle by 4-HNE...... 170 6.4.6 Modulation of Heat Shock Response by 4-HNE...... 170 6.5 Modulation of NMDA Receptor by 4-HNE...... 171 6.6 Modulation of Gene Expression by 4-HNE...... 172 6.7 Modulation of Peroxisomes by 4-HNE...... 173 6.8 Modulation of Nucleic Acid Metabolism by 4-HNE...... 173 6.9 Modulation of Phospholipid Metabolism by 4-HNE...... 175 6.10 Modulation of Blood–Brain Barrier by 4-HNE...... 175 6.11 4-HNE in Neurological Disorders...... 176 6.11.1 4-HNE in Alzheimer Diseases...... 176 6.11.2 4-HNE in Parkinson Disease...... 177 6.11.3 4-HNE in Amyotrophic Lateral Sclerosis...... 178 6.11.4 4-HNE in Prion Diseases...... 179 6.11.5 4-HNE in Ischemia...... 179 6.11.6 4-HNE in Spinal Cord Injury...... 180 6.11.7 4-HNE in Traumatic Brain Injury...... 181 6.11.8 4-HNE in Kainic Acid Neurotoxicity...... 183 6.12 4-Hydroxyhexenal (4-HHE)...... 183 6.13 Conclusion...... 184 References...... 184 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived Lipid Mediators in the Brain...... 193 7.1 Introduction...... 193 7.2 Generation of IsoPs in the Brain...... 194 7.3 Degradation of IsoPs in the Brain...... 195 7.4 IsoPs and Signal Transduction Processes...... 198 7.5 IsoPs in Neurodegenerative Diseases...... 202 7.6 IsoPs in Neurotraumatic Diseases...... 204 7.7 NPs in Neurological Disorders...... 204 7.8 Isoketals (Isolevuglandins) in Neurological Disorders...... 206 7.9 Neuroketals in Neurological Disorders...... 207 7.10 Isofurans in Neurological Disorders...... 208 7.11 Neurofurans in Neurological Disorders...... 209 7.12 IsoPs in Kainic Acid Neurotoxicity...... 209 7.13 Conclusion...... 210 References...... 211 8 Ceramide and Ceramide 1 Phosphate in the Brain...... 217 8.1 Introduction...... 217 8.2 Synthesis of Ceramide and Ceramide 1-phosphate in the Brain...... 221 8.3 Degradation of Ceramide and Ceramide 1-phosphate in the Brain...... 227 Contents xxi

8.4 Roles of Ceramide and Ceramide 1-phosphate in the Brain...... 228 8.5 Ceramide in Neurological Disorders...... 229 8.5.1 Ceramide in Ischemic Injury...... 231 8.5.2 Ceramide in Alzheimer Disease...... 232 8.5.3 Ceramide in Parkinson Disease...... 234 8.5.4 Ceramide in Amyotrophic Lateral Sclerosis...... 234 8.5.5 Ceramide in Multiple Sclerosis...... 235 8.5.6 Ceramide in HIV-1...... 235 8.5.7 Ceramide in Batten Disease...... 236 8.5.8 Ceramide in Major Depressive Disorders...... 236 8.5.9 Ceramide in Kainic Acid Neurotoxicity...... 236 8.6 Conclusion...... 237 References...... 238 9 Sphingosine and Sphingosine 1 Phosphate in the Brain...... 245 9.1 Introduction...... 245 9.2 Sphingosine Kinases in the Brain...... 248 9.3 Sphingosine 1 Phosphate Receptors in the Brain...... 251 9.4 Sphingosylphosphorylcholine in the Brain...... 254 9.5 FTY720 and Its Neurochemical Effects...... 255 9.6 Sphingosine 1 Phosphate in Neurological Disorders...... 257 9.6.1 Sphingosine 1 Phosphate in Ischemia...... 257 9.6.2 Sphingosine 1 Phosphate in Traumatic Brain and Spinal Cord Injuries...... 259 9.6.3 Sphingosine 1 Phosphate in Alzheimer Disease...... 259 9.6.4 Sphingosine 1 Phosphate in Multiple Sclerosis...... 260 9.6.5 Sphingosine 1 Phosphate in Kainic Acid Neurotoxicity...... 261 9.7 Conclusion...... 261 References...... 262 10 Cholesterol and Hydroxycholesterol in the Brain...... 267 10.1 Introduction...... 267 10.2 Synthesis of Cholesterol and Hydroxycholesterols in the Brain...... 269 10.3 Degradation of Cholesterol in the Brain...... 275 10.4 Roles of Hydroxycholesterols in the Brain...... 277 10.4.1 Hydroxycholesterols in Neural Cell Differentiation...... 278 10.4.2 Hydroxycholesterols in Exocytosis...... 279 10.4.3 Hydroxycholesterols in Apoptosis...... 279 10.5 Cholesterol and Hydroxycholesterols in Neurological Disorders...... 281 10.5.1 Cholesterol and Hydroxycholesterol in Alzheimer Disease...... 282 10.5.2 Cholesterol and Hydroxycholesterol in Parkinson Disease...... 284 xxii Contents

10.5.3 Cholesterol and Hydroxycholesterol in Huntington Disease...... 285 10.5.4 Cholesterol and Hydroxycholesterols in Niemann-Pick Type C...... 286 10.5.5 Cholesterol and Hydroxycholesterol in Multiple Sclerosis...... 287 10.5.6 Hydroxycholesterol in Traumatic Brain Injury...... 288 10.5.7 Cholesterol and Hydroxycholesterols in Cerebrotendinous Xanthomatosis...... 288 10.6 Cholesterol and Hydroxycholesterols in Kainic Acid Neurotoxicity...... 289 10.7 Conclusion...... 290 References...... 291 11 Perspective and Direction for Future Studies on Lipid Mediators...... 299 11.1 Introduction...... 299 11.2 Lipid Mediators in the Brain...... 300 11.3 Interactions Among Phospholipid-, Sphingolipid-, and Cholesterol-Derived Lipid Mediators in the Brain...... 303 11.4 Detection and Levels of Lipid Mediators in Neurological Disorders by Lipidomics...... 306 11.5 Modulation of Lipid Mediators by Diet...... 308 11.6 Conclusion...... 309 References...... 310

Index...... 315 Abbreviations

AD Alzheimer disease ALS Amyotrophic lateral sclerosis ARA Arachidonic acid BDNF Brain-derived neurotrophic factor C1P Ceramide 1 phosphate Cer Ceramide COX Cyclooxygenase DHA Docosahexaenoic acid EPOX Epoxygenase HD Huntington disease

Ins-1,4,5-P3 Inositol-1,4,5-trisphosphate LOX Lipoxygenase LTs Leukotrienes LX Lipoxin

NPD1 Neuroprotectin D1 PD Parkinson disease PGs Prostaglandins PKC Protein kinase C

PLA2 Phospholipase A2 PLC Phospholipase C PLD Phospholipase D PlsCho Choline plasmalogen PlsEtn Ethanolamine plasmalogen PtdCho Phosphatidylcholine PtdEtn Phosphatidylethanolamine PtdH Phosphatidic acid

xxiii xxiv Abbreviations

PtdIns Phosphatidylinositol

PtdIns(4,5)P2 Phosphatidylinositol 4,5-bisphosphate PtdIns4P Phoshatidylinositol 4-phosphate PtdSer Phosphatidylserine ROS Reactive oxygen species

RvD1 Resolvin D1

RvD2 Resolvin D2

RvE1 Resolvin E1

RvE2 Resolvin E2 S1P Sphingosine 1 phosphate Sph Sphingosine TXs Thromboxanes Chapter 1 Metabolism and Roles of Eicosanoids in Brain

1.1 Introduction

Eicosanoids are signaling molecules generated through enzymic oxidation of arachidonic acid (ARA, 20:4n-6) by cyclooxygenases (COX-1 and 2) (O’Banion, 1999; Vane et al., 1998; Phillis et al., 2006), lipoxygenases (LOX) (Kuhn and O’Donnell, 2006; Kim et al., 2008), and epoxygenases (EPOX) (Phillis et al., 2006; Spector, 2009). Eicosanoids include prostaglandins (PGs), leukotriene (LTs), lipoxins (LXs), and thromboxanes (TXs), as well as hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatetraenoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) (Figs. 1.1 and 1.2). Numerous eicosanoids have been detected in the nervous system in neurons, astrocytes, cerebral vascular endothelial cells, and cerebrospinal fluid (O’Banion, 1999; Schaad et al., 1991; Simmons et al., 2004; Toda and Okamura, 1993; Vila, 2004; Werz, 2002; Wolfe and Horrocks, 1994). Eicosanoids produce a wide range of biological actions including potent effects on inflammation, vasodilation, vasoconstriction, apoptosis, and immune responses. Neural membrane phospholipids are enriched in ARA and docosahexaenoic acid (DHA, 22:6n-3), which are exclusively located at the sn-2 position of glycerol moiety. ARA is released mainly by the action of cytosolic phospholipase A2 (cPLA2) (Farooqui et al., 2000a), and DHA is hydrolyzed by plasmalogen-selective- phospholipase A2 (PlsEtn-PLA2) (Hirashima et al., 1992; Farooqui, 2010a) (Fig. 1.3). Free ARA is either reincorporated in neural membrane glycerophospholipids by reacylation reactions or oxidized by enzymic and nonenzymic mechanisms into various oxygenated metabolites (Farooqui et al., 2000a, b; Lee et al., 2004; Rapoport, 1999), which play important roles, not only in regulating signal transduction and gene transcription processes, but also in inducing and maintaining the acute inflammatory responses, oxidative stress, and neurodegeneration (Wolfe and Horrocks, 1994). In contrast, DHA is metabolized by 15-LOX-like enzyme into resolvins and neuroprotectins. These metabolites are collectively known as docosanoids. They not only antagonize the effects of eicosanoids (Fig. 1.3), but also modulate leukocyte

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 1 DOI 10.1007/978-1-4419-9940-5_1, © Springer Science+Business Media, LLC 2011 2 1 Metabolism and Roles of Eicosanoids in Brain ae O HO OH COOH COOH

HO OH OH b HO f OH COOH COOH

HO OH HO OH OH c OH COOH

g HO OH COOH d O COOH

OH C5H5

Fig. 1.1 Chemical structures of prostaglandins, leukotrienes, and lipoxins. PGE2 (a), PGF2a (b),

LTB4 (c), LTA5 (d), LXA4 (e) LXB4 (f), and epi-LXA4 (g) trafficking as well as downregulating expression of cytokines (Hong et al., 2003;

Marcheselli et al., 2003). ARA releasing and oxidizing enzymes (PLA2, COX, LOX, and EPOX) occur in several isoforms in brain tissue. Although little is known about the interplay among the isoforms of PLA2, COX, LOX, and EPOX enzymes, but it is becoming increasingly evident that interactions between cPLA2 and sPLA2 and among glutamate, cannabinoid, and dopamine receptors take place during prostaglandin synthesis in neural and non-neural cells (Shinohara et al., 1999; Phillis et al., 2006). It is likely that eicosanoid synthesis through COX, LOX, and EPOX enzymes may involve different pools of ARA that may be coupled to distinct PLA2 isoforms at different cellular and subcellular levels (Farooqui et al., 2006; Ueno et al., 2001).

The action of PLA2 on phospholipid also generates 1-alkyl-2-lyso-sn-glycero-3- phosphocholine. This metabolite is the immediate precursor for platelet activating factor (PAF), a potent inflammatory mediator (Fig. 1.3). PAF not only modulates transcription factors and gene expression, but is also involved in stimulation and modulation of PLA2, PLC, and PLD, and COX activities (Farooqui et al., 2008). Besides generating eicosanoids, COX, LOX, and EPOX-catalyzed reactions also produce reactive oxygen species (ROS). ROS include oxygen-free radicals 1.1 Introduction 3

O O O O

OH OH

CH3 CH3

5,6-EET 8,9-EET

OH OH

CH3 CH3

O O 11,12-EET 14,15-EET

OH OH O OH OH O OH OH

CH3 CH3

5,6-DHET 8,9-DHET

Fig. 1.2 Chemical structures of epoxyeicosatetraenoic acids (EETs)

(superoxide radicals, hydroxyl, and alkoxyl radicals), and peroxides (hydrogen peroxide and lipid hydroperoxide). At low levels, ROS function as signaling intermediates in the regulation of fundamental cell activities such as growth and adaptation responses. At higher concentration, ROS contribute to neural membrane damage when the balance between reducing and oxidizing (redox) forces shifts toward oxidative stress (Fig. 1.3). The other biological targets of ROS may be membrane proteins, unsaturated lipids, and DNA (Berlett and Stadtman, 1997). The reaction between ROS and proteins or unsaturated lipids in the plasma membrane leads to a chemical cross-linking of membrane proteins and lipids and a reduction in membrane unsaturation. The depletion of unsaturation in membrane lipids is associated with decreased membrane fluidity and decreased activity of membrane-bound enzymes, ion-channels, and receptors (Farooqui et al., 2008). Nonenzymic peroxidation of ARA and DHA produces 4-hydroxynonenals (4-HNE) and 4-hydroxyhexenal (4-HHE), respectively. These reactive aldehydes are important mediators of brain damage because they form covalent bonds with lipids, proteins, and nucleic acids producing disruption of important cellular functions (Esterbauer et al., 1991; Farooqui and Horrocks, 2006). In addition, free radical- mediated nonenzymic oxidation of ARA produces isoprostanes (Roberts et al., 1998), 4 1 Metabolism and Roles of Eicosanoids in Brain

2+ Ca A2 Extracellular A1 R1 PlsEtn PtdCho R2 PM Reacylation + Reacylation Intracellular cPLA2 PlsEtn-PLA- 2

+ ARA LXs Lyso-PtdCho DHA + Lyso-PlsEtn 15-LOX Cox-1 & 2 15-LOX 15-LOX 5-LOX

PAF PGE LTs EETs 2 RESOLVINS NEUROPROTECTINS -

+ NEUROINFLAMMATION RESOLUTION NEUROINFLAMMATION

+ -

NEURODEGENERATION

Fig. 1.3 Hypothetical diagram showing the interactions among ARA and DHA-derived enzymic and nonenzymic products. Agonist (A1 and A2), receptors (R1 and R2), plasma membrane (PM), phosphatidylcholine (PtdCho), ethanolamine plasmalogen (PlsEtn), lysophosphatidylcholine (Lyso-PtdCho), ethanolamine lysoplasmalogen (Lyso-PlsEtn), platelet activating factor (PAF), cal- 2+ cium (Ca ), cytosolic phospholipase A2 (cPLA2), plasmalogen-selective phospholipase A2 (PlsEtn-

PLA2), cyclooxygenase-1 and 2 (COX-1 and 2), 4-hydroxynonenal (4-HNE), and reactive oxygen species (ROS) isofurans and isoketals, whereas nonenzymic lipid mediators of DHA include neuroprostanes, neurofurans, and neuroketals (Bazan, 2009; Farooqui and Horrocks, 2007). All these mediators are reliable indices of oxidative stress in vivo.

1.2 Multiplicity of Cyclooxygenases, Lipoxygenases, and Epoxygenases in the Brain

Cyclooxygenase (COX) catalyzes the conversion of ARA into prostaglandin G2

(PGG2) followed by the hydroperoxidation to prostaglandin H2 (PGH2), which is subsequently converted to PGs (PGE2, PGI2, PGF2a), and thromboxane A2 (Fig. 1.4). PGs are involved in many processes including fever, sensitivity to pain, sleep, inflammation, and oxidative stress, and are the target of aspirin-like drugs. LOXs catalyze the biosynthesis of LTs and LXs, from ARA (Fig. 1.5), and cytochrome P450 (CYP) epoxygenases convert ARA into four EET regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET (Fig. 1.6). Agonist PGE 2

EP PtdIns-4,-- 5-P2 Receptor PtdCho

G PLC 2+ Ca cPLA2 InsP + Lyso-PtdCho 3 DAG lipase ATP AC cAMP DAG ARA COX PAF Mobilization of Ca2+ from intracellular stores PGH2 PGE2 PKA TXA2 +

PGF2 TXB 2 PGD2 PGI2 PKC 2-AG

Autocrine/paracrine signaling Intracellular signalin g Intracellular signalin g CELLULAR RESPONSE

Fig. 1.4 Generation of prostaglandins from ARA and their interactions with EP receptors. Phosphatidylcholine (PtdCho), lysophosphatidylcholine (Lyso-PtdCho), cytosolic phospholipase

A2 (cPLA2), arachidonic acid (ARA), cyclooxygenase (COX), phospholipase C (PLC), diacylglycerol (DAG), protein kinase C (PKC), Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), inositol 1,4,5-trisphosphate (InsP3), protein kinase A (PKA), adenylate cyclase (AC), cyclic-AMP (cAMP), 2-arachidonylglycerol (2-AG)

PtdCho PtdIns-4,5-- P2 Receptor

2+ cPLA2 Ca PLC Lyso-PtdCho + InsP DAG-lipase 3 2-AG ARA DAG 5-LOX

15(S)-HETE 15-HPETE 15-LOX 5-HPETE 15- 12-LOX H2O PKC 12-HPETE LXA4 LXB4 LTA4 LTB4

Glutathione

LTC4LTC4 12-HETE + Glutamate

LTE4 LTD4

CELLULAR RESPONSE

Fig. 1.5 Generation of leukotrienes from ARA. Phosphatidylcholine (PtdCho), lysophosphatidylcholine (Lyso-PtdCho), cytosolic phospholipase A2 (cPLA2), arachidonic acid (ARA), lipoxygenase (LOX), hydroxyeicosatetraenoic acid (HETE), phospholipase C (PLC), diacylglycerol (DAG), protein kinase C (PKC), Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), inositol 1,4,5- trisphosphate (InsP3), and 2-arachidonylglycerol (2-AG) 6 1 Metabolism and Roles of Eicosanoids in Brain

Glycerophospholipid Receptor

2+ cPLA2 Ca

Omega-terminalOmega- terminal ARA Midchain HETES HETESHETES

CYP2C CYP2J BETA OXIDATION AUTOCRINE FUNCTION Modulation of : EET Receptors Ion channels Signal transduction Transcription factors ELONGATION Regulatory proteins Epoxide PARACRINE FUNCTION hydrolase

DHET

Fig. 1.6 Generation of epoxyeicosatetraenoic acids from ARA. Cytosolic phospholipase A2

(cPLA2), arachidonic acid (ARA), epoxygenases (CYP2C and CYP2J), cytochrome P450 (CYP), hydroxyeicosatetraenoic acid (HETE), epoxyeicosatetraenoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs)

1.2.1 Cyclooxygenases (COXs)

COXs are heme-containing bifunctional N-glycosylated proteins that catalyze the synthesis of eicosanoids. COXs contain two catalytic centers. COX center facilitates the bisoxygenation and cyclization of ARA to form the hydroperoxy arachidonate metabolite PGG2 and the other peroxidase center that reduces PGG2 to PGH2. Detailed mechanistic studies have indicated that a carbon-centered pentadienyl radical at C-11 and a carbon-centered radical at C-8 as the two radical species may lead to endoperoxide formation during the COX center-catalyzed reaction and a tyrosyl radical derived from Tyr385 during the peroxidase center-catalyzed reaction. Both activities are functionally coupled with each other. The overall reaction is initiated by the oxidation of the heme group of the peroxidase reaction by low levels of a hydroxyperoxide (PGG2) to generate a radical intermediate that start with the

COX reaction (Jiang et al., 2004). PGH2 is a precursor to several prostaglandins,

thromboxanes (TXA2), and prostacyclins (PGI2) (Fig. 1.4). Protein turnover experiments demonstrate that COX-2 is much more susceptible to degradation than COX-1 (Mbonye et al., 2008). In murine NIH/3T3 fibroblasts, COX-2 has a half-life of approximately 2 h as a result of degradation by the cytosolic 26S proteasome under conditions in which COX-1 is very stable (t1/2 > 12 h) (Mbonye et al., 2006). The degradation of COX-2 protein occurs through two independent pathways. 1.2 Multiplicity of Cyclooxygenases, Lipoxygenases, and Epoxygenases in the Brain 7

One pathway begins by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. Post-translational N-glycosylation of Asn-594 is modulated by a 27-amino acid instability motif (27-IM). The second pathway for COX-2 protein degradation starts with substrate-dependent suicide inactivation. Suicide-inactivated protein is then catabolized. Although the biochemical steps and mechanisms have not been determined, but substrate-dependent break down is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases (Mbonye et al., 2008; Wada et al., 2009). Based on detailed investigation, it is suggested that the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled with the rate of substrate turnover (Mbonye et al., 2008). Three forms of COX enzymes (COX-1, COX-2, and COX-3) have been described in brain. COX-1 is constitutively expressed in brain, and is responsible for the physiological production of prostaglandins (Phillis et al., 2006). It is involved in several homeostatic processes and therefore called a “housekeeping” enzyme. Very low levels of COX-2 are detectable in brain, but this enzyme is induced by inflammatory- mediators such as IL-1a/b, TNF-a, IFN-g, lipopolysaccharide (LPS), epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, and onco- genes (v-src and v-ras), (Hoffmann, 2000). COX-1 and COX-2 resemble each other in structure and amino acid sequence. Both isoforms are homodimers. They transform ARA into PG and contain similar active sites. However, there are two important structural differences between COX-1 and COX-2. The active site of COX-2 is larger and more accommodating than that of COX-1 and COX-1 displays negative allosterism at low concentrations of ARA. This property may be responsible for greater eicosanoid production by COX-2 when ARA concentration is low. ARA is the preferred substrate for COX-1 and COX-2. However, these enzymes also oxidize g-linolenic acid and eicosapentaenoic acid (EPA) (Smith and Song, 2002; Serhan et al., 2004). Thus, enzymic oxidation of EPA by COX-2 produces the 3-series of prostaglandins and thromboxanes and the 5-series of leukotrienes. These eicosanoids have different biological properties than the corresponding analogs produced by the metabolism of ARA. For example, TXA3 is less active than TXA2 in aggregating platelets and constricting blood vessels (Calder and Grimble, 2002; James et al., 2000). COX-1 and COX-2 have been cloned (DeWitt and Smith, 1988; Yokoyama and Tanabe, 1989). The COX-2 gene is 8.3 kb, whereas the COX-1 gene is much larger (22 kb) (Vane et al., 1998) (Table 1.1). The COX-2 gene contains several regulatory sites, such as the cyclic AMP response element, IL-6 response element, AP-2, nuclear factor-kB (NF-kB), Sp-1, PEA-3, GATA-1, and glucocorticoid response element (Wu, 1995). In contrast, the COX-1 gene contains Sp-1, Ap-2, NF-IL-6, GATA-1, and a shear stress response element sites. The location of these sites differs considerably in COX-1 gene than COX-2 gene. COX-1 does not respond to NF-kB as intensely as COX-2. The mRNA for COX-1 is approximately 2.8 kb, while COX-2 mRNA is approximately 4.0 kb. Homology between COX-1 and COX-2 enzymes at cDNA and amino acid levels may be responsible for similar structural and kinetic properties (Smith et al., 2000; Rouzer and Marnett, 2009). Although the 8 1 Metabolism and Roles of Eicosanoids in Brain Peng et al., 2 00 8 Ochi and Goto, 2 00 Chandrasekharan et al., 2 00 Carrasco et al., 2 00 5 Minghetti, 2 00 4 ; Snipes et al., 5 Minghetti, 2 00 4 ; Snipes et al., 5 Minghetti, 2 00 4 ; Snipes et al., 5 Snipes et al., 2 00 5 Minghetti, 2 00 4 ; Snipes et al., 5 Reference COX-1 nor COX-2 COX-1 Unknown Unknown Inhibited//no effect – Resembles neither Fever and pain Fever 60 – Constitutive COX-3 Inhibited (IC50, 80 nM) Inhibited (IC50, 65 m M) No effect Inhibited Resembles COX-1 Inflammation stress Oxidative 68 Chromosome 1 Constitutive and inducible Constitutive COX-2 Inhibited (IC50, 9 m M) Inhibited (IC50, 28 nM) No effect No effect Resembles COX-2 Housekeeping 68 Chromosome 9 Constitutive COX-1 Molecular properties of neural and non-neural preparations of COX-1, COX-2, and COX-3 COX-2, Molecular properties of neural and non-neural preparations COX-1, DuP-697 (5-Bromo-2-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl)thiophene) : Modified from Phillis et al. ( 2 00 6 ) Source DuP-697 FR122047 Effect of tylenol Effect Effect of vioxx Effect Amino acid sequence Role Molecular mass (kDa) Chromosomal location Expression in brain Table 1.1 Table Property 1.2 Multiplicity of Cyclooxygenases, Lipoxygenases, and Epoxygenases in the Brain 9 conformation of substrate binding sites in the catalytic regions between COX-1 and COX-2 enzymes are similar, but not identical. COX-1 contains Val at the 434 and 523 positions, whereas COX-2 has Ile at positions 434 and 523. It is proposed that these differences in amino acid sequences result in larger and more flexible substrate and inhibitor binding sites in COX-2 than in COX-1 (Kurumbail et al., 1996). Amino acid sequences of COX-1 and COX-2 also differ from each other at the N and C termini. COX-2 lacks a 17 amino acid sequence at the N-terminus but has an extra 18 amino acid sequence at the C-terminus. In spite of these differences, these proteins catalyze the same reactions. COX-1 deletion attenuates, whereas COX-2 deletion increases the neuroinflammatory response, blood–brain barrier permeability, and leukocyte recruitment during lipopolysaccharide (LPS)-mediated innate immune activation (Aid et al., 2010). Immunocytochemical studies indicate that in rat and ovine brain, COX-1 and COX-2 immunoreactivities are present in discrete neuronal populations in different areas of cerebral cortex, midbrain, and hippocampus. COX-1 immunoreactivity is enriched in midbrain, pons, and medulla (Breder et al., 1995), whereas COX-2 immunoreactivity prevails in neurons and glial cells of hippocampus, hypothalamus, and amygdala (Yamagata et al., 1993; O’Banion and Olschowka, 1999). In neurons, astrocytes, and microglial cells, COX-2 immunoreactivity is also localized to the perinuclear regions (Tomimoto et al., 2000). The expression of COX-2 is markedly increased in microglial cells after intraperitoneal administration of lipopolysaccharide (LPS), whereas neuronal COX-2 remains unchanged (Elmquist et al., 1997). In microglial cells, COX-2 expression and ability to release PGE2,

TXA2 and TXB2 upon stimulation by LPS is several times higher than in astrocytes, but lower than in peripheral macrophages (Giulian et al., 1996). COX-2 induction in microglia by proinflammatory stimuli is apparently similar to peripheral macrophages and plays important roles in inflammatory and immune responses. COX-3 is a splice variant of COX-1. COX-3 is a glycoprotein product of the COX-1 gene with retention of a highly structured, G+C-rich intron 1 in its mRNA. COX-3 mRNA encodes a 127 amino acid protein with no similarity with known COX sequences (Kis et al., 2006). In canine brain, COX-1 intron 1 consists of a 93-bp sequence, and translation of this novel approximately 5.2-kb COX-3 RNA message introduces a hydrophobic 31 amino acid sequence (RECDPGARWGIF LASWWSLECQAQPLILSSA) into the signal peptide of COX-1, generating a catalytically active 65-kDa membrane-bound COX. In human brain, COX-1 intron 1 consists of a 94-bp core sequence, and because it is out of frame, alternative RNA splicing events, probably mediated by complex intron 1RNA secondary structures generate a functional COX-3 RNA and protein (Cui et al., 2004). Comparison among amino acid sequences of COX-1, COX-2, and COX-3 indicates that COX-1 (602 aa residues) and COX2 (604 aa residues) are of the same length, whereas COX-3, a putative cyclooxygenase-3 from house mouse has considerably shorter sequence (127 aa residues). COX-3 is deletion of the COX-2 but not of the COX-1 gene. So many gaps in COX-3 sequence make it difficult to have proper comparison. Thus, COX-3 has several different features in its amino acid sequence compared with COX-1 and COX-2: including (1) length, (2) too many gaps, and (3) branch distance (Fig. 1.7). To get a clear picture, it would be interesting to compare these sequences Fig. 1.7 Alignment was produced by ClustalW (2.0.12), a general purpose multiple sequence alignment program (Thompson et al. 1994), for amino acid sequences of three cyclooxygenases. Amino acid sequences were identified by NCBI DATA bank accession number. (a) Cyclooxygenase 1 (Rattus norvegicus), COX1 (accession no. (#) AAA03465) was aligned with prostaglandin G/H 1.2 Multiplicity of Cyclooxygenases, Lipoxygenases, and Epoxygenases in the Brain 11 with cyclooxygenases from other species, including humans. COX-3 mRNA has also been cloned and sequenced from rat cerebral endothelial cells. Sequence analysis indicate that the 98-bp intron-1 of COX-1 gene remains unprocessed in COX-3, inducing a frameshift mutation and a 127-amino acid open reading frame with no sequence similarity with COX-2 (Snipes et al., 2005). This retention of intron 1 in COX-1 inserts 30–34 amino acids depending on the mammalian species. This retention may change protein folding and active site conformation. Pharmacologically, COX-3 is selectively inhibited by acetaminophen and some nonsteroidal anti- inflammatory drugs (Chandrasekharan et al., 2002; Davies et al., 2004). It is likely that blockage of COX-3 may represent a primary central mechanism by which above drugs decrease pain and fever (Chandrasekharan et al., 2002; Davies et al., 2004). Highest expression of COX-3 mRNA occurs in choroid plexus and spinal cord followed by pituitary gland, hypothalamus, hippocampus, medulla, cerebellum, and cortex. In cerebrovascular system, the highest expression of COX-3 mRNA occurs in brain microvessels followed by major brain arteries. The expression pattern of COX-3 mRNA in the rat CNS primarily relates to the vascular density of a given region (Kis et al., 2003, 2004). Rat COX-3 is a cytosolic glycoprotein. Amino acid analysis also shows that COX-3 protein has a very basic character with a pI value of 12.40. In addition to the abundance of basic amino acids, the COX-3 protein is also very rich in proline (11.81%). The occurrence of high levels of proline argues against the formation of extensive a-helical domains and for the possible formation of b-turns as well as the formation of antiparallel b-sheets (Snipes et al., 2005). Collective evidence suggests that while COX-2 is a major player in generating prostaglandins under pathological conditions, COX-3 may also play ancillary roles in membrane-based COX signaling.

1.2.2 Lipoxygenases (LOXs)

LOXs convert ARA into hydroxyperoxyeicosatetraenoic acids, followed by their conversion into hydroxyeicosatetraenoic acids, leukotrienes, lipoxins, and hepoxilins (Phillis et al., 2006). LOXs are categorized with respect to their positional specificity of ARA dioxygenation into 5-LOX, 8-LOX, 12-LOX, and 15-LOX (Fig. 1.5). The substrate specificity of LOXs depends on the isoform. The human 5-LOX oxidizes free C20-fatty acids (ARA and EPA) but hardly oxygenates C18-derivatives or esterified fatty acids. In contrast, most 12/15-LOXs oxidize a variety of polyenic

Fig. 1.7 (continued) synthase 2 (Rattus norvegicus) COX2 (accession no. (#) NP_058928); and putative cyclooxygenase 3 (Mus musculus) COX3 (accession no. (#) AAT09993). Amino acid residues in red with an asterisk (*) correspond to fully conserved region. Amino acid residues in green with a symbol (:) correspond to amino acid residues in similar groups. Amino acid residues in yel- low with a symbol (∙) correspond to semiconserved substitution (similar shapes). (b) A phylogram was constructed by this program. The branch length is proportional to the number of evolutionary events that took place along this branch, representing the amount of evolutionary divergence 12 1 Metabolism and Roles of Eicosanoids in Brain fatty acids regardless of their chain length and these enzymes also oxygenate ester lipids even if they are incorporated in biomembranes and lipoproteins (Kuhn and O’Donnell, 2006). 5-LOX and 12-LOX are most common LOXs found in the brain with their mRNA present in rat cortical neurons, astrocytes, and oligodendrocytes (Bendani et al., 1995). They predominantly generate 5- and12-hydroxyeicosatetraenoic acid (5-HETE and 12-HETE) and some 15-HETE has also been detected. Since LOX-catalyzed pathways can provide both constrictor and dilator, influencing the cerebral vasculature, they are capable of affecting the cerebral circulation in a comparable manner to COX-1 and 2 pathways (Phillis et al., 2006). Among LOXs, 5-LOX and 12-LOX are generally considered procarcinogenic, while 15-LOX-2 suppresses carcinogenesis. The 5-LOX pathway may also contribute to the mechanisms of neural plasticity ranging from neurogenesis and neural differentiation (Wada et al. 2007) to regulation of synaptic plasticity (Chabot et al., 1998; Ménard et al., 2005). For example, metabolites derived from 5-LOX activity have been implicated in the expression of long-term depression in hippocampal slices (Chabot et al., 1998). In addition, 5-LOX is the key enzyme in the biosynthesis of anti- inflammatory lipoxins. LOX enzymes induce structural and metabolic changes in cells during a wide variety of physiological and pathological processes, such as differentiation, angiogenesis, carcinogenesis, inflammation, and atherogenesis. LOX are nonheme, iron-containing dioxygenases that insert molecular oxygen into ARA (Rådmark and Samuelsson, 2009). Their nomenclature is based on the position of oxygen insertion (carbon 5, 8, 12, or 15 of the aliphatic chain) and stereo- configuration (R versus S) of the resulting product hydroperoxyeicosatetraenoic acid (HPETE) (Phillis et al., 2006). LOXs have a molecular mass of 75–78 kDa. They contain a single polypeptide chain, which is folded into two domains (Brash, 2001). Like non-neural tissues, three forms of LOX are present in brain. They include 5-LOX, 12-LOX, and 15-LOX. The 5-LOX gene is highly conserved across species. It consists of 14 exons and 13 introns, and contains a promoter region that encompasses consensus regions for transcription regulators of the Egr, Sp, NF-kB, GATA, myb, and AP families. 12-LOX is the predominant LOX isoform in the CNS and its mRNA is present in rat cortical neurons, oligodendrocytes, and astrocytes (Bendani et al., 1995). 12-LOX catalyzes the stereospecific incorporation of molecular oxygen into the C-12 position of ARA to generate 12-HPETE, which is reduced by cellular glutathione peroxidase to 12-HETE (Li et al., 1997). 12-LOX has been cloned and characterized from rat brain (Watanabe et al., 1993). Rat brain 12-LOX contains six conserved histidines and displays marked resemblance with porcine leukocyte 12-LOX (71%) and to human 15-lipoxygenase (75%), but has less identity with human platelet 12-LOX (59%) or rat leukocyte 5-LOX (41%). 5-LOX and 15-LOX have also been sequenced from several non-neural sources (Dixon et al., 1988; Matsumoto et al., 1988). They have a molecular mass of 78 kDa and share considerable homology with 5-LOX and 15-LOX from soybean. LOXs also catalyze a dehydration reaction, generating an unstable epoxide intermediate, leukotriene A4 (LTA 4). LTA4 is metabolized to LTB4 by LTA4 hydrolase or to LTC4 by conjugation of glutathione at the sixth carbon by the action of LTC4 synthase. LTA4 is the precursor for the family of cysteinyl leukotrienes (cysLTs: LTC4, LTD4, and LTE4) (Wolfe and Horrocks, 1994) (Fig. 1.5). 1.2 Multiplicity of Cyclooxygenases, Lipoxygenases, and Epoxygenases in the Brain 13

In the brain, 5-LOX is localized in the cytoplasm. Receptor-mediated stimulation of 5-LOX results in translocation of this enzyme from cytosol to the plasma membrane and nuclear envelope, where 5-LOX transforms ARA into 5(S)-HpETE with the concerted interaction of 5-LOX- activating protein (FLAP), a 18-kDa resident integral protein that functions as a transfer protein facilitating the binding of ARA to 5-LOX. FLAP is essential for leukotriene C4 (LTC4) synthesis (Manev et al.,

2000a). Although FLAP shows homology with LTC4 synthase and other microsomal glutathione transferases, but it has no enzymic activity itself. It is suggested that FLAP acts as a scaffolding protein for the assembly of other enzymes involved in the leukotriene synthetic pathway at the nuclear envelope of leukocytes (Sampson, 2009). The regulation of LTs production occurs at various levels, including expression of 5-LOX, translocation of 5-LOX to the perinuclear region, and phosphorylation to either enhance or inhibit the activity of 5-LOX. In addition, the synthesis of

LTs is also supported by the translocation of cPLA2 to the perinuclear region. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s-dependent EPOX (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Gamma-glutamyl transpeptidase converts LTC4 into LTD4 and a membrane-bound dipeptidase that converts LTD4 into LTE4 before w-oxidation (Murphy and Gijon, 2007). These metabolic conversions of the primary leukotrienes are critical for terminating their biological activity. By blocking the formation of LTs, FLAP inhibitors may act as broad-spectrum leukotriene-modifier drugs that may have a wide range of therapeutic applications. 5-LOX also contains a Src homology 3 (SH3) binding motif, which facilitates the interaction of 5-LOX protein with growth factor receptor-bound protein 2 (Grb2) (Lepley et al., 1996).

1.2.3 Cytochrome P450 Epoxygenases (EPOXs)

The cytochrome P450 (CYP) epoxygenase enzymes CYP2J and CYP2C catalyze the epoxidation of ARA to EETs, which are rapidly hydrolyzed to DHETs by soluble epoxide hydrolase (sEH) (Zeldin, 2001) (Fig. 1.6). CYP epoxygenase-derived EETs possess potent vasodilatory, anti-inflammatory, and angiogenic effects. In addition, EETs not only retard endothelial cell activation and leukocyte adhesion via attenuation of nuclear factor-kappaB activation, but inhibit hemostasis, protect against ischemia-reperfusion injury, and promote endothelial cell survival via modulation of multiple cell signaling pathways (Zeldin, 2001). In the brain, EETs are produced by astrocytes and the vascular endothelium and are involved in the control of cerebral blood flow (CBF). It is also reported that epoxygenases and sEH are present in perivascular vasodilator nerve fibers innervating the cerebral surface vasculature. Ischemic preconditioning is accompanied by the expression of cytochrome P450 2C11 epoxygenase (CYP2C11) in the brain, and that pharmacological inhibition and genetic deletion of sEH increases EETs, leading to protection against stroke-induced 14 1 Metabolism and Roles of Eicosanoids in Brain brain damage (IIiff et al., 2007). Double-labeling experiments indicate that CYP2C11- and sEH-IR are predominantly colocalized with neuronal nitric oxide synthase-IR within perivascular nerve fibres. Significant colocalization for CYP2C11 and sEH also occurs with the parasympathetic markers vasoactive intestinal peptide and choline actetyltransferase, in addition to the sensory fibre markers calcitonin gene- related peptide and substance P. No colocalization is observed for either CYP2C11 or sEH with the sympathetic nerve markers dopamine b-hydroxylase or neuropeptide Y. The presence of enzymes involved in production and inactivation of EETs within extrinsic parasympathetic and sensory vasodilator fibres suggests a novel role for EETs in the neurogenic control of cerebral arteries (IIiff et al., 2007). Based on various studies, it is proposed that EETs contribute not only to the regulation of the cerebral blood flow and protection from ischemic injury, but also inhibit inflammation, release peptide hormones, and therefore are crucial for cerebrovascular homeostasis. The ability of various types of neural and non-neural cells to produce and respond to EETs-mediated effects suggests that EETs signaling may be an important integrator of cell–cell communication in the CNS, coordinating cellular responses across different cell types. Under pathophysiological conditions, such as cerebral ischemia, EETs protect neurons, astroglia, and vascular endothelium, thus preserving the integrity of cellular networks unique to and essential for proper CNS function (Iliff et al., 2010). In addition, EETs are also involved in the secretion and action of insulin and the metabolism of carbohydrates and lipids. The RhoA/Rho kinase (ROCK) signaling pathway is downstream to ARA metabolism and is reported to mediate metabolic brain dysfunctions in insulin resistance. It is proposed that pharmacological manipulation of EETs may be a useful therapeutic approach for hypertension, diabetes mellitus, and the metabolic syndrome (Mustafa et al., 2009). EETs can act like long-chain fatty acids and bind to fatty-acid-binding proteins and nuclear peroxisome-proliferator-activated receptors (PPARg and PPARa). In fact, all four EETs and their metabolite DHET can stimulate PPAR/RXR heterodimer binding to a peroxisome proliferator response element (Cowart et al., 2002; Fang et al., 2006). Activation of peroxisome proliferator-activated receptor alpha (PPARa) by fatty acids and fibrates results in the induction of sEH, the enzyme that degrades EET into the dihydroxyeicosatrienoic acids (DHETs). In contrast, the CYP2C epoxygenases, which are responsible for EET formation, are repressed after fibrate treatment. It is proposed that P450 eicosanoids interact with PPARa and modulate PPARa-mediated gene expression (Ng et al., 2007). In endothelial cell cultures, the overexpression of EPOX not only upregulates endothelial nitric oxide synthase (eNOS), but also activates enzymes of PtdIns3-kinase/Akt pathways. Not only eNOS inhibitors, but also PtdIns3-kinase/Akt signaling pathway inhibitors, can prevent this upregulation by EPOX, suggesting that both eNOS and PtdIns3-kinase/Akt pathways may mediate the angiogenic effects of EETs. EPOX gene transfection activates the MARK pathway in endothelial cells. Collectively, these studies suggest that EPOX-derived EETs modulate angiogenesis via a nitric oxide-dependent mechanism as well as via activation of PtdIns 3 kinase and MARK pathways (Wang et al., 2005). 1.3 Eicosanoids and Their Receptors in Brain 15

1.3 Eicosanoids and Their Receptors in Brain

Eicosanoids are released outside of the cells immediately after their synthesis. They act through eicosanoids receptors that are located on plasma and nuclear membranes. These receptors modulate signal transduction pathways and gene transcription. Among eicosanoids, PGs are potent autocrine and paracrine lipid mediators that contribute appreciably to physiologic and pathophysiologic responses in the CNS and

PNS (Table 1.2). Among 12 PGs, the most potent are PGD2, PGE2, and PGF2. PGE2 mediate their signaling through four distinct G protein-coupled receptors, EP1, EP2,

EP3, and EP4, (Sugimoto and Narumiya, 2007) which are encoded by different genes and differ in their responses to various agonists and antagonists and differentially expressed on neuronal and glial cells throughout the central nervous system. Activation 2+ + 2+ of EP1 receptors disrupts Ca homeostasis by impairing Na --Ca exchange, a key mechanism by which neurons cope with excess Ca2+ accumulation after an excito- 2+ toxic insult. Thus, EP1 receptors contribute to neurotoxicity by augmenting the Ca dysregulation underlying excitotoxic neuronal death (Farooqui et al., 2008). EP2 activation is involved in microglial-mediated paracrine neurotoxicity as well as suppression of microglia internalization of aggregated neurotoxic peptides (Cimino et al.,

2008). The activation of EP2 receptor leads to BDNF secretion through stimulation of cyclic AMP-dependent signaling involving cAMP-dependent protein kinase (PKA). The catalytic subunit of PKA stimulates gene transcription through the phosphorylation of cAMP-response element-binding (CREB) protein.This signaling may contribute to neurotoxicity or neuroprotection in microglial cells and astrocytes. EP3 receptor signaling not only involves the inhibition of adenylyl cyclase via Gi activation, but is also associated with Ca2+-mobilization through Gbg from Gi. Along with Gi activation, the EP3 receptor can stimulate cAMP formation via G(s) activation. At least three isoforms of EP3 are generated through alternative RNA splicing from a single gene and differ only in the efficiency of G protein activation and in the specificity of coupling to G proteins. Among EP receptors, EP4 utilizes phosphatidylinositol

3-kinase (PtdIns3K) as well as PKA. In addition, EP4 receptor activates the extracellular signal-regulated kinases (ERKs) 1 and 2 by way of PtdIns3K leading to the induction of early growth response factor-1 (EGR-1), a transcription factor tradition- ally involved in wound healing and inflammatory processes (Cimino et al., 2008).

Studies on knockout mice and selective drugs indicate that PGE2 mediates fever through EP3, while both EP1 and EP3 are involved in ACTH release. Some cells and tissues express all four receptors, whereas others express some or no receptors. It is becoming increasingly evident that EPs play different roles in brain tissue, such as controlling impulsive behaviors under psychological stress, but the exact involvement and contribution of each in inflammation is not yet completely clear, and might differ between species, organs, and pathologies. Collective evidence suggests that PGs produced by various cell types in brain may integrate multiple stress stimuli through PGE receptor subtypes for adaptive responses (Furuyashiki and Narumiya, 2009). In addition, brain tissue also contains PGF receptor (FP), PGI receptor (IP), and TXA receptor (TP), which may contribute to neurotransmitter release, sleep, and vasodilation and vasoconstriction of cerebral vessels in the brain. 16 1 Metabolism and Roles of Eicosanoids in Brain al., ; Vane et Vane 2 00 0 ; al., ; Smith et Smith 1 99 8 ; 1 99 8 Powell and Rokach, 2 00 5 Powell Chandrasekharan et al., 2 00 Minghetti, 2 00 4 ; Smith et al., 0 ; Minghetti and Levi, Minghetti, 2 00 4 ; Minghetti and Levi, Minghetti, 2 00 4 ; Simmons et al., Chiang et al., 2 00 5 et al., 2 00 5 2 00 9 ; Wang Spector, et al., 2 00 7 Nigam Pfister et al., 2 00 3 Funk, 1 99 6 ; Maccarrone et al., 2 00 Reference receptors, receptors 4 4 , receptors 2 , and EP , and EP 3 3 , EP , EP 2 2 and CysLT 1 , EP , EP TXA receptor 1 1 EP EP g PPAR CysLT ALX, LXA receptors a receptor PPAR Unknown Unknown Receptor neurotransmitter release, and synaptic plasticity apoptosis, and gene expression trafficking, apoptosis, gene and T-cell expression flow, ion transport, vasorelaxation as well ion transport, flow, effects anti-inflammatory and profibrinolytic nociception Fever and pain Fever Homeostasis, vasodilation, vasoconstriction, vasoconstriction, Homeostasis, vasodilation, inflammation, vasoconstriction, Vasodilation, Cell proliferation inflammation, monocyte Vasoconstriction, Gene expression, angiogenesis, cerebral blood Gene expression, Ca2+ release, neuro-modulation Vasodilation Resolution, proliferation, differentiation, Resolution, proliferation, differentiation, Role 2 , TxA 2 4 , PGI , LTE Roles of eicosanoids in brain tissue 2 a 2 4 2 , PGJ , PGF , PGI , LTD 2 2 2 2 4 15-epi-LXA4 PGA LTC PGE PGD EETs Hepoxilins Trioxilins LXA4, LXB4, PGE Table 1.2 Table Eicosanoids 1.3 Eicosanoids and Their Receptors in Brain 17

Dehydration of the cyclopentane ring of PGE2, PGE1, and PGD2 transforms them into cyclopentenone prostaglandins PGA2, PGA1, and PGJ2. PGJ2 is further metabolized into d12-PGJ2 and 15-deoxy-d12,14-PGJ2 (15d-PGJ2). Many of these metabolites possess potent anti-inflammatory, antineoplastic, and antiviral activities (Straus and Glass, 2001). The actions of above cyclopentenone prostaglandins are not mediated through any G protein receptors, but rather caused the bioactivity of these compounds, which results from their interaction with other cellular target proteins.

15-d-PGJ2 is a reactive cyclopentenone eicosanoid formed from the dehydration of cyclooxygenase (COX)-derived PGD2. This compound possesses an a,b-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via Michael addition. Due to its reactivity, 15-d-PGJ2 has been proposed to modulate inflammatory and apoptotic processes (Hardy et al., 2010). Furthermore, 15-deoxy-d12,14-PGJ2 is a high affinity ligand for the nuclear receptor PPARg and modulates gene transcription by binding to this receptor. Unlike other PGs, which elicit their neurochemical effects through the interactions with specific G-protein-coupled receptors (GPCRs), 15-d-PGJ2 exerts its biochemical effects through interaction with key intracellular targets. As stated above, some of its activities may be mediated through the nuclear receptor

PPAR-g (Forman et al., 1995). Activation of PPAR-g by 15-d-PGJ2 is caused by covalent binding of 15-d-PGJ2 to a critical cysteine residue in the PPARg ligand binding pocket (Shiraki et al., 2005). In addition, 15-d-PGJ2 also block the inflammatory response in a number of cell types, including macrophages, by retarding NF-kB-mediated gene expression via covalent modification of a critical cysteine residue in IkB kinase and the DNA-binding domains of NF-kB subunits (Pérez-Sala et al., 2002). 15-d-PGJ2 is also associated with resolution of inflammation by enhancing apoptosis in activated macrophages through a p38 mitogen-activated protein kinase (MAPK)-dependent increase in ROS production (Hortelano et al.,

2000). Furthermore, 15-d-PGJ2 enhances the antioxidant response pathway via covalent modification of reactive cysteine residues in Keap 1, leading to Nrf2 activation and induction of antioxidant proteins, including HO-1, peroxiredoxin 1, g-GCL, and heat shock protein 70 (Itoh et al., 2004). Finally, 15-d-PGJ2 has been reported to inhibit proliferation of vascular smooth muscle cells by inducing cell cycle arrest (Miwa et al., 2000). Other activities of the cyclopentenone prostaglandins are facilitated by the reactive a,b-unsaturated carbonyl group located in the cyclopentenone ring. NF-kB and its activating kinase (IkB kinase inhibition and blockade of NF-kB nuclear binding) are major targets for the anti-inflammatory activity of 15d-PGJ2, which blocks NF-kB-induced transcriptional activation by PPARg-dependent and independent molecular mechanisms (Fig. 1.8).

Other cyclopentenone prostaglandins, such as d7-PGA1 and d12-PGJ2, show potent antitumor activity. These compounds cause cell cycle arrest or apoptosis of tumor cells depending on the cell type and treatment conditions (Straus and Glass, 2001). In addition cyclopentenone prostaglandins form covalent adducts with thiol residues within specific signaling proteins, resulting in modulation of redox-sensitive cell signaling pathways. Biotinylated 15-deoxy-Delta12,14-PGJ2 was more efficient than biotinylated PGA1 in forming adducts with components of the transcription factors 18 1 Metabolism and Roles of Eicosanoids in Brain

Growth and differentiation

Ligand for PPARy Redox alterations

Cytoskeletal dysfunction 15d-PGJ2 Antioxidant response

Antiinflammatory Apoptosis

Turnover of proteins

Fig. 1.8 Roles of 15d-PGJ2 in neural and non-neural tissues

NF-kB and activator protein-1 (AP-1). The presence of GSH differentially modulates the formation of protein-cyclopentenone prostaglandins adducts. Another signaling pathway, which is potently activated by 15d-PGJ2 is the Keap1-Nrf2-ARE pathway. As stated above, in cytoplasm, phosphorylation of Kelch-like ECH-associated protein 1 (Keap1) results in the dissociation and translocation of Nrf2 into the nucleus, where it binds to an antioxidant response element (ARE). Through transcriptional induction of ARE-bearing genes that encode antioxidant-detoxifying proteins, Nrf2 activates cellular rescue pathways against oxidative injury, inflammation/immunity, apoptosis, and carcinogenesis. ARE-driven genes include direct antioxidants (glutathione peroxidases), thiol metabolism-associated detoxifying enzymes (glutathione S-transferase), stress-response genes (hemeoxygenase), and others (proteasome subunit beta type-5). Collective evidence suggests that 15d-PGJ2 may protect cells from endogenous and exogenous stresses as well as anti-inflammatory effects (Gayarre et al., 2007; Kansanen et al., 2009).

As stated above, LTs consist of dihydroxy leukotriene LTB4 and the cysteinyl leukotrienes LTC4, LTD4, and LTE4. Neurochemical effects of LTs are transduced through 7-transmembrane G-protein-coupled receptors. The two arms of the LT pathway interact with distinct receptors called as BLT receptors (activated by LTB4) and CysLT receptors (activated by the cysteinyl-LTs), respectively (Back, 2008).

The BLT receptors are named as BLT1 and BLT2 for the high and low affinity receptor subtypes, respectively. These receptors activated by the cysteinyl-LTs are characterized by their sensitivity to available antagonists, and are referred as CysLT1 and

CysLT2 (Back, 2008). 1.3 Eicosanoids and Their Receptors in Brain 19

Lipoxins

Inhibition of Inhibition of chemokine & nociception cytokine gene expression

Reduction Inhibition of PMN In edema infiltration Inhibition of inflammation

Fig. 1.9 Roles of lipoxins in neural and non-neural tissues

Lipoxins (LXs), a group of trihydroxytetraene eicosanoids, are generated by the action of lipoxygenases on hydroperoxyeicosatetraenoic acid (HPETE) and hydroxyeicosatetraenoic acid (HETE). LXs include 5S,6R,15S-trihydroxy- (7E,9E,11Z,13E)-tetraenoic acid (LXA4), 5S,14R,15S-trihydroxy-6E,8Z,10E,12E- eicosatetraenoic acid (LXB4), 15 epi-LXA4, and 15 epiLXB4. They selectively regulate actions on neutrophils and monocytes. LXs also inhibit edema, pain, and inflammation (Fig. 1.9). They are generated through cell–cell interactions by the process of transcellular biosynthesis (Marcus, 1986). LXs (LXA4) also modulate the expression of many PDGF-induced genes, including transforming growth factor-b1, fibronectin, thrombospondin, matrix metalloproteinase 1, and several collagens. These genes are closely associated with matrix accumulation and profibrotic change (Rodgers et al., 2005). Mammalian non-neural cells have three major routes of transcellular biosynthesis of LXs (Romano, 2010). The first pathway of LX biosynthesis involves the interaction of platelets with PMN within the vascular lumen. In this pathway, LX biosynthesis starts with the release of the epoxide intermediate LTA4 (formed by 5-LOX in activated leukocytes), which is then converted by the platelet

12-LOX to LXA4 and LXB4 (Serhan and Sheppard, 1990; Serhan and Romano,

1995). The second pathway of transcellular LXA4 biosynthesis involves the sequential interaction of a 15-LOX with a 5-LOX. This LXA4 formation route takes place mainly in tissues in which endothelial and epithelial cells expressing 15-LOX can interact with 5-LOX-containing leukocytes (Chiang et al., 2005). The third pathway of LX biosynthesis is initiated by aspirin, which acetylates COX-2 and switches its catalytic activity from COX to 15-LOX. Following this change, PG biosynthesis is inhibited and COX-2 transforms arachidonic acid to 15(R)-HETE. 15(R)-HETE is subsequently transformed by activated leukocytes possessing 5-LOX to a new series of carbon-15 epimers of LXA4 that carry their 15 alcohol in the R configuration

(15-epi-LXA4) (Clària and Serhan, 1995; Serhan, 2005; Chiang et al., 2005). 20 1 Metabolism and Roles of Eicosanoids in Brain

Biosynthesis of LX in the tissue not only depends on cells and enzymes present therein, but also on other factors such as cytokines (Serhan, 2005). For example, interleukin 4 (IL-4) and IL-13, putative negative regulators of inflammatory and immune responses, promote transcellular LX generation through enhanced expression of 15-LOX in blood monocytes and epithelial cells (Munger et al., 1999). IL-3 upregulates 5-LOX transcript (Murakami et al., 1995) while IL-1b, IL-6, and TNF-a induce COX-2, thus potentially contributing to the formation of ATLs in vivo (Parente and Perretti, 2003). The initial phase of the acute inflammatory response is accompanied by the synthesis of proinflammatory mediators followed by a second phase in which lipoxins with proresolution activities may be generated. LXs facilitate resolution not only by modulating key steps in leukocyte trafficking and preventing neutrophil-mediated acute tissue injury, but also by stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages (Kantarci et al., 2003; Yacoubian and Serhan, 2007). They also reduce vascular permeability. Collective evidence suggests that a key event in resolution is the temporal “switch” in the lipid mediator class from proinflammatory

(PGE2 and PGD2) to anti-inflammatory (lipoxins) eicosanoids. Drugs that disrupt this switch may delay the onset of resolution (Yacoubian and Serhan, 2007). The occurrence of aspirin-triggered LXs (ALX) has emerged as potential antifibrotic mediator that may influence profibrotic cytokines and matrix-associated gene expression in response to growth factors (Maderna and Godson, 2009). In neural and non-neural tissues, LXs and aspirin-triggered LX induce their actions at least through two major mechanisms including (a) the activation of high affinity ALXR and low affinity LXA, and (b) interaction of subclasses of cysteinyl peptide-LTs receptor. Direct activation of the lipoxin receptor mediates anti-inflammatory and proresolution activities whereas interactions with CysLT and growth-factor receptors retard angiogenesis, and mesangial cell proliferation and fibrosis. In addition,

LXA4 also interact with the nuclear receptor aryl hydrocarbon receptor, which triggers expression of suppressor of cytokine signaling 2 in LX-stimulated dendritic cells (Serhan and Levy, 2003; Serhan, 2005; Chiang et al., 2005). Collective evidence suggests that LXs and aspirin-triggered LXs (ATLs) are “braking signals” molecules in inflammation, limiting the entrance of leukocytes to the site of inflammation through inhibition of neutrophil and eosinophil trafficking. In vivo studies with stable LX and ATL analogs have established that these eicosanoids also possess potent immunoregulatory properties. A LXA4 receptor has been cloned (Romano et al., 2007). It belongs to the family of chemotactic receptors and clusters with formyl peptide receptors on chromosome 19. Therefore, it is denominated formyl peptide receptor-like 1 (FPRL1). This receptor binds with high affinity and stereoselectivity LXA4 and ATL. It also recognizes a variety of peptides, synthetic, endogenously generated, or disease associated, but with lower affinity compared to

LXA4. For this reason, this receptor has been renamed ALX (Romano et al., 2007).

In general, TXA2 is a potent vasoconstrictor and produces vasospasm, whereas

PGI2 has opposing effects (Phillis et al., 2006). PGs, LTs, and TXs are involved in many processes including fever, sensitivity to pain, sleep, inflammation, oxidative stress, and neurodegeneration (Table 1.1). TXs act through TXs receptors (TPs), which are widely distributed in the brain tissues. Like other eicosanoid receptors 1.3 Eicosanoids and Their Receptors in Brain 21

TPs are coupled with the seven-transmembrane G-protein-coupled receptor (GPCR). A single gene on chromosome 19p13.3 may be responsible for the expression of two separate TP isoforms: TPa receptor, which is broadly expressed in numerous tissues and a splice variant known as TPb, which may have a more limited tissue distribution (Huang et al., 2004). TPa and TPb differ from each other in their C-terminals. In addition, TPa and TPb are coupled with PLC and RhoGEF domain of Rho/Rac/Cdc4 like GTPase through different G proteins. They undergo hetero-dimerization, resulting in changes in intracellular traffic and receptor protein conformations (Nakahata, 2008). This signaling modulates a broad range of cellular responses including phosphoinositide metabolism, calcium redistribution, cytoskeletal arrangement, integrin activation, kinase activation, and the subsequent nuclear signaling events involved in DNA synthesis, cell proliferation, differentiation, cell survival, and cell death (Huang et al., 2004). Generation of EETs inhibits vascular smooth muscle migration, decreases inflammation, inhibits platelet aggregation, and decreases adhesion molecule expression, therefore representing an endogenous protective mechanism against atherosclerosis. The other effects of EETs include inhibition of endothelial activation and leukocyte adhesion through the attenuation of nuclear factor-kB activation, inhibition of hemostasis, protection against myocardial ischemia-reperfusion injury, facilitation of endothelial cell survival via modulation of multiple cell signaling pathways, and angiogenesis (Deng et al., 2010). Among EETs, 5,6- and 8,9-EET are powerful and selective angiogenic lipids that provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) into less active dihydroxyeicosatrienoic acids (DHET). EETs are important for maintaining the homeostasis and in responding to stress. It is not clear whether these effects are mediated through the action of EETs on a universal receptor or through a mechanism involving second messengers (Kaspera and Totah, 2009). Recent studies indicate that EETs/DHETs represent novel autoregulatory agents that directly modulate the actions of COX- derived eicosanoids following ARA mobilization (Behm et al., 2009). The vasodilatory effects of EET are largely through their ability to activate endothelial NO synthase (eNOS) and NO release (Hercule et al., 2009). The occurrence of 2-(11,12-epoxyeicosatrienoyl)glycerol (2–11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2–14,15-EG) (Fig. 1.10) in the kidney and spleen and brain has also been reported (Chen et al., 2008). These metabolites (2-11,12-EG and 2-14,15-EG) interact and activate cannabinoid (CB) receptor subtypes, CB1 and CB2 with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals (Chen et al., 2008), suggesting that 2-11,12-EG and 2-14,15-EG may act as a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This observation also indicates that ARA-derived 2-11,12-EG and 2-14,15-EG can activate G-protein-coupled CB receptors, indicating that there may be a relationship between the cytochrome P450 enzyme system and the endocannabinoid receptor system (Chen et al., 2008). In addition, in brain the oxidation of anandamide by cytochrome P450 (P450) enzymes generates 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), a potent, stable, and selective CB2 22 1 Metabolism and Roles of Eicosanoids in Brain

O O OH OH O O OH OH

O O 2-11, 12-EG 2-14, 15-EG

HO H HO H O N OH OH N

O O

5,6-EET-EA 5,6-DHET-EA

OH OH O O OH OH

O O 2-11,12-EG Ether 2-14,15-EG Ether

Fig. 1.10 Chemical structures of EET derivatives agonist (Fig. 1.10), which modulates signaling pathways within the endocannabinoid system (Snider et al., 2009). These results once again support the view that there may be a functional link between the cytochrome P450 enzyme system and the endocannabinoid receptor system. The synthesis of eicosanoids under physiological conditions is involved in signal transduction processes related to normal cell function. In contrast, the production of eicosanoids under pathological conditions is associated with neuroinflammation, oxidative stress, modulation of cerebrovascular blood flow, and neurodegeneration. Their active production by circulating cells such as platelets and leukocytes induces alterations in the microcirculation and ultimately to CNS dysfunction (Phillis et al., 2006).

1.4 Interplay Among COX, LOX, and EPOX-derived Products,

and Relationship to Upstream PLA2 Isoforms

Although mRNA, enzymic activities, and immunoreactive of COX, LOX, and EPOX are found in several regions of the brain tissue, but very little is known about their interactions with each other and linkages to upstream ARA releasing enzymes

(PLA2 PLC, PLD isozymes) and downstream. PGE synthases, LT synthases, and 1.4 Interplay Among COX, LOX, and EPOX-derived Products, and Relationship… 23

TX synthases in brain tissue (Bosetti et al., 2004; Bosetti and Weerasinghe, 2003; Murakami and Kudo, 2004; Tanioka et al., 2000; Yan et al., 2005; Farooqui et al., 2007b; Farooqui, 2009). Several PGE synthases isozymes have been identified including membrane-associated PGE synthases, whose expression is induced by proinflammatory stimuli and cytosolic PGE synthases that are expressed constitutively. The conversion of PGH2 to other bioactive products, including PGD2, PGF2a,

PGI2 (prostacyclin), and TxA2, via specific synthases is also biologically important (Narumiya et al., 1999). The efficiency of coupling between isoforms of COX, LOX, EPOX, and specific PG, LT, LX, and TX synthases may be modulated by their spatial and temporal compartmentalization and by the amount of ARA released by

PLA2 isoform at a moment when PG, LT, and TX synthesis is taking place (Ueno et al., 2001). These processes not only depend on the presence of regulatory cofac- tors and interfacial binding of PLA2, COX, LOX, and EPOX isoform to membrane glycerophospholipids, which may differ according to neural cell type, stimuli, secretory processes, and subcellular distributions, but also on levels of n-6 fatty acids present in the diet, which modulate levels of glycerophospholipid molecular species in the brain tissue. The reaction rates of COX, LOX, and EPOX with the various fatty acids and molecular species of glycerophospholipids have been reported to be quite different. For example, for LOXs, setting the rate of linoleic acid oxygenation 100%, linoleic acid-containing glycerophospholipids are oxygenated with a rate of about 20%, and biomembranes with about 5%. The oxygenation rate of low-density lipoprotein is even lower (1–2%). This suggests that oxidation rates of free fatty acids and glycerophospholipid substrate strongly depend on substrate preparation (particularly for biomembranes and lipoproteins) and on the composition of the assay system (inclusion of detergents) (Kuhn and O’Donnell, 2006; Phillis et al., 2006). In addition, substrate concentration may strongly impact the absolute catalytic activities of these enzymes. Although multiplicity of COX, LOX, and EPOX enzymes in brain tissue provides diversity in their function and the specificity of various isoforms in the regulation of enzymic activity in response to a wide range of extracellular and intracellular signals, but the interpretation of results becomes very complex when one considers the different rates of expression of isoforms of PLA2, COX, LOX, and EPOX and downstream PG, LT, and TX synthases in different stages of disease pathology (Hoozemans et al., 2008). For example in an early stage of AD, when low-fibrillar Ab deposits are present and only very few neurofibrillary tangles are seen in the cortical areas, COX-2 is elevated in neurons. The increased neuronal COX-2 expression parallels and colocalizes with the expression of cell cycle proteins. COX-1 is primarily expressed in microglia, which are associated with fibrillar bA deposits. This suggests that in AD brain COX-1 and COX-2 are involved in inflammatory and regenerating pathways, respectively (Hoozemans et al., 2008). COX-2 and 5-LOX pathways work in parallel in the regulation of cell proliferation, differentiation, and angiogenesis. Furthermore, it is also suggested that neuronal and astrocytic 5-LOX, 12-LOX, and 15-LOX and EPOX isozymes differ considerably in responses to exogenous stimuli (Kwon et al., 2005). The complexity of cross-talk or interplay becomes obvious when one considers the coupling mechanisms of various isoforms 24 1 Metabolism and Roles of Eicosanoids in Brain of COX, LOX, and EPOX with different receptors at cellular and subcellular levels and tries to relate them to neuronal and glial cell functions (Bosetti et al., 2004). Some isoforms of COX, LOX, and EPOX are constitutively expressed while others are inducible in response to cytokines and growth factors (Farooqui, 2009). The isoforms of COX, LOX, and EPOX may not function interchangeably but act in parallel to transducer signals. It is likely that various isoforms of COX, LOX, and EPOX are coupled with different forms of EP, LT, TX, and LX receptors through different coupling mechanisms and act on different cellular pools of ARA located in different subcellular organelles of various types of neural cells. COX, LOX, and EPOX compete for ARA. Thus, generation of PGs may reduce the synthesis of LTs, and LX biosynthesis reduces LT formation. This suggests that there may be an inverse relationship between LT and LX biosynthesis (Serhan, 2005; Chiang et al., 2005). Different coupling mechanisms may regulate these isoforms, generating common second messengers. Coordination and integration of these second messengers in various subcellular compartments is necessary for optimal functioning of signal transduction processes. In vitro studies suggest that COXs and 12/15-LOX products not only act as coactivators of peroxisomal proliferator activating-receptors (PPAR), but also regulate cytokine expression and generation, and modulate gene expression related to resolution steps of neuroinflammation. Collectively, these studies suggest that interactions among PLA2, COX, LOX, and EPOX-generated metabolites at plasma membrane, cytoplasmic and nuclear membrane levels may provide neural cells and brain tissue with great versatility in ensuring that arachidonic acid is efficiently utilized in brain tissue (Bosetti et al., 2004; Farooqui et al., 2007b; Farooqui, 2010b). It is tempting to speculate that coordinated cross talk, not only among COX, LOX, and EPOX isozymes, but also with upstream PLA2 isozymes and downstream PGE synthases, is essential for maintaining normal neuronal and glial cell growth. In the nuclear membrane and nucleus, COX, LOX, and EPOX-mediated signaling has the advantage over plasma membrane signaling in that second messengers generated by these enzymes during differentiation may directly interact with nuclear factors producing physiological responses and morphological changes. In the brain tissue, endogenous activities of COX, LOX, and EPOX isoforms may depend not only on the structural, physicochemical, and dynamic properties of neural membranes from neuronal and glial cells, but also on their metabolic state (Bosetti et al., 2004; Farooqui, 2009). The ability of COX, LOX, and EPOX isoforms to orchestrate complex PGs, LTs, LXs, and EETs-mediated physiologic processes reflects an elaborate interplay among various receptors. Their levels not only modulate initiation, amplification, or dampening of inflammatory responses, but also influence the magnitude, duration, and nature of subsequent immune responses. The activation of COX, LOX, and EPOX isoforms at a subcellular level in neural cells is the rate-limiting step for the production of above lipid mediators. Therefore, an efficient regulation of COX, LOX, and EPOX isozymes is very important for normal brain function. Under pathological situations changes in activities of COX, LOX, and EPOX produces high levels of PGs, LTs, TXs, and LXs, which not only overstimulate their receptors, but may produce other harmful effects in neural 1.5 Roles of Eicosanoids in the Brain 25 membranes and nuclei. As stated above, the regulation of COX, LOX, and EPOX activities is complex and mediated by several factors including translocation, feedback regulation, release of cytokines and growth factors and mechanisms such as gene expression and cross talk among isoforms of these enzymes at cellular and subcellular levels. To understand the contribution of isoforms of COX, LOX, and EPOX in physiological and disease processes, a systematic approach will be necessary to identify second messengers and signaling pathways by which each isoform couples to its receptors in neuronal and glial cells at various subcellular levels (Farooqui, 2009, 2010b).

1.5 Roles of Eicosanoids in the Brain

Eicosanoids are released in response to a variety of physiological and pathological stimuli and function to maintain the body homeostasis. Eicosanoids are not stored in neural cells. Because of their amphiphilic nature, they can cross cell membranes and leave the cell in which they are synthesized to act on neighboring cells. In the brain tissue, neurons and glial cells produce PGs, LTs, and LXs whereas cerebral microvessels and the choroid plexus mainly synthesize TX. Synthesis of eicosanoid is markedly increased during inflammation. LTs not only contribute to the inflammation, but also increase the microvascular permeability, and are potent chemotactic agents. LXs are associated with the resolution of inflammation, stimulation of nonphlogistic phagocytosis of apoptotic cells by macrophages, and EETs modulate ion transport and gene expression, producing vasorelaxation as well as anti-inflammatory and profibrinolytic effects (Phillis et al., 2006). Eicosanoids act through eicosanoid receptors, which are typically G protein-coupled receptors with seven transmembrane segments that have an extracellular amino terminus and an intracellular carboxyl terminus. These receptors are involved in the generation of cyclic AMP, diacylglycerol, and phosphatidyl 1,4,5-trisphosphate and the modulation of calcium ion influx. Collective evidence suggests that eicosanoids play important roles in neural function including sleep induction (PGD2), long-term potentiation, spatial learning and synaptic plasticity (PGE2), induction of neuroinflammation

(PGE2), and resolution of inflammation (lipoxins) (Farooqui,2 009). However, some PGs act neuroprotectively by elevating intracellular cAMP levels in neurons. These molecules also function as anti-inflammatory molecules by reducing the synthesis of nitric oxide and blocking the production of proinflammatory cytokines.

1.5.1 Eicosanoids in Neuroinflammation

Neuroinflammation is a protective process that isolates the damaged brain tissue from uninjured area, destroys affected cells, and repairs the extracellular matrix. Inflammatory response is aimed to neutralize or eliminate the causative inflammatory 26 1 Metabolism and Roles of Eicosanoids in Brain factors and ultimately bring about the healing of the lesion by recruiting lymphocytes, monocytes, and macrophages of the hematopoietic system and certain glial cells of the CNS (Minghetti, 2005). Neuroinflammation is orchesterated by microglia and astrocytes. These cells are on “high-alert” for inflammatory triggers and are closely associated with the assembly and activation of the inflammasome, a cytoplasmic caspase-1 activating and self-oligomerizing signaling complex with molecular mass of greater than 700 kDa (Chakraborty et al., 2010). Inflammasome orchestrate the activation of the precursors of proinflammatory caspases, which in turn cleave the precursor forms of interleukin-1b, IL-18, and IL-33 into their active forms; the secretion of which lead to marked stimulation of PLA2, COX, LOX, and EPOX isoforms and production of ROS, proteinases, and complement proteins leading to a potent inflammatory response. Alterations in expression of inflammasome mediators may facilitate neurodegeneration in neurotraumatic and neurodegenerative diseases (Farooqui, 2010c). Based on this information, it is proposed that modulation of inflammasome machinery may be a better combating strategy than suppressing all inflammation in most neuroinflammatory situations (Farooqui et al., 2007a, b; Chakraborty et al., 2010; Farooqui, 2010c). Several anti-inflammatory drugs of various chemical ingredients have been shown not only to repress the microglial activation, but to exert neuroprotective effects in neurotraumatic and neurodegenerative situations. Neuroinflammatory response also involves the recruitment of polymorphonuclear leukocytes (PMN) from the blood stream into brain tissue. This PMN migration is a coordinated multistep process involving chemotaxis, adhesion of PMN to endothelial cells in the area of inflammation, and diapedesis (Farooqui et al., 2007a). PMN facilitate the elimination of invading anti- gens by phagocytosis and release free radicals and lytic enzymes into phagolyso- somes. This is followed by a process called resolution which is a turning off mechanism by neural cells to limit tissue injury. Collective evidence suggests that neuroinflammation involves several converging mechanisms responsible for sensing, transducing, amplifying, and turning off mechanisms that involve the participation of eicosanoids. Some PGs and LTs produce proinflammatory effects while others induce anti-inflammatory effects (Farooqui et al., 2006; Farooqui, 2010c). During the inflammatory reaction, synthesis of proinflammatory eicosanoids

(PGE2, PGF2, and LTB4) brings about the onset of neuroinflammation, secretion of proinflammatory cytokines, amplifies and maintains the inflammatory responses, and generation of LXs along with the synthesis of resolvins and protectins, breaking the intensity of neuroinflammation by reducing neutrophil entry to the neuroinflammation site, and stimulating the uptake of apoptotic polymorphonuclear leukocytes by macrophages (Yacoubian and Serhan, 2007; Lawrence and Gilroy, 2007; Serhan, 2005) (Fig. 1.3). The onset of neuroinflammation involves the expression and stimulation of iPLA2 with the generation of PGE2 and LTB4 through COX-2, and LOX reactions, respectively. The amplification and maintenence of neuroinflammation is associated with cPLA2 and sPLA2-mediated release and COX and LOX-mediated oxidation of ARA. Finally, the cPLA2, sPLA2, and PlsEtn-PLA2 coupled release of ARA and COX and LOX-mediated generation of LXs, pro resolving prostaglandin, PGD2 and synthesis of DHA-derived resolvins and protectins 1.5 Roles of Eicosanoids in the Brain 27 may be responsible for the resolution of neuroinflammation (Yacoubian and Serhan, 2007; Lawrence and Gilroy, 2007; Serhan, 2005). In addition, eicosanoids also serve as autocrine factors regulating platelet aggregation, vascular tone, and edema.

PLA2, COX-2, and LOX inhibitors have been used to treat acute inflammation and pain in various animal models of inflammation-mediated pain (Farooqui, 2010c).

1.5.2 Eicosanoids in Neurodegeneration

Neurodegeneration is a complex, progressive, and multifaceted process that results in neural cell dysfunction and death in the brain tissue. In brain tissue, neural cell death occurs either through apoptosis or necrosis. The necrotic cell death is characterized by the passive cell swelling, intense mitochondrial damage, rapid loss of ATP, alterations in neural membrane permeability, high calcium influx, glutathione depletion, and disruption of ion homeostasis. This type of cell death leads to membrane lysis and release of intracellular components that induce inflammatory reactions. In contrast, apoptotic cell death usually involves the participation of caspases, a group of endoproteases with specificity for aspartate residues in protein. Apoptotic cell death is accompanied by cell shrinkage, dynamic membrane blebbing, chromatin condensation, DNA laddering, loss of phospholipids asymmetry, low ATP levels, and mild calcium overload (Sastry and Subba Rao, 2000; Farooqui et al., 2004; Farooqui, 2009). Thus, apoptosis and necrosis are two extremes of a wide spectrum of cell death processes with different mechanistic and morphological features. However, there is considerable overlap in terms of some common mediators and signal transduction processes, which cannot be separated. Collective evidence suggests that PLA2, COX, LOX, and EPOX-derived eicosanoids along with caspases are involved in necrotic and apoptotic cell deaths. Apoptosis is dependent not only on an adequate ATP level but also on glutathione levels and necrotic cell death is associated with marked reduction in ATP (Farooqui, 2009). It is becoming increasingly evident that under pathological conditions, stimulation of isoforms of PLA2, COX, and LOX result in the accumulation of PGE2, a major ARA-derived PG, closely associated with apoptotic and necrotic types of cell deaths (Farooqui, 2009). Cytokines interleukin-1b (IL-1b) induces COX-2 mRNA and protein synthesis and the release of PGE2 in the human neuroblastoma cell line SK-N-SH (Farooqui and Horrocks, 2007). In addition, an uncontrolled ARA cascade sets the stage for the increased production of ROS and ROS-mediated damage to membrane proteins, ion channels, and receptors. The action of 12-LOX produces lipid hydroperoxides. These hydroperoxides are known to inhibit reacylation of phospholipids in neuronal membranes (Farooqui and Horrocks, 2007). Free radical scavengers and inhibitors of PLA2, and p38 mitogen-activated protein kinase (MAPK) reduce IL-1b-induced synthesis of COX-2, suggesting that upregulation of COX-2 may play an important role in neurodegeneration. Similarly in mutant SOD model of ALS, PGE2 signaling via the EP2 receptor functions in regulating the 28 1 Metabolism and Roles of Eicosanoids in Brain

expression of a cassette of proinflammatory genes. Inhibition of EP2 signaling may represent a novel strategy to downregulate PGE2-mediated inflammatory response in the ALS (Liang et al., 2008).

As stated above, dehydration of PGD2 generates the bioactive cyclopentenone prostaglandins of the J2 series (Pierre et al., 2009). 15d-PGJ2 is a specific potent ligand for the peroxisome proliferator activator receptor-gamma (PPARg). It blocks cell growth and induces apoptosis in a number of different cancer cells (Cho et al.,

2006). 15d-PGJ2-mediated ROS generation and cell death can be retarded by the antioxidant N-acetylcysteine. 15d-PGJ2 treatment also induces mitochondrial dysfunction, but the 15d-PGJ2-mediated cell death is not blocked by caspase inhibitors

(Cho et al., 2006). Taken together, these observations suggest that 15d-PGJ2 triggers cell death through a caspase-independent mechanism and ROS generation and disruption of mitochondrial membrane potential play an important role in the

15d-PGJ2-mediated cell death in A172 human glioma cells (Cho et al., 2006).

1.5.3 Eicosanoids in Nociception (Pain State)

Pain is a complex process involving peripheral and central mechanisms (Svensson and Yaksh, 2002). Injury to brain and spinal cord is often accompanied by induction of spontaneous pain, hyperalgesia (increased responsiveness to noxious stimuli), and allodynia (painful responses to normally innocuous stimuli). Peripheral and central mechanisms of pain are associated with inflammation and upregulation of brain and spinal cord COX-2 with a subsequent increase in central PGE2 levels associated with the development of hyperalgesia (Svensson and Yaksh, 2002).

Furthermore, peripheral administration of PGI2 and PGE2 produces pain-like behavior in rats (Taiwo and Levine, 1990). COX-2 inhibitors not only prevent the increase in COX-2 activity, but also lower the intensity of allodynia. Collective evidence suggests that in inflamed brain and spinal cord, upregulation of COX-2 and increase in

PGI2 and PGE2 levels contribute both to the inflammation itself and to pain hypersensitivity, acting on the peripheral terminals of nociceptors. Studies in models of “sharp” rapidly transmitted pain (hot-plate) and slowly developing, diffuse pain (writhing) with COX-isozyme-deficient mice indicate that COX-1 and COX-3 (not COX-2) play an important role in chronic pain and inflammation in dorsal root ganglia (Ballou et al., 2000). Collective evidence suggests that all isoforms of COX enzymes are involved in pain transmission processes in brain and spinal cord.

1.5.4 Eicosanoids in Synaptic Plasticity

Neuronal plasticity is a fundamental property of brain involved in adequate interactions of neuronal cells with dynamic environment. One of the most throughly investigated forms of the neuronal plasticity is a long-term potentiation (LTP), a phenomenon associated with learning and memory processes. COX-1 and

COX-2-mediated generation of PGE2, PGF2a, and PGD2 modulates the synaptic 1.6 Eicosanoids in Neurotraumatic Diseases 29 activity and efficacy by exerting their paracrine effect through pre-and postsynaptic glutamate receptors and their autocrine effect through intraneuronal and glial second messengers (Piomelli, 1994). Selective COX-2 inhibitors significantly decrease postsynaptic membrane excitability, back-propagating dendritic action potential- associated Ca2+ influx, and LTP induction in hippocampal dentate granule neurons, while COX-1 inhibitors are ineffective, supporting the view that COX-2 plays a major role in the induction of synaptic plasticity and LTP (Chen et al., 2002).

COX-2 inhibitors block the induction of LTP. The addition of PGE2, but not

PGD2 or PGF2a, reverses COX-2-mediated suppression of LTP. These studies suggest that PGE2 is the effector of COX-2-induced synaptic plasticity. Collective evidence suggests that endogenous PGE2 dynamically regulates membrane excitability, synaptic transmission, and plasticity and that the PGE2-mediated synaptic modulation is supported through the involvement of cAMP-PKA and PKC pathways in rat hippocampal CA1 pyramidal neurons.

1.6 Eicosanoids in Neurotraumatic Diseases

Neurotraumatic diseases include ischemia, spinal cord trauma, and traumatic brain injury. These injuries arise from very different kinds of initial insult to CNS and PNS. Ischemia is a metabolic insult caused by severe reduction or blockade in cerebral blood flow. This blockade decreases oxygen and glucose delivery to brain tissue and results in the build-up of potentially toxic products in brain (Farooqui et al., 2004; Farooqui, 2010b). In contrast, mechanical impact and shear forces cause spinal cord trauma and traumatic brain injury (McIntosh et al., 1998; Farooqui, 2010b). Spinal cord trauma and traumatic brain injury consists of two broadly defined components: a primary component, attributable to the mechanical insult itself, and a secondary component, attributable to the series of systemic and local neurochemical and pathophysiological changes that occur in brain and spinal cord after the initial traumatic insult (Klussmann and Martin-Villalba, 2005). The primary injury leads to a rapid deformation of brain and spinal cord tissues, resulting in the rupture of neural cell membranes, release of intracellular contents, and disruption of blood flow and breakdown of the blood–brain barrier. In contrast, secondary injury to brain and spinal cord is characterized not only by the release of glutamate from intracellular stores (Sundström and Mo, 2002), but also by excitotoxicity, inflammation, oxidative stress, and overexpression of cytokines (Hayes et al., 2002; Ahn et al., 2004). Collective evidence suggests that secondary injury induces edema, neutrophil infiltration, production of a range of inflammatory mediators, and apoptosis. Among neural cells, neurons and oligodendrocytes are equally susceptible to spinal cord trauma or traumatic brain injury and astrocytes and microglia become activated. Recent studies on improved understanding of the pathological changes occurring in oligodendrocytes following spinal cord trauma or traumatic brain injury indicate that the death of oligodendrocytes play a vital role in axonal demyelination of axons, which impairs the conductive capacity of surviving axons (Wu and Ren, 2009). Thus, secondary injury-mediated spinal cord or brain damage that occurs within 30 1 Metabolism and Roles of Eicosanoids in Brain

Table 1.3 Activities of COX, LOX, EPOX, and levels of eicosanoids in neurotraumatic diseases COX, LOX, Eicosanoid Disease and EPOX activities levels Reference Ischemia Increased Increased Nogawa et al., 1997; Tomimoto et al., 2000; 2002; IIliff and Alkayed, 2009 Spinal cord trauma Increased Increased Resnick et al., 1998 Traumatic brain injury Increased Increased Cernak et al., 2001 Epilepsy Increased Increased Marcheselli and Bazan, 1996

hours to days after spinal cord trauma or traumatic brain injury contributes significantly to neurodegeneration, sensory loss, and other functional disabilities.

1.6.1 Eicosanoids in Ischemic Injury

Ischemic injury is accompanied with upregulation of COX-2, and LOX immunoreactivities in neurons and endothelial cells in rodents in acute cerebral infarction models (Iadecola et al., 1999; Tomimoto et al., 2000) (Table 1.3). Intense COX-2 immunoreactivity is observed in the glial cytoplasm following ischemic injury (Tomimoto et al., 2000). In normal human brain, COX-1 immunoreactivity is expressed in the microglia, but weakly in the neurons. COX-1-immunoreactivity in microglia is markedly increased following ischemic damage and hypoxemia. In contrast, normal human brain does not show COX-2 immunoreactivity. However, COX-2 is induced robustly in the neuronal cell bodies and dendrites during the acute stages of focal ischemic damage, and then subsided at the subacute stages (Tomimoto et al., 2002). These COX-2-immunoreactive neurons accumulated in the peri-infarct regions, but not in the distant regions. Furthermore, in focal ischemic damage COX-2 is upregulated in the microglia. Like COX-2, neuronal immunostaining for 5-LOX is also upregulated occasionally during hypoxemia and focal ischemic damage (Tomimoto et al., 2002). Glial cells immunoreactivity for 5-LOX appears in the foci of the ischemic damage, with small blood vessels being infil- trated by 5-LOX-immunoreactive mononuclear leukocytes. Collectively, these studies suggest that COX-1 and COX-2 are differentially regulated depending on the cell type and ischemic damage, and that vascular 5-LOX may accelerate the migration of leukocytes and augment the blood–brain barrier permeability (Tomimoto et al., 2002). Transient ischemic attack also induces ischemic tolerance by a mechanism temporally linked to the upregulation of EPOX (P450 2C11) (Alkayed et al., 2002). EETs, which are generated through EPOX catalyzed reaction, exhibit a wide array of potentially beneficial actions in stroke, including vasodilation, neuroprotection, promotion of angiogenesis and suppression of platelet aggregation, oxidative stress, and postischemic inflammation (IIliff and Alkayed, 2009).The increase in COX-2 and 5-LOX, EPOX activities is coupled with the 1.6 Eicosanoids in Neurotraumatic Diseases 31 accumulation of PGs, LTs, and EETs in the mammalian brain during and after cerebral ischemic injury (Table 1.3) (Gaudet et al., 1980; Moskowitz et al., 1984; Nakano et al., 1990). Increase in PGs and LTs is also noticed in cerebrospinal fluid (Aktan et al., 1991).

1.6.2 Eicosanoids in Spinal Cord Trauma

Spinal cord trauma is accompanied by increased expression of COX-2 and induction of 6-keto-PGF1, and TXB2 2 h following injury (Resnick et al., 1998; Harada et al., 2006) (Table 1.3). COX-2 levels peak at 48 h following spinal cord trauma. Selective inhibition of COX-2 activity with SC58125 not only results in neuroprotection from spinal cord trauma, but also improves mean BBB scores in injured animals. RT-PCR studies indicate that COX-2 transcription in the spinal cord starts to increase within 30 min, peaks at 3 h after the spinal cord trauma (Adachi et al., 2005). Western blotting analysis demonstrates that the deglycosylated COX-2 protein is significantly increased 6 h after the spinal cord trauma. Similarly, in severe clip compression model of spinal cord trauma in the rat, the expression of COX-2 and formation of 8-OHdG and protein carbonyl groups are markedly increased after spinal cord trauma while APE/Ref-1 expression is decreased (Bao et al., 2004). Anti-CD11d mAb treatment clearly attenuates COX-2 expression and 8-OHdG and protein carbonyl formation and rescues APE/Ref-1 expression after spinal cord trauma, demonstrating that anti-CD11d mAb treatment significantly reduces intraspinal free radical formation after spinal cord trauma, thereby reducing protein and DNA oxidative damage (Bao et al., 2004). Accumulating evidence suggests that COX-2 mRNA and protein expression are induced by spinal cord trauma, and that selective inhibition of COX-2 or CD11d mAB improves functional outcome following experimental spinal cord injury. Among PGs, 15d-PGJ2, a metabolite of PGD2 has anti-inflammatory actions and its administration significantly improves the sensory and locomotor functions following spinal cord trauma. Similarly, 5-LOX, FLAP, and CysLT1 mRNAs are markedly increased in spinal cord microglia following spinal cord trauma. The increase in 5-LOX is blocked by p38 mitogen-activated protein kinase inhibitor. Continuous intrathecal infusion of the 5-LOX inhibitor or BLT1 and CysLT1 receptor antagonists retards mechanical allodynia mediated by spared nerve injury. It is proposed that the increase of LT synthesis in spinal cord microglia mediated by p38 MAPK plays a role in the generation of neuropathic pain following spinal cord trauma (Okubo et al., 2010).

1.6.3 Eicosanoids in Traumatic Brain Injury

Like spinal cord trauma, traumatic brain injury (TBI) initiates a central inflammatory response, which is accompanied by stimulation in COX and LOX activities, increase in levels of PGs (PGE1, 6-keto-PGF1a, and PGF2a) not only in cerebral 32 1 Metabolism and Roles of Eicosanoids in Brain

cortex, but also generation of ROS in brain tissue of ROS in brain tissue, plasma, and CSF (Phillis et al., 2006) (Table 1.3). These elevations in PGs are sustained at up to 30–60 min post injury. Elevations in leukotriene levels also occur in brain after concussive brain injury (Kunz et al., 2002; Phillis et al., 2006).

Detailed investigations have shown that elevation in PGs and LC4 (LTC4, LTD4,

LTE 4, and LTB4) following traumatic brain injury occurs in the cerebral cortex, hippocampus, and CSF in rats. It lasts for 1–2 h and is coupled with persistent accumulation of microglial cells, neutrophils, and macrophages expressing COX-1, COX-2, and LOX (Dhillon et al., 1996; Schwab et al., 2001, 2002; Schuhmann et al., 2003). The administration of MK-886 prevents the release of LTs and reduces brain lesion volumes (Farias et al., 2009). It is proposed that increases in leukotriene levels may be related to tissue edema, leukocyte infiltration, macrophage accumulation, microglial activation, and disruption of the blood–brain barrier.

1.6.4 Eicosanoids in Epilepsy

Epileptic seizures in rat and human brain produce markedly alterations in ARA levels in focal regions compared to nonfocal regions of the cerebral cortex (Bazan 2+ et al., 2002; Bazan, 1971) with increase in Ca influx, stimulation of PLA2 and COX-2 along with the release of PGs and LTs (Bazan et al., 1986). Thus, spontaneous seizures produce elevation in PGD2 levels in the cerebral cortex, hippocampus, and striatum and of an LTC4-like substance in the cortex (Simmet et al., 1988). These areas are involved in the generation and propagation of seizures. Similarly, the formation of immunoreactive PGF2a and leukotriene-like activity is observed in the brains of spontaneously convulsing gerbils (Simmet et al., 1988). Administration of dexamethasone, a PLA2 inhibitor, attenuated bicuculline-induced free fatty acid accumulation in a dose-dependent manner (Bazan et al., 1986). In the genetically epilepsy-susceptible E1 mouse model, expression of COX-2 in the hippocampus is increased after an epileptic seizure, and indomethacin, a COX-2 inhibitor, shortens the duration from seizure onset to full recovery (Okada et al., 2001). Collective evidence suggests that epileptic seizures induce upregulation of COX-2 and 5-LOX resulting in the increased synthesis of PGs and LTs. The increase in PGs and LTs can be blocked by selective inhibitors of COX-2 and 5-LOX.

1.7 Eicosanoids in Neurodegenerative Diseases

Neurodegenerative diseases are characterized by the site-specific premature and slow death of specific neuronal populations and synapses in brain and spinal cord along with development of brain dysfunction in skilled movements, decision making, cognition, and memory (Graeber and Moran, 2002; Soto and Estrada, 2008). These diseases include Alzheimer disease (AD), Parkinson disease (PD), Huntington 1.7 Eicosanoids in Neurodegenerative Diseases 33

Table 1.4 Activities of COX, LOX, EPOX, and levels of eicosanoids in neurotraumatic diseases COX, LOX, Disease and EPOX activities Eicosanoid levels Reference AD Increased Increased Bazan et al., 2002; Manev et al., 2000b PD – – – PD animal/cell Increased Increased Phillis et al., 2006; culture models Bharath et al., 2002; Przybyłkowski et al., 2004 ALS Increased Increased Yasojima et al., 2001 CJD Increased Increased Minghetti et al., 2002; Minghetti and Pocchiari, 2007 HIV Increased Increase Griffin et al., 1994

disease (HD), amyotrophic lateral sclerosis (ALS), and prion diseases. It is not known when and how do neurodegenerative diseases actually start and how long does it take for neuropathological changes to appear. The most important risk factors for neurodegenerative diseases are old age, positive family history, unhealthy life style, and exposure to toxic environment (Farooqui and Farooqui, 2009; Farooqui, 2010b). In general neurodegenerative diseases are characterized by their onset in old age, extensive neurodegeneration, synaptic dysfunction, and the accumulation of intracellular or extracellular cerebral deposits of misfolded protein aggregates with a b-sheet conformation, such as b-amyloid (Ab) in AD, a-synuclein in PD, huntingtin in HD, elevation in membrane-associated oxidative stress in ALS resulting in genetic abnormalities (Cu/Zn-superoxide dismutase mutations) (Farooqui and Horrocks, 2007; Farooqui, 2010b) and accumulation of advanced glycation end-products (AGEs) (Miranda and Outerio, 2010).

1.7.1 COX, LOX, EPOX, and Eicosanoids in AD

AD, the most common form of dementia, is characterized by the loss of neurons in nucleus basalis and hippocampus, accumulation of b-amyloid, formation of neurofibrillary tangles, oxidative stress, neuroinflammation, decline in cognitive responses, and consequent memory loss. Alterations in COX and LOX pathways in AD have been extensively investigated in brain tissue from AD patients and age-matched controls (Bazan et al., 2002; Manev et al., 2000b; Qin et al., 2003; Hoozemans and O’Banion, 2005). A marked increase of COX-1 and COX-2 expression and immunoreactivity in cerebral cortex and hippocampal regions of AD brains correlates with the number of senile plaques, neuronal atrophy, and increased levels of PGE2 found in AD (Table 1.4) (Pasinetti and Aisen, 1998; Qin et al., 2003). Alterations in the expression of COX-1 and COX-2 have been reported to occur in the different stages of AD pathology. In an early stage, when low-fibrillar bA deposits are present 34 1 Metabolism and Roles of Eicosanoids in Brain and only very few neurofibrillary tangles are observed in the cortical areas, COX-2 is increased in neurons. The increased expression of neuronal COX-2 parallels and colocalizes with the expression of cell cycle proteins. COX-1 is primarily expressed in microglia, which are associated with fibrillar bA deposits. It is suggested that in AD brain COX-1 and COX-2 are associated with inflammatory and regenerating pathways, respectively (Hoozemans et al., 2008). Furthermore, COX-generated product, PGH2, rapidly rearranges in aqueous solution and is converted to levuglandins E2 and D2. These g-ketoaldehydes are highly reactive and rapidly adduct to accessible amine groups on macromolecules, particularly the epsilon-amine of lysine residues on proteins. The immediate LG-lysine adducts are themselves reactive, and can covalently crosslink proteins. PGH2, acting via levuglandins, accelerates the formation of the type of oligomers of Ab, which contribute to neurotoxicity and cell death (Boutand et al., 2005). The neuroprotective effects of COX-1 and COX-2 inhibitors (NSAID) strongly support the view that upregulation of these enzymes in AD is detrimental to neuronal survival. COX-1 and COX-2 potentiate b-amyloid peptide formation through mechanisms that involve g-secretase activity

(Qin et al., 2003). Interplay between increased PGE2 synthesis and g-secretase activity not only regulate b-amyloid peptide accumulation but also modulate neuroinflammation in AD brain (Gasparini et al., 2004). Detailed investigations reveal that

PGE2-mediated production of Ab involves EP4 receptor-mediated endocytosis of

PS-1 followed by activation of the g-secretase, as well as EP2 receptor-dependent activation of adenylate cyclase and PKA; both of which are important in the inflammation-mediated progression of AD (Hoshino et al., 2009). Furthermore, COX-2 expression is also involved in the regulation of cell cycle activity, and cell cycle abnormalities are associated with the pathogenesis of AD. Re-entry into the cell cycle may underlie COX-2-mediated neuronal damage in AD (Xiang et al., 2002). Studies on 5-LOX-targeted gene disruption on the amyloid phenotype of a transgenic mouse model of AD (Tg2576) indicate that Ab deposition in the brains of Tg2576 mice lacking 5-LOX is decreased by 64–80% compared with Tg2576 controls. This decrease parallels a similar significant reduction in bA levels. Absence of 5-LOX has no effect on amyloid-b precursor protein (APP) levels and processing, or Ab degradation. Furthermore, in vitro studies indicate that 5-LOX activation or increase in leukotrienes levels stimulates Ab deposion, whereas 5-LOX inhibition reduces Ab deposition. These observations support the view that 5-LOX activity is closely associated with the modulation of g-secretase activity and pathogenesis of AD-like amyloidosis (Firuzi et al., 2008). Upregulation of 5-LOX expression also occurs in various regions of older rat brain compared to younger animals (Manev and Manev 2004). Collectively, these studies suggest that 5-LOX protein is upregulated in AD hippocampus, where it is primarily associated with neurofibrillary structures and Ab-containing plaques. In addition, Ab peptide fibrilization and tau overphosphorylation themselves activate LOX activity, creating a vicious cycle of pathological cascades, neuroinflammation, and oxidative stress that may perpet- uate neuronal degeneration and loss of synapses in AD (Ikonomovic et al., 2008). At present, no information is available on EPOX activity and expression in AD brain. In vitro studies indicate that LOX inhibitors, nordihydroguaiaretic acid, 1.7 Eicosanoids in Neurodegenerative Diseases 35

AA861, and baicalein, and the EPOX inhibitors, SKF25A and metyrapone prevent cell death in neuronal cultures, suggesting that LOX and EPOX pathways may also be involved in neurodegeneration (Kwon et al., 2005).

1.7.2 COX, LOX, EPOX, and Eicosanoids in PD

The gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta results in PD (Jenner and Olanow, 2006). Loss of these neurons causes pathological changes in neurotransmission in the basal ganglia motor circuit. Although the cause of dopaminergic neuronal death in PD is not fully understood, but accumulation of a-synuclein, oxidative stress, and neuroinflammation is closely associated with the pathogenesis of PD (Farooqui, 2010b). At present, nothing is known about the expression of COX-2 in patients with PD. Recent RT-PCR studies indicate that in mice COX-1 mRNA expression does not change following N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration, but COX-2 gene and protein expression in striatum increases from the 3rd to the 7th and 14th days, and diminishes on the 21st day. Production of PGs is augmented only briefly after MPTP treatment, but does not correlate with increased COX-2 mRNA and COX-2 protein production, suggesting that the increase in COX-2 expression does not follow the acute stage of cell death suggesting that COX-2 does not contribute to neurons death following MPTP administration (Przybyłkowski et al., 2004). Nothing is known about the expression of LOX and EPOX in patients with PD or in animal models of PD.

1.7.3 COX, LOX, EPOX, and Eicosanoids in Amyotrophic Lateral Sclerosis

ALS causes progressive loss of motor neurons leading to muscle loss, paralysis, and death from respiratory failure. Although the cause and molecular mechanism associated with pathogenesis of ALS remains unknown, but it is becoming increasingly evident that multiple pathophysiological mechanisms may be associated with the loss of motor neuron. These mechanisms include oxidative stress, mitochondrial impairment, protein aggregation, axonal dysfunction, and alterations in mutant superoxide dismutase expression, cytoskeletal disorganization, glutamate cytotoxicity, inflammation, and apoptotic cell death (Farooqui and Horrocks, 2007). The sporadic form of ALS is characterized by a prominent neuroinflammatory component, upregulation of COX-2 mRNA, and oxidative stress (Yasojima et al., 2001). A similar upregulation of COX-2 mRNA and increase in expression of CD40 also occurs in SOD1 transgenic mice and G93A mice, a transgenic mouse model of ALS at the onset of ALS (Almer et al., 2001). The increase in expression of CD40 and COX-2 occurs in reactive microglia and astrocytes. It is proposed that in ALS the 36 1 Metabolism and Roles of Eicosanoids in Brain upregulation of CD40 in reactive glial cells contribute to motor neuronal loss via the induction of COX-2 and treatment of ALS with COX-2 inhibitors provides neuroprotective effects (Pompl et al., 2003). Although upregulation of COX-2 and PGE2 levels may not be the root cause of ALS, alterations in these parameters may be responsible for the induction and maintenance of inflammation during the progression of ALS. At present, information on LOX and EPOX expression and their activities is not available for the ALS brain and spinal cord.

1.7.4 COX, LOX, EPOX, and Eicosanoids in Creutzfeldt-Jakob Disease (CJD)

CJD is the most prevalent prion disease in humans. It is characterized by neuronal loss, astrocytosis, and spongiform degeneration, and deposition of abnormal extracellular b-helix-rich prion protein (PrPsc) (Brown, 1999; Prusiner, 2001). This abnormal protein is an isoform of a normally occurring a-helix-rich prion protein (PrPc) probably associated with synaptic function, regulation of circadian rhythms, and copper transport (Brown, 1999; Prusiner, 2001). RT-PCR and Western blotting studies indicate that both COX-1 and COX-2 are significantly increased in brains from CJD patients compared to age-matched controls (Deininger et al., 2003). In CSF from sporadic and familial CJD, and in brain homogenates of scrapie-infected mice, the increase in COX-1 and COX-2 activities is accompanied by a several-fold increase in concentrations of PGE2 and F2-isoprostane 8-epi-prostaglandin F2a (8-epi-PGF2a) compared to age-matched controls (Minghetti et al., 2000) (Table 1.4). Similar results on PGE2 levels have been obtained in CSF of CJD variants (Minghetti et al., 2002). These findings suggest that cyclooxygenase activity may participate in the inflammatory process and have a role in CJD pathogenesis. In experimental model of prion diseases, increased COX-2 immunoreactivity is specifically localized to microglial cells and reactive astrocytes. The increase in immunoreactivity is not only related with elevation in COX-2 mRNA and protein levels, but also with the progression of disease. The induction of COX-2 parallels a substantial increase in the brain PGE2 levels. Only few scattered COX-1-positive microglia-like cells are detected in the brain suggesting that COX-2 is the major isozyme that may contribute to neuroinflammation in prion diseases (Minghetti and Pocchiari, 2007; Kim et al., 2007). Enrichment of ARA metabolism also occurs in a cell culture model of prion diseases. Thus, the treatment of primary cerebellar granule neuronal cultures with prion peptide (PrP106-126) causes an enhancement of ARA metabolism through the activation of 5-LOX pathway (Stewart et al., 2001). Nordihydroguaiaretic acid and caffeic acid, the inhibitors of 5-LOX prevent PrP106-126-mediated increase in ARA metabolism and neurodegeneration. These inhibitors also retard the PrP106- 126-mediated caspase-3 activation and annexin V binding associated with 5-LOX- mediated apoptosis. In contrast, indomethacin, a COX-1 and COX-2 inhibitor, and baicalein, a 12-LOX inhibitor, have no affect on PrP106-126-mediated neurotoxicity in cerebellar granule neuronal cultures (Stewart et al., 2001). References 37

1.8 Conclusions

Eicosanoids are lipid mediators that are derived from ARA, which is located at the sn-2 position of glycerol moiety of neural membrane phospholipids. In response to stimuli, ARA is mobilized from phospholipid pools and metabolized by enzymes, COX, LOX, and EPOX to form PGs, LTs, TXs, LXs, and EETs. The production of these lipid mediators is tightly regulated and coordinated in a cell-specific manner. Each mediator interacts with its receptor and performs regulatory and homeostatic functions not only in the proliferation, differentiation, and synaptogenesis, but also in the onset and resolution of inflammation, immune responses, gene expression, and tissue repair. In addition, eicosanoids act as second messengers in learning and memory and contribute to vascular function by regulating cerebral blood flow. Although it may not be the primary cause, but in neurotraumatic as well as neurodegenerative diseases the stimulation and upregulation of COX, LOX, and EPOX isozyme activities and the generation of excessive amounts of prostaglandins, leukotrienes, thromboxanes, and epoxyeicosatrienoic products may result in neuroinflammation, and oxidative stress-mediated neurodegeneration, which are closely associated with the pathogenesis of neurotraumatic and neurodegenerative diseases.

References

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2.1 Introduction

Neuroprotectins and resolvins are subfamilies of endogenous oxygenated metabolites derived from n-3 or w-3 fatty acids (eicosapentaenoic acid, 20:5, EPA and docosahexaenoic acid, 22:6, DHA) (Serhan et al., 2000, 2002; Hong et al., 2003; Marcheselli et al., 2003). EPA not only modulates neuronal activity, but also participates in bal- ancing the immune function by reducing arachidonic acid (20:4, ARA) level on cell membrane and prostaglandin E2 (PGE2) synthesis (Farooqui, 2009). DHA is a major structural membrane fatty acid. At the subcellular level, highest concentration of DHA is found in synaptic membranes followed by mitochondria and microsomes (Scott and Bazan, 1989). The presence of DHA in neural membranes significantly alters many basic membrane properties, including acyl chain order and fluidity, elas- tic compressibility, phase behavior, permeability, fusion, flipflop, and protein activity. DHA also plays important roles in gene expression, and neurotransmission, associated with serotonin, norepinephrine and dopamine receptors. This may be related to the etiology of mood and cognitive dysfunction in neuropsychiatric and neurodegenerative diseases (Farooqui, 2009). In addition, treatment of hippocampal neuronal cultures with DHA reduces glutamate-mediated neurotoxicity (Wang et al., 2003). Changes in the polyunsaturated fatty acid (PUFA) composition of neuronal membranes may lead not only to alterations in the physical characteristics of the neural membrane, but also functional changes in the activity of receptors and other proteins embedded in the membrane glycerophospholipid bilayer. Both of these processes alter physicochemical membrane properties. Beyond their structural importance in neural and retinal membranes, DHA and EPA are involved in the synthesis and functions of neurotransmitters, and in the modulation of the immune system (Farooqui, 2009, 2010a). Neuronal membranes contain phospholipid pools that are reservoirs for the synthesis of specific lipid messengers on neuronal stimulation or injury. These messengers in turn participate in signaling cascades that can either promote neurodegeneration or neuroprotection. DHA and EPA-derived metabolites also improve

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 49 DOI 10.1007/978-1-4419-9940-5_2, © Springer Science+Business Media, LLC 2011 50 2 Recent Development on the Neurochemistry of Docosanoids

Modulation of growth and differentiation

Modulation of learning Modulation of enzymes and memory and ion channels

Modulation of transcription n-3 Polyunsaturated Generation of pro-and anti- factors and gene expression fatty acids inflammatory lipid mediators

Modulation of Inhibition of neurotransmission oxidative stress

Stabilization of neural membranes

Fig. 2.1 Role of polyunsaturated fatty acids in brain biological functions such as signal transduction, ion channeling and ligand binding to nuclear receptors (Fig. 2.1). Collective evidence suggests that dietary PUFAs regulate both eicosanoids and proinflammatory cytokine production. DHA and EPA are anti- inflammatory while ARA (n-6 fatty acids) and its metabolites are proinflammatory. Inappropriate amounts of dietary n-6 and n-3 fatty acids may lead to abnormal inflammatory and immune responses because of their abundance in the brain. An adequate ratio of n-6 and n-3 fatty acids may promote a healthier balance between n-6-and n-3 PUFA-derived lipid mediators, which may maintain optimal neural membrane function (Farooqui, 2009).

2.2 Importance of n-3 Polyunsaturated Fatty Acids in the Brain n-3 polyunsaturated fatty acids are essential for brain growth and development. They play an important role throughout life, as critical modulators of neuronal function and regulation of neuroinflammatory and oxidative stress-mediated mechanisms in the normal CNS during aging and chronic neurological diseases. Inadequate levels of DHA in brain during development and old age induce cognitive deficits such as memory loss and learning disability in experimental animals. Thus, the inadequacy of DHA in neural membranes may contribute to cholinergic, dopaminergic, and glutameterigic receptor dysfunction in synapses associated with the hippocampal neurons, and growing evidence suggests that low levels of DHA in the brain are associated not only with neurotraumatic, neurodegenerative and neuropsychiatric diseases, but also with peroxisomal disorders (Farooqui, 2009). DHA, the 2.2 Importance of n-3 Polyunsaturated Fatty Acids in the Brain 51 major n-3 fatty acid found in neurons, has taken on a central role as a target for therapeutic intervention in neurotraumatic, neurodegenerative, and neuropsychiatric diseases (Farooqui, 2009, 2010a). DHA acts as a ligand for the PPAR-g and the RXR receptors. In conjunction with these receptors, which act as transcription factors, DHA modulates various neurochemical processes that maintain cellular homeostasis by modulating lipid metabolism, neural cell differentiation, and apoptosis (Farooqui, 2009). DHA downregulates protein kinase C, Ras, and NF-kB; activates the Jak/Stat pathway, and sustains phosphorylation of EGFR. DHA attenuates the transcription of NF-kB-dependent genes. Thereby the COX-2/PGE2-dependent generation of pro-angiogenic vascular endothelial growth factor and levels of antiapoptotic bcl-2 and bcl-X(L) are reduced. Eicosanoid-independent pro-apoptotic pathways include enhanced lipid peroxidation, modulation of mitochondrial calcium homeostasis, and enhanced production of reactive oxygen species (ROS) as well as activation of p53. In brain, DHA also restores levels of cerebellar phospho (p)-AKT, phospho-extracellular regulated kinase (p-ERK) and phospho-c-Jun N-terminal kinase (p-JNK) supporting their role in down-regulation of neuronal apoptosis (Sinha et al., 2009). n-3 PUFAs also quench gene expression of cyclooxygenase-2 and other enzymes, thereby diminishing the formation of proinflammatory eicosanoids. In addition, n-3 PUFAs scavenge of free radicals, which diminish inflammatory response and oxidation of lipoprotein particles, notably low-density lipoproteins. DHA also suppresses insulin/neurotrophic factor signaling deficits, neuroinflammation, and oxidative damage that may contribute synaptic loss and neuronal dysfunction in old age and demented subjects (Cole and Frautschy, 2010). Finally, DHA increases brain levels of neuroprotective brain-derived neurotrophic factor and reduces the (n-6) fatty acid arachidonate and its oxidative metabolites. The cross-talk among these molecular processes has distinct neuroprotective effects not only through the stabilization of neural membranes, modulation of ion channels and receptors, but also through inhibition of inflammatory processes and generation of anti-inflammatory lipid mediators (Farooqui et al., 2007; Farooqui, 2009). Dietary intake of ARA, EPA, and DHA results in incorporation of these PUFAs in neural membranes. EPA is readily incorporated into membrane phospholipids with a considerable amount present in mitochondrial cardiolipin. The incorporation of EPA in mitochondrial membranes is accompanied by a decrease in mitochondrial membrane potential, and increase in lipid peroxide and ROS generation (Colquhoun, 2009). DHA incorporates into ethanolamine and serine glycerophospholipids, which are located in the inner leaflet of lipid bilayer. The ethanolamine glycerophospholipids include phosphatidylethanolamine and ethanolamine and choline plasmalogens. These lipids are closely associated not only with the stability of synapse and functioning of various receptors but also with membrane fluidity and permeability (Farooqui et al., 2008), whereas DHA-enriched phosphatidylserine (PtdSer) is an essential cofactor for the activation of several proteins including, protein kinase C, Raf-1 kinase, Akt, which translocate from cytoplasm to the membrane for their activation, supporting the view that translocation of these kinases may target signaling events modulated by the DHA-mediated neuronal specific increase of PtdSer (Kim et al., 2010). PtdSer also modulates activities of diacylglycerol kinase, nitric 52 2 Recent Development on the Neurochemistry of Docosanoids oxide synthase, and Na+, K+-ATPase (Ikemoto et al., 2000; Farooqui, 2009).

Ester-linked ARA, EPA, and DHA are mobilized by PLA2 to generate free ARA, EPA, and DHA (Hirashima et al., 1992; Farooqui and Horrocks, 2007; Green et al., 2008; Farooqui, 2010b), which can act as substrates for cyclooxygenase and lipoxygenase to produce lipid mediators (eicosanoids, resolvins, and protectins/neuroprotectins) (Nieves and Moreno, 2006; Cherian, 2007; Farooqui and Horrocks, 2007). Compared to DHA, levels of EPA in brain tissue are quite low. This may be due to rapid b-oxidation of EPA following its uptake by the brain tissue (Chen et al., 2009a). In rat hepatocytes L-carnitine, a long chain fatty acid mitochondrial matrix transporter, not only increases b-oxidation of EPA, but only marginally elevates the oxidation of ARA and alleviates competitive inhibition of ARA-dependent PGE2 synthesis and COX-2 expression by EPA. It is suggested that L-carnitine modulates the competition between ARA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA. This suggests that the beneficial effects of n-3 PUFA, especially EPA, are modulated by the cellular oxidation capacity (Du et al., 2010). Supplementation of diet containing EPA not only antagonizes the synthesis of

PGE2 from ARA, but also reduces IL-1b-mediated increase in levels of PGE2, elevation in corticosterone, and upregulation in nerve growth factor expression in olfactory bulbectomized rat model of depression (Song et al., 2004). Furthermore, IL-1b-mediated suppression of the antiinflammatory cytokine IL-10 is also blocked by the ethyl-EPA treatment. Collectively, these results indicate that ethyl-EPA treatment has beneficial effects on neurotraumatic, neurodegenerative, and neuropsychiatric (cognitive and affective disorders) in which inflammation and oxidative stress play a critical role (Song et al., 2004, 2009; Farooqui, 2009).

2.3 EPA-Derived Lipid Mediators in the Brain

The lipid mediators derived from EPA include 3-series of prostaglandins and thromboxanes, 5-series of leukotrienes, and E series resolvins (Resolvin E1 or RvE1) (Serhan et al., 2000, 2002). The oxidized metabolites of EPA possess antiinflammatory and antiproliferative effects (Table 2.1). The oxidation of EPA by COX and LOX enzymes results in the production of 3-series of prostaglandins and thromboxanes and the 5-series of leukotrienes (Fig. 2.2). These eicosanoids have different biological properties than the corresponding analogs generated by the oxidation of

ARA. For example, TXA3 is less active than TXA2 in aggregating platelets and constricting blood vessels (Calder and Grimble, 2002; Calder, 2009). In addition to generating less active lipid mediator, EPA exerts its effects on other aspects of inflammation like leukocyte chemotaxis and inflammatory cytokine production. Some of these effects are likely due to changes in nuclear factor-kB-mediated gene expression, e.g. adhesion molecule in microglia, astrocytes, and in visceral inflammatory and immune cells. In contrast, recent studies on the effects of prostaglandins (PGE2 and PGE3) and leukotrienes (LTB4 and LTB5) on endothelium permeability and mononuclear adhesion and migration across endothelial cell 2.3 EPA-Derived Lipid Mediators in the Brain 53

Table 2.1 Activities of resolvins and protectins/neuroprotections/maresin in neural and non- neural systems Parameter Resolvins and protectins Reference Aggregation Antiaggregatory Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009 Thrombotic activity Antithrombotic Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009 Inflammation Antiinflammatory Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009 Lipid status Hypolipidemic Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009 Cell death nature Antiapoptotic Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009 Effect on excitotoxicity Antiexcitotoxic Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009 Pain sensitivity Antinociceptive Serhan et al., 2000, 2002, 2009; Serhan, 2005; Farooqui, 2009

Diet

EPA

PLA2? J3-IsoP F3-IsoP

(Antiinflammatory) RvE2 Free EPA RvE1 (Antiinflammatory) COX TXA3 LTB5 5-LOX Proinflam- Proinflam- LT (5-series) PGE matory LTC5 PG (3-series) 3 matory

LTD5 15-HPEPE 12-HPEPE PGI3

15-HEPE 12-HEPE

Activation of Nrf2

Fig. 2.2 Generation of EPA-derived lipid mediators in neural and non-neural tissues. Phospholipase

A2 (PLA2); 5-lipoxygenase (5-LOX); resolving E1 (RvE1); resolving E2 (RvE2); 3-series of prostaglandins and thromboxanes (PGE3 and PGI3); 5-series of leukotrienes (LTB5, LTC5, and LTD5); 15-hydroperoxy-5,8,11,13,15-eicosapentaenoic acid (15-HPEPE); 12-hydroperoxy-5,8,11,13, 12-eicosapentaenoic acid (12-HPEPE); 12-hydroxy-5,8,10,14-eicosatetraenoic acid (15-HETE), and 15-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE); cyclopentenone-IsoP molecules- derived from EPA (J3-IsoP); F-ring IsoP-like compounds derived from EPA (F3-IsoPs); and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) 54 2 Recent Development on the Neurochemistry of Docosanoids

cultures indicate that PGE3 produces more pronounced effect on trans-endothelial

Evans Blue-albumin (EBA) permeability than PGE2, and these effects are antago- nized by EP1 and EP2 antagonists (Moreno, 2009). LTB4 and LTB5 produce a slight effect on EBA extravasation. Collective evidence suggests that the enhancement of endothelial permeability in the presence of polymorphonuclear (PMN) cells is modulated by interplay between leukotrienes and prostaglandins (Moreno, 2009; Yin et al., 2007). n-3 PUFAs also decrease the production of the classic inflammatory cytokines tumor necrosis factor, interleukin-1, and interleukin-6 and the expression of adhesion molecules involved in inflammatory interactions between leukocytes and endothelial cells. These latter effects may occur by eicosanoid-independent mechanisms, including modulation of the activation of transcription factors involved in inflammatory processes. The anti-inflammatory actions of long chain n-3 fatty acid-induced effects may be of therapeutic use in conditions with an acute or chronic inflammatory component. EPA and ARA compete for the same COX enzymes (Zhao et al., 2004; Phillis et al., 2006), but the rate of oxidation of EPA is only 10% of the ARA. Although EPA significantly inhibits COX-1-mediated oxidation of ARA (Wada et al., 2007; Schmitz and Ecker, 2008), the oxidation of ARA by COX-2 is only modestly inhibited by EPA. EPA-derived eicosanoids antagonize the pro-inflammatory effects of ARA-derived eicosanoids. EPA and ARA are ligands/modulators for the nuclear receptors NF-kB, PPAR and SREBP-1c, which modulate various genes of inflammatory signaling and lipid metabolism. In addition, the n-3/n-6 PUFA ratio of neural membranes and microdomains strongly influences membrane function and numerous cellular processes such as cell death and survival (Wada et al., 2007; Schmitz and Ecker, 2008; Yin et al., 2007). EPA is a precursor for resolvins E series (Arita et al., 2006, 2007), including resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) and resolvin E2 (15S,18R-dihydroxy-EPE) (Fig. 2.3). EPA is oxidized to 18R-hydroxyeicosapentaenoic acid (18R-HEPE) by endothelial cell cyclooxygenase-2 (COX-2). Aspirin acetylates COX-2 and the acetylated enzyme no longer produces prostaglandins, but can still convert EPA to 18R-HEPE. During cell–cell interactions, 18R-HEPE is released to neighboring leukocytes for subsequent conversion by 5-LOX to RvE1 via a 5(6) epoxide-containing intermediate. RvE1 is present in human whole blood, and its levels can be increased by ingestion of aspirin

(Serhan et al., 2000; Arita et al., 2006, 2007). RvE1 is transformed into several metabolic products, including 20-hydroxy-RvE1, 20-carboxy-RvE1, 19-hydroxy-RvE1,

18-oxo-RvE1 and 10,11-dihydro-RvE1 by human PMNs and whole blood as well as in murine inflammatory exudates, lungs, spleen, kidney, and liver (Seki et al., 2009,

2010). Among these products, 20-carboxy-RvE1, 18-oxo-RvE1, and 10,11-dihydro-

RvE1are essentially biologically inactive and may serve as inactive biomarkers of

RvE1 metabolism in vivo. In contrast, 20-hydroxylated product of RvE1 has some of the activity of RvE1 suggesting that more metabolites of RvE1 are generated during inflammatory response.

RvE1 and RvE2 produce potent anti-inflammation/pro-resolution effects in vivo

(Arita et al., 2006) via specific G protein-coupled receptors (see below). RvE1 suppresses the activation of NF-kB by tumor necrosis factor-a (TNF-a) through 2.3 EPA-Derived Lipid Mediators in the Brain 55

OOH OH

COOH EPA 5-LO HO 18-hydropxy-EPE 5S-hydroperoxy-18-hydroxy-EPE

HOOC Reduction OH O COOH

COOH 5S-(6)-epoxy- Enzymatic epoxidation 18-HEPE Resolvin E2 HO HO OH

Resolvin E1

HO OH

COOH

Suppression of NF-kB Modulation of Akt phosphorylation Prevention of pain & Inflammation, promotion of resolution

Fig. 2.3 Synthesis of RvE1 and RvE2 from EPA and proposed roles of RvE1 in brain. Dietary EPA is converted to RvE1 by the action of 5-lipoxygenase (5-LOX). After aspirin treatment, RvE1 synthesis occurs even in the absence of inflammation. Aspirin inactivates COX-2 but allows the synthesis of the intermediate 18R-hydroxy-EPA which is converted to RvE1 through the action of 5-LOX.

In nonneural cells, RvE1 interacts with ChemR23 receptor (a specific seven-transmembrane G protein–coupled receptor) and inhibits inflammation by down-regulating NF-kB activation and blocking the generation of proinflammatory mediators (Adapted from Serhan et al., 2008) binding to human polymorphonuclear leukocyte (PMN) (Arita et al., 2007).

Intrathecal RvE1 injection blocks spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and TNF-a. RvE1 mediates its anti-inflammatory activity not only by decreasing neutrophil infiltration, paw edema, but also by inhibiting the expression of proinflammatory cytokine (Xu et al., 2010). In addition, RvE1 not only abolishes transient receptor potential vanilloid subtype-1 (TRPV1)- and TNF-a-mediated excitatory postsynaptic current increases, but inhibits TNF-a-mediated N-methyl-d-aspartic acid (NMDA) receptor hyperactivity in spinal dorsal horn neurons via inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway (Xu et al., 2010). It is suggested that resolvins may normalize the spinal synaptic plasticity associated with pain hypersensitivity (Fig. 2.4). In addition, resolving E1 also prolongs the survival of solid organ transplants (Levy et al., 2010). 56 2 Recent Development on the Neurochemistry of Docosanoids

Series D and E resolvins

Inhibition of Inhibition of nociception hypersensitivity

Inhibition of PMN Modulation of infiltration Synaptic plasticity Inhibition of inflammation

Fig. 2.4 Roles of resolvins in neural and non-neural tissues

RvE1 mediates its actions through specific seven-membrane spanning G protein- coupled receptors, which are expressed on dendritic cells and monocytes. These receptors are called as ChemR23 receptors (Ohira et al., 2010). RvE1 modulates the phosphorylation of Akt in a time - and dose-dependent manner in human ChemR23- transfected Chinese hamster ovary cells (Ohira et al., 2010). RvE1 not only stimulates the phosphorylation of Akt, but also phosphorylates a downstream 30-kDa protein, which is identified as a ribosomal protein S6. The phosphorylation is blocked by a phosphatidylinositol 3-kinase (PtdIns 3K) inhibitor (wortmannin) and an ERK inhibitor (PD98059) but not by a p38-MAPK inhibitor (SB203580). Recent studies on RvE1 metabolome indicate that RvE1 is metabolized to several novel products by human polymorphonuclear leukocytes and whole blood as well as in murine inflammatory exudates, spleen, kidney, and liver. The new RvE1-derived products include 19-hydroxy-RvE1, 20-carboxy-RvE1, and 10,11-dihydro-RvE1.

The metabolomic profiles of RvE1 were species-, tissue-, and cell type-specific

(Hong et al., 2008). Direct comparisons between RvE1 and RvE1-derived products indicate that 10,11-dihydro-RvE1, 18-oxo-RvE1, and 20-carboxy-RvE1 display reduced bioactivity in vivo. At concentrations as low as 1 nM, RvE1 stimulates macrophage phagocytosis. In addition, RvE1 enhances phagocytosis of zymosan A by human macrophages, which are inhibited by PD98059 and rapamycin (mTOR inhibitor). Collectively, these studies indicate that RvE1 initiates direct activation of ChemR23 and signals receptor-dependent phosphorylation. These phosphorylation- signaling pathways identified for RvE1 receptor–ligand interactions emphasize the importance of endogenous pro-resolving agonists in resolving acute inflammation (Ohira et al., 2010). + The oxidation of RvE1 by NAD -dependent hydroxyprostaglandin dehydrogenase to the oxo product (18-oxo-RvE1) results in inactivation of RvE1 activity. Based on detailed pharmacological investigations on the effect of RvE1, it is suggested that 2.3 EPA-Derived Lipid Mediators in the Brain 57

the binding of RvE1 to BLT1 receptor mediate the resolution of inflammation (Arita et al., 2006, 2007). RvE2 is synthesized from EPA by human PMNs and produces similar actions as RvE1. When given together, the protective actions of RvE1 and

RvE2 are additive at low doses, suggesting distinct receptors for RvE2 and RvE1

(Tjonahen et al., 2006). Collective evidence suggests that both RvE1 and RvE2 contribute to the beneficial actions of EPA in human diseases. Rv E1 and RvE2 dramatically reduce dermal inflammation, peritonitis, dendritic cell migration, and interleukin production.

Similarly, in experimental model of pneumonia in mice, RvE1 significantly decreases lung tissue levels of several proinflammatory chemokines and cytokines, including IL-1b, IL-6, HMGB-1, MIP-1a, MIP-1b, keratinocyte-derived chemokine, and MCP-1, in a manner independent of the anti-inflammatory mediators IL-10 and lipoxin A4. In addition, animals treated with RvE1 show a marked improvement in survival suggesting that RvE1 administration may represent a novel therapeutic strategy for acute lung injury and pneumonia (Seki et al., 2010).

Non-enzymic oxidation of EPA generates cyclopentenone-IsoP (A3/J3-IsoPs)

(Brooks et al., 2008). Like ARA-derived IsoPs, the A3/J3-IsoPs are formed in situ esterified in phospholipids and are then released in the free form by a PAF hydrolase like PLA2. It is suggested that A3/J3-IsoPs are generated after rearrangement of their endoperoxides precursors to molecules with E- and d-prostane rings that subsequently dehydrate. The oxidation products of EPA activate the Nrf2 transcription factor (a member of the basic leucine-zipper NF-E2 family) that induces antioxidant responses in reactive electrophiles-mediated induction of gene transcription (Gao et al., 2006, 2007). These genes include glutathione S-transferase, NADPH:quinone oxidoreductase 1, UDP-glucuronosyltransferase, gamma-glutamate cysteine ligase, and hemeoxygenase-1. They mediate detoxification and/or to exert antioxidant functions, thereby, protecting cells from genotoxic damage. The non-enzymic products of EPA interact with the ubiquitin ligase adaptor protein Keap1 (a BTB-Kelch protein), the direct inhibitor of Nrf2, initiating Keap1 dissociation from Cullin3 to yield Nrf2 translocation and activation causing induction of Nrf2-directed gene expression. Binding of Keap1 to Nrf2 directs the ubiquitination and proteasome- dependent degradation of Nrf2. Inhibition of Nrf2 ubiquitination results not only in Nrf2 accumulation, but also in increased ARE-directed gene expression, and an enhanced ability to respond to oxidant stress (McMahon et al., 2003). It is proposed that molecules containing an a,b-unsaturated carbonyl moiety, specifically J3-IsoPs, may be responsible for eliciting this biological activity (Gao et al., 2007). Thus, both enzymic and non-enzymic oxidation products of EPA modulate signal transduction processes associated with anti-inflammatory responses in neural and non-neural systems. In addition, EPA is also metabolized by cytochrome P450 (CYP2C/2J)-isoforms, which convert EPA into 17,18-epoxyeicosatetraenoic (17,18-EEQ) acid. These omega-3 epoxides are highly active as antiarrhythmic agents. They suppress Ca2+- induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations (Arnold et al., 2010). Dietary EPA/DHA supplementation of rats results in substantial replacement of ARA by EPA in membranes from 58 2 Recent Development on the Neurochemistry of Docosanoids heart, kidney, liver, lung, and pancreas, with less pronounced effects in brain. Changes in fatty acids are accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the epoxyeicosatrienoic acids (EETs):17, 18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) ratio from 87:0:13 to 27:18:55 in the heart). Collectively, these studies demonstrate that CYP-enzymes efficiently convert EPA to novel epoxy- and hydroxy-metabolites that may mediate some of the beneficial cardiovascular effects of dietary omega-3 fatty acids (Arnold et al., 2010).

2.4 DHA-Derived Lipid Mediators in the Brain

DHA is a precursor of D-series resolvins. The action of 15-LOX-like enzyme converts endogenous DHA into 17S-hydroperoxy-DHA (17S-H(p)DHA). This biosynthetic intermediate is transformed into several bioactive compounds, including resolvin

D1-D6 (RvD1, RvD2, RvD3, RvD4, RvD5, and RvD6) (Figs. 2.5 and 2.6). There are

Diet

DHA-PlsEtn

(Proinflammatory)( Acyltransferase 2 Acyl-CoA NP NF NKNK 4-HHE4-HHE

Lyso-PlsEtn Non-enzymic PlsEtn-PLA oxidation

15-LOX 15-LOX ProtectinsProtectins ResolvinResolvinss (docosatriens)(docosatriens) DHADHA (RvD seies)seies) (Anti-inflammatory) (Anti-inflammatory)

RvD1 RvD2 RvD3 RvD4 RvD5 RvD6

Fig. 2.5 Generation of enzymic and non-enzymic lipid mediators from DHA. Plasmalogen

(PlsEtn); Plasmalogen-selective PLA2 (PlsEtn-PLA2); lyso-ethanolamine plasmalogen (lyso- PlsEtn); neuroprostane (NP); neurofuran (NF); neuroketal (NK); 4-hydroxyhexenal (4-HHE); Resolvin D (RvD) 2.4 DHA-Derived Lipid Mediators in the Brain 59

Phospholipids containing DHA

PlsEtn-PLA2

DHA Asp:COX-2

AT-protectin D1 17R-hydroperoxy-DHA 4S-hydroperoxy-17R-hydroxy-DHA

7S-hydroperoxy-17R-hydroxy-DHA 4S(5)-epoxy-17R-hydroxy-DHA

7S(8)-epoxy-17R-hydroxy-DHA

COOH OH OH HOOC HO COOH OH OH COOH OH OH OH OH HO HO HO

Aspirin Aspirin Aspirin Aspirin triggered resolvin D 1 triggered resolvin D2 triggered resolvin D3 triggered resolvin D4 AT-RvD 1 AT-RvD2 AT-RvD3 AT-RvD4

Suppression of PMN infiltration Downregulation of NF-KB Promotion of resolution Inhibition of apoptosis Neuroprotection following ischemia

Fig. 2.6 Generation of resolvins D series from DHA in non-neural cells. DHA is released from

PtsEtn by plasmalogen-selective PLA2. Free DHA is metabolized in aspirin-COX-2 dependent manner into ATRvD1, ATRvD2, ATRvD3, and ATRvD4 through 7S(8)-epoxy-17R-hydroxy-DHA formation. Similar synthesis may occur in neural cells (Adapted from Serhan, 2005; Serhan et al., 2008) also aspirin-triggered forms of D series resolvins (AT-Rv), which are also enzymically derived from DHA through a pathway with sequential oxygenation initiated by aspirin-acetylated COX-2 (Serhan and Chiang, 2008). For the biosynthesis of AT-RvDs, DHA is initially transformed into 17R-hydroxy-DHA. The stereochemistry at carbon 17 is maintained, so each AT-RvD carries a 17R alcohol group configuration, resulting in a distinct series of compounds. The complete stereochemistry of

RvD1 (7S,8R,17S,-trihydroxy-4Z,9E,11E,13Z,15E,9Z-docosahexaenoic acid) and 60 2 Recent Development on the Neurochemistry of Docosanoids

AT-RvD1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid has also been studied) (Serhan, 2005; Ariel and Serhan, 2007). These lipid mediators not only antagonize the effects of eicosanoids, but also modulate leukocyte trafficking and down-regulate the expression of cytokines in glial cells. They possess potent anti-inflammatory, neuroprotective and pro-resolving properties (Hong et al., 2003; Marcheselli et al. 2003; Serhan, 2005). Biosynthesis of neuroprotectin D1 (NPD1) involves the release of DHA by PlsEtn-PLA2 or from PtdSer by Ptdser-selective PLA2 or cPLA2. Free DHA undergoes lipoxygenation and epoxidation followed by hydrolysis to form NPD1. Thus far, binding sites for

NPD1 have been identified in retinal pigment epithelium cells and polymorphonuclear cells (Niemoller and Bazan, 2010) (Fig. 2.7). Like DHA, docosapentaenoic acid n-6 (DPAn-6; 22:5) also generates resolvins called 17S-HDPAn-6 (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid) and 10S,17S-HDPAn-6 (10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E-pentaenoic acid). These resolvins like DHA-derived D series resolvins produce potent anti-inflammatory effects (Dangi et al., 2010). However, DPA-derived metabolites are not strong inhibitors of cytochrome-P450 enzymes. In brain tissue, initiation, intensification, and resolution of inflammation requires a distinct micro-environments composed of astrocytes, microglia endothelial cells, and highly specialized extracellular matrix (ECM) components of neural cells. At the conclusion of the inflammatory response, above neural cells also contribute to the process resolution by (a) the withdrawal of survival signals, (b) the normalization of chemokine gradients, and (c) the synthesis of lipoxins, resolvins and neuroprotectins inducing resolution programs that allow infiltrating cells to undergo apoptosis. The dysregulation of lipoxin, resolvin, and neuroprotectin may lead to persistent chronic inflammation, which can be remarkably stable.

2.4.1 17S D Series Resolvins

Resolvins act through specific receptors found in neural and non-neural cells. They are called as resolvin D receptors (resoDR1) (Serhan et al., 2006a, b; Serhan et al.

2008a, b). ResoDR1 induce potent anti-inflammatory and immunoregulatory activities. D series resolvin not only block the production of pro-inflammatory mediators, but also regulate the trafficking of leukocytes cells at the sites of inflammation (Serhan et al., 2008; Hong et al., 2003) and inhibit the expression of cytokines and modulate inflammation. Studies on characterization of ResoDR1 are currently in progress.

2.4.2 Protectins and Neuroprotectin

15-LOX converts DHA into protectin D1 through epoxide intermediate with epoxy group at the 16(17) position. The synthesis of PD1 with complete stereochemistry 2.4 DHA-Derived Lipid Mediators in the Brain 61

PhospholipidsPhospholipids containing DHA DHA

cPLA2 PlsEtn-PLA2

DHA

15-LOX H(O)O

COOH

17S-hydroperoxy-DHA O2 Second oxygenation Reduction

Reduction HO

10S, 17S-dihydroxy-DHA COOH O

COOH

17S-hydroxy-DHA 16(17)-epoxy-DHA

Enzymic hydrolysis

OH COOH

Neuroprotectin D1 OH

AntiapoptoticAntiapoptotic Bcl-2Bcl-2 proteins proteins Anti-inflammatory Inhibition of NF-KB Suppression of PMN infiltration

Fig. 2.7 Generation of neuroprotection D1 from DHA in brain. DHA is hydrolyzed from PlsEtn by PlsEtn-PLA2. It undergoes lipoxygenation, epoxygenation and hydrolysis to generate NPD1 (Adapted from Serhan et al., 2008 and Marcheselli et al., 2010) 62 2 Recent Development on the Neurochemistry of Docosanoids

Diet

EPAEPA ARAARA DHADHA

Aspirin COX-2 COX-2 COX-2 5-LOX 15-LOX Aspirin 15-LOX 5-LOX COX-2 3-series PGs RvERvE 3-series PGs 1 1 −- −- 3-Series3-series TXs TXs RvD1, RvD2, RvD3 RvERvE2 RvD1, RvD2, RvD3 AT-RvD1-D4 NPD1 2 5-Series5-series LTs LTs RvD , RvD , RvD AT-RvD1-D4 NPD1 RvD44, RvD5,5 RvD6 6 −- −- −-

HighHigh levelslevels of 2-2-serieseriess PGs,TX2,PGs,TX2, 4-series 4-series LTsLTs

OxidativeOxidative stress InflammatioInflammationn −- −-

ApoptosisApoptosis

Fig. 2.8 Regulation of inflammation, oxidative stress, and apoptotic cell death by resolvins and neuroprotections

of PD1 has been established and confirmed by total organic synthesis, showing that PD1 is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (Serhan et al., 2000, 2002, 2006b; Hong et al., 2003; Marcheselli et al., 2003). The reaction sequence of biosynthesis for PD1 via the epoxide intermediate distinguishes it from the formation of the double dioxygenation product 10S,17S-dihydroxy-

DHA. PD1 is more potent than DHA in neuroprotective action. In sharp contrast,

PD1 positional isomers, including 4S,17S-diHDHA or 7S,17S-diHDHA, display less potent and non-selective actions. The occurrence of PD1 has also been reported in brain, where it is called as neuroprotectin D1 (10R, 17S-dihydroxy-docosa-

4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid, NPD1) (Hong et al., 2003). Tritium- 3 labeled NPD1 (H]-NPD1) binds to ARPE-19 cells with high-affinity d(K

31.3 ± 13.1 pmol/mg of cell protein). The stereospecific NPD1 interactions with these cells provide potent protection against oxidative stress-mediated apoptosis 3 (Fig. 2.8). H-NPD1/PD1 also shows specific and selective high affinity binding with isolated human neutrophils (Kd approximately 25 nM). Neither resolvin E1 nor 3 lipoxin A4 compete for H-NPD1/PD1 specific binding with human neutrophils.

Collectively, these results indicate that stereoselective specific binding of NPD1/PD1 with retinal pigment epithelial cells as well as human neutrophils may occur through specific receptors in both the immune and visual systems (Marcheselli et al., 2010). Although the isolation and characterization of 10(S),17(S)-dihydroxy-docosahexa- 4Z,7Z,11E,13Z,15E,19Z-enoic acid, a main dihydroxy conjugated triene derived 2.4 DHA-Derived Lipid Mediators in the Brain 63 from the lipoxygenation of DHA has also been reported, but nothing has been reported on its interactions with neural and non-neural cells. It is an isomer of protectin/neuroprotectin D1 (PD1/NPD1) and has been named PDX (Chen et al., 2009b). This metabolite inhibits human blood platelet aggregation at submicromolar concentrations. Like resolvins, the neuroprotectins block the infiltration of PMN (Serhan et al., 2006b) (Figs. 2.4 and 2.7). They down-regulate the expression of cytokines in the glial cells (Hong et al., 2003; Serhan et al., 2006b). NPD1 reduces retinal and corneal injury (Mukherjee et al., 2004; Gronert et al., 2005) and produces neuroprotective effect in ischemic injury (Marcheselli et al., 2003). Similarly, NPD1 promotes neural cell survival via the induction of antiapoptotic and neuroprotective gene- expression programs that suppress Ab42-mediated neurotoxicity in Alzheimer disease (AD) (Lukiw et al., 2005; Bazan, 2009a, b). DHA and NPD1 protect synapses and decrease the number of activated microglia in the hippocampal system (Pomponi et al., 2008). Although the molecular mechanisms associated with the above processes are not fully understood. However, it is becoming increasingly evident that

NPD1 not only inhibits IL-1b-stimulated expression of COX-2, but also regulates apoptotic signaling at the level of mitochondria, inducing the release of cytochrome c and activating effector enzyme, caspase-3 (Table 2.2). In addition in rat-infused with Ab, DHA and its oxidative metabolites attenuate elevation in levels of lipid peroxides and ROS in the cerebral cortex and the hippocampus, indicating that DHA and its metabolites facilitate neuroprotection by down-regulating g-secretase activity, an enzyme that liberates Ab from soluble amyloid precursor protein-a (Lukiw et al., 2005). Furthermore, soluble amyloid precursor protein-a stimulates the synthesis of NPD1 (Lukiw et al., 2005; Bazan, 2009a, b). Activities of key PLA2

(PlsEtn-PLA2 and cPLA2) and 15-LOX, enzymes required for NPD1 biosynthesis, are markedly increased in the AD hippocampal CA1 region (Farooqui et al., 1997, 2006; Farooqui, 2010b; Bazan, 2009a). Collective evidence suggests that DHA and its oxidative metabolites limit the generation and accumulation of the Ab peptide, which is closely associated with the pathogenesis of AD. DHA and its metabolites also suppress several signal transduction pathways induced by Ab, including two major kinases that phosphorylate the microtubule-associated protein tau and promote neurofibrillary tangle pathology (Farooqui, 2009). It is recently shown that in macrophages DHA is also metabolized through a 14-LOX pathway resulting in generation of 7,14-dihydroxydocosa-4Z,8,10,12, 16Z,19Z-hexaenoic acid. This metabolite is called as maresin (MaR1) (Fig. 2.9). This metabolite not only terminates PMN infiltration, but also stimulates macrophage phagocytosis. An isomer of MaR1, 7S,14S-diHDHA, acts less potently than MaR1. This suggests that these DHA-derived metabolites may be stereoselec- tively regulate catabasis and facilitate arrival of tissues to homeostasis (Serhan et al., 2009). In retina, epithelium-derived factor acts as an agonist and induces the synthesis of NPD1 and thus promoting NPD1-mediated paracrine and autocrine signal transduction processes. Also, DHA and epithelium-derived factor not only synergistically activates NPD1 generation and antiapoptotic protein expression, but also 64 2 Recent Development on the Neurochemistry of Docosanoids Seki et al., 2 00 9 Seki et al., 2 00 9 et al., 2 00 7 ; Bazan, 009a, b et al., 2 00 7 ; Bazan, 009a, b Serhan et al., 2 000, 00 ; Seki 9 Serhan et al., 2 000, 00 ; Seki 01 0 Serhan et al., 2 000, 00 ; Seki 01 0 Serhan et al., 2 000, 00 ; Serhan, 8 Serhan, et al. 2 000, 00 ; Bazan, 009a, b Serhan et al., 2 000, 00 ; Serhan, 8 Serhan, et al., 2 000, 00 ; Mukherjee Reference Serhan et al., 2 000, 00 ; Mukherjee caspase-3; induction of neuroprotective gene-expression caspase-3; induction of neuroprotective caspase-3; induction of neuroprotective gene-expression caspase-3; induction of neuroprotective caspase-3; induction of neuroprotective gene-expression caspase-3; induction of neuroprotective Inhibition of proinflammatory cytokine expression cytokine Inhibition of proinflammatory expression cytokine Inhibition of proinflammatory expression cytokine Inhibition of proinflammatory Inhibition of proinflammatory cytokine expression cytokine Inhibition of proinflammatory Decrease in proapoptotic Bcl-2 expression and inhibition of Decrease in proapoptotic Bcl-2 expression expression cytokine Inhibition of proinflammatory Decrease in proapoptotic Bcl-2 expression and inhibition of Decrease in proapoptotic Bcl-2 expression Molecular mechanism Decrease in proapoptotic Bcl-2 expression and inhibition of Decrease in proapoptotic Bcl-2 expression Beneficial Beneficial Beneficial Beneficial Beneficial Beneficial Beneficial Effect of resolvin/ Effect neuroprotectin Beneficial Effects of resolvins and neuroprotectins in animal models neural non-neural diseases Effects disease degeneration Asthma Bacterial pneumonia Acute lung injury Inflammatory bowel Inflammatory bowel Inherited retinal Periodontal disease Alzheimer disease Table 2.2 Table Animal model Stroke 2.4 DHA-Derived Lipid Mediators in the Brain 65 a

COOH COOH

OH OH OH HO b

HO d COOH COOH OH

OH HO OH

Fig. 2.9 Chemical structures of docosahexaenoic acid and docosanoids. 16,17S-docosatriene (a); 10,17S-docosatriene (b); maresin (c); and 4S5,17S-resolvin (d). These metabolites inhibit inflammation by retarding the actions of eicosanoids down-regulates proapoptotic Bcl-2 protein expression and activation of caspase 3 during oxidative stress (Mukherjee et al., 2007). NPD1 also promotes AKT translocation and activation and interacts with PPAR-gamma family of ligand-activated nuclear receptors, which may be involved in various aspects of neuroinflammation and neurodegeneration (Palacios-Pelaez et al., 2010; Niemoller and Bazan, 2010;

Farooqui, 2010a, c). Receptors for NPD1 have not been characterized in brain tissue, but their occurrence has been suggested (Hong et al., 2003; Marcheselli et al., 2003;

Mukherjee et al., 2004). Thus, NPD1-mediated regulation targets upstream events of brain cell apoptosis and modulation of neuroinflammatory signaling promote the cellular homeostasis, and restoration of brain damage through above-mentioned mechanisms. This is tempting to speculate that the generation of DHA-derived resolvins, neuroprotectins, maresin, and synthesis of ARA-derived lipoxins may be internal neuroprotective mechanisms that block neuroinflammation and apoptosis- mediated brain damage caused by neurotraumatic and neurodegenerative diseases (Serhan, 2005; Bazan, 2009a, b; Farooqui, 2010b). Investigators are using lipidomics, proteomics and genomics techniques to identify and determine levels of ARA, EPA and DHA-derived lipid mediators

(F2-isoprostanes, eicosanoids, lipoxins, resolvins, and NPD1) (Serhan et al., 2006b, 66 2 Recent Development on the Neurochemistry of Docosanoids

2008; Ariel and Serhan, 2007; Brooks et al., 2008; Bazan, 2009a, b) and developing diagnostic test in cerebrospinal fluid (CSF) from patients with acute neural trauma, neurodegenerative, and neuropsychiatric diseases. The use of proteomic strategies for characterizing resolvin and neuroprotectin metabolizing enzymes in subcellular organelles of human brain and CSF may provide new information on properties and therapeutic targets. Resolvin and neuroprotectin synthesizing and catabolizing enzymes not only control the levels of these lipid mediators in normal and diseased brain, but also modulate physiology and pathology of chronic brain diseases. Collectively, these studies suggest that combining lipidomics, proteomics, and genomics techniques may greatly enhance the existing knowledge of molecular mechanism associated with homeostasis between inflammation inducing mediators (eicosanoids and platelet activating factor) and inflammation blocking lipid mediators (lipoxins, resolvins, and neuroprotectins) in neural trauma, neurodegenerative and neuropsychiatric diseases (Serhan et al., 2006b, 2008; Brooks et al., 2008; Bazan, 2009a, b; Farooqui, 2010a). Information about the temporal and spatial positioning of inflammation inducing and inflammation blocking pathways in brain may not only be dictated by anti-inflammatory drugs but also by the diet. The Western diet has extremely high levels of omega-6 fatty acids with ARA to DHA of about 20:1. The Paleolithic diet on which human beings have evolved, and lived for most of their existence, had a ratio of 2–1:1, and was high in fiber, rich in fruits, vegetables, lean meat, and fish (Simopoulos, 2002, 2008; Cordain et al., 2005). The high intake of ARA enriched food in Western diet not only elevates levels of eicosanoids and platelet activating factor, but also upregulates the expression of proinflammatory genes including genes for cytokines (TNF-a and IL-1b), secretory phospholipase A2, cyclooxygenase-2, and nitric oxide synthase . In contrast, consumption of DHA-enriched diet has anti-inflammatory effects that are partly mediated by repression of genes that code for pro-inflammatory cytokines. Since Western diet is low in DHA and high in ARA, it is linked to many chronic visceral diseases as well as neurodegenerative and neuropsychiatric disorders (Simopoulos, 2002, 2008; Cordain et al., 2005; Farooqui, 2009). Enrichment of DHA in Western diet may improve inflammation and oxidative stress in neurotraumatic and neurodegenerative diseases not only through the effects of DHA on physicochemical properties of neural cell membranes, but also through modulation of genes and generation of resolvins Ds and neuroprotectins. It is worth noting that in AD transgenic mice dietary DHA restores cerebral blood volume, reduces Ab deposition, and ameliorates Ab pathology (Green et al., 2007). Although molecular mechanisms associated with above processes are not fully understood, recent studies indicate that in 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture,

NPD1 not only down-regulates Ab42-mediated expression of the pro-inflammatory enzyme COX-2 (Fig. 2.10), but also reduces the expression of B-94, a TNF-a- inducible pro-inflammatory element resulting in inhibition of apoptosis. Moreover,

NPD1 suppresses Ab42 peptide shedding by down-regulating b-secretase-1 (BACE1) while activating the a-secretase ADAM10 and up-regulating sAPPa, thus shifting the cleavage of bAPP holoenzyme from an amyloidogenic into the non- amyloidogenic pathway (Zhao et al., 2011). It is also shown that anti-amyloidogenic 2.4 DHA-Derived Lipid Mediators in the Brain 67

Excitotoxicity Neuroprotection Glu PtdCho PlsEtn NMDA-R

+ + Ca2+ PlsEtn-PLA cPLA2 2

ARA - CO DHA + X- 2 Lyso-PtdCho /5- LO X sAPPα ROS Eicosanoids 15-LOX

PAF ADAM10 + IKB/NFKB Inflammation -

NPD1 βAPP IKB Oxidative - stress BACE1 - - - Aβ42 COX-2 NF-KB-RE - sPLA2 iNOS - Transcription of genes TNF-α Apoptosis IL-1β NUCLEUS IL-6 PPARγ

Fig. 2.10 Hypothetical diagram showing anti-inflammatory actions of neuroprotectin 1D , production of Ab from holo-bAPP, and generation of ARA-derived lipid mediators in the brain. Glutamate (Glu); N-Methyl-d-aspartate receptor (NMDA-R); phosphatidylcholine (PtdCho); ethanolamine plasmalogen (PlsEtn); cytosolic phospholipase A2 (cPLA2); arachidonic acid (ARA); reactive oxygen species (ROS); plasmalogen-selective phospholipase A2 (PlsEtn-PLA2); 15-lipoxygenase

(15-LOX); platelet activating factor (PAF); neuroprotectin D1 (NPD1); ADAM (a-secretase); peroxisome proliferator-activated receptor g (PPARg); amyloid precursor protein (APP); holo-bAPP (bAPP holoenzyme); nuclear factor kappa B (NF-kB); tumor necrosis factor-alpha (TNF-a); interleukin-1beta (IL-1-b); interleukin-6 (IL-6); inducible nitric oxide synthase (iNOS); secretory phospholipase A2 (sPLA2); cyclooxygenase-2 (COX-2)

effects of NPD1are mediated in part through activation of the PPARg receptor, while

NPD1induced stimulation of non-amyloidogenic pathways is PPARg-independent.

In summary, NPD1 potently down-regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, supporting the potential of this lipid mediator in rescu- ing human brain cells in early stages of neurodegenerations (Zhao et al., 2011). In spite of above laboratory observations, to date studies on the dietary intervention of n-3 PUFA in AD have so far failed. It is proposed that long-term DHA supplementation may not only restore signal transduction processes associated with behavioral deficits and learning activity, but also produce several neuroendocrinological and immunological effects on brain tissue, which may be beneficial in neurotraumatic, neurodegenerative, and neuropsychiatric diseases (Farooqui, 2010a, c). 68 2 Recent Development on the Neurochemistry of Docosanoids

2.5 Conclusion

EPA and DHA-derived lipid mediator resolvins and protectins/neuroprotectins regulate immune systems by modulating signal transduction processes associated with neuroinflammation and neurodegeneration. EPA-derived E-series resolvins

(i.e., RvE1 and RvE2) and DHA-derived D-series resolvins (RvD1 and RvD2) have potent anti-inflammatory and pro-resolution properties. They retard excessive inflammatory responses and promote resolution by enhancing clearance of apoptotic cells and debris from inflamed brain tissue. These actions may underlie the beneficial effects of EPA and DHA in human health and neurotraumatic and neurodegenerative diseases. Aspirin initiates resolution by triggering biosynthesis of specific epimers of resolvins and protectins. In addition to their synthesis in resolution, these lipid mediators also display potent protective roles in neural systems, liver, lung, and eye. Potent actions of resolvins and protectins in models of chronic human disease indicate that down-regulation in resolution pathways may contribute to the pathogenesis of many chronic neurodegenerative and visceral diseases.

References

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3.1 Introduction

Lysophospholipids are metabolic intermediates in glycerophospholipid metabolism. In unstimulated neural cells, lysophospholipids occur at low levels (0.5–3%). They are transiently synthesized during the remodeling of glycerophospholipids (Farooqui et al., 2000a). Lysophospholipids interact with neural membrane’s lipid and enzyme components and modulate activities of membrane-associated enzymes and growth factors. The metabolism of lysophospholipid facilitates translocation of Toll-like receptor 4 to membrane and this process regulates inflammatory responses (Jacson et al., 2008a). Neural membrane lysophospholipids include lysophosphatidylcho- lines (lyso-PtdCho), lysophosphatidylethanolamines (lyso-PtdEtn), lysohosphati- dylserines (lyso-PtdSer), lysophosphatidylinositols (lyso-PtdIns), lysocardiolipin

(Lyso-Ptd2Gro), lysoplasmalogens (lyso-PlsEtn and lyso-PlsCho), and lysophosphatidic acid (lyso-PtdH) (Fig. 3.1). Many lysophospholipids are nonbilayer- forming lipids, which at low concentration form micelles, but at higher concentration, they tend to form cylindrical hexagonal phases (Fuller and Rand, 2001). At high concentrations, lysophospholipids also alter membrane permeability, and disturb osmotic equilibrium. In lipid bilayers, lysophospholipids modulate the gating kinetics of several membrane channels including gramicidin and TRPC5 calcium channels (Lundbaek and Andersen, 1994; Flemming et al., 2005). Lysophospholipids stabilize dimer formation between two peptide helices in apposing monolayers, forming an open channel. In neural cells, lysophospholipids are produced through the action of phospholipases A1 (PLA1), phospholipases A2 (PLA2), and plasmalogen-selective phospholipase A2 (PlsEtn-PLA2) on PtdCho, PtdEtn, PtdSer, and plasmalogens (Farooqui and Horrocks, 2004, 2007). Lysophospholipids are quantified and separated from native phospholipids by high-performance liquid chromatography, liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS), and electrospray ionization mass spectrometry (Lesnefsky et al., 2000; Han et al., 2001; Taguchi, 2009). Other lysophospholipids such as lyso-phosphatidic acid

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 73 DOI 10.1007/978-1-4419-9940-5_3, © Springer Science+Business Media, LLC 2011 74 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids… abO H C O C R 2 H2C O CH CH R

HO C H HO C H O O OOCH CH NH H2C P 2 2 2 H C O P O CH CH NH - 2 2 2 2 O O- cdO

H2C O C R H2C OCH CH R HO C H CH3 HO C H O O CH3 + + H C O P O CH2 CH2 N CH3 2 H C OPO CH2 CH2 N CH3 - 2 O CH - 3 O CH3 efO O H C OCR 2 H2C OCR

HO C H HO C H O O - OPOH H2C OPOCH2 CH COO H2C - + O O- NH3

Fig. 3.1 Chemical structures of various lysophospholipids. Lyso-phosphatidylethanolamine (a); lyso- plasmenyethanolamine (b); lyso-phosphatidylcholine (c); lyso-choline plasmalogen (d); and lyso-phosphatidylserine (e); and lyso-phosphatidic acid (f)

(lyso-PtdH) and lyso-platelet-activating factor (lyso-PAF) are also generated in mammalian brain. Their metabolism has been described elsewhere (Farooqui and Horrocks, 2007).

3.2 Deacylation/Reacylation Cycle (Land’s Cycle) and Its Importance

Deacylation/reacylation cycle (Land’s cycle) is an important mechanism that introduces polyunsaturated fatty acids into neural membrane phospholipids by effectively bypassing the de novo synthesis of the entire phospholipid molecule. It plays a critical role in regulating availability of ARA for eicosanoid production. The deacylation arm of Land’s cycle involves phospholipase A2 (PLA2) activity, whereas reacylation arm utilizes acyl-CoA synthetase, acyl-CoA: lysophospholipid acyltransferase, and acyl-CoA hydrolase activities. These enzymes occur in multiple forms and have been purified and characterized from neural and non-neural tissues 3.2 Deacylation/Reacylation Cycle (Land’s Cycle) and Its Importance 75

(Sun and MacQuarrie, 1989; Farooqui et al., 2000a). In the deacylation/reacylation cycle, multiple forms of acyltransferases and PLA2 activities control glycerophospholipid composition and the availability of free ARA and DHA (Farooqui et al., 2000a, b). Lysophosphatidylcholine acyltransferases modulate inflammatory responses to lipopolysaccharide and other microbial stimuli (Jackson et al., 2008a, b). Specific inhibition of lysophosphatidylcholine acyltransferase downregulates inflammatory cytokine production in monocytes and epithelial cells by preventing translocation of Toll-like receptor (TLR4) into membrane lipid raft domains, indicating the existence of new regulatory mechanisms that not only facilitate the innate immune responses to microbial molecular patterns, but also provide a basis for the anti- inflammatory activity observed in many glycerophospholipid-derived metabolites (Jackson et al., 2008a, b; Farooqui, 2009). Lysophospholipid acyltransferases,

Phospholipipases A2, C, and D (PLA2, PLC, and PLD), cyclooxygenases, and lipoxygenases are key enzymes that regulate cellular responses to a variety of stimuli through the generation of lipid mediators that modulate oxidative stress and inflammation in brain (Farooqui et al., 2000a, b; Farooqui, 2009). Regulation or manipulation of lysophospholipid acyltransferases, PLA2, PLC, and PLD, cyclooxygenases, and lipoxygenases may thus provide important mechanisms for novel antioxidant and anti-inflammatory therapies for neurotraumatic and neurodegenerative diseases (Jackson et al., 2008b; Farooqui, 2009, 2010a). Collectively, these studies suggest that in neural membranes, the deacylation--reacylation cycle maintains a balance between free and esterified fatty acids, resulting in low levels of arachidonic acid and lysophospholipids. In addition, deacylation--reacylation cycle is necessary for not only normal membrane integrity and function, but also for the optimal activity of the membrane-bound enzymes, receptors, and ion channels involved in normal signal-transduction processes.

3.2.1 Acyl-CoA Synthetases in the Brain

Enzymes catalyzing the thioesterification of fatty acids with coenzymeA and generating activated acyl-CoA derivatives are called as acyl-CoA synthetases (ACS). These enzymes not only play a fundamental role in lipid metabolism, but also contribute to lipid homeostasis (Fig. 3.2). The products of the ACS enzyme reaction (acyl-CoAs) are required for complex lipid synthesis, energy production via b-oxidation, protein acylation and fatty-acid dependent transcriptional regulation (Farooqui et al., 2000a). Activation of fatty acids is a two-step process generating an acyl-AMP intermediate in the first step, and in the second step the enzyme facilitates the exchange of AMP with CoA to produce the activated acyl-CoA. Limited information is available on ACS in brain (Soupene and Kuypers, 2008). Some isoforms of ACS are involved in the activation of short-chain fatty acids while others are associated with the activation of long-chain fatty acids. The activation of ARA and DHA involves long-chain acyl-CoA synthetases (ACSL) in mammalian brain. The incubation of [1-14C]ARA with brain microsomes in the presence of ATP, CoA, and 76 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids…

Ca2+ Extracellular A R Phospho lipid s Ca2+ Phospho lipids phosphotransferase Intracellular ARA-CoA Lyso- PLA phospholipid 1 DAG Arachidonyl-CoA acyltrarnsferase synthase Acyl-CoA hydrolase PLA2 ARA Free fatty acid

1-acyl-lysophospholipid 2-acyl-lysophospholipid

Lysophospholipase Lysophospholipase

COX LOX EPOX Glycerophosphodiester CTP Phosphodiesterase

Phosphobase CDP-base PGs, LTs, TXs, CTP: cytidylyltransferase LXs, EETs Base Kinase Phosphatase

Base

Fig. 3.2 Acylation/deacylation cycle for the incorporation and release of arachidonic acid in the brain. Agonist (A); Receptor (R); arachidonic acid (ARA); arachidonyl-Coenzyme A (ARA-CoA); phospholipase A2 (PLA2); phospholipase A1 (PLA1); prostaglandins (PGs); Leukotrienes (LTs); thromboxanes (TXs); lipoxins (LXs); epoxyeicosatetraenoic acids (EETs); diacylglycerol (DAG); cyclooxygenase (COX); lipoxygenase (LOX); epoxygenase (EPOX); and acyl-CoA hydrolase

14 MgCl2 results in the formation of [1- C]arachidonyl-CoA. The omission of ATP or CoA results in a 98% decrease in enzymic activity, indicating the absolute requirement of ATP and CoA for the acyl-CoA synthetase reaction (Reddy et al., 1984; Reddy and Bazan, 1984). The addition of unlabeled ARA and DHA inhibits the enzymic activity with Ki values of 31 mM for both fatty acids. Based on various kinetic parameters and effect of inhibitors, it is suggested that a single acyl-CoA synthetase may be associated with the activation of long-chain fatty acids. In contrast, other investigators have reported the occurrence of several isoforms of long- chain acyl-CoA synthetase, namely ACSL1, ACSL2, ACSL3, ACSL4, ACSL5, and ACSL6 in brain tissue (Marszalek et al., 2005; Van Horn et al., 2005). Among above isoforms, ACSL3 and ACSL6 are the predominant ACSL isoforms in brain. These isoforms have been cloned from rat brain. In a direct competition assay with palmitic acid, all the polyunsaturated fatty acids competitively retard the activation of palmitic acid. Upregulation of ACSL results in rapid synthesis of arachidonyl-CoA and docosahexaenoyl-CoA in brain. Intracellular concentrations of acyl-CoAs are controlled by the balance among acyl-CoA synthesizing enzymes, acyl-CoA utilizing enzymes (acyl-CoA: lysophospholipid acyltransferase), and fatty acid metabolizing enzymes (acyl-CoA hydrolases) (Farooqui et al., 2000a; Corkey et al., 2000). 3.2 Deacylation/Reacylation Cycle (Land’s Cycle) and Its Importance 77

The intracellular levels of free unbound acyl-CoA esters are tightly regulated by feedback inhibition of the acyl-CoA synthetase, and are buffered by specific acyl- CoA binding proteins. In addition to being intermediates in fatty acid metabolism in brain, acyl-CoAs also modulate ion fluxes, vesicle trafficking, protein phosphorylation, long-term potentiation in the hippocampus (Zhang et al., 2000), synaptic vesicle formation (Schmidt et al., 1999), and gene expression (Faergeman and Knudsen, 1997).

3.2.2 Acyl-CoA: Lyso-phospholipid Acyltransferase in the Brain

The transfer of acyl group from acyl-CoA to lysophospholipid acceptor is catalyzed by acyl-CoA: lysophospholipid acyltransferase, which is located in microsomal fraction and can be solubilized from bovine brain microsomes using Miranol (Baker and Chang, 1981; Deka et al., 1986). Three hundred fold purification of acyl-CoA: lysophospholipid acyltransferase from bovine brain microsomes is achieved by DEAE-cellulose, Blue-2-agarose, and Matrex green chromatographies with an overall recovery of 6.0% (Deka et al., 1986). The purified enzyme moves as a single protein band (molecular mass of 43 kDa) on SDS-PAGE. The purified enzyme is specific for lyso-PtdCho. Enzyme shows less specificity toward the acyl-CoA derivatives. Among various acyl-CoAs, enzyme shows preference for arachidonyl- CoA. High concentrations of arachidonoyl-CoA inhibit the enzymic activity. Differences in the rate of acylation of different lysophospholipid subclasses of glycerophospholipids by human brain acyl-CoA: lysophospholipid acyltransferase have also been reported (Ross and Kish, 1994). The rate with lysophosphatidylinositol (lysoPtdIns) is highest, followed by lysoPtdCho and LysoPtdSer. In contrast, acylation of lysoPtdEtn is barely detectable. Based on various kinetic and metabolic studies, lysoPtdCho acyltransferase may compete with acyl-CoA hydrolase and lysophospholipase for acyl-CoA and lysoPtdCho, respectively (Ross and Kish, 1994). At least two families of lysophospholipid acyltransferases have been identified in neural and non-neural tissues. Acyl-CoA lysocardiolipin acyltransferase 1 (ALCAT1) is initially identified as a microsomal lysocardiolipin acyltransferase and acyl-CoA: lysophosphatidylethanolamine acyltransferase 2 (LPEAT2) is predominantly expressed in brain (Cao et al., 2004, 2008). ALCAT1 plays an important role in cardiolipin remodeling. In addition, ALCAT1 possesses acyltransferase activities toward lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG) (Cao et al., 2008). Human embryonic kidney 293 (HEK293) cells overexpressing human ALCAT1 display significant increase in LPI acyltransferase (LPIAT) and LPG acyltransferase (LPGAT) activities with several fatty acyl-CoAs. The affinity of ALCAT1 toward LPI and LPG depends on fatty acyl-CoA (Zhao et al., 2009). The reduction in the expression of ALCAT1 in Hela cells significantly lowers 6 LPIAT and LPGAT activities, but has no effect on ALCAT1 activity. Based on structural and functional studies, it is suggested that critical amino acids D168 and L169 within ALCAT1 may play an important role in lysophospholipid substrate binding 78 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids…

(Zhao et al., 2009). LPEAT shows significant acyl-CoA-dependent acyltransferase activity toward 1-O-alkenyl-lysophosphatidylethanolamine, lysophosphatidylgly cerol, 1-O-alkyl-lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylcholine, but lacks appreciable acylating activity toward glycerol 3-phosphate, lysophosphatidic acid, lysophosphatidylinositol, and diacylglycerol, indicating that multiple but selective roles of LPEAT2 as an enzyme are involved in phospholipid remodeling. Although LPEAT2 recognizes a broad range of medium and long-chain fatty acyl-CoA, its activity is not affected by Ca2+. When overexpressed in mammalian cells, LPEAT2 is localized to the endoplasmic reticulum. Treatment with LPEAT siRNA in HEK293T cells significantly reduces LPEAT and 1-alkenyl-LPEAT activities, but produces no effect on other lysophospholipid acylating activities (Cao et al., 2008). Inhibition of LPCAT retards TNF-a expression and secretion by preventing the translocation of TLR4 into membrane lipid raft domains in MM6 cells (Jackson et al., 2008a), suggesting that LPCAT may regulate the assembly of a functional LPS receptor complex in the membrane by controlling membrane PtdCho composition. Indeed, upregulation of LPCAT activity, by cytokines, such as IFN-a, increases cellular responses to LPS by stimulating the assembly of the LPS receptor (Jackson et al., 2008a). Collective evidence suggests that ALCAT1 and LPEAT2 play an important role in the biosynthesis of cardiolipin and ethanolamine-containing phospholipids in neural and non-neural tissues. Acyl- CoA: lysophospholipid acyltransferase-mediated remodeling of glycerophospholipids may be involved in maintaining membrane asymmetry and diversity, which modulates membrane fluidity and curvature. Presence of multiple forms of acyl- CoA: lysophospholipid acyltransferases and their genes encoding these activities explain the occurrence of more than 100 glycerophospholipids molecular species in brain and other mammalian tissues (Yamashita et al., 1997; Cao et al., 2008).

3.2.3 Phospholipases A2 (PLA2) in the Brain

PLA2s constitute a superfamily of enzymes that catalyze the hydrolysis of unsaturated fatty acids from the sn-2 position of glycerol moiety of neural membrane phospholipids.

These enzymes are classified into two groups: (a) intracellular PLA2 and (b) extracellular PLA2. The intracellular PLA2 are further divided into (a) cytosolic

PLA2 (cPLA2), (b) calcium-independent PLA2 (iPLA2), and (c) plasmalogen-selective PLA2 (PlsEtn-PLA2), whereas extracellular PLA2 includes secretory PLA2

(sPLA2) and PAF acetylhydrolases (Hirabayashi et al., 2004; Ohto et al., 2005;

Kolko et al., 2006; Farooqui and Horrocks, 2007; Ong et al., 2010). cPLA2 (mol. mass 85 kDa) is present in the cytosol, and is activated by mM concentrations of Ca2+ and protein serine/threonine kinases. Calcium is not required for the catalytic activity of cPLA2, but it is important for the interaction of this group of enzymes with the phospholipid membrane (Farooqui et al., 1997). iPLA2 is also localized in the cytosol (mol. mass 80 kDa). Calcium is not required for its activity. Although 3.2 Deacylation/Reacylation Cycle (Land’s Cycle) and Its Importance 79

the precise function of the iPLA2 is not known, it may be associated with tissue developments and hormone secretion. sPLA2 (mol mass 18–20 kDa) contains multiple disulfide (S-S) bonds. It is stimulated by mM concentrations of Ca2+, and its function is primarily extracellular. PAF acetylhydrolase inactivates PAF and hydrolyzes oxidized phospholipids to yield lysophospholipids (Farooqui and Horrocks, 2007). Molecular biology and cloning studies have resulted in isolation and

characterization of many paralogs, splice variants, and multiple forms of cPLA2, iPLA2, and sPLA2 in neural and non-neural tissues. cPLA2 paralogs include cPLA2a, cPLA2b, cPLA2g, cPLA2d, cPLA2e, and cPLA2z. iPLA2 splice variants include iPLA2a, iPLA2b, and iPLA2g. Multiple forms of sPLA2 include sPLA2- IB, sPLA2-

IIA, sPLA2-IIC, sPLA2-IID, sPLA2-IIE, sPLA2-IIF, sPLA2-III, sPLA2-V, sPLA2-X, sPLA2-XIIA, and sPLA2-XIIB (Hirabayashi et al., 2004; Ohto et al., 2005; Kolko et al., 2006; Farooqui and Horrocks, 2007; Ong et al., 2010). Paralogs, splice variants, and multiple forms of PLA2 superfamily differ from each other in enzymic properties, tissue distribution, cellular and subcellular localizations, and role in various physiological and pathophysiological conditions (Farooqui and Horrocks, 2007; Ong et al., 2010). In addition to hydrolyzing fatty acids from the sn-2 position of glycerol moiety of neural membrane phospholipids, PLA2 isoforms also have other biochemical activities. Thus, in lungs acidic iPLA2 activity has nonselenium glutathione peroxidase (NSGPx) activity (Chen et al., 2000) and cPLA2 from several sources also shows lysophospholipase and 7-hydroxycoumarin esterase activities

(Leslie, 1991; Murakami et al., 1997). Among paralogs of cPLA2, cPLA2g (mol. mass 61 kDa) has 30% sequence identity with its ortholog, cPLA2a. cPLA2g not only contains a potential prenylation motif at its C terminus, but also displays PLA2, lysophospholipase, coenzyme A (CoA)-independent transacylation and lysophospholipid dismutase (LPLase/transacylase) activities. This observation indicates that cPLA2 paralogs are involved in fatty acid remodeling of phospholipids as well as in the clearance of toxic lysophospholipids in cells (Yamashita et al., 2005). Collectively these studies suggest that members of PLA2 superfamily constitute a complex signal transduction network that not only maintains cross-talk among excitatory amino acid, dopamine, retinoid, cannabinoid, cytokine, and growth factor receptors through the generation and modulation of lipid mediators, but also contribute to enzymic and nonenzymic transacylation reaction. This cross-talk among above receptors and tight regulation of all PLA2 isoforms is essential for maintaining normal neural cell function. Upregulation of various PLA2 isoforms and splice variants is associated with the degradation of neural membrane phospholipids and generation of arachidonic acid and docosahexaenoic-derived lipid metabolites that have been implicated in fundamental neural cell functions including neurotransmitter release, LTP and LTD induction, the auditory startle reflex and sensorimotor gating and vacuous chewing movements nociception, neuroinflammation, oxidative stress, and neurodegeneration (Farooqui, 2009). Collective evidence suggests that members of PLA2 superfamily are involved in the synthesis of lipid mediators that have been implicated in fundamental cellular responses, including growth, differentiation, adhesion, migration, secretion, and apoptosis (Farooqui and Horrocks, 2007; Farooqui, 2009; Ong et al., 2010). 80 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids…

3.2.4 Long-Chain Acyl-CoA Hydrolase or Thioesterases in the Brain

Long-chain acyl-CoA hydrolases or thioesterases catalyze the hydrolysis of CoA esters to free coenzyme A (CoASH) and carboxylic acids (Hunt and Alexson, 2002). Among various brain regions, pons, medulla, and midbrain have higher activity of long-chain acyl-CoA hydrolase than in the cerebral cortex and caudate nucleus (Yamada et al., 1996). At the cellular level, long-chain acyl-CoA hydrolase is localized to neurons (Yamada et al., 1996). In addition to various nuclei, some neuronal cells, such as mitral cells in the olfactory bulb, pyramidal cells in the cerebral cortex, and Purkinje cells in the cerebellum, are also immunostained with antilong-chain acyl-CoA hydrolase antibody (Yamada et al., 1996). At the subcellular level, long- chain acyl-CoA hydrolases are localized in endoplasmic reticulum, cytosol, mitochondria, and peroxisomes. In developing brain, microsomal long-chain acyl-CoA hydrolase activity decreases sharply during the first 20 days after birth, whereas the acyl-CoA synthetase activity remains high indicating that long-chain acyl-CoA are needed for the synthesis of neural membrane phospholipids in neural membranes during the intense period of myelination. Brain long-chain acyl-CoA hydrolase has been purified and characterized from the rat and human brain cytosols (Lin et al., 1984; Yamada et al., 1996, 1999). The native enzyme has molecular mass of 104 kDa. It is made up of subunits of 36 kDa. The enzyme shows high activity with long-chain acyl- CoAs. With palmitoyl-CoA as substrate, the rat brain enzyme shows maximal velocity of 262 nmol/min/mg and Km of 5.7 mM and human brain shows maximal velocity of 295 nmol/min/mg and Km of 6.4 mM. It also hydrolyzes other long-chain acyl- CoAs (C8–18). Rat brain long-chain acyl-CoA hydrolase shows very high activity toward arachidonoyl-CoA (ARA-CoA). Arachidonic acid (ARA) and not its CoA ester is the substrate for enzymes in the production of prostaglandins, leukotrienes, and thromboxanes. Rat brain long-chain acyl-CoA hydrolase is inhibited by p-chlo- romercuribenzoate, lysophospholipids, palmitoyl carnitine, and bovine serum albumin. Metal ions such as Mn2+, Mg2+, and Ca2+ also inhibit enzymic activity (Lin et al., 1984; Yamada et al., 1996; Broustas and Hajra, 1995). Rat brain long-chain acyl-CoA hydrolase is inactivated by diethyl pyrocarbonate, an active center histidine-reacting agent. There is a lot of similarity in amino acid sequence of rat and human brain long- chain acyl-CoA hydrolase. The human brain long-chain acyl-CoA hydrolase cDNA encodes a 338-amino acid sequence, which is 95% identical to that of a rat homolog. The human brain long-chain acyl-CoA hydrolase gene spans about 130 kb and comprised nine exons, and was mapped to 1p36.2 on the cytogenetic ideogram. Recently several acyl-CoA hydrolases have been identified in liver peroxisomes, where they act as auxiliary enzymes in a- and b-oxidation in this organelle and are regulated by peroxisome proliferator-activated receptors (PPARs) (Hunt and Alexson, 2002). The physiological roles of long-chain acyl-CoA hydrolases remain elusive. These enzymes play a key role in regulating and maintaining normal levels of deacylation/reacylation cycle associated lipid metabolite such as acyl-CoA, CoASH, and free fatty acid levels. These metabolites are closely associated with the regulation potential for many physiological and pathological processes in the brain. 3.2 Deacylation/Reacylation Cycle (Land’s Cycle) and Its Importance 81

O O

R2C O CR1 H2C OC R3 O + R2 C O C H HO C H O O O POCH CH NH O O CH CH NH H2C 2 2 2 H2C P 2 2 2 O O

O O

H C O CR H2C O CR 2 1 O 3 + HO C H R2 C O C H O O O POCH CH NH O POCH CH NH H2C 2 2 2 H2C 2 2 2 O O

Fig. 3.3 CoA-dependent and CoA-independent transacylation reactions

3.2.5 CoA-independent Reacylation in the Brain

Brain microsomes contain a CoA-independent transacylation system, which transfers fatty acids from diacyl glycerophospholipids to various glycerophospholipids in the absence of any cofactor (MacDonald and Sprecher, 1991; Yamashita et al., 1997). The only fatty acids transferred by this system are C20 and C22 polyunsaturated fatty acids. Diacyl-glycerophosphocholine is the most preferred substrate (Fig. 3.3). Human brain homogenates possess the ability to transfer fatty acids from lysoPtdCho to lysoPtdEtn but not to lysoPtdSer or lysoPtdIns (Ross and Kish, 1994). Recent studies indicate that purified recombinant cPLA2g catalyzes an acyltransferase reaction from one molecule of lyso-PtdCho to another, forming PtdCho. Lyso-PtdCho or lyso-PtdEtn can act as acyl donor and acceptor. No acyl group transfer is observed with lyso-PtdSer, lyso- PtdIns, and lyso-PtdH. PtdCho and PtdEtn also act as weak acyl donors (Yamashita et al., 2009). Acyl group transfer is controlled by reaction conditions, such as addition of lyso-PtdH/ PtdH, pH above 8, and increase in temperature. These parameters can change the balance between lysophospholipase and transacylation activities, suggesting that lysophospholipase/transacylation activities of cPLA2g may be regulated by various factors. As lysophospholipids are known to accumulate in ischemic injury and induce alterations in brain function, the cPLA2g may have a protective role through clearance of lysophospholipids by its transacylation activity (Yamashita et al., 2009). 82 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids…

3.3 Effects of Lysophospholipids on Neural Membrane Metabolism

As stated above, due to their detergent-like properties high levels of lysophospholipids are potentially toxic to cells (Weltzien, 1979; Farooqui et al., 1997). The levels of lysophospholipids are strictly controlled. High levels of lysophospholipids under pathological situations perturb neural membrane structure, affect many membrane- bound enzymes related to signal transduction, and cause cell lysis (Farooqui and Horrocks, 2006, 2007).

3.3.1 Lysophosphatidylcholine (Lyso-PtdCho)

Lyso-PtdCho is an amphiphilic and proinflammatory phospholipid, which is synthesized in brain through the action of PLA1 and PLA2 on PtdCho and is metabolized by lysophospholipases and acyltransferases. The incorporation of lyso-PtdCho in neural membranes perturbs the orderly packed glycerophospholipid molecules in the lipid bilayer. Lyso-PtdCho not only interacts directly with ion channels and neurotransmitter receptors, but also modulates their activity indirectly through the modulation of neural membrane fluidity (Farooqui and Horrocks,2 007) (Fig. 3.4). Injections of lyso-PtdCho into brain not only produce acute inflammatory demyelination at the injection site, but also promote blood–brain barrier breakdown, and interstitial edema around the injection site (Lovas et al., 2000; Ousman and David, 2000, 2001; Degaonkar et al., 2002, 2005). Similarly, the treatment of cerebellar slices with lyso-PtdCho markedly affects their integrity and also causes demyelination in vitro (Birgbauer et al., 2004). In spheroid cultures, repeated exposure to lyso-PtdCho for a week results not only in 30% loss of MBP protein concentration and sharp decrease in 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase activity with partial remyelination after a week of recovery (Vereyken et al., 2009). The number of dividing cells is increased after lyso-PtdCho exposure. Only oligodendrocytes undergo cell division. Microglia, astrocytes, and neurons are not affected by lyso-PtdCho exposure. This suggests that lyso-PtdCho toxicity is specific for myelin and oligodendrocytes. Addition of cholesterol and simvastatin to cell cultures reduces Lyso-PtdCho toxicity, suggesting that lyso-PtdCho acts through altering membrane composition (Vereyken et al., 2009). Thus, spheroid culture model for demyelination with potential for remyelination offers a good model not only for testing drugs, but also studying molecular mechanisms of remyelination (Vereyken et al., 2009). Injections of lyso-PtdCho in mice result in statistically significant higher concentrations of 5-hydroxytryptamine (5-HT) and Norepinephrine (NA) in the blood (Itokwa et al., 2007). As stated above, Lyso-PtdCho interacts with neurotransmitter, cannabinoid, and growth factor receptors (Fig. 3.5). Thus, lyso-PtdCho interferes 3.3 Effects of Lysophospholipids on Neural Membrane Metabolism 83

Deramification of microglia

Intensification of Transportation of DHA pain

Modulation of Lyso - Modulation of enzyme receptor function phosphatidylcholine activities

Modulation of exocytosis Modulation of ion - channels

Regulation of cytokine and Chemokine expression

Fig. 3.4 Roles of lyso-phosphatidylcholine in brain

Lyso-phosphatidylcholine

Modulation of Modulation of heparin Cannabinoid receptors Binding receptor

Modulation of Modulation of PDGF NMDA receptor Receptors Modulation of dopaminergic Receptors

Fig. 3.5 Modulation of various receptors by lyso-phosphatidylcholine with dopaminergic neurotransmission and promotes the induction of bradykinesia in rats (Lee et al., 2005). Lyso-PtdCho may act as a ligand to its own receptors (Zhu et al., 2001). It is likely that modulation of dopamine receptor function by lyso-PtdCho may be due to an interaction between dopaminergic receptor and 84 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids… lyso-PtdCho receptor (Lee et al., 2004; Soga et al., 2005). The occurrence of G protein-coupled lyso-PtdCho receptor G2A has been recently reported in T cells, where G2A contributes to the recruitment of T cells at sites of inflammation (Ghasemlou et al., 2007; Osmers et al., 2009). Since demyelinating diseases can be associated with painful sensory phenom- ena such as tactile allodynia and hyperalgesia, studies have been performed of the effect of lyso-PtdCho in animal model of pain. Intracerebroventricular injections of lyso-PtdCho result in increased behavioral responses and allodynia in carrageenan- induced acute and chronic models of inflammatory pain (Vahidi et al., 2006), suggesting that generation of lyso-PtdCho may be closely associated with the modulation nociceptive processes in neurons (Ji et al., 2003). The administration of

PLA2 inhibitors blocks behavioral responses and allodynia (Yeo et al., 2004). In contrast, injections of lyso-PtdEtn have no significant effect on behavioral responses. Lyso-PtdCho also inhibits N-methyl-d-aspartate (NMDA) receptor responses, both in nucleated patches taken from cultured neurons and in cells expressing recombinant NMDA receptors (Fig. 3.5). This inhibition is reversible, voltage independent, and stronger at nonsaturating doses of agonist. It is not linked to the charge on the polar head and is not mimicked by lyso-PtdH or PtdCho (Casado and Ascher, 1998), suggesting that the lyso-PtdCho effect requires insertion into the lipid bilayer. In brain, lyso-PtdCho also regulates exocytosis and inflammation by modulating membrane-bound enzymes (protein kinases, nitric oxide synthase and cyclooxygenase-2, CTP-phosphocholine cytidylyltransferases), receptors, and ion channels, the transcription of cytokines, chemokines, MMP, adhesion molecule, and platelet aggregation (Oishi et al., 1988; Yuan et al., 1996; Murugesan et al., 2003). Lyso-PtdCho promotes the transformation of ramified resting microglia consist- ing of a small cell body and long processes with secondary branching into the activated deramified and amoeboid microglia, which actively participate in the maintenance of brain immune function. Although the morphological transformation of ramified microglia into the amoeboid microglia is observed under a wide variety of in vitro as well as in vivo conditions (Raivich et al., 1999; Schilling et al., 2004a), the precise molecular mechanism of deramification in the microglia remains largely unknown. It is proposed that lyso-PtdCho promotes microglial activation through the modulation of P2X7R signaling (Takenouchi et al., 2007). Thus, in a mouse microglial cell line (MG6) and primary microglial cell cultures, lyso-PtdCho induces 2+ 2+ the sustained increase in the intracellular Ca (Cai ) through P2X7 nucleotide recep- 2+ tor (P2X7R) channels activated by ATP or BzATP. This increase in (Cai ) is blocked by P2X7R antagonists, brilliant blue G and oxidized ATP. G2A, a receptor for lyso- PtdCho, is expressed in MG6 cells, but not involved in promoting the effect of lyso- 2+ PtdCho on the P2X7R-mediated change in (Cai ). Furthermore, lyso-PtdCho increases the P2X7R-associated formation of membrane pores and the activation of p44/42 mitogen-activated protein kinase supporting the view that lyso-PtdCho may regulate microglial functions in the brain by enhancing the sensitivity of P2X7R (Takenouchi et al., 2007). The activated microglial cells migrate to area of injured brain tissue 3.3 Effects of Lysophospholipids on Neural Membrane Metabolism 85 and they engulf and destroy microbes and cellular debris (Gehrmann et al., 1995; Farooqui, 2009). The role of microglia in brain may not always be beneficial. Uncontrolled microglial activation and the subsequent excessive neuroinflammation may contribute to a variety of the CNS pathologies including neurodegenerative diseases (Farooqui et al., 2007). Thus, activation of microglia has to be strictly regulated, and the apoptotic elimination of activated microglia is thought to be one mechanism of the microglial self-regulation (Farooqui, 2009). The deramification of microglial cells can be blocked by inhibition of nonselective cation channels and K+-Cl− cotransporters. Lyso-PtdCho not only stimulates cell motility, but facilitates the release of proinflammatory cytokines. Docosahexaenoic acid (DHA) is an essential fatty acid required for the normal function of several tissues, especially the brain (Farooqui, 2009). Lyso-PtdCho is a preferred carrier of DHA to the brain (Bernoud et al., 1999; Picq et al., 2010), although the pathways of the formation of DHA-containing lysophospholipids in plasma have not been delineated. It is recently proposed that endothelial phospholipase A1 may be responsible for the generation of DHA lysophospholipids in plasma (Chen and Subbaiah, 2007). Substrate specificities studies with deuterium-labeled phospholipids with different polar head groups, as well as DHA-enriched natural phospholipids indicate that recombinant endothelial phospholipase A1 shows the polar head group specificity in the order of PtdEtn > PtdCho > PtdSer >PtdH.

Within the same phospholipid class, the recombinant endothelial phospholipase A1 shows preference for the species containing DHA at the sn-2 position, and is inactive in the hydrolysis of phospholipids containing an ether linkage. Since cells of blood–brain barrier secrete endothelial phospholipase A1, it is suggested that enzyme may play an important role in the delivery of DHA lysophospholipid carriers to the brain (Chen and Subbaiah, 2007). Collectively, studies mentioned above support the view that in brain, at low concentrations lyso-PtdCho may have multiple effects ranging from membrane fusion, exocytosis, signal transduction to gene expression, and DHA transport into the brain.

3.3.2 Lyso-phosphatidylethanolamine (Lyso-PtdEtn)

Lyso-PtdEtn synthesis has been reported to occur in rat brain synaptosomes (Iwata et al., 1986). Lyso-PtdEtn synthesis is markedly stimulated by calcium ion. The addition of veratridine to synaptosomal preparation stimulates the accumulation of lyso- PtdEtn. This observation suggests that sodium channels along with enhanced calcium influx may be closely associated with the enhanced synthesis of lyso-PtdEtn (Iwata et al., 1986). In SK-OV3 ovarian and OVCAR-3 ovarian cancer cells lyso-PtdEtn- 2+ mediated increase in Cai is inhibited by U-73122. Moreover, pertussis toxin (PTX) 2+ almost completely blocks the lyso-PtdEtn-mediated increase in Cai indicating the involvement of PTX-sensitive G-proteins. In addition, lyso-PtdEtn also stimulates chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. Based 86 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids… on detailed investigation, it is suggested that LPE stimulates a membrane bound receptor, different from well-known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells (Park et al., 2007).

3.3.3 Lyso-phosphatidylserine (Lyso-PtdSer)

Very little is known about the metabolism of lyso-PtdSer in brain tissue, but significant information on lyso-PtdSer is available on mast cells, leukocytes, and platelets. The deacylation of PtdSer by PLA1 and PLA2 generates 2-acyl-1-lyso- sn-GroPSer and 1-acyl-2-lyso-sn-GroPSer, respectively (Bruni et al., 1982; Inoue et al., 1989; Nagai et al., 1999; Hosono et al., 2001; Aoki et al., 2002; Kim et al., 2008; Nakamura et al., 2010). The addition of lyso-PtdSer to leukocytes and mast cells results in its rapid incorporation. In the presence of phorbol esters, lyso- PtdSer is rapidly converted into PtdSer by an acyl-CoA: lyso-PtdSer acyltransferase (Mietto et al., 1987). Lyso-PtdSer is an autocoid and immunological regulator, which dramatically augments the degranulation of rat peritoneal mast cells (RPMCs) (Bruni et al., 1988; Sugo et al., 2006). This effect is mediated by a lysoPtdSer receptor. Lyso-PtdSer also induces a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in human GPR34-expressing Chinese hamster ovary (CHO/hGPR34) cells. These cells do not respond to other structurally related phospholipids (Sugo et al., 2006). Similar concentrations of PtdSer induce RPMC degranulation as well as the activation of GPR34 suggesting that activation of GPR34 is closely associated with both these processes and GPR34 is a functional mast cell lyso-PtdSer receptor. It is well known that mast cells are located throughout the human body and exposure to allergen results in their stimulation via the immunoglobulin E (IgE) receptor (Fc(epsilon)RI) to release several proinflammatory mediators such as tumor necrosis factor-a (TNF-a), reactive oxygen and nitrogen species such as ROS, NO, proteases, and lipid-derived mediators (Ozben and Erdogan, 2008; Theoharides and Kalogeromitros, 2006). Lyso-PtdSer-mediated stimulation of mast cells is closely associated with the development of allergic inflammation through the release of cytokines (IL-b1, interleukin (LTE4)), which modulate vasodilatation, bronchoconstriction, cellular chemotaxis, and increase vascular permeability (Theoharides and Kalogeromitros, 2006). Lyso-PtdSer also increases intracellular calcium ions in L2071 mouse fibroblast cells. U-73122 blocks this increase, but pertussis toxin does not, suggesting that lyso-PtdSer stimulates calcium signaling through the activation of PLC coupled G-protein coupled receptor (Park et al., 2006). The lyso-PtdH receptor antagonist, VPC 32183, has no effect on lyso-PtdSer-mediated calcium mobilization, indicating that lyso-PtdSer binds to its own receptor. In L2071 cells, lyso-PtdSer interacts with extracellular signal-regulated protein kinase (ERK) and p38 kinase. Pertussis toxin blocks the activation of both kinases, indicating the association of pertussis toxin-sensitive G-proteins in the stimulatory process. In L2071 cells, 3.3 Effects of Lysophospholipids on Neural Membrane Metabolism 87

PtdSer stimulates chemotactic migration. Pertussis toxin completely inhibits this process, indicating the involvement of pertussis toxin-sensitive Gi protein(s). This chemotaxis of L2071 cells induced by lyso-PtdSer is also dramatically retarded by LY294002, a potent and selective inhibitor of phosphoinositide 3-kinases and by PD98059, a selective noncompetitive inhibitor of the MAPK pathway. Collective evidence suggests that lyso-PtdSer stimulates at least two different signaling cascades: one involving a pertussis toxin-insensitive but phospholipase C-dependent intracellular calcium ion increase, and the other a pertussis toxin- sensitive chemotactic migration mediated by phosphoinositide 3-kinase and ERK (Park et al., 2005, 2006). Studies on lyso-PtdSer and lyso-PtdH-mediated increase in intracellular calcium in mouse bone marrow-derived mast cells (BMMC), and rat C6 glioma and human HCT116 colon cancer cells indicate that increases in calcium due to LPS and LPA can be blocked by PTX, U-73122 and 2-APB. This indicates that both lipids stimulate calcium signaling via G proteins (Gi/o types), PLC activation, and subsequent InsP3 generation. It is also reported that Ki16425 completely blocks lyso-PtdSer-mediated Ca2+ response in all cell types, but that the effect of VPC32183 varies from complete inhibition in BMMC and C6 glioma cells to partial inhibition in HCT116 cells. It is concluded that lyso-PtdSer increases intracellular Ca2+ through Ki16425/VPC32183-sensitive G protein-coupled receptors

(GPCR), G protein, PLC, and InsP3 in mouse BMMC, rat C6, and human HCT116 cells (Kim et al., 2008).

3.3.4 Lyso-phosphatidylinositol (Lyso-PtdIns)

In neural membranes, two mechanisms generate lyso-PtdIns; one is associated with

PLA1-mediated deacylation of PtdIns and the other involves the reverse reaction of lyso-PtdIns acyltransferase (Ueda et al., 1993; Yamashita et al., 2003). In synaptic membranes, lyso-PtdIns is catabolized by a lyso-PtdIns-specific PLC (Kobayashi et al., 1996). Lyso-PtdIns interacts with GPR55 receptor, a novel ligand for the orphan G protein-coupled receptor 55, which promotes Rho-dependent signaling. Additional events downstream of GPR55 include activation of ERK-MAP kinase and Ca2+ release from stores, as well as the induction of a number of transcription factors (Pertwee, 2007; Nevalainen and Irving, 2010). Although GPR55 has very little sequence homology with the CB1 or CB2 cannabinoid receptors, it clearly interacts with certain cannabinoid ligands. However, the physiological significance of this signaling remains elusive. In PC12 cell lyso-PtdIns induces exocytosis, which depends not only on the cellular cholesterol content, but also on the integrity of lipid rafts in the PC12 cell membranes (Ma et al., 2010). Depletion of cholesterol by preincubating cells with the cholesterol chelator results in the attenuation of exocytosis. Lyso-PtdIns- mediated exocytosis also depends on calcium ions. The depletion of calcium by 88 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids… thapsigargin treatment blocks the lyso-PtdIns-mediated exocytosis. Collective evidence suggests that lyso-PtdIns-mediated exocytosis depends on the integrity of the lipid raft and intracellular calcium (Ma et al., 2010). In non-neural cells, lyso- PtdIns not only stimulates the degradation of phosphoinositide and mobilization of cytosolic Ca2+, but also enhances arachidonic acid release, indicating that it activates both PLC and PLA2 activities. The effects of lyso-PtdIns can be distinguished from those of the well-known mitogen lyso-PtdH, which modulates the signaling pathways differently. These results suggest that the mitogenic activity of lyso-

PtdIns may involve the activation of PLC and PLA2 and are relatively specific for ras-transformed cells (Falasca et al., 1995). Addition of ionophore A23187 to H-Ras-transformed fibroblasts results in a 10-fold increase in the levels of this lysolipid in the extracellular medium (Falasca et al., 1998), suggesting the stimulation of PLA2 activity. In H-Ras-transformed fibroblasts, extracellular lyso-PtdIns breaks down rapidly into inositol 1:2-cyclic phosphate. It is proposed that the formation and release of lyso-PtdIns may function as an autocrine mechanism, which may contribute to the Ras-dependent stimulation of cell growth (Falasca et al., 1998). Collective evidence demonstrates that in non-neural tissues lyso-PtdIns not only performs autocrine function, but also contributes to CoA-dependent transacylation system, which modulates polyphosphoinositide homeostasis in rat liver microsomes (Yamashita et al., 2003).

3.3.5 Lyso-cardiolipin (Lyso-Ptd2Gro)

Cardiolipin (diphosphatidyl glycerol) is almost exclusively located in the inner mitochondrial membrane, where it is synthesized from phosphatidylglycerol and cytidinediphosphate-diacylglycerol. After primary synthesis, the mature acyl chain composition of lyso-Ptd2Gro is achieved by at least two remodeling mechanisms (Houtkooper and Vaz, 2008; Paradies et al., 2009). This phospholipid is not only intimately associated with mitochondrial bioenergetic processes, but also plays active roles in apoptosis and mitochondrial membrane dynamics (Farooqui, 2009; Paradies et al., 2009). During apoptotic cell death, Ptd2Gro interacts with two major components of proapoptotic machinery – tBid and cytochrome c. The binding of Ptd2Gro to cytochrome c unfolds the protein and that the complex functions as a peroxidase, catalyzing Ptd2Gro peroxidation essential for the release of proapoptotic factors, whereas binding with tBid not only activates proapoptotic protein, Bak and Bax, but also orchestrates apoptosis, supporting changes in mitochondrial electeron transport (Tyurin et al., 2007). At the molecular level during apoptosis TNFR1/ Fas promotes the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria, where it mediates the destabilization of the mitochondrial bioenergetic homeostasis. These changes produce mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition of the adenosine diphosphate 3.3 Effects of Lysophospholipids on Neural Membrane Metabolism 89

(ADP)-stimulated respiration and phosphorylation rate. tBid mediates the disruption of mitochondrial homeostasis, blocks the overexpression of Bcl-2 and

Bcl-XL. In mitochondrial membranes, Ptd2Gro induces inhibits state-3 respiration by modulating the activity of the ADP/ATP translocator (Gonzalvez et al., 2005). In the cytosol, the released cytochrome c mediates the allosteric activation of apoptosis-protease activating factor 1, which is required for the proteolytic maturation of caspase-9 and caspase-3. Collectively, these studies demonstrate that interactions of tBid with lyso-Ptd2Gro ‘primes’ the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of above-mentioned apoptogenic factors (Esposti et al., 2003; Garrido et al., 2006).

3.3.6 Lyso-ethanolamine and Choline Plasmalogens (Lyso-PlsEtn and Lyso-PlsCho)

Hydrolysis of ethanolamine and choline plasmalogens (1-O-alk-1 eny-2-acyl-sn- glycero-3-phosphoethanolamine and 1-O-alk-1 eny-2-acyl-sn-glycero-3-phosphocholine) by plasmalogen-selective phospholipase A2 (PlsEtn-PLA2) generates lyso-PlsEtn and lyso-PlsCho (Farooqui and Horrocks, 2001; Farooqui, 2010b) (Fig. 3.6). In neural membranes, lyso-PlsEtn and lyso-PlsCho are either rapidly incorporated through reacylation to maintain normal levels of plasmalogens or hydrolyzed by an enzyme called lysoplasmalogenase into fatty aldehyde and sn-glycero-3-phosphoethanolamine or sn-glycero-3-phosphocholine (Jurkowitz- Alexander et al., 1989). Lysoplasmalogens can also be transformed into an acetylated platelet-activating factor analog either by a CoA-independent transacetylase activity or through acetyl transferase activity (Lee, 1998). Lysoplasmalogens are amphiphilic molecules, which due to their detergent-like properties regulate physicochemical properties and neural membrane integrity not only by interacting with membrane-bound structural proteins, but also by modulating activities of enzymes. Their interactions with neural membranes increase membrane fluidity, which not only affect permeability, but also modulate activities of various enzymes, such as cAMP-dependent protein kinase (PKA) and Na+, K+- ATPase (Williams and Ford, 1997; Han and Gross, 1991). The activation of PKA by lysoplasmalogens suggests that lysoplasmalogenase may play an important role in turning off signal transduction processes involving PKA-mediated phosphorylation. At the plasma membrane level, synthesis of lyso-PlsEtn may induce alterations in permeability resulting in influx of external calcium, which not only facilitates the translocation and activation of downstream enzymes (cyclooxygenases and lipoxygenases) that convert ARA into eicosanoids (Phillis et al., 2006). In addition, lyso-PlsEtn or lyso-PlsCho-mediated alterations in membrane permeability may allow influx of external calcium, which may influence activities of calcium-dependent enzymes. By altering the action potential 90 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids…

1-Alkyl-GroPCho Lyso-platelet activating factor 1-Alkyl-2-acyl-GroPCho

• Generates PAF analogs AcylCoA:lysophospholipis acyltransferase • Increases membrane fluidity • Activates PKA

R CH2 C H C O CH CH R H H C O CH CH R O 2 2 R C O C H HO C H O O PlsEtn-PLA2 OPO CH CH NH OPO CH CH NH H2C 2 2 2 H2C 2 2 2 O O Ethanolamine plasmalogen Lyso-ethanolamine plasmalogen

O RCCH H C OH Lysoplasmalogenase 2 2 H Aldehyde HO C H O OPO CH CH NH H2C 2 2 2 O sn-Glycero-3-phosphoethanolamine

Fig. 3.6 Metabolism of plasmalogen and lysoplasmalogen in brain. Platelet activating factor (PAF); protein kinase A (PKA)

and inducing depolarization, high levels of lyso-PlsEtn or lyso-PlsCho in heart tissue alter the action potential and produce spontaneous contraction, which may induce and maintain arrhythmic activity in the heart muscle (Caldwell and Baumgarten, 1998).

3.3.7 Lyso-phosphatidic Acid (Lyso-PtdH)

Lyso-phosphatidic acid (lyso-PtdH, 1-O-acyl-2-hydroxy-sn-glycerol-3-phosphate) is the simplest phospholipid that acts as a growth-factor. It is not only an intermediate in the de novo phospholipid biosynthesis, but also an extracellular lipid agonist. Lyso-PtdH is synthesized by the glycerophosphate acyltransferase-mediated acylation of sn-glycerol-3-phosphate by acyl CoA in the membranes of endoplasmic reticulum and mitochondria (Das and Hajra, 1989; Pagès et al., 2001). Lyso-

PtdH is also synthesized by the action of PtdH-specific PLA2 on phosphatidic acid (PtdH) (Thomson and Clark, 1995). In addition, action of PLD on lyso-PtdCho generates lyso-PtdH (Pagès et al., 2001; Moolenaar et al., 2004). A plasma enzyme called autotaxin (ATX) is responsible for the most of lyso-PtdH synthesis in our 3.3 Effects of Lysophospholipids on Neural Membrane Metabolism 91 bodies. ATX converts lysophospholipids such as lyso-PtdCho to Lyso-PtdH by its lysophospholipase D activity (Sugimoto et al., 2006; Xie and Meier, 2004; Okudaira et al., 2010). Lyso-PtdH is catabolized by several enzymes. Thus lyso- PtdH is converted to PtdH by a lyso-PtdH-acyltransferase (Pagès et al., 2001). This enzyme is localized in microsomes and plasma membranes. It is crucial for de novo synthesis of glycerophospholipids. Lyso-PtdH can also be converted into monoacylglycerol by lysophosphatidate phosphohydrolase (lysophosphatidic acid phosphatase, LPP) (Brindley et al., 2002). The dephosphorylation of lyso-PtdH is the major pathway that terminates signaling processes mediated by lyso-PtdH. Expression of LPPs on internal membranes regulates not only lyso-PtdH-mediated signaling, but also signaling associated with different lipid phosphate. It is likely that different LPPs may perform distinct roles, based on integrin binding, their locations, and their abilities to metabolize different lipid phosphates in vivo (Brindley and Pilquil, 2009). In neural cells, lyso-PtdH produces neurochemical effects, including stimulation of the release of noradrenaline from cerebral cortical synaptosomes, inhibition of glutamate uptake by astrocytes, elevation of neuronal intracellular calcium, and stimulation of dopamine release from PC12 cells (Tigyi et al., 1996a, b; Holtsberg et al., 1997; Ye et al., 2002; Moolenaar et al., 2004; Anliker and Chun, 2004). Lyso-PtdH retards b-adrenergic-mediated shape change and enhances forskolin- mediated increase in cyclic AMP levels in glial cells (Koschel and Tas, 1993; Kreps et al., 1993). Lyso-PtdH contributes to the induction of growth cone collapse and neurite retraction in neuroblastoma, PC12, and primary neuronal cell cultures. Lyso-PtdH acts through the activation of the Rho/ROCK and the phosphatidylinositol 3-kinase/Akt pathways, preventing neuronal differentiation (Dottori et al., 2008). Lyso-PtdH also induces the reversal of stellation in astrocytes (Ramakers and Moolenaar, 1998). Lyso-PtdH modulates tight-junction permeability of brain- derived endothelial cells and its high concentration disrupts blood–brain barrier function (Tigyi et al., 1995; Schulze et al., 1997). It increases the migration of murine microglial cells through the activation of Ca2+-activated K+ currents (Schilling et al., 2004b). Collective evidence indicates that lyso-PtdH acts as an intracellular messenger in a paracrine/autocrine manner (Xie et al., 2002). It modulates neurite retraction, cytoskeleton reorganization, neurogenesis, neural migration, myelination, apoptosis, and neurotransmitter release (Fukushima, 2004) (Fig. 3.7). Lyso-PtdH modulates above neurochemical response through six types of 7-transmembrane G-protein-coupled receptors called Lyso-PtdH receptors (Fig. 3.8). These receptors have an apparent mol mass of 38–40 kDa. Lyso-PtdH receptors are classified into at least six groups namely LPA1, LPA2, LPA3, LPA4,

LPA 5, and LPA6 (Okudaira et al., 2010; Chun, 1999; Fukushima et al., 2001). Based on their primary structures, Lyso-PtdH receptors are divided into two families.

LPA 1 (EDG2), LPA2 (EDG4), and LPA3 (EDG7) belong to the endothelial differentiation gene (EDG) family. These receptors share high sequence homology to S1P receptors and cannabinoid receptors (CB1 and CB2). In contrast, LPA4 (P2Y9/

GPR23), LPA5 (GPR94), and LPA6 (P2Y5) fall under the P2Y receptor family, whose members target nucleotides rather than lysophospholipids. The brain 92 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids…

PAF PAF receptor signaling Myelination

PLA2 PtdCho Lyso-PtdCho Neurite retraction

PLD Lyso-PLD Cytoskeleton reorganization

PtdH-specific PLA2 PtdH Lyso-PtdH Lyso-PtdH-R Differentiation Acyltranferase

Neuritogenesis DAG Kinas e PtdH phosphatas e DAG-lipase Lyso-PtdH phosphatase Apoptosis DAG 2-AG Cannabinoid receptor signaling MAG-lipase

ARA COX-2 Neural cell migration Eicosanoids Eicosanoid receptor signaling

Fig. 3.7 Metabolism and roles of lysophosphatidic acid in brain. Phosphatidylcholine (PtdCho); lysophosphatidylcholine (lyso-PtdCho); lysophosphatidic acid (lyso-PtdH); phosphatidic acid (PtdH); diacylglycerol (DAG); 2-arachidonylglycerol (2-AG); platelet activating factor (PAF); phospholipase A2 (PLA2); phospholipase D (PLD); and lyso-phospholipase D (lyso-PLD)

expresses relatively high levels of LPA4. In B103 neuroblastoma cell cultures,

lyso-PtdH produces growth cone collapse and neurite retraction through G12/13- RhoA-Rho-associated kinase (ROCK) activation (Lee et al., 2007) and in human embryonic stem cells LPA4 modulates inhibition of neurosphere formation and neuronal differentiation (Dottori et al., 2008). Emerging evidence suggests that the binding of lyso-PtdH to its receptors activates multiple signal transduction pathways, including those initiated by the small GTPases Ras, Rho, and Rac. Another important target of lyso-PtdH is the peroxisome proliferator-activated receptor gamma (PPARg), which not only plays an essential role in regulating lipid and glucose homeostasis and cell proliferation, but also modulating apoptosis and inflammation (Tigyi, 2010). These responses have a direct impact on human diseases, particularly neurological disorders, diabetes, atherosclerosis, and cancer. It is activated by unsaturated acyl species of lyso-PtdH (Tsukahara et al., 2006). Studies on LPA receptor knockout mice indicate that LPA signaling may be involved in neurological disorders, cardiovascular diseases, angiogenesis, reproduction, cancer progression, and neuropathic pain (Okudaira et al., 2010). 3.4 Lyso-phospholipids in Neurotraumatic and Neurodegenerative Diseases 93

A Lyso-PtdH

PtdChoPtdCho NMD-AR

PLA 2 Ca2+ ATX/Lyso-PLD G12/13 ARA + Lyso-PtdCho Gi

RHO PtdIns 3K SOS

MAG PtdIns-4,5-P2 AKT Rho kinase + TIAM1 RAS ARA + 2+ PKC activation [Ca ]i Survival Stress fiber formation - RAC ERK + Cell rounding

Eicosanoids Cell spreading - DNA synthesis - Migration Viability

Fig. 3.8 Lyso-PtdH-mediated signaling in brain. Agonist (A); N-methyl-D-aspartate (NMDA-R) Phosphatidylcholine (PtdCho); lyso-phosphatidylcholine (lyso-PtdCho); arachidonic acid (ARA); autotaxin (ATX); lyso-phospholipase D (lyso-PLD); phospholipase C (PLC); phosphatidylinositol

4,5-bisphosphate (PtdIns 4,5-P2); inositol 1,4,5-trisphosphate (InsP3); protein kinase C (PKC); diacylglycerol (DAG); monoacylglycerol (MAG); serine/threonine protein kinases (AKT); small GTPases (Rac and Rho); and salt-overly sensitive pathway (SOS)

3.4 Lyso-phospholipids in Neurotraumatic and Neurodegenerative Diseases

Neurotraumatic diseases (stroke, spinal cord trauma, and traumatic head injury) are characterized by glutamate-mediated overstimulation of NMDA receptor, activation of PLA2 and PLC, and increased degradation of neural membrane phospholipids resulting in enhanced production of free fatty acids and lysophospholipids, which under pathological conditions are either converted into platelet activation factor or hydrolyzed by lysophospholipases (Farooqui and Horrocks, 2007; Farooqui et al., 2007). Under oxidative stress (anaerobic conditions) lysophospholipids are transformed into LO radicals, which abstract a hydrogen radical from another lysophospholipid molecule. The radical thus formed adds oxygen and decomposes into a 2-oxolysophospholipid and a HOO radical (protonated superoxide). HOO radical in turn abstracts hydrogen atoms from other molecules and produces H2O2 (Spiteller, 2010). In contrast, in neurodegenerative diseases (AD) glutamate is not 94 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids… released, but significantly lowers the expression of NR2A and NR2B transcripts in susceptible regions of AD brain is observed (Bi and Sze, 2002; Hynd et al., 2004). This process results in an overactivation of glutamate receptors in a tonic rather than a phasic manner (Parson et al., 2007). This continuous mild activation may lead to neuronal damage through the stimulation of Ca2+-dependent enzymes related to lipid, protein, and nucleic acid metabolism. Ca2+-dependent enzymes associated with glycerophospholipid, sphingolipid, and cholesterol metabolism modulate levels of glycerophospholipid-, sphingolipid-, and cholesterol-derived lipid mediators (Farooqui and Horrocks, 2007).

3.4.1 Lyso-phospholipids in Ischemic Injury

Ischemic injury in the brain is accompanied by the stimulation of isoforms of PLA2, massive release of free fatty acids, and increase in levels of lysophosphatidylcholine, which inhibits CTP: phosphocholine cytidylyltransferase, an enzyme that modulates PtdCho synthesis (Adibhatla et al., 2004). In contrast to the membrane damaging effects of lysophospholipids (Blondeau et al., 2002) in transient model of global ischemia, intravenous injections of lyso-PtdCho facilitate the survival of CA1 pyramidal neurons, even when the treatment is started 30 min after 15-min global ischemia. In contrast, lysophosphatidic acid produces no protection. Several mechanisms may be associated with neuroprotective effects of lyso-PtdCho. Lyso-PtdCho may either produce beneficial effects by modulating 2P-domain K+ channels (Blondeau et al., 2002), or may act through its anti-inflammatory effects. Treatment of RAW 264.7 cells with 2-docosahexaenoyl lyso-PtdCho decreases LPS-induced formation of nitric oxide (NO), tumor necrosis factor-a (TNF-a), or IL-6 by inhibiting

LTC 4 synthesis (Hung et al., 2010). Furthermore, intravenous administration of 2-(17-hydroperoxydocosahexaneoyl)-lyso-PtdCho more effectively blocks zymosan A-induced plasma leakage than 2-docosahexaenoyl lyso-PtdCho. This observation suggests that 2-(17-hydroperoxydocosahexaneoyl)-lyso-PtdCho, a product from oxygenation of 2-docosahexaenoyl-lyso-PtdCho by 15-lipoxygenase (LOX), may be an active metabolite, intimately responsible for anti-inflammatory effects of 2-docosahexaenoyl-lyso-PtdCho. In addition, 2-docosahexaenoyl-lyso-PtdCho is more efficient than 1-docosahexaenoyl-lysoPC or docosahexaenoic acid (DHA) as substrate for 15-lipoxygenases such as soybean LOX-1, leukocyte 12/15-LOX, and human 15-LOX-2. Collectively, these studies suggest that 2-docosahexaenoyl-lyso- PtdCho and its oxygenation products may exert anti-inflammatory action after oral administration (Hung et al., 2010).

Although several isoforms of PLA2, including cPLA2, sPLA2, and circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) or platelet-activating factor acetylhydrolase are activated in brain following ischemic injury, but Lp-PLA2 has emerged as a novel biomarker for cerebrovascular and cardiovascular diseases

(Zalewski et al., 2009; Mannheim et al., 2008). Lp-PLA2 is a 45-kDa protein with 3.4 Lyso-phospholipids in Neurotraumatic and Neurodegenerative Diseases 95

441 amino acids that is distinct from other members of the phospholipase A2 family in that it is calcium independent. It is produced by inflammatory cells, co-travels with circulating low-density lipoprotein (LDL), and hydrolyzes phospholipids containing oxidatively fragmented residues at the sn-2 position (oxidized phospholipids; OxPLs) into proinflammatory lyso-PtdCho and oxidized nonesteri- fied fatty acids in LDL. OxPLs accumulate in the artery wall and are closely associated with vascular inflammation and atherosclerosis involved in cerebrovascular and cardiovascular diseases. Plasma levels of OxPLs present on apolipoprotein B-100 particles (OxPL/apolipoprotein B) are not only involved in coronary artery, carotid, and peripheral arterial disease, but also strongly correlated with lipoprotein levels (Tsimikas et al., 2007). Selective inhibition of Lp-PLA2 with darapladib (an inhibitor under development and testing by GlaxoSmithKline plc) retards the development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine (Wilensky et al., 2008). Darapladib markedly inhibits plasma and lesion

Lp-PLA2 activity and decreases the levels of lyso-PtdCho in the lesions. Detailed studies on coronary gene expression indicate that darapladib exerts a general anti- inflammatory action by reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment not only produces a considerable decrease in plaque area, but also induces notable decrease in necrotic core area. Collective evidence suggests that selective inhibition of Lp-PLA2 retards the progression and formation of advanced coronary atherosclerotic lesions. In addition Darapladib also stabilizes these dangerous plaques by decreasing the size of the core and reducing the number of inflammatory-immune cells present within the plaque (Wilensky et al., 2008). Levels of lyso-PtdH are increased in cerebral ischemia (Sun et al., 1992; Tigyi et al., 1995). Aspirin taken for 1 month significantly lowers lyso-PtdH level in stroke patients (n = 142) (2.41 ± 1.03 mM/L) compared with that before taking acetylsalicylate (4.06 ± 1.03 mM/L (Li et al., 2008)). There is a close relationship between increased plasma lyso-PtdH levels and platelet activation. As mentioned above, high levels of lyso-PtdH induce neurite retraction, a process associated with neurodegeneration. It is proposed that lyso-PtdH acts through Rho-mediated signaling to induce contraction- related cytoskeleton changes to reorganize astrocytic morphology (Tigyi et al., 1996a, b; Ramakers and Moolenaar, 1998).

3.4.2 Lyso-phospholipids in Alzheimer Disease

AD is characterized by accumulation of aggregated Ab peptide and neurofibrillary tangles composed of hyperphosphorylated t protein. Experimental evidence indicates that Ab accumulation precedes and drives t aggregation (Oddo et al., 2003; Octave, 2005).

In AD, increase in activities of PLA2 isoforms and lysophospholipases is accompanied by elevation in phosphodiesters, phosphomonoesters, fatty acids, 96 3 Metabolism, Roles, and Involvement of Lyso-glycerophospholipids… prostaglandins, isoprostanes, 4-hydroxynonenals, and other lipid mediators (Table 8.2) (Farooqui and Horrocks, 2006, 2007). Very little is known about levels of lysophospholipids in Alzheimer disease (AD). Determination of lyso-PtdCho in CSF of AD patients and subjects with subjective memory complaints without dementia by tandem mass spectrometry indicate that lyso-PtdCho levels and the lyso-PtdCho/PtdCho ratio are significantly decreased in CSF of AD patients compared to controls. The decrease in lyso-PtdCho levels may be due to increase in lysophospholipase activity in AD patients (Farooqui et al., 1990). The lower lyso- PtdCho/PtdCho ratio in CSF of patients with AD indicates that alterations in the metabolism of choline-containing phospholipids in the brain in AD may be closely associated with membrane alterations in AD (Mulder et al., 2003). Physicochemical and pathological consequences of decrease in lyso-PtdCho/ PtdCho ratio may cause alterations in membrane fluidity and permeability, alterations in ion homeostasis, and induction of oxidative stress (Farooqui and Horrocks, 2007; Farooqui, 2009). Hyperphosphorylation and accumulation of tau in neurons (and glial cells) is one of the major pathologic hallmarks in AD and other tauopathies, such as Pick disease (PiD), progressive supranuclear palsy, corticobasal degeneration, and argyrophilic grain disease (Ferrer et al., 2005). Hyperphosphorylation of tau is modulated by several kinases that phosphorylate specific sites of tau in vitro. Based on the effect of kinase inhibitors, involvement of glycogen synthase kinase-3 (GSK-3) has been proposed. Phosphorylation of GSK-3 at Ser9 inactivates GSK-3 in the majority of neurons with neurofibrillary tangles and dystrophic neurites of senile plaques of AD, and in Pick bodies. Although detailed investigations have not been performed, lyso-PtdH-mediated activation of GSK-3 occurs in the Rho pathway and may represent an important link between microtubule and microfilament dynamics in AD (Sayas et al., 1999, 2002).

3.4.3 Lyso-phospholipids in MS and EAE

Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination and axonal transaction resulting in reversible and irreversible neurological deficits. The pathophysiology of MS is illusive, but may involve multifactorial immune- mediated demyelination (Steinman et al., 2002). Activities and expression of isoforms of PLA2 (cPLA2 and iPLA2 as well as sPLA2) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS in rodents. As stated above, PLA2 isozyme generated arachidonic acid via cyclooxygenases and lipoxygenases is metabolized into protaglandins and leukotrienes, which contribute to neuroinflammation (Calder,2 003). The other product of PLA2 isozyme catalyzed reaction, lyso-PtdCho, also facilitates neuroinflammation and demyelination by upregulating the expression of proinflammatory cytokines and chemokines and initiating the synthesis of platelet activating factor (Ousman and David, 2000, 2001). References 97

3.4.4 Lyso-phospholipids in NCL

Infantile-onset Neuronal Ceroid Lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder caused by mutations in the gene encoding palmitoyl-protein thioesterase 1 (PPT1), an enzyme responsible for the removal of a palmitate post- translational modification from an unknown set of substrate proteins (Zhang et al., 2007). PPT1-knockout (PPT1-KO) mice brain containing increased levels of lyso-

PtdCho caused the activation of cPLA2. Age-dependent increase in lyso-PtdCho levels in the PPT1-KO mouse brain positively correlates with elevated expression of the genes characteristically associated with phagocytes. It is proposed that increased synthesis of lyso-ptdCho in the PPT1-KO mouse brain is at least one of the mechanisms that promote phagocyte infiltration in the pathophysiology of INCL (Zhang et al., 2007).

3.5 Conclusion

Action of PLA1 and PLA2 generates lysophospholipids in the brain. In addition, lysophospholipids are also generated through the transacylation reactions between phospholipids. In normal brain, lysophospholipids are not only involved in the modulation of receptor-mediated neurotransmitter (dopamine, glutamate, and acetylcholine) release and regulation of activities of enzymic activities (protein kinases, nitric oxide synthases, and cytidylyltransferases), but also in transport of docosahexaenoic acid through the blood–brain barrier, microglial cell activation and deramification, neuroinflammation, and modulation of exocytosis and in transcription of cytokines and chemokines. In neurotraumatic and neurodegenerative diseases, the accumulation of lysophospholipids promotes oxidative stress, demyelination, and neural cell injury. In neural membranes, their levels are controlled not only by their reacylation, but also through activities of lysophospholipases, as well as PLA2. In neural and non-neural cells, lyso-glycerophospholipids also induce morphological changes in neural membranes including membrane fusion and lysis. They also induce secretion of hormones, growth factors, and other lipid mediators that modulate the homeostasis and dynamics of membrane glycerophospholipids.

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4.1 Introduction

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent biologically active mediator that is involved in intracellular and extracellular communication. PAF belongs to a family of biologically active and structurally related alkyl phospholipids produced by neural and non-neural cells (neural cells, macrophages, platelets, endothelial cells, mast cells, and neutrophils). PAF not only induces platelet aggregation, but also enhances histamine and serotonin release resulting in vasodilation and enhancement in vascular permeability, increase in eosinophil and neutrophil motility, and degranulation producing edema, hyper- emia, itching, and pain (Farooqui et al., 2008). PAF is normally present in the mammalian tissues in picomolar concentrations and is found both in the cytosol and body fluids including blood plasma, cerebrospinal fluid, urine, and amniotic fluid (Lynch and Hensen, 1986). PAF-synthesizing enzymes (phospholipase A2 and acetyltransferase) are modulated by MAP kinase signaling pathways (Farooqui et al., 2008) whereas PAF-hydrolyzing enzyme (PAF-acetyl hydrolase) is regulated by proinflammatory cytokines such as LPS, TNF-a, IL-1, IL-8, and IFN-a. Thus, levels of PAF are tightly regulated both by PAF synthesizing enzymes

(plasmalogen-phospholipase A2 and acetyltransferase) and PAF hydrolyzing enzymes (PAF-acetylhydrolases) (Farooqui et al., 2008). In brain tissue, PAF may be associated with neural cell migration, gene expression, calcium mobilization, noniception, and long-term potentiation (Fig 4.1). PAF also modulates neural plasticity (Moriguchi et al., 2010). In cultured hippocampal neurons, PAF increases autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of synapsin I and myristoylated alanine-rich protein kinase C substrate (MARCKS). In hippocampal neurons, PAF exposure also increases protein kinase Ca (PKCa) autophosphorylation and extracellular signal-regulated kinase (ERK) phosphorylation. Collective evidence suggests that PAF induces synaptic facilitation through activation of CaMKII, PKC, and ERK in the hippocampal CA1 region (Moriguchi et al., 2010). Although enzymes associated with

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 107 DOI 10.1007/978-1-4419-9940-5_4, © Springer Science+Business Media, LLC 2011 108 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement…

Excitotoxicity

Gene expression

Calcium mobilization

Platelet activating factor BBB permeability

Long-term potentiation

Apoptosis

Immune function

Fig. 4.1 Roles of platelet-activating factors in the brain

PAF synthesis have not been purified and fully characterized from brain tissue, many reports on PAF synthesis in mammalian brain have been published (Francescangeli et al., 2000; Goracci et al., 2009). In neural cells PAF is not stored, but synthesized either spontaneously or under appropriate stimulation. In neuronal and glial cell cultures, acetylcholine (ACh) dramatically stimulates PAF synthesis, and addition of cholinergic receptor antagonist, atropine, produces the inhibition of PAF synthesis (Sogos et al., 1990). PAF may be involved in interactions between astroglial cells and neurons. Astrocytic PAF mediates neurotrophic signaling from astrocytes to injured neurons. It is suggested that interplay between PAF and the neurotrophic receptor may be involved in regenerative processes in the brain tissue. In brain, physiological activity of PAF is not limited to its proinflammatory function, neurotoxicity, apoptosis, and blood–brain barrier permeability, but also associated with neurotrophic effects (Fig. 4.1). In non-neural cells, PAF is not only involved in neutrophil adhesion, chemotaxis, allergic reactions, and reproduction, but also in increased vascular permeability, vasodialation, and circulatory system disturbances (Chao and Olson, 1993; Honda et al., 2002; Bazan, 2003).

4.2 PAF Biosynthesis in the Brain

Three different pathways are responsible for PAF synthesis in mammalian tissues (Honda et al., 2002; Snyder, 1995). De novo synthesis is a minor pathway responsible for the synthesis of basal levels of PAF for maintaining normal functions under physiological conditions. The remodeling pathway is a major pathway of PAF generation. High levels of PAF are synthesized from a specific subclass of PtdCho that 4.2 PAF Biosynthesis in the Brain 109

CH Acetyl-CoA CH 2 O-CH2-(CH2)n-CH3 2 O-CH2-(CH2)n-CH3 acetyltransferase O DHAP HO CH + Acetyl-CoA CH -C -O CH O 3 O

O P OH OPOH CH2 CH2 O O

1-O-alkyl-glycero-3-phosphate 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate

Phosphohydrolase

CH O-CH -(CH )n-CH CH O-CH -(CH )n-CH O 2 2 2 3 O 2 2 2 3 CH -C-O CH Cholinephosphotransferase CH -C-O CH 3 O 3

O PO-choline OH CH2 CH2 O CDP CDP-Choline PAF 1-O-alkyl-2-acetyl-sn-glycerol Cytidylytransferase

Phosphocholine+ CTP

Fig. 4.2 De novo synthesis of platelet-activating factor in the brain contains an ether bond, rather than an ester bond at the sn-1 position of the glycerol backbone (Snyder, 1995; Bazan, 2003) in response to glutamate release and stimulation in cytokine expression (tumor necrosis factor-a, interferon-g, and interleukin-1) under pathological conditions (Sogos et al., 1990; Prescott et al., 2000; Kunievsky and Yavin, 1994). Under oxidative stress, oxidative breakdown of PtdCho also results in generation of minor oxidized phospholipids, which can produce PAF- like activity in neural and non-neural cells. This minor pathway is called as oxidative fragmentation pathway of PAF synthesis.

4.2.1 De Novo Synthesis of PAF

In resting state, neural cells synthesize by the de novo synthesis. Three enzymes namely 1-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP): acetyl-CoA acetyltransferase, 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase, and dithiothreitol (DTT)-insensitive 1-alkyl-2-acetyl-sn-glycerol: CDP-choline phosphotransferase (Heller et al., 1991; Snyder, 1995) are associated with the de novo synthesis of PAF (Fig. 4.2). In this pathway, a 1-Alkyl-2-lyso-sn-glycero-3-phosphate (alkyllyso- GP): acetyl-CoA acetyltransferase transforms 1-O-alkyl-glycero-3-phosphate into 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate in the presence of acetyl-CoA (Lee et al., 1986; Baker and Chang, 1993). Specific activity ofalkyl-lyso-GP:acetyl-CoA 110 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement… acetyltransferase in nuclear fraction of cerebral cortex is three times those of the microsomal fraction (Baker and Chang, 1996). This enzyme shows optimal activity between pH 8–9 and is inhibited by MgATP or oleoyl CoA. The alkyl-lyso-glycerol phosphate acetyl-CoA acetyltransferase is distinguished from the nuclear lyso-PAF acetyltransferase by a greater sensitivity to MgATP inhibition (Baker and Chang, 1996, 1998, 2000). Bovine serum albumin or the fatty acyl CoA synthetase inhibitor (Triacsin C) prevents MgATP inhibition. Addition of exogenous free fatty acids promotes the MgATP-induced inhibition. Oleoyl CoA, in the absence of MgATP, also inhibits alkyl-lyso-GP:acetyl-CoA acetyltransferase suggesting that MgATP stimulates the conversion of nuclear free fatty acids to fatty acyl CoA. Fatty acyl CoA may directly inhibit nuclear alkyl-lyso-GP:acetyl-CoA acetyltransferase (Baker and Chang, 2002). 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate is then dephosphorylated into 1-O-alkyl-2-acetyl-sn-glycerol by 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase (Lee et al., 1988). Very little information is available on brain 1-alkyl- 2-acetyl-sn-glycero-3-phosphate phosphohydrolase. This phosphohydrolase shows no notable substrate selectivities with regard to variations in alkyl chain length (C16:0 versus C18:0) at the sn-1 position or short-chain acyl groups (C2:0 to C6:0, with the exception of C3:0) at the sn-2 position of the glycerol moiety. In the brain, the enzymic activity of 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase is 30- to 90-fold higher than alkyl-lyso-GP:acetyl-CoA acetyltransferase (Lee et al., 1988). 1-O-Alkyl-2-acetyl-sn-glycerol is converted into PAF by a DTT-insensitive microsomal 1-alkyl-2-acetyl-sn-glycerol: CDP-choline phosphotransferase. Although this enzyme occurs in brain, 1-alkyl-2-acetyl-sn-glycerol: CDP-choline phosphotransferase has not been purified and characterized from brain tissue. Availability of CDP-choline is the rate-limiting step in the PAF synthesis (Snyder, 1997; Gimenez and Aguilar, 2001). Therefore, factors that regulate cytidylyltransferase activity play an important role in modulating the levels of PAF in various tissues. For example, fatty acids (Blank et al., 1988; Vallari et al., 1990) and intracellular levels of CDP-choline (Lee et al., 1990) have been shown to upregulate PAF biosynthesis through the de novo synthesis. Brain PAF-synthesizing 1-alkyl-2- acetyl-sn-glycerol: CDP-choline phosphotransferase not only shows similar subcellular localization, Mg2+ requirement, and Ca2+ inhibitory properties, but also has similar thermal inactivation profiles as the kidney medulla enzyme (Goracci and Francescangeli, 1991; Francescangeli et al., 2000). It shows optimal activity at pH 8.0, and Km and Vmax values for 1-alkyl-2-acetylglycerol were 42 mM and 3.0 nmol/min/mg protein, respectively (Goracci and Francescangeli, 1991). Subcellular distribution studies in 15-day old rabbit cerebral cortices indicate that PAF-synthesizing cholinephosphotransferase shows highest specific activity in microsomal fraction. The microsomal fraction is subfractioned into rough and smooth microsomal fractions (Baker and Chang, 1993). Rough microsomal sub- fraction has higher specific activity than smooth microsomal fraction. PAF- synthesizing cholinephosphotransferase is inhibited by Triton X-100 (Francescangeli et al., 2000). 4.2 PAF Biosynthesis in the Brain 111

H C O CH (CH ) CH H C O CH (CH ) CH 2 2 2 n 3 O 2 2 2 n 3 2+ PLA2 Ca CH3 (CH ) CH2 C O C OH HO C OH 2 n O O O P O CH CH N(CH ) OP O CH CH N(CH ) H2C 2 2 3 3 H2C 2 2 3 3 O Fatty acid O

Acetyl-CoA Acyltransferase Acetyltransferase

Acyl-CoA

Acetic acid H C 2 O CH2 (CH2)n CH3 H2C O CH2 (CH ) CH3 O 2 n HO C OH CH3 C O C OH O Acetylhydrolase O OP O CH CH N(CH ) H2C 2 2 3 3 OP O CH CH N(CH ) H2C 2 2 3 3 O O

Lyso-PAF PAF

Fig. 4.3 Remodeling pathway for the synthesis of platelet-activating factor in brain and inflammatory cells

4.2.2 Remodeling Pathway for PAF Synthesis

The remodeling pathway involves a structural modification of preexisting ether- linked phospholipids that serve as structural components of membranes (Stafforini et al., 1987; Snyder et al., 1996). The remodeling pathway occurs primarily in inflammatory cells (Fig. 4.3). In this pathway, arachidonic acid is hydrolyzed from 1-O-alkyl-2-arachidonyl-sn-glycero-3-phosphocholine by cytosolic phospholipase

A2 (cPLA2), resulting in the generation of 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF) (Rubin et al., 2005). Lyso-PAF is acetylated by acety- CoA: 1-O-alkyl-2-lysophosphatidylcholine acetyltransferase to produce PAF. This enzyme is localized in microsomes. Acetyl CoA: lyso-PAF acetyltransferase has been purified and cloned from non-neural tissues (Shindou et al., 2007). The enzyme has molecular mass of 60 kDa. Addition of detergents to the reaction mixture results in total inactivation of the enzymic activity. Glycerol stabilizes the enzyme activity. DTT and mercaptoethanol significantly inhibit acetyl CoA: lyso-PAF acetyltransferase, indicating that there are disulfide bridges present in the enzyme. Tyrosine kinase inhibitors and calmodulin antagonists inhibit acetyl CoA: lyso-PAF acetyltransferase activity in H2O2-stimulated cells, suggesting that tyrosine kinase and calcium/calmodulin-dependent protein kinase may be associated with regulation of acetyltransferase activity (Tosaki et al., 2007). Similarly, in human neutrophils, 112 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement…

PlsEtn Recepto r

PlsEtn-PLA2 ARA or DHA

COX, Lyso-PlsEtn PakCho LOX 15-LOX PGs, LTs, Txs and LXs

PlsEtn Lyso-PAF Resolvins and neuroprotectins

PAF Lyso-PAF hydrolase acetyltransferase

PAF Cellular response and modulation of neural cell function

Fig. 4.4 Conversion of plasmalogen into platelet-activating factor in neural and non-neural cells.

Plasmalogens are hydrolyzed by PlsEtn-PLA2 into lyso-PlsEtn. Transacylation of lysoPlsEtn (lysoplasmalogen) with PakCho (alkylacyl-glycerol-3-phosphocholine) results in the production of lyso-PAF. Acetylation of lyso-PAF by acetyl-CoA: 1-alkyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase results in synthesis of PAF acetyltransferase activity is modulated by mitogen-activated protein kinases, namely the p38 kinase (Nixon et al., 1999). Thus non-neural acetyl CoA: lyso-PAF acetyltransferase activity is modulated by phosphorylation/dephosphorylation process. In the endothelial cells, acetyl CoA: lyso-PAF acetyltransferase is not only regulated by cytosolic Ca2+ and H+, but through the activation of protein kinases C and A (Heller et al., 1991). Collective evidence suggests that acetyl CoA: lyso-PAF acetyltransferase activity is regulated by phosphorylation/dephosphorylation processes. PAF can also be synthesized from plasmalogens. Thus, endothelin-mediated stimulation of brain PlsEtn-PLA2 decreases PlsEtn levels, and evokes the generation of PAF (Collado et al., 2003). The second step of PAF production requires the conversion of lyso-PAF to PAF by the enzyme acetyl CoA: lyso-PAF acetyltransferase (Fig. 4.4). Both of these enzymes are activated by post-translational phosphorylation (Prescott et al., 1990; Baker et al., 2002). The transfer of the acetyl group from PAF to lysoplasmalogen in a CoA-independent manner is catalyzed by PAF:lysoplasmalogen transacetylase in HL-60 cells, endothelial cells, and a variety of rat tissues (Karasawa et al., 1999; Lee et al., 1992). The purified enzyme catalyzes the transacetylation of the acetyl group not only from PAF to lysoplasmalogen forming plasmalogen analogs of PAF, but also to sphingosine producing

N-acetylsphingosine (C2-ceramide) (Karasawa et al., 1999). In addition, this enzyme 4.2 PAF Biosynthesis in the Brain 113

PAF-R Arachidonyl- 1-O-Alkyl-2- arachidonyl- PtdCho Lyso-PtdEtn PtdIns-4,5-P PtdIns-4,5-P PtdCho 3 2 Gi PtdIns3K cPLA2 Akt Akt S473 + ARA P ERK cPLA2

P38 MARK COX, PtdEtn Lyso-PAF LOX

Acetyl- transferase Bcl-2 Cell survival Cell death

Eicosanoids PAF

Cellular response and modulation of neural cell function

Fig. 4.5 PAF receptor-mediated modulation of cellular responses. Lysophosphatidyethanolamine (lyso-PtdEtn); phosphatidylethanolamine (PtdEtn); lyso-platelet-activating factor (lyso-PAF); platelet-activating factor (PAF); lysophosphatidyethanolamine (lyso-PlsEtn); cytosolic phospholipase A2 (cPLA2); cyclooxygenase (COX); 15-lipoxygenase (15-LOX); arachidonic acid (AA); docosahexaenoic acid (DHA); extracellular-signal-regulated protein kinase (ERK); and p38 mitogen-activated protein kinase (p38 MAPK); G protein (Gi); plus sign indicates activation, and minus sign indicates inhibition has PAF-acetylhydrolase activity in the absence of lipid acceptor molecules. Collective evidence suggests that PAF-dependent transacetylase is an enzyme that modifies the cellular functions of PAF through generation of other diverse lipid mediators (Karasawa et al., 1999). The addition of exogenous lysoplasmalogens or thrombin to endothelial cell cultures markedly stimulates the synthesis of PAF in a CoA-independent manner

(McHowat et al., 2001). This suggests that plasmalogen-selective PLA2, an enzyme that hydrolyzes plasmalogen into lysoplasmalogen, may modulate PAF synthesis (Fig. 4.5). Based on these observations, it has been proposed that cross-talk (interplay) may occur between plasmalogen and PAF metabolism in vivo. This cross- talk may be involved in fine-tuning not only PAF-receptor-induced biological responses, but also generation and maintenance of levels of other lipid mediators such as arachidonic acid-derived eicosanoids and docosahexaenoic acid-derived docosanoids through the receptor-mediated plasmalogen degradation of plasmalogens (Farooqui and Horrocks, 2007). In non-neural cells, activation of the PAF receptor (PAF-R) modulates PtdIns 3-kinase signaling, resulting in the conversion of PtdIns 3,4,5- trisphosphate into PtdIns-4,5-bisphosphate by Akt, a process 114 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement… closely associated with the activation of mitochondria and caspase-mediated apoptotic cell death (Fig. 4.5) (Lu et al., 2008). The effect of PAF-R activation is mimicked by LY-294002, an inhibitor of PtdIns 3-kinase. Conversion of PtdIns- 4,5-bisphosphate into PtdIns 3,4,5-trisphosphate results in the inhibition of mitochondrial depolarization inducing cell survival.

4.2.3 Oxidative Fragmentation Pathway for PAF Synthesis

The third pathway for the synthesis of PAF involves oxidative fragmentation of PtdCho. Oxidation of 1-O-alkyl-2-arachidonyl-sn-glycerophosphocholine results in the generation of many species of 1-O-alkyl phospholipids with different short-chain substituents at the sn-2 position. These 1-O-alkyl phospholipids interact with PAF-R and mediate a variety of biological effects (Stafforini et al., 1996; Prescott et al., 2000). The fragmentation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by ozonolysis generates 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (Smiley et al., 1991; Prescott et al., 2000). This phospholipid stimulates human neutrophils at submicromolar concentrations, and its effects are blocked by specific PAF receptor antagonists (WEB2086, L659989, and CV3988) (Smiley et al., 1991). Similarly, oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine with soybean lipoxygenase results in the production of 15-hydroperoxy derivatives, which are further oxidized to numerous fragmented phospholipids, some of which interact and activate polymorphonuclear leukocytes. The hydrolysis of these derivatives with PAF acetylhydrolase blocks their biologic activity. Based on these observations, the occurrence of oxidative fragmentation pathway of PAF analog production has been proposed in non-neural tissues.

4.3 Catabolism of PAF in the Brain

PAF is hydrolyzed by an enzyme called PAF acetylhydrolase. This enzyme selectively removes short acyl chains (C2 to C9) from the sn-2 position of PAF (Fig. 4.6), and has no activity with acyl chains longer than C9. PAF structural analogs are also broken down by PAF-acetylhydrolase. These analogs competitively inhibit PAF-acetylhydrolase activity (Stafforini et al., 1997). PAF receptor antagonists block PAF-acetylhydrolase activity. This is in contrast to cPLA2 and sPLA2 that 2+ 2+ require Ca . cPLA2 uses Ca for binding to phospholipid substrate, which is 2+ located in neural membranes, whereas sPLA2 needs mM concentrations of Ca for catalysis (Farooqui et al., 2006). Besides short chain phospholipids, PAF- acetylhydrolase also degrades short-chain diacylglycerols, triacylglycerols, and acetylated alkanols (Tselepis and Chapman, 2002; Min et al., 2001). Unlike cPLA2 and sPLA2, PAF-acetyl hydrolase degrades PAF, its analogs, and short- chain oxidized phospholipids in a calcium-independent manner (Gelb et al., 2000). 4.3 Catabolism of PAF in the Brain 115

CH OR CH2OR 2 O CH COCH PAF acetylhydrolase HOCH 3 O O O + CH COH H COPOCH CH N(CH ) 3 H2COPOCH2CH2N(CH3)3 2 2 2 3 3 O O (PAF) (Lyso-PAF) (Acetate)

CH OR CH OCH CH R 2 Neutral lipid 2 2 2 O acetylhydrolase O H HOC H + CH3CO C CH3COH

H2COH H2COH

(1-Alkyl-2-acetyl-sn-glycerol) (Alkylglycerol) (Acetate)

Fig. 4.6 Hydrolysis of PAF and 1-alkyl-2-acetyl-sn-glycerol by PAF-acetylhydrolase

The structure of PAF-acetylhydrolase is similar to neutral lipases and serine esterases. PAF-acetylhydrolase contains a serine/aspartate/histidine catalytic triad whose linear orientation and spacing are consistent with the a/b hydrolase conformation of other lipases and esterases. At least three PAF-acetyl hydrolases occur in mammalian tissues. These enzymes have different physicochemical and kinetic properties, and are encoded by different genes. Two PAF-acetylhydrolases, Type I, and Type II, are intracellularly found in brain and other visceral organs, and the third PAF-acetylhydrolase is extracellular that is found in plasma (Arai et al., 2002; Tjoelker and Stafforini, 2000; Arai, 2002; Stafforini, 2009). Although Type I and Type II are serine-dependent enzymes, their ability to hydrolyze PAF is quite different. Type I PAF-acetylhydrolases are found in two forms (Ia and Ib isoforms) in brain tissue. Ia is a heterodimeric isoform composed of a1 and a2 subunits, whereas Ib isoform is composed of a1, a2, and b subunits, and is coupled with G protein. Ib isoform is abundantly found in brain tissue

(Hattori et al., 1996). The majority of adult tissues, except the brain express only a2 subunit. Type I PAF-acetylhydrolases are expressed in macrophages and in tissues containing a high content of inflammatory cells (Derewenda and Derewenda,1 998; Arai, 2002; McMullen et al., 2000). Type II PAF-acetylhydrolase is a monomer of 40 kDa with 392 amino acid residues. PAF acetylhydrolases contain a Gly-X-Ser-X-Gly motif that is characteristic of lipases and serine esterases. The purified type I and II PAF acetylhydrolases show similar activity against PAF and oxidatively modified phosphatidylcholine, but do not hydrolyze phosphatidylcholine or phosphatidylethanolamine with two long- chain acyl groups (Hattori et al., 1993, 1996; Manya et al., 1998, 1999). These enzymes differ from each other in primary sequence, tissue distribution and localization, subunit composition, and substrate specificities. 116 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement…

Plasma PAF-acetylhydrolase has been purified from several sources using multiple column chromatographic procedures. SDS-PAGE indicates that molecular mass varies from 43 to 63 depending on animal species (Karasawa et al., 2003). Plasma PAF acetylhydrolase shares 43% homology with type II PAF acetylhydrolase. The binding of purified enzyme with concanavalin A suggests that plasma PAF acetylhydrolase is a glycoprotein. Determination of carbohydrates in purified enzyme indicates that it contains N-linked heterogenous sugar chains (9 kDa) containing sialic acid. Treatment of human plasma PAF-acetylhydrolase with peptide N-glycosidase F results in production of lower molecular mass peptide as indicated by the changes in mobility on the SDS-PAGE. This observation again supports N-glycosylated nature of human plasma PAF-acetylhydrolase. Intracellular type II and plasma PAF acetyl hydrolases have high affinity for esterified2 F -isoprostanes, a series of prostaglandin-like compounds formed in vivo through nonenzymic peroxidation of arachidonic acid, but the rate of esterified F2-isoprostane hydrolysis is slower compared with the rate of hydrolysis of other substrates hydrolyzed by these enzymes (Stafforini et al., 2006; Stafforini, 2009). Accumulating evidence suggests that PAF-acetyl hydrolases are unique serine esterases that transform PAF into lyso- PAF (Blank et al., 1981). The levels of PAF-acetyl hydrolase are critical for the regulation of the circulatory PAF and its physiological and pathological activities (Tjoelker and Stafforini, 2000). Some forms of PAF-acetylhydrolases are specific for PAF while others act on variety of PAF analogs. Thus, PAF-acetylhydrolases act as general scavengers of glycerophospholipids species, which may accumulate inappropriately under pathological conditions (Tjoelker and Stafforini, 2000). In brain tissue, PAF and its metabolism are involved in neuroprotection, inhibition of inflammation, modulation of long-term potentiation, modulation of gene expression, and neuronal migration (Umemura et al., 2007; Tjoelker and Stafforini, 2000; Bate et al., 2004; Bazan, 2003; Grassi et al., 1998) (Fig. 4.1).

4.4 PAF Receptors in the Brain

As mentioned above, PAF may be associated with neural cell migration, gene expression, calcium mobilization, noniception, and long-term potentiation in brain tissue (Fig. 4.1). PAF interacts with specific receptors called as PAF receptors (PAF-Rs). These receptors have been cloned and characterized from neural and non-neural tissues (Honda et al., 1991). PAF-Rs consist of seven transmembrane helices, which are coupled with G proteins such as Gao, Gai, Gbg, and Gaq (Honda et al., 1991; Clark et al., 2000). PAF-R mRNA has been detected in hypothalamus, medulla-pons, olfactory bulb, hippocampus, cerebral cortex, spinal cord, thalamus, and cerebellum in rat brain. In situ hybridization and Northern blotting studies indicate that mRNA for PAF-R is expressed in both neurons and glial cells (Bito et al., 1992; Marcheselli et al., 1990; Mori et al., 1996; Ishii et al., 1996).

Stimulation of PAF-Rs results in activation of phospholipases (PLA2, PLC, and PLD), GTPases, and kinases (Table 4.1) (Maclennan et al., 1996; Mori et al., 1996; 4.4 PAF Receptors in the Brain 117

Table 4.1 PAF-mediated stimulation of enzymic activities in neural and non-neural cells Enzyme Effect Reference

Phospholipase A2 Stimulation Izumi and Shimizu, 1995 Phospholipase C Stimulation Izumi and Shimizu, 1995 Phospholipase D Stimulation Izumi and Shimizu, 1995 Adenylate cyclase Inhibition Izumi and Shimizu, 1995 PtdIns-3-kinase Stimulation Izumi and Shimizu, 1995 MAP-kinase, protein tyrosine kinase Stimulation DeCoster et al., 1998 Stimulation Izumi and Shimizu, 1995 Cyclooxygenase-2 Stimulation Bazan et al., 1991 Metalloproteinase-9 Stimulation Ottino et al., 2005 Caspase-3 Stimulation Hostettler and Carlson, 2002

PAF-R Extracellular 1-O-alkyl-2-arachidonyl-sn- PtdIns-4,5-P PM glycerophosphocholine 2 Gi Gq Intracellular + ? cPLA2 PAF PtdIns MEKK PKC DAG + InsP3 ARA kinase Remodeling pathway

Ca2+ mobilization PKC Raf-1

MAPKK Eicosanoids

MAPK

Cellular response

Fig. 4.7 PAF receptor-mediated modulation of kinases and gene expression in brain. Phosphatidylinositol 3-kinase (PtdIns kinase); protein kinase Cz (PKCz); mitogen-activated protein kinase kinase kinase (MEKK); protein kinase C (PKC); product of oncogene c-raf (raf-1). This protein functions in the MARK/ERK signal transduction; mitogen-activated protein kinase kinase (MARKK); mitogen-activated protein kinase (MARK); platelet activating factor (PAF); and platelet activating factor receptor (PAF-R)

Honda et al., 2002; Kornecki and Ehrlich, 1991; Clark et al., 2000). Activation of

PLA2, PLC, and PLD liberates arachidonic acid, which is converted into eicosanoids through the action of cyclooxygenases and lipoxygenases (Fig. 4.7). PAF-mediated stimulation of polyphosphoinositide turnover generates diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (InsP3) inducing protein kinase C activation and 118 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement… elevation in intracellular calcium levels (Izumi and Shimizu, 1995; Ishii and Shimizu, 2000). PAF activates guanylate cyclase, which generates cGMP; and inhibits adenylate cyclase, which lowers cAMP levels. These processes modulate phosphorylation of many proteins through the activation of various kinases including MAP-kinases, phosphatidylinositol 3-kinase, and tyrosine kinases (Chao and Olson, 1993; Honda et al., 2002; Ishii and Shimizu, 2000). These enzymes are closely associated with modulation of gene expression in neuronal and glial cells (Bazan et al., 1994). In addition, PAF-R activation is not only involved in the elevation of calcium via calcium channel opening as well as from intracellular stores (Izumi and Shimizu, 1995; Ishii and Shimizu, 2000), but also in upregulation of nerve growth factor expression and generation of several lipid mediators, processes that are closely associated with neurodegeneration as well as neuroprotection in brain tissue.

4.5 PAF in Neurological and Visceral Disorders

As stated above, PAF is not stored, but synthesized by activated neural cells (neurons, astrocytes, oligodendrocytes, and microglial cells) as well as non-neural cells (platelets, inflammatory and endothelial cells) on cellular demand by remodeling, de novo synthesis, and oxidative fragmentation pathways. De novo synthesis is not affected by the external stimuli. In response to neural injury, convulsions, and oxidative stress, the remodeling and oxidative fragmentation pathways are activated in neural and non-neural cells. Treatment of neural cells with neurotransmitters such as dopamine, acetylcholine, and glutamate results in PAF synthesis in a calcium- dependent manner (Sogos et al., 1990). Microglia, which express functional PAF receptors to a high level, show a marked chemotactic response to PAF. Microglia derived from PAF-receptor-deficient mice do not show chemotactic response (Aihara et al., 2000). Thus, PAF functions as a key messenger in neuron-microglial interactions. On exposure to oxidative stress, neural and non-neural cells generate large quan- tities of oxidatively fragmented phospholipids or PAF-like lipids from neural and non-neural membrane phospholipids in brain regions, where the density of PAF receptors and PAF acetylhydrolase activity is very low. This may result in accumulation of PAF and PAF-like molecules, which may induce excitotoxic events and abnormal signal transduction processes, resulting in inflammation and oxidative stress associated with neural and non-neural diseases (Farooqui et al., 2007, 2008).

4.5.1 PAF in Neurological Disorders

PAF and PAF-like molecules accumulate during ischemia and traumatic brain injury, (TBI) and spinal cord injury (SCI) (Farooqui et al., 2008) (Fig. 4.8) and PAF receptor antagonists provide neuroprotection in the gerbil brain ischemia/reperfusion model by decreasing glutamate-mediated polyunsaturated fatty acid release 4.5 PAF in Neurological and Visceral Disorders 119

Cyto + Glu

1-O-alkyl-2-arachidonyl- sn-phosphocholine NMDA-R cPL A

Cytokine-R Gi + 2+ + Ca 2 Degradation ROS ARA Remodeling IκK pathway IκB-P p65 p50 Eicosanoids Lyso-PAF NF-κ B

Neuroinflammation Increased levels NF-κ B RE of PAF

TNF-α,IL-1β IL-6 & proinflammatory enzymes

Miller-Dieker HIV Stroke TBI and SCI Prion diseases MS Bacterial Nucleus lissencephaly miningitis

Neurological diseases

Fig. 4.8 PAF receptor-mediated alterations in lipid mediators and association of platelet-activating factor with neural and non-neural diseases. Nuclear factor-kappa B (NF-kB); Nuclear factor-kappa B response element (NF-kB-RE); tumor necrosis factor-a (TNF-a); interleukin-b1 (IL-b1); cytosolic phospholipase A2 (cPLA2); reactive oxygen species (ROS); N-methyl-d-aspartate receptor (NMDA-R); arachidonic acid (ARA); traumatic brain injury (TBI); spinal cord injury (SCI); multiple sclerosis (MS); and human immunodeficiency (HIV)

and restoring cerebral blood flow (Panetta et al., 1987; Belayev et al., 2009). During ischemic injury and epileptic seizures, the rates of PAF synthesis and breakdown no longer maintain a modulated PAF pool size resulting in increased PAF levels due to remodeling pathway. Thus, PAF accumulates during ischemia-reperfusion injury, and is involved in the activation of platelets, neutrophils, and proinflammatory signaling. At higher concentrations, PAF not only becomes proinflammatory messenger, but also activates COX-2 expression as well as several early response genes that encode transcription factors (Squinto et al., 1989; Bazan et al., 1994). PAF receptor antagonists provide sustained neuroprotection that may be valuable in designing the stroke therapy. In TBI and SCI, most of the damage that occurs in brain and spinal cord tissues is due to secondary injury caused by glutamate, Ca2+ overload, and oxidative stress. High levels of PAF can be detected in the rat brain after cold-induced local brain injury (Tokutomi et al., 2001). Brain damage caused by head injury can be partially prevented by BN-52021, a PAF antagonist. PAF also contributes to the secondary damage, following SCI. It is proposed that excitotoxicity, oxidative stress, and 120 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement… increased expression of cytokine induction may contribute to spinal cord trauma (Hostettler and Carlson, 2002). Treatment with PAF antagonist, WEB 2170, 15 min prior to injury significantly decreases mRNA levels for cytokines, and oxidative stress at 6 h post injury. These results suggest that PAF modulates the induction of proinflammatory cytokine after spinal cord trauma (Hostettler and Carlson, 2002). Increase in PAF levels is closely associated with neuroinflammation in epileptic seizures, Miller-Dieker lissencephaly, bacterial meningitis, multiple sclerosis, prion diseases, and HIV replication associated with AIDS dementia complex (Fig. 4.8) (Feuerstein, 1996; Farooqui et al., 2008). In addition, alterations in PAF have been reported to occur in Miller-Dieker lissencephaly, a neurodevelopmental disease characterized by thick cerebral cortex formed without its usual folds (Hattori et al., 1994; Walsh, 1998). As stated above, brain contains significant amounts of PAF- acetylhydrolase isoform Ib, an oligomeric protein complex, which contains three types of subunits: two homologous (63% identity) 26 kDa catalytic subunits (a1 and a2) harboring PAF-acetylhydrolase activity, and the 45 kDa b-subunit (LIS1), a product of the causal gene for Miller-Dieker lissencephaly (McMullen et al., 2000). During fetal development, the preferentially expressed a1-subunit forms a homodimer, which binds to a homodimer of LIS1, whereas in adult organisms a1/a2 and a2/a2 dimers, also bound to dimeric LIS1, are the prevailing species. It is reported that a1 expression is restricted to actively migrating neurons in rats and that switching of catalytic subunits from the a1/a2 heterodimer to the a2/a2 homodimer takes place in these cells during brain development, suggesting that PAF acetylhydrolase plays a role(s) in neuronal migration (Manya et al., 1998). The consequences of this ‘switching’ may contribute to the pathogenesis of Miller-Dieker lissencephaly (McMullen et al., 2000). Expression of LIS1 in the dentate gyrus modulates PAF-acetyl hydrolase activity (Shmueli et al., 1999). Reduction in LIS1 protein levels found in lissencephaly patients may render animals more susceptible to seizures. Overexpression of LIS-1 has been reported to interfere with cell division, with alterations in chromosome attachment to the mitotic spindle and on the interaction of astral microtubules with the cell cortex. LIS1/b also plays an important role in the induction of nuclear movement and control of microtubule organization. Although it is suggested that mutations in LIS-1 may produce a lissencephalic phenotype either by interfering with the movement of neuronal nuclei within extending processes, or by interference with the division cycle of neuronal progenitor cells in the ventricular and subventricular zones of the developing nervous system, the role of PAF and PAF-acetylhydrolase in the pathophysiology still remains controversial (Vallee et al., 2000; Arai, 2002). In animal models of lissencephaly, PAF agonists and antagonists modulate migration of cerebral granule and hippocampal cells indicating that PAF and PAF-like molecules may act like neural cell migration stop signal (Adachi et al., 1997; Bix and Clark, 1998). Alzheimer disease (AD), the cause of one of the most common types of dementia, is a brain disorder characterized by the formation of two main protein aggregates: senile plaques and neurofibrillary tangles. It is accompanied by the production and accumulation of the Ab1-42 peptide (Ab), loss of synapses, dementia, and gradual cognitive decline. PAF has also been implicated in the neuronal damage in Alzheimer disease (AD) (Table 4.2). The mechanisms linking neural cell injury to 4.5 PAF in Neurological and Visceral Disorders 121

Table 4.2 Involvement of PAF in neurological disorders Effect of PAF Neurological disorder PAF levels antagonist Reference Ischemia Increased Beneficial Belayev et al., 2009 Traumatic brain injury Increased Beneficial Tokutomi et al., 2001 Spinal cord injury Increased Beneficial Hostettler and Carlson, 2002 Alzheimer disease Increased Beneficial McLamon et al., 2005 Prion diseases Increased Beneficial Bate et al., 2010 Multiple sclerosis Increased Beneficial Edwards and Constantinescu, 2009; Kihara et al., 2008 HIV-mediated dementia Increased Beneficial Tsoupras et al., 2008 Meningitis Increased Beneficial Maclennan et al., 1996; Townsend and Scheld, 1994 Neuropathy Increased Not known Noris et al., 1993 Subarachnoid hemorrhage Increased Hirashima et al., 1994 Migraine Increased Sarchielli et al., 2004

PAF synthesis are fully understood. However, it is well known that microglia from 2+ 2+ AD patients have significantly higher (20%) basal Ca [Ca ]i relative to microglia from nondemented patients (McLamon et al., 2005). In addition, microglia from AD patients show diminished amplitudes (reduction of 61%) of SOC-mediated Ca2+ entry induced by PAF and prolonged time courses (increase of 60%) of ATP responses with respect to microglia from nondemented patients (McLamon et al., 2005). These observations support the view that significant abnormalities are present in Ca2+-mediated signal transduction in microglia isolated from AD patients. It is likely that PAF receptor-mediated mobilization of calcium through calcium channels and intracellular stores may contribute to the stimulation of calcium-dependent enzymes, resulting in neural injury (Clark et al., 2000; Farooqui et al., 2008). Other mechanisms of PAF-mediated neurotoxicity may involve Ab. Treatment of hippocampal neurons with Ab reduces the amounts of synaptophysin, a presynaptic membrane protein that is essential for neurotransmission, indicating synapse damage. Pretreatment of cortical or hippocampal neuronal cultures with PAF antagonists, ginkgolides A or B protects against Ab-mediated loss of synaptophysin. This protective effect is observed with nanomolar concentrations of ginkgolides. It is suggested that PAF antagonists may protect against the synaptic damage and the cognitive loss seen during the early stages of AD (Bate et al., 2008). Similarly, Ginkgo biloba extract EGb761 (quercetin and ginkgolide B) also protects against Ab-induced neurotoxicity by blocking Ab-induced cell apoptosis, ROS production, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt signaling pathways (Shi et al., 2009). Collectively, these studies suggest that PAF plays an important role in inflammation, oxidative stress, and neurodegeneration in AD and PAF antagonists protect against PAF-R-mediated neurotoxicity. Studies on the effect of PAF in 1-methyl-4-phenylpyridinium (MPP+), a toxin used for producing Parkinson disease-like neurochemical changes, in PC12 cells 122 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement… indicate that PAF modulates MPP+-mediated apoptotic cell death in concentration- dependent manner. Treatment with lower concentration (0.75 mM) of PAF significantly attenuates the MPP+-induced toxicity by increasing Bax levels, decreasing Bid and Bcl-2 levels, and altering mitochondrial membrane potential. These processes result in the release of cytochrome c and subsequent caspase-3 activation (Ha and Lee, 2010). The inhibitory effect of PAF does not involve nuclear factor-kappaB (NF-kB) activation. In contrast, PAF at the concentrations more than 2.5 mM are not only toxic, but produce additive effect on the MPP+ toxicity. It is suggested that PAF at low concentrations may not be toxic and may retard the MPP+ toxicity by suppressing the apoptosis-related protein activation. In contrast, higher concentrations of PAF may produce an additive toxic effect against the MPP+ toxicity by increasing apoptosis-related protein activation. Prion diseases are accompanied by the accumulation of abnormal isoforms of cellular prion protein (PrPC), motor dysfunctions, synaptic loss, dementia, spongiosis, astroglyosis, and neurodegeneration (Farooqui, 2010). Pretreatment of cell cultures with PLA2 inhibitors (AACOCF3, MAFP, and aristolochic acids) protects against PrP82-146-mediated synapse degeneration. Synapse degeneration is increased following the addition of a specific PLA2 activating peptide (PLAP) and the addition of PrP82-146. Activation of PLA2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B, Hexa- PAF, and CV6029) protect against synapse degeneration induced by PrP82-146 and PLAP (Bate et al. 2010). These results support the view that PrP82-146-mediated activation of cytoplasmic PLA2 may elevate levels of PAF and PGE2. The increase in PAF and PGE2 causes synapse degeneration. It is proposed that PLA2 inhibitors and PAF antagonists may cross the blood–brain barrier and protect against the synapse degeneration observed in prion diseases (Bate et al., 2010). PAF plays a critical role in experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Levels of PAF in the spinal cord of EAE mice and cerebrospinal fluid of MS patients are increased (Kihara et al., 2008). This increase in PAF is coupled with the increased activities of PLA2 and acetyl- CoA:lyso-PAF acetyltransferase (LysoPAFAT) in the SC of EAE mice compared with those of naive mice and correlate with disease severity (Kihara et al., 2008).

Although levels of other isoforms of cPLA2, sPLA2, and LysoPAFAT transcripts are upregulated in the spinal cord of EAE mice, PAF acetylhydrolase activity is unchanged during the course of EAE. Collectively, these studies suggest that PAF accumulation in the spinal cord of EAE mice is profoundly dependent on the group cPLA2/LysoPAFAT activities present in the infiltrating macrophages and activated microglia (Kihara et al., 2008). PAF also plays an important role in the pathogenesis of several AIDS manifestations such as AIDS dementia complex, Kaposi’s sarcoma, and HIV-related neph- ropathy. PAF antagonists have been studied in these conditions with promising results (Tsoupras et al., 2008). Increase in PAF levels is observed in the internal jugular venous blood of migraine patients. PAF levels are decreased at the end of the migraine attack, reaching levels significantly lower than those measured before migraine attack (Sarchielli et al., 2004). PAF levels are also increased in multiple 4.5 PAF in Neurological and Visceral Disorders 123

Table 4.3 PAF levels in visceral diseases Visceral diseases PAF levels Reference Asthma Increased Tsukioka et al., 1996 Systemic lupus erythematosus Increased Tetta et al., 1990 Juvenile rheumatoidarthritis Increased Tselepis et al., 1999 Acute myocardial infarction Increased Lupia et al., 2003 Sepsis Increased Mathiak et al., 1997 Crohn disease Increased Hocke et al., 1999 Ulcerative colitis Increased Hocke et al., 1999 sclerosis, and viral and bacterial infections (Edwards and Constantinescu, 2009). Administration of PAF antagonists has beneficial effect in animal models of multiple sclerosis (Kihara et al., 2008).

4.5.2 PAF in Visceral Disorders

PAF is closely associated with the pathogenesis of bronchial asthma (Table 4.3). It not only induces key pathogenic features of asthma, but also influences the activity of cells involved in immune-inflammatory process. Apart from its known potent ability to activate platelets, PAF administration can mimic some of the abnormalities observed in asthma, including bronchoconstriction, bronchial hyper responsiveness, and gas exchange impairment, which may be mediated by leukotrienes acting as secondary mediators of some PAF effects. PAF may therefore serve as a possible direct target for antiasthmatic drugs. Surprisingly, PAF receptor antagonists have not been useful in providing beneficial clinical effects. It is suggested that combining the effect of PAF antagonists with antagonists of other mediators associated with the pathogenesis of asthma may improve clinical efficacy (Kasperska- Zajac et al., 2008). In systemic lupus erythematosus (SLE), PAF levels are elevated not only in plasma during the most active phases of the disease (Tetta et al., 1990), but also in monocytes of SLE patients compared with inactive patients and controls (Bussolati et al., 2000) . This is due to decrease in PAF-acetylhydrolase activity. Enhanced PAF may be responsible for the increase in immunoglobulin production by B lymphocytes in SLE patients (Smith and Shearer, 1994; Mazer et al., 1991). Juvenile rheumatoid arthritis (JRA) is the most common childhood chronic systemic autoimmune inflammatory disease characterized by progressive joint damage. Patients with active JRA have lower plasma PAF-acetylhydrolase activity and higher plasma triglyceride levels than age-matched control subjects (Tselepis et al., 1999). This may result in increased levels of PAF and inflammatory cytokines (Table 4.3). In addition, patients with active JRA show low levels of HDL2 and HDL3. These alterations suggest that active JRA is associated with partial loss of the anti-inflammatory activity of plasma Apo B- and Apo A-I-containing lipoproteins (Tselepis et al., 1999). 124 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement…

Sepsis is a serious and complex clinical syndrome caused by an overly active host response to infection. PAF is associated with the pathology of endotoxic shock, and the blood PAF levels are increased during endotoxemia and that the administration of PAF antagonists in animals protects them from the deleterious effects of endotoxin (Giral et al., 1996). The clinical trials of PAF receptor antagonists have failed to demonstrate efficacy in diseases such as septic shock, asthma, and pancrea- titis (Mathiak et al., 1997). Levels of PAF are also increased in stool of patients with ulcerative colitis and Crohn disease (Hocke et al., 1999). PAF antagonists may be used for the treatment of inflammatory bowel diseases.

4.5.3 PAF in Kainic Acid Neurotoxicity

Kainic acid (KA) is a cyclic and nondegradable analog of glutamate. KA causes neuronal loss in specific striatal and hippocampal areas of the brain after intraventricular and intracerebral injections. KA-induced neurotoxicity and epileptogenesis is accompanied by increase in cPLA2 activity, elevation in arachidonic acid oxidation products, and alterations in localization of PAF-R mRNA. Although PAF-R mRNA is normally expressed by neurons and microglia in rat hippocampus, KA-mediated neurotoxicity restricts the expression of PAF-R mRNA to apoptotic neurons and glial cells (Bennett et al., 1998). PAF along with excitatory amino acids increase mitogen-activated protein (MAP) kinases in neurons and glial cells. Treatment of hippocampal neuronal cultures with PAF and KA indicate that MAP kinase activity is significantly increased (De Coster et al., 1998). Extracellular signal- regulated kinase, c-Jun N-terminal kinase, and p38 kinases are also increased by kainate or PAF in hippocampal neurons and non-neural cells (Margues et al., 2002). The activation of kinases is blocked by the KA receptor antagonists CNQX and PAF receptor antagonist, BN 50730. The PAF receptor antagonist, BN 50730 also retards KA-induced activation. CNQX has no effect on PAF activation of the kinases, indicating that PAF is downstream of kainate activation (De Coster et al., 1998). These results indicate that PAF and KA activate similar kinase pathways. PAF acts downstream of the kainate receptor, and excessive stimulation of PAF receptors contribute to neurodegeneration in PAF-mediated neurotoxicity (De Coster et al., 1998).

4.6 Molecular Mechanism Associated with PAF-Mediated Neural Injury

As stated above, PAF-Rs are coupled with the activation of PLA2, PLC, and PLD. Interactions of PAF with its receptors not only enhance the breakdown of PtdCho, PlsEtn, and PtdIns but also mobilizes calcium (Clark et al., 2000; Honda et al., 2002), resulting in generation of many lipid mediators including, arachidonic acid, generation of arachidonic acid-derived prostaglandins, leukotrienes thromboxanes lipoxins, liberation of diacylglycerol, and inositol 1,4,5-trisphosphate (InsP3). 4.7 Conclusion 125

PAF- induced elevation in calcium is not only responsible for neuronal growth cone collapse (Rehder et al., 1992), but also for the activation of guanylate cyclases, which produce cGMP. This second messenger is involved in phosphorylation of various proteins through the activation of various kinases including MAP-kinases, phosphatidylinositol 3-kinase, and tyrosine kinases (Chao and Olson, 1993; Honda et al., 2002). Excessive production of above lipid mediators and phosphorylation of neural protein may cause neural injury. In addition, PAF facilitates the generation of ROS, which interact with NFkB. In the nucleus NFkB mediates the transcription of many genes implicated in inflammatory and immune responses. These genes include COX-2, intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-selectin, TNF-a, IL-1b, IL-6, sPLA2, inducible nitric oxide synthase (iNOS), and matrix metalloproteinases (MMPs). In neural membrane oxidized phospholipids, the length of the sn-2 carbon chain dictates affinity for PAF-R. Phospholipid species with sn-2 acetyl group show the highest affinity (Carolan and Casale, 1990; Ishii et al., 1997), whereas with higher carbon chain length show lower affinity. Substituting phosphoethanolamine for phosphocholine substantively reduces binding (O’Flaherty et al., 1994). The sn-1 ether linkage is not required for PAF agonist and PAF-R interactions, but does increase potency several hundred-fold (Prescott et al., 2000). The sn-1 carbon chain length and degree of unsaturation may modulate the activation of different PAF-R signaling pathways (Carolan and Casale, 1990). These PAF isoform-specific differences may explain observations that PAF-R expression augments chemotherapeutic cytotoxicity yet provide protection cells TNF-a, TRAIL, and 1-O-hexadecyl-2- acetyl-sn-glycero-3-phosphocholine (C16-PAF)-induced toxicity (Li et al., 2003). It is recently shown that the length of the sn-1 carbon chain of PAF dictates the molecular mechanism of neurodegeneration in PAF-R−/−, PAF-R+/+ and null mice. Both −/− C16-PAF and C18-PAF are toxic to neurons that lack PAF-R (PAF-R ), but this toxicity is initiated by different cell death pathways (Ryan et al., 2008). Exposure of

PAF-R-deficient neurons to 16C -PAF results in a signaling cascade through the activation of executioner caspase 7, whereas C18-PAF-mediated cell death is accompanied by caspase-independent pathway. PAF-mediated neurodegeneration is also modulated by PAF-R expression. Ectopic or endogenous PAF-R expression protects neurons from C16-PAF-mediated neurodegeneration, whereas PAF-R expression transforms C18-PAF-mediated neurodegeneration from a caspase-independent to a caspase-dependent signaling pathway. Collective evidence suggests that carbon chain length may dictate the molecular mechanism underlying the apoptotic cell death in cerebellar granule neurons.

4.7 Conclusion

PAF is a phospholipid mediator released from activated neural and non-neural cells that promotes pathophysiologic inflammation. PAF interacts with specific receptors called as PAF receptors (PAF-Rs). These receptors have been cloned and characterized from neural and non-neural tissues. PAF-Rs consist of seven transmembrane 126 4 Platelet-Activating Factor in Brain: Its Metabolism, Roles, and Involvement… helices, which are coupled with G proteins. Stimulation of PAF-Rs is linked with the activation of PLA2, PLC and PLD, GTPases, and kinases. Activation of various phospholipases liberates arachidonic acid, which is converted into eicosanoids through the action of cyclooxygenases and lipoxygenases. These processes are closely associated with PAF-mediated neural injury. Levels of PAF are modulated by the activities of PAF-synthesizing and PAF-degrading enzymes. Levels of PAF are markedly increased in neurological disorders (ischemia/reperfusion injury, traumatic brain injury, spinal cord injury, Alzheimer disease, and Miller-Dieker lissencephaly) and visceral diseases (asthma, systemic lupuserythematosus, juvenile rheumatoid arthritis, acute myocardial infarction, sepsis, and Crohn disease). Neither the cell types nor mechanism responsible for the synthesis of PAF and its target cells have been fully identified. In animal models of above neural and visceral diseases, administration of PAF antagonists result in beneficial effects, but clinical trials of PAF antagonists in humans have failed. Thus, more studies are required on the synthesis of better PAF antagonists and randomize clinical trials in humans.

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5.1 Introduction

The cannabinoid system-mediated signaling involves specific G protein-coupled receptors (CB1, CB2, and CB3), exogenous (marijuana-derived cannabinoids), endogenous cannabinoids ligands (2-arachidonoylglycerol, anandamide, noladin, virodhamine, and N-arachidonyl-dopamine), and a machinery associated with the synthesis and degradation of endocannabinoid. The endocannabinoid system is associated with the critical phase for cerebral development, regulating the release and action of different neurotransmitters and influencing fundamental developmental processes such as neuronal cell proliferation, migration, differentiation, morphogenesis, and synaptogenesis (Harkany et al., 2008). Among cannabinoid receptors, CB1 receptors are mainly expressed in the central and the peripheral nervous systems (nerve terminals, spinal dorsal horn, and periaqueductal gray), where they not only participate in the inhibition of neurotransmitter release, but also in induction and maintenance of synaptic efficacy (Kano et al., 2009). Among various brain regions, CB1 receptors are found at highest concentrations in the hippocampus, neocortex, basal ganglia, cerebellum, and anterior olfactory nucleus (Glass et al., 1997). Moderate receptor levels are also present in the basolateral amygdala, hypothalamus, and the periaqueductal gray matter of the midbrain (Glass et al.,

1997). CB2 receptors are abundantly detected in immune and glial cells and brainstem area of the brain where they are associated with the modulation of cytokine release (Pertwee and Ross, 2002; Ahluwalia et al., 2002; Pertwee, 2005; Van Sickle et al., 2005).

Both CB1 and CB2 genes encode a seven-transmembrane-domain protein belonging to the Gai protein-coupled receptor family (Munro et al., 1993). CB1 and CB2 receptors have been cloned from several sources. Although based on electrophysiological evidence, the occurrence of a third receptor (“CB3” or Anandamide receptor) in brain and in endothelial tissues has been reported (Breivogel et al., 2001), but its cloning, expression, and characterization have not yet been accomplished. There is 44% similarity in amino acid sequence of CB1 and CB2 receptor proteins. CB1 and

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 133 DOI 10.1007/978-1-4419-9940-5_5, © Springer Science+Business Media, LLC 2011 134 5 Cannabinoids in the Brain: Their Metabolism, Roles…

O O O O N CN Me NH OMe OMe N I N Me OMe O I N Cl N

LY 320135 Cl AM281 O AM 630 (CB1) (CB1) (CB1)

O O O N N Me Me NH 3 NH NH

N N N 5 N N Cl N Cl 1 I Cl Cl Me

Me Cl Cl SR 144528 SR 141716A AM 251 (CB2) (CB1) (CB1)

Fig. 5.1 Chemical structures of some cannabinoid receptor agonists

CB2 differ considerably in their amino acid sequence and tertiary structures. CB1 receptor has four clusters of potential cAMP-dependent protein kinase and Ca2+- calmodulin-dependent protein kinase sites. These clusters are conserved across the human, rat, and mouse receptor proteins. There is a single potential protein kinase

C site that is also conserved in these CB1 receptors. Although human CB2 receptor shares 81% amino acid identity with rat and mouse, unlike CB1 receptor there is no

PKC site in CB2 receptors. Cannabinoid receptor agonists interact with CB1 and

CB2 receptors and modulate a variety of neurochemical responses, such as analgesia, hypothermia, spontaneous activity, hypotension, modulation of Ca2+, inhibition of adenylate cyclase, and modulation of long-term potentiation and nociception (Beltramo, 2009; Varvel et al., 2009; Wolf et al., 2010). Considerable progress has been made on biology, pharmacology, and genetics of cannabinoid receptors. Thus, the genomic location of the human cannabinoid receptor gene (CB1) has been mapped to chromosome 6q14-q15 (Hoehe et al., 1991). The mouse CB1 and CB2 receptor genes are located in the proximal arm of chromosome 4 (Onaivi et al.,

2002). CB1 and CB2 receptor genes in rat have been mapped to chromosome 5. The bovine CB1 receptor gene has been mapped to chromosome 9q22 (Pfister-Genskow et al., 1997; Basavarajappa, 2007). These investigations on pharmacology of cannabinoid receptors have led to the development of CB1- and CB2-selective agonists and antagonists. Cannabinoid receptor synthetic agonists and antagonists include

CP-55,940, WIN-55,212-2, SR141716A, AM251, AM281 and LY320135 (CB1- selective), and SR144528 and AM630 (CB2-selective) (Figs. 5.1 and 5.2). The development of selective antagonists and the creation of genetically engineered 5.1 Introduction 135

O OH

OH N Me O

N Me Me HO O WIN 55,212-2 CP 55940

11 Me O Me 9 OH OH N 1 H Me 1' Me O 3 R-(+)-Methanandamide

O OH N H Palmitoyl-EA (CB2 only)

Fig. 5.2 Structures of some cannabinoid receptor antagonists

mice lacking CB1 (Ledent et al., 1999) or CB2 (Buckley et al., 2000) have enabled investigators to determine relative contribution of each cannabinoid receptor to neurochemical and pharmacological effects of cannabinoids. Thus, numerous compounds have been synthesized with the goal of separating psychotropic activity from analgesic/anti-inflammatory action. Among the most promising is 1¢,1¢-dimethylheptyl-THC-11-oic acid (ajulemic acid, AJA) (Fig. 5.1) (Burstein and Zurier, 2009). Oral administration of low-dose AJA suppresses joint inflammation and tissue injury in rats with adjuvant arthritis. Treatment of human cells with AJA not only reduces generation of interleukin (IL)-1b, but suppresses matrix metalloproteinases, and increases apoptosis of human T lymphocytes. In addition, AJA also reduces production of IL-6 by human monocyte-derived macrophages (Burstein and Zurier, 2009). Although in the adjuvant arthritis model, the occurrence of synovial inflammation has been reported in AJA-treated rats, it does not progress, and articular cartilage degradation and bone erosion do not occur as it happens in rats treated with placebo (Stebulis et al., 2008). Collective evidence suggests that resolution of inflammation takes place earlier in AJA-treated animals compared to placebo group. At the molecular level, addition of AJA to human synovial cells in vitro not only increases the generation of PGJ2, an eicosanoid that facilitates resolution of inflammation, but also enhances the production of lipoxin

A4, an eicosanoid which facilitates the resolution of inflammation (Stebulis et al., 2008; Burstein and Zurier, 2009). 136 5 Cannabinoids in the Brain: Their Metabolism, Roles…

In the immune system, endocannabinoids are associated with several functions. They modulate T- and B-lymphocytes proliferation and apoptosis, macrophage- induced death of sensitized cells, inflammatory cytokine generation, immune cell activation by inflammatory stimuli, chemotaxis, and inflammatory cell migration

(Klein, 2005). CB2 receptors mediate immunosuppressive effect of endocannabinoids in immune cells through the inhibition of the cAMP/protein kina- seA (PKA) pathway (by decreasing the expression of cAMP-responsive genes). Endocannabinoids also paticipate at the nuclear level, where they modulate phosphorylation of IkB that enhances the transcription of several apoptotic genes modulated not only by NF-kB, but also by peroxisome proliferator-activated receptor gamma (PPAR-g)-dependent inhibition of nuclear factor of activated T cells (NF- AT). Endocannabinoids interfere with cell cycle through the activation of p21waf-1/ cip-1 and induction of G1/S phase arrest (Malfitano et al., 2006).

5.2 Cannabinoid Receptor-Mediated Signaling in the Brain

CB1 receptor-mediated signal transduction occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, inhibit voltage-gated Ca2+ channels, activate inward rectifying K+ channels, activate mitogen-activated protein kinases (MAPK), focal adhesion kinase, phosphatidylinositol 3-kinase, and c-Jun N-terminal kinase, which regulate nuclear transcription factors (Howlett et al., 2010) (Fig. 5.3). CB1 activation evokes a transient Ca2+ elevation in a phospholipase C (PLC)-dependent manner through either Gi/o or Gq proteins (Sugiura et al., 1997). CB1 receptors, like other GPCRs, interact with several other proteins including GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization), and the novel cannabinoid receptor-interacting proteins (CRIP(1a/b)). These proteins play important roles in the regulation of intracellular trafficking, desensitization, downregulation, signal transduction, and constitutive activity of CB1 receptor (Smith et al., 2010). Activated and phosphorylated CB1 receptor also bind with b-arrestin molecules, which promotes the formation of signaling complexes and participates in the regulation of G-protein-coupled receptors (GPCR) signaling. As stated above, brain CB1 receptors are involved in regulating synaptic functions, memory, and motor learning. In addition, activation of CB1 receptor inhibits type 5-HT3 ion channels (Barann et al., 2002), modulates the production of nitric oxide (Howlett and Mukhopadhyay, 2000), alters sodium channel conductance (Nicholson et al., 2003) and activates the Na+/H+ exchanger (Bouaboula et al., 1999).

The activation of CB2 receptor is associated with upregulation of the expression of key genes involved in lipid synthesis (e.g., PPAR transcription factors and some of their target genes) (Dobrosi et al., 2008). These processes involve CB2 receptor- coupled signaling, which involves the MAPK pathway. This signaling is not only blocked by specific agonists/antagonists of2 CB receptor, but also by RNA interference. Because cells with “silenced” CB2 have significantly suppressed basal lipid synthesis, it is suggested that human sebocytes (highly specialized and 5.3 Occurrence and Synthesis of Endocannabinoids in the Brain 137

Glu CB2

+ CB1 PtdIns-4,5-P ARA-PtdCho /N a 2 + channel K NMDA-R + K ATP G G cPLA PtdIns3K Akt Ca2+ PLC

2 AC Ca2+ DAG InsP 3 p38 Raf1 ARA + Lyso-PtdCho PKA cAMP PKC JNK -- ERK + Ca2+ ROS PAF

cPLA CREB 2 ELK-1 c-fos c-Jun CREB

NF-κB

? ? Krox-24 ? ?

NUCLEUS Gene transcription

Fig. 5.3 CB1 receptor-mediated signaling. Adenylyl cyclase (AC); cAMP-dependent protein kinase (PKA); protein kinase C (PKC); phospholipase C (PLC); diacylglycerol (DAG); -130-Cas

(CA); arachidonyl-phosphatidylcholine (ARA-PtdCho); cytosolic phospholipase A2 (cPLA2); arachidonic acid (ARA); reactive oxygen species (ROS); nuclear factor-kappa B (NF-kB); cAMP response element-binding (CREB); member of ETS oncogene family (ELK1); proto-oncogene (c-fos and c-Jun); Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases sebum-producing epithelial cells) may utilize a paracrine–autocrine, endogenously active, CB2-mediated endocannabinoid signaling system for positively regulating lipid production and apoptosis (Dobrosi et al., 2008). CB2 receptors are found in microglial cells, not in astrocytes (Nunez et al., 2004). They are upregulated in response to inflammation and chronic pain (Maresz et al., 2005). CB2 receptor activation represents a very promising therapeutic target in neural and visceral inflammatory states, where there is immune activation and motility dysfunction (Maresz et al., 2005).

5.3 Occurrence and Synthesis of Endocannabinoids in the Brain

The natural ligands of CB1 and CB2 receptors are endogenous, lipid-like substances called endocannabinoids, which include arachidonoyl ethanolamide or anandamide and 2-arachidonoylglycerol (2-AG) (Sugiura, 2009). They mimic 138 5 Cannabinoids in the Brain: Their Metabolism, Roles…

H C OH O 2 O CO CH OH N H H2C OH

2-Arachidonyl glycerol Anandamide H C 2 OH O H2 C OCH NH2 O OH H2C

Noladin ether Virodhamine

OH OH O OH O N H N H

Docosatetraenoyl ethanolamide N-Arachidonyldopamine

Fig. 5.4 Chemical structures of endocannabinoids several pharmacological effects of d-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. 2-AG acts as a full agonist, whereas anandamide is a partial agonist for CB1 and CB2 receptors. In addition, other ARA containing endogenous molecules, such as virodhamine (O-arachidonyl-ethanolamine), noladin, and arachidonyldopamine have been reported to occur in brain (Fig. 5.4). The presence of N-palmitoylethanolamine, N-oleoylethanolamine, and N-stearoylethanolamine has also been reported in human, rat, and mouse brain where they retard the degradation of 2-AG and anandamide (Mechoulam et al., 1998, 2002). In the brain, endocannabinoids act as neuromodula- tors or retrograde messengers, which inhibit the release of various neurotransmitters. In the peripheral and neural tissues, endocannabinoids are associated with the modulation of proteins and nuclear factors involved in cell proliferation, differentiation, and apoptosis, as paracrine or autocrine mediators supporting the view that endocannabinoids play a role in the control of cell fate (Guzman et al., 2001). 2-AG and anandamide are short-lived mediators, which are synthesized through two distinct pathways (Fig. 5.5). Termination of 2-AG and anandamide-mediated signaling involves not only the transport of endocannabinoids from the extracellular to the intracellular space, but also intracellular degradation by either through their hydrolysis by MAG-lipase and fatty acid amide hydrolase, or oxidation catalyzed by cyclooxygenases/lipoxygenases (Di Marzo, 2006a; Farooqui, 2009a). Degradation of 2-AG and anandamide not only augments cellular uptake and termination of extracellular signaling, but also modulates the intracellular signaling events of these two endocannabinoids. Collective evidence suggests that endocannabinoids are neuromessengers, which are synthesized on demand and released from postsynaptic 5.3 Occurrence and Synthesis of Endocannabinoids in the Brain 139

CB1 Receptor +

/Na 1, 2-Arachidonyl-PtdCho + PtdEtn K channe l NMDA-R

G + - ATP 2+ 1 Ca 3 PKA 2 cAMP 1-Lyso-2- arachidonyl- N-Arachidonyl- PtdCho PtdEtn 1-Acyl-2-arachidonylglycerol 2 5

4 2-arachidonyl- Anandamide glycerol 8 6 7 2-arachidonyl- 9 PtdH

ARA + Glycerol ARA + Ethanolamine

Fig. 5.5 Pathway showing the generation of anandamide and 2-arachidonylglycerol in brain. Phosphatidylcholine (PtdCho); phosphatidylethanolamine (PtdEtn); protein kinase A (PKA); Cannabinoid receptor1 (CB1) N-methyl-D-aspartate receptor (NMDA-R); N-acyltransferase (1); phospholipase C (2); Adenylyl cyclase (3); diacylglycerol lipase (4); N-acylphosphatidylethanolamine-specific phospholipase D (5); monoacylglycerol lipase (6); and amidase (7). Activation of receptors coupled to the phosphatidylinositol-specific phospholipase C and diacylglycerol lipase pathway leads to increases in 2-AG production neurons to restrain the release of classical neurotransmitters from presynaptic terminals. This retrograde signaling occurs at dopaminergic, GABAergic, and glutamatergic synapses and is associated with short and long-term synaptic plasticity involved in a variety of brain functions, including anxiety, fear, and mood (Gaetani et al., 2009; Farooqui, 2009a).

5.3.1 Metabolism of 2-Arachidonylglycerol in the Brain

Transfer of ARA from sn-1 position of 1,2-arachidonyl-PtdCho to the N-position of PtdEtn generates 1-lyso-2-arachidonyl-PtdCho and N-arachidonyl-PtdEtn. This reaction is catalyzed by a Ca2+-dependent, membrane-associated N-acyltransferase (Fig. 5.5). 1-Lyso-arachidonyl-PtdCho is converted to 2-AG by PLC. Another pathway for the generation of 2-AG involves the hydrolysis of 1,2-arachidonyl- PtdCho by PLC, followed by the action of DAG-lipase on 1-acyl-2-arachidonylglycerol. 2-AG interacts with CB1 and CB2 receptors and act as neurotransmitter or neuromodulator in brain, immune and cardiovascular systems. Its levels in the brain are 170-fold higher than anandamide (Stella et al., 1997). As stated above, stimulation of both receptors inhibit cAMP formation via Gi/o proteins, and activate mitogen-activated-protein kinase (Childers and Breivogel, 1998). Unlike tradi- tional neurotransmitters, ARA-derived endocannabinoids are not stored in vesicles. 140 5 Cannabinoids in the Brain: Their Metabolism, Roles…

PGE2-GE

15-HETE-GE Induction of neurite outgrowth 15-LOX

COX-2 Activation of K+ channel EPOX 2-(14,15-EET)-GE 2-Arachidonyl glycerol Full agonist for CB1 receptor

Onset of dementia

12-LOX 12-HETE-GE Notch mRNA2 Presenilin 2 MAG-lipas e

Glycerol + Arachidonate

Fig. 5.6 Degradation and role of 2-arachidonylglycerol in brain. Cyclooxygenase-2 (COX-2); prostaglandin E2-glyceryl ester (PGE2-GE); 15-lipoxygenase (15-LOX); 15- hydroperoxyeicosatetraenoic acid glyceryl ester (15-HETE-GE); 12-lipoxygenase (12-LOX); 12- hydroperoxyeicosatetraenoic acid glyceryl ester (12-HETE-GE); epoxygenase (EPOX); glyceryl ester (2-(14,15-EET)-GE; and monoacylglycerol lipase (MAG-lipase)

They are generated from ARA-containing glycerophospholipids within the neural membranes. 2-AG is hydrolyzed by monoacylglycerol lipase (MAG-lipase) into arachidonic acid and glycerol. This enzyme is heterogeneously distributed in the rat brain with the highest levels observed in regions expressing CB1 receptors, such as the cortex, thalamus, hippocampus, and cerebellum (Dinh et al., 2002, 2004). Inhibition of monoacylglycerol lipase by 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5- yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) reduces neuropathic pain through distinct receptor-mediated mechanisms and may present good targets for the development of analgesic drugs (Kinsey et al., 2009). Considering the involvement of endocannabinoids and cannabinoid receptors in numerous physiological processes monoacylglycerol lipase is now considered as a promising target for not only the treatment of pain and inflammatory disorders, but also cancer research. 2-AG is also metabolized by cyclooxygenase-2 (COX-2), which oxygenates

2-AG to generate prostaglandin glyceryl esters (PGE2-GE or PGE2-G) (Fig. 5.6) 2+ (Sang and Chen, 2006; Sang et al., 2007). PGE2-G mediates the mobilization of Ca in RAW264.7 cells at pm to nm levels. The mobilization of Ca2+ is concomitant with 2+ a transient elevation of PtsInsP3 levels. Ca mobilization can be blocked by the

PtdInsP3 receptor antagonist, TMB-8 (Nirodi et al., 2004). Although depletion of extracellular Ca2+ reduces, it does not eliminate the response and is consistent with an initial release of Ca2+ from intracellular stores followed by capacitative entry. Depletion of Ca2+ from endoplasmic reticulum by pretreatment of cells with thapsigargin retards the PGE2-G response. Intraplantar administration of PGE2-G stimulates behavioral sensitivity to thermal and mechanical stimulation in a manner likely to be independent of cannabinoid and prostanoid receptors. These effects are only partially reversed by coadministration of PGE2-G with a cocktail of prostaglandin receptor antagonists. PGE2-G stimulates hippocampal glutamatergic transmission and induces neuronal death through caspase-3 activation. Stimulation of CB1 receptor elevates levels of PGE2-G and this elevation is not inhibited by SR141716, a CB1 5.3 Occurrence and Synthesis of Endocannabinoids in the Brain 141

GPR55

PGE2-EA Notch mRNA1 15-HETE-EA 15-LOX COX-2 PPARs

EPOX 5,6 8,9 11,12 14,15- Anandamide CB1 EETs-EA

CB2 12-LOX FAAH 12-HETE-EA TRPV1 Ethanolamine + Arachidonate

Presenilin 1

Fig. 5.7 Degradation of role of anadamide in brain. Cyclooxygenase-2 (COX-2); prostaglandin

E2-ethanolamide (PGE2-EA); 15-lipoxygenase (15-LOX); 15- hydroperoxyeicosatetraenoic acid ethanolamide (15-HETE-EA); 12-lipoxygenase (12-LOX); 12- hydroperoxyeicosatetraenoic acid ethanolamide (12-HETE-EA); epoxygenase (EPOX); ethanolamide (2-(14,15-EET)-EA; and monoacylglycerol lipase (MAG-lipase) receptor antagonist. This observation suggests that multiple mechanisms may contribute to the increase in PGE2-G levels. NMDA receptor antagonists have been shown to inhibit PGE2-G-mediated neurotoxicity (Sang and Chen, 2006; Sang et al., 2007). 2-AG-derived prostaglandins do not bind or interact with known prostaglandin receptors. They probably exert their effects through a unique and previously uncharacterized receptor (Guindon and Hohmann, 2008, 2009). In a macrophage cell line, PGE2-G mediate their effect through their interactions with NF-kB in a concentration-dependent manner; high doses of PGE2-G increase and low doses of

PGE2-G reduce the activation of NF-kB (Hu et al., 2008). It is proposed that endogenous COX-2-derived 2-AG metabolites enhance nociception through a mechanism that may involve NF-kB-mediated signal transduction. Their effect is opposite to that of ARA-derived prostaglandin on inhibitory synaptic transmission, and alterations in COX-2 activity may have significant impact on endocannabinoid signaling in hippocampal synaptic activity (Sang and Chen, 2006). Metabolism of 2-AG by 12-lipoxygenase (12-LOX) and 15-lipoxygenase (15- LOX) generates 12-hydroperoxyeicosatetraenoic acid glyceryl ester (12-HETE-GE) and 15- hydroperoxyeicosatetraenoic acid glyceryl ester (15-HETE-GE), respectively (Moody et al., 2001; Kozak et al., 2002) (Fig. 5.6). These metabolites mediate their biological activities via established receptors, including the cannabinoid receptors, peroxisome proliferator-activated receptor (PPAR-a), and transient receptor potential vanilloid receptor (TRPV1) (Kozak et al., 2002; Di Marzo, 2006a). In vitro, cytochrome P450 enzymes have also been implicated in metabolizing 2-AG (Snider et al., 2007). Oxidative metabolism of both of 2-AG by microsomal P450 epoxygenases produces a diverse array of oxygenated products (Figs. 5.6 and 5.7), including epoxides (epoxyeicosatetraenoic acids). 142 5 Cannabinoids in the Brain: Their Metabolism, Roles…

5.3.2 Metabolism of Anandamide in the Brain

N-arachidonoyl-phosphatidylethanolamine (N-arachidonyl-PtdEtn), the precursor for anandamide, is synthesized by the enzymic transfer of arachidonic acid in the sn-1 position of a phosphatidylcholine to the amide group of a phosphatidylethanolamine (Fig. 5.5). This reaction is catalyzed by a calcium-independent N-acyl-transferase (NAT) (Jin et al., 2009). N-arachidonyl-PtdEtn is transformed into anandamide by N-acylphosphatidyl-ethanolamine-specific PLD, a member of the metallo- b-lactamase family, which specifically hydrolyzesN -acylphosphatidylethanolamine among glycerophospholipids, and appears to be constitutively active (Di Marzo et al., 1996; Ueda et al., 2005). Recombinant N-acylphosphatidylethanolamine PLD hydrolyzed various N-acylphosphatidylethanolamines, including the anandamide precursor N-arachidonoylphosphatidylethanolamine at similar rates, but is inactive with phosphatidylcholine and phosphatidylethanolamine (Okamoto et al., 2004). Involvement of N-acylphosphatidyl-ethanolamine-specific PLD in the biosynthesis of ananadamide has been put in doubt because N-acylphosphatidyl-ethanolamine- specific PLD knockout mice do not show any deficit in anandamide synthesis suggesting that multiple enzymic mechanisms may contribute to the generation of anandamide (Leung et al., 2006). Fatty acid amide hydrolase, a postsynaptic membrane-bound enzyme that regulates levels of anandamide by hydrolyzing it into arachidonic acid and ethanolamine, terminates anandamide-mediated signaling in neurons and astrocytes (Di Marzo, 2006b). Like 2-AG, anandamide is also oxidized by COX-2, 12-LOX, and 15-LOX resulting in the generation of protstaglandin E2 ethanolamide (PGE2-EA), 12-HETE ethamolamide (12-HETE-EA) and 15-HETE ehanolamide (15-HETE-EA), respectively (Fig. 5.7). These metabolites are significantly more stable metabolically than free acid PGs, suggesting that COX-2 action on endocannabinoids may provide oxygenated lipids with sufficiently long half-lives to act as systemic mediators or prodrugs (Kozak et al., 2004). Cytochrome P450 enzymes have also been implicated in metabolizing anandamide (Snider et al., 2007). Action of Epoxygenase on anandamide generates a diverse array of oxygenated products including epoxides (epoxyeicosatetraenoic acids) (Snider et al., 2007). These metabolites have been reported to interact with PPAR-a and mediate many neurochemical activities (Di Marzo, 2006a, b). Anandamide is also an endogenous agonist for the TRPV1 (Smart et al., 2000) and the gastrin-releasing peptide 55 receptor (Pertwee, 2005; Lauckner et al., 2008). TRPV1 receptors are expressed in nociceptive sensory neurons and can detect/ respond to noxious mechanical, thermal (heat), and chemical (capsaicin) stimuli (Piomelli, 2001; Caterina et al., 1997; Ross, 2003). The administration of anandamide directly into the hippocampus, increases rapid-eye-movement (REM) sleep in a dose-dependent manner during the dark but not during the light phase of the cycle. The increase of REMs can be blocked by the

CB1 antagonist, AM251. This effect is specific for the hippocampus since anandamide administration in the surrounding cortex does not elicit any change in REMs. These results support the idea of a direct relationship between hippocampal activity and sleep mechanisms by means of anandamide (Rueda-Orozco et al., 2010). 5.4 Interplay Among Cannabinoid, Glutamate, and Dopamine Receptors… 143

5.4 Interplay Among Cannabinoid, Glutamate, and Dopamine Receptors in the Basal Ganglia

It is becoming increasingly evident that coordinated interplay (cross-talk) among various receptors is essential for normal synaptic function. This interplay involves various neurotransmitters, neuropeptides, growth factors, and lipid mediators including eicosanoids, docosanoids, endocannabinoids, and platelet-activating factor and their receptors (Farooqui, 2009b). Thus, the presence of CB1 receptors in several GABAergic and glutamatergic synapses within the basal ganglia as well as the presence of TRPV1 receptors in nigrostriatal dopaminergic neurons enables endocannabinoids to directly modulate the release of GABA and glutamate. In addition, the preferential presynaptic location of these receptors makes it likely that endocannabinoids mainly control presynaptic events, such as the synthesis, release, or reuptake of glutamate and dopamine (Gerdeman and Fernández-Ruiz, 2008). This suggestion is supported by studies not only on the administration of cannabinoids combined with agonists or antagonists for GABA and glutamate receptors but also by electrophysiological and neurochemical analysis studies (Köfalvi et al., 2005). Based on these studies, it is proposed that an important function of cannabinoid signaling within the basal ganglia circuitry is to modulate GABAergic and glutamatergic signaling at synapses (Fig. 5.8) (Gerdeman and Fernández-Ruiz, 2008). Furthermore, anandamide has been shown to directly block certain presynaptic events in dopaminergic synapses within the striatum by acting through TRPV1 receptors located in nigrostriatal dopaminergic neurons (de Lago et al., 2004). Furthermore, dopamine transmission is also modulated through interactions with G protein/adenylyl cyclase

Phospholipids

2-Arachidonylglycerol or anandamide

CB1-mediated alterations in GABA- CB1-mediated alterations CB1or TRPV1-mediated alterations ergic neurotransmission in glutamatergic in dopaminergic neurotransmission neurotransmission Decrease in GABA release Decrease in dopamine Stimulation of GABA uptake Decrease in glutamate release synthesis and release

Synaptic communication

Inhibition of motor activity

Fig. 5.8 Modulation of GABA-, glutamate-, and dopamine-neurotransmission by endocannabinoids 144 5 Cannabinoids in the Brain: Their Metabolism, Roles…

signal transduction mechanisms shared by both CB1 and D1/D2 dopamine receptors

(Di Marzo et al., 2000). Interactions and cross-talk between CB1 receptors and dopamine D2 receptors modulate synaptic activity within nigrostriatal neuronal networks.

In addition, CB1 receptors are localized preferentially in the nigrostriatal circuitry, where their ability to modulate GABA-mediated neurotransmission coincides with the capacity of GABA- and dopamine-containing neurons to produce endocannabinoids (Köfalvi et al., 2005). In vitro studies indicate that at subsaturating agonist concentrations endocannabinoid and dopamine signaling promote the heterodimerization of CB1 receptors and D2 receptors (Kearn et al., 2005) and not only stimulate cAMP production, but also enhances ERK1/2 activity. If present in vivo,

CB1–D2-receptor heterodimers may be involved in the control of motor behavior through phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) (Andersson et al., 2005). Phospho-DARPP-32-mediated Ca2+ influx is known to facilitate Ca2+-dependent endocannabinoid production and control of neuronal activity in the basal ganglia. Collective evidence suggests that interplay among endocannabinoids, glutamate, and dopamine signaling systems may influence neuronal development, modulate motor activity, and is necessary for the maintenance of normal synaptic function. Apparent heterogeneity of various receptors in brain warrants more studies on the organizational, spatial, and temporal constraints of endocannabinoid signaling at the cellular and neuronal network levels.

5.5 Endocannabinoids and Neurological Disorders

As stated above, CB1 receptors are expressed in neurons, whereas some CB2 receptors are found mostly in microglia, which during injury and neurodegeneration become activated and overexpress cannabinoid receptors. The overexpression of cannabinoid receptors and alterations in endocannabinoid levels have been reported to occur in neurotraumatic diseases (ischemia, spinal cord trauma, and traumatic brain injury) and neurodegenerative diseases, such as Alzheimer disease (AD), multiple sclerosis (MS), amyotropic lateral sclerosis (ALS), Parkinson disease (PD), and Huntington chorea (HD). In addition, cannabis has been used for centuries for its pain-relieving properties. The main active ingredient of cannabis,

9-tetrahydrocannabinol, induces antinociceptive effects by binding to CB1 and CB2 receptors. In particular CB2 receptors have emerged as an interesting target for chronic pain treatment as demonstrated by several studies on inflammatory and neuropathic preclinal pain models (Anand et al., 2009; Beltramo, 2009). The molecular mechanisms involved in CB2 receptor-mediated analgesia are still controversial, but it is becoming increasingly evident that cannabinoids modulate pain either through their effect on inflammatory cells and/or on nociceptors and spinal cord relay centers. In addition, cannabinoids also produce muscle relaxation, immunosuppression, antiallergic effects, sedation, improvement of mood, stimulation of appetite, antiemesis, lowering of intraocular pressure, bronchodilation, and neuroprotective and antineoplastic effects. The immunomodulatory effects of endocannabinoids are 5.5 Endocannabinoids and Neurological Disorders 145

mediated through CB2 receptors on glia and microglia. It is proposed that overexpression of CB1 and CB2 receptors and alterations in endocannabinoid levels may be initial attempt of brain tissue to counteract harmful effects of excitotoxicity and inflammation and provide a balanced metabolism during the pathogenesis of AD, MS, ALS, PD, and HD (Bisogno and Di Marzo, 2010). Collective evidence suggests that the endocannabinoid system protects neurons against glutamate excitotoxicity and neuroinflammation in both in vitro and in vivo models of neurotraumatic and neurodegenerative diseases (Bisogno and Di Marzo, 2010).

5.5.1 Endocannabinoids in Neurotraumatic Diseases

Ischemic injury is accompanied by the appearance of CB2-expressing cells and accumulation of N-acylethanolamines at the lesion site in the rat brain (Berger et al., 2004; Ashton et al., 2007; Degn et al., 2007). Levels of 2-AG are markedly increased at 4 h, but returned to basal level 12 h after induction of ischemia (Degn et al., 2007). No changes are observed in anandamide levels in this model of ischemic injury. It is suggested that N-acylethanolamines and 2-AG may be involved in regulation of neuroprotection during focal cerebral ischemia. In mouse cerebral cortex, focal cerebral ischemia greatly increases levels of palmitoylethanolamide, only moderately increases anandamide, and has no effect on 2-arachidonoylglycerol levels (Franklin et al., 2003). Palmitoylethanolamide potentiates anandamide-induced microglial cell migration, without affecting other steps of microglial activation, such as proliferation, particle engulfment, and nitric oxide production. This potentiation of microglial cell migration by palmitoylethanolamide is associated with the reduction in cAMP levels. It is proposed that palmitoylethanolamide acts through

CB2-linked Gi/o-coupled receptors (Franklin et al., 2003).

In a mouse model of ischemic stroke, CB2 agonist (JWH-133) reduces the infarct size measured 3 days after onset of ischemia. The CB2 activation not only decreases the number of neutrophils in the ischemic brain, but also reduces the activity of myeloperoxidase and inhibits adherence of neutrophils to brain endothelial cells (Murikinati et al., 2010). In addition, JWH-133 also interferes with the migration of neutrophils, which is mediated by the endogenous chemokine CXCL2 through activation of the MAP kinase p38. This effect on neutrophils may be responsible for the neuroprotection mediated by JWH-133. Thus, by activating p38 in neutrophils, CB2 agonists inhibit neutrophil recruitment to the brain and protect against ischemic brain injury. Collective evidence suggests that exogenous and endogenous cannabinoids provide neuroprotection in a variety of in vitro and in vivo models of ischemic injury through different mechanisms, including prevention of excitotoxicity by cannabinoid

CB1-mediated inhibition of glutamatergic transmission, reduction of calcium influx, antioxidant activity, activation of the phosphatidylinositol 3-kinase/protein kinase B pathway, induction of phosphorylation of extracellular-regulated kinases and expression of transcription factors and neurotrophins, lowering of cerebrovasoconstriction, and induction of hypothermia (van der Stelt and Di Marzo, 2005). The release of endocannabinoids during ischemic injury may constitute a protective response. 146 5 Cannabinoids in the Brain: Their Metabolism, Roles…

Spinal cord trauma is accompanied by mechanical insult to the spinal cord followed by secondary injury, which contributes to the spread of the damage and neurological deficits through the release of glutamate, influx of calcium, secretion of cytokines, generation of high levels of lipid mediators, and apoptotic cell death (Farooqui, 2010). During early stages of spinal cord trauma, levels of anandamide and palmitoylethanolamide are increased along with upregulation of the synthesizing enzyme, NAPE-phospholipase D and a downregulation of the degradative enzyme fatty acid amide hydrolase (FAAH) in spinal cord lesion (Garcia-Ovejero et al., 2009). In delayed stages of spinal cord trauma, levels of 2-AG are increased along with a strong upregulation of the synthesizing enzyme diacylglycerol lipase-a that is expressed by neurons, astrocytes, and infiltrating immune cells. The monoacylglycerol lipase activity is also moderately increased in the lesion but only 7 days after spinal cord trauma. Administration of a single dose of 2-AG early after spinal cord trauma reduces lesion expansion (Arevalo-Martin et al., 2010). 2-AG treatment also preserves the white matter around the epicenter of the injury site by protecting myelin and reducing oligodendrocyte loss. In addition, 2-AG also prevents the myelin damage and delays oligodendrocyte loss induced at 10 mm from the epicenter. Interestingly, the early protective action of 2-AG is maintained 28 days after injury when the lesion size is still smaller, and the preservation of white matter is better in 2-AG-treated animals. Thus, generation of endogenous cannabinoid may be a protective mechanism for spinal cord trauma (Arevalo-Martin et al., 2010). Like spinal cord trauma, traumatic brain injury (TBI) is accompanied by the release of glutamate, calcium influx, upregulation of cytokines, accumulation of harmful mediators, and apoptotic cell death leading to brain damage and dysfunction (Farooqui, 2010). Neurochemical changes in TBI also lead to significant increase in 2-AG levels (Panikashvili et al., 2001). Administration of synthetic 2-AG to mice after TBI reduces brain edema, decreases infarct volume, and reduces hippocampal cell death compared with controls resulting in better clinical recovery in a dose-dependent manner. Administration of 2-AG with 2-acyl-glycerols significantly enhances functional recovery. The beneficial effect of 2-AG can be attenuated by SR-141761A, an antagonist of the CB1 cannabinoid receptor −/− (Panikashvili et al., 2001). CB1 receptor knockout mice CB1 following TBI show minor spontaneous recovery after 24 h compared to wild-type (WT) mice. −/− Administration of 2-AG in the CB1 mice does not improve neurological performance and edema formation (Panikashvili et al., 2005). In addition, 2-AG retards 3- to 4- fold increase in nuclear factor-kB (NF-kB) transactivation, after 24 h −/− after TBI in the WT mice, while TBI has no effect on NF-kB in the CB1 mice (Panikashvili et al., 2005). Studies on the effect of anandamide on primary cell culture indicate that this endocannabinoid also binds to vanilloid (VR1) receptors and induces cell death. Intracerebroventricular administration of anandamide induces sustained cerebral edema, regional cell loss, and impairment in long-term cognitive function (Cernak et al., 2004). These effects are mediated, in part, through interactions between anadamide and vanilloid receptor (VR1) as well as through calpain-dependent mechanisms, but not through CB1 receptors or caspases. These studies also indicate that 5.5 Endocannabinoids and Neurological Disorders 147

Neuroprotective effects of endocannabinoid

Mutiple sclerosis HIV associated diseases

Parkinson disease Amyotrophic lateral sclerosis

Alzheimer disease

Fig. 5.9 Neuroprotective effects of endocannabinoids in neurodegenerative diseases central administration of anandamide also significantly upregulates genes involved in proinflammatory/microglial-related responses (Cernak et al., 2004). Accumulating evidence suggests that endo- and exocannabinoids may produce both neuroprotective and neurotoxic effects in animal models of spinal cord trauma and traumatic brain injury.

5.5.2 Endocannabinoids in Neurodegenerative Disorders

Cannabinoid-based drugs produce beneficial effects in basal ganglia disorders, including PD and HD (Fernandez-Ruiz, 2009) (Fig. 5.9). Beneficial effects include the alleviation of specific motor symptoms, namely choreic movements with cannabinoid receptor type 1 (CB1)/transient receptor potential vanilloid type 1 agonists in HD and bradykinesia with CB1 antagonists and tremor with CB1 agonists in PD. Several cannabinoid agonists have been used for the treatment of HD and PD in various animal models. Degeneration of dopaminergic neurons in animal model of PD is reduced by agonists of CB1, CB2, and non-CB1 or non-CB2 receptors – an effect may be associated with modulation of interactions between glial cells and neurons (Lastres-

Becker et al., 2005). CB1 receptors, however, also exert detrimental effects on dopamine cell survival by potentiating the toxic effects of the TRPV1 agonist, capsaicin (Kim et al., 2005). It is thus likely that endocannabinoids such as anandamide, which activates both TRPV1 and CB1 receptors (Di Marzo, 2006a) might contribute to PD pathophysiology by favoring apoptosis of dopamine neurons. Collective evidence indicates that cannabinoids delay the progression of these diseases and protect neurons via (a) a reduction in neuroinflammation through the activation of CB2 receptors, which are located in glial cells, (b) a normalization of glutamate homeostasis then limiting excitotoxicity, and (c) an antioxidant effect exerted by cannabinoid receptor- independent mechanisms (Pazos et al., 2008; Garcia-Arencibia et al., 2009). 148 5 Cannabinoids in the Brain: Their Metabolism, Roles…

In AD, Ab-mediated brain damage is accompanied by endocannabinoids released from neurons and glial cells resulting in elevation in Erk activity and induction of brain-derived neurotrophic factor (BDNF) and activation of CB1-mediated neuroprotective pathways (Huestis et al., 2001; Marsicano et al., 2003). In addition, endocannabinoids modulate the release of inflammatory mediators in microglia through cannabinoid CB2 receptors. It should be noted that activated microglial cells in AD express significant levels of cannabinoid CB2 receptors supporting the view that endocannabinoids may trigger CB2-dependent proinflammatory cytokine release and neuroinflammation, a process closely associated with the pathogenesis of AD (Maresz et al., 2005; Benito et al., 2003). Induction of endocannabinoid-mediated neuroinflammation in AD is also supported by high activity of FAAH in astrocytes surrounding neurite plaques. FAAH may produce ARA through the hydrolysis of AEA and 2-AG and eicosanoids derived from ARA may enhance neuroinflammation. Surprisingly intracerebroventricular administration of the synthetic cannabinoid (WIN55,212-2) to rats prevents Ab-induced microglial activation, cognitive impairment, and loss of neuronal markers (Fig. 5.9). Other synthetic cannabinoids (HU-210 and JWH-133) prevent Ab-mediated activation of cultured microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-a release; these effects are independent of the antioxidant action of cannabinoid compounds and are also induced by a CB2-selective agonist (Table 5.1). Moreover, cannabinoids abrogate microglia-mediated neurotoxicity after Ab addition to rat cortical cocultures (Ramirez et al., 2005). It is proposed that cannabinoid receptors may be associated with the pathology of AD and that cannabinoids may exert beneficial effects on neurodegenerative process in AD (Ramirez et al., 2005). In G93A-SOD1 mutant mice (animal model ALS), administration of nonselective cannabinoid partial agonists prior to, or upon, symptom appearance minimally delays disease onset and prolongs survival through undefined mechanisms. In

Table 5.1 Therapeutic potentials of cannabinoids for neurological diseases Endocannabinoids/agonists/ Disease antagonists Reference MS Dexanabinol (HU-211) Cabranes et al., 2005; Croxford, 2003 HD CB1/vanilloid agonists Micale et al., 2007 AD HU-210, WIN55,212-2, and Micale et al., 2007; Rivers and Ashton, JWH-133, CB2 agonists 2010; Ramirez et al., 2005 ALS CB2 agonist (AM-1241) Shoemaker et al., 2007 PD CB1 antagonists; vanilloid Fernandez-Ruiz, 2009; Giuffrida and type 1 agonists McMahon, 2010 Ischemia Dexanabinol (HU-211) Croxford, 2003 TBI Dexanabinol (HU-211) Eljaschewitsch et al., 2006; Croxford, 2003 SCI 2-Arachidonylglycerol Arevalo-Martin et al., 2010 Depression AM251, AM404, HU210 Vinod and Hungund, 2006 HIV wasting disease Dexanabinol (HU-211) Croxford, 2003 Motor dysfunction CB1 and CB2 agonists Fernandez-Ruiz, 2009 5.6 Use of FAAH and MGL Inhibitors for Inflammatory Pain 149

G93A-SOD1 mutant mice ALS progression is accompanied by dramatic upregulation of CB2 receptor mRNA in spinal cords. More importantly, daily injections of the selective CB2 agonist AM-1241 started at the onset of ALS increases the survival interval after disease onset by 56%. It is suggested that CB2 agonists may slow motor neuron degeneration and preserve motor function, and represent a novel therapeutic modality for treatment of ALS (Fig. 5.9) (Shoemaker et al., 2007). Similarly, in hSOD1G93A mice genetic ablation of fatty acid amide hydrolase not only exerts robust anti-inflammatory and neuroprotective effects, but also causes a delay in the progression of ALS (Raman et al., 2004; Bilsland et al., 2006). CB2 receptor- mediated augmentation of endocannabinoids levels results in a neuroprotective effect in hSOD1G93A mice (Table 5.1). In contrast, activation of the CB1 receptor produces a negative influence on motor neuron survival (Bilsland et al., 2006). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, decrease in cannabinoid signaling is accompanied by alterations in levels of anandamide and 2-arachidonoylglycerol in motor-related regions (striatum, midbrain) along with motor disturbances in the basal ganglia (Cabranes et al., 2005). Cannabinoid-based drugs, AM404, arvanil, and OMDM2 reduce the magnitude of the neurological impairment in EAE rats (Fig. 5.9), whereas VDM11 does not produce any effect. The beneficial effects of AM404 are reversed by blocking TRPV1 receptors with capsazepine, but not by blocking CB1 receptors with SR141716, thus indicating the involvement of endovanilloid mechanisms in these effects (Cabranes et al., 2005). Cannabinoids can alleviate tremor and spasticity in EAE, and clinical trials of the use of these compounds for these symptoms are in progress (Croxford, 2003). Studies on the levels of endocannabinoids (ECs) in cerebrospinal fluid samples from MS patients indicate that levels of AEA are increased in the CSF from relapsing MS patients, but levels of 2-AG are not affected (Centonze et al., 2007). Peripheral lymphocytes of these patients also higher levels of AEA, an effect associated with increased synthesis and reduction in degradation of this endocannabinoid. Increased synthesis, reduced degradation, and increased levels of AEA were also detected in the brains of EAE mice in the acute phase of the disease, possibly accounting for its antiexcitotoxic action in this disorder (Centonze et al., 2007). This observation is tempting to speculate that the AEA and 2-AG are differentially engaged in CNS inflammatory processes. Although AEA and 2-AG share many pharmacological actions, they may be associated with differential regulatory mechanisms involved in pathological events (Chevaleyre et al., 2006). Collectively, these studies support the view that in MS patients and in animal models of MS, stimulation of CB1 and of CB2 receptors produces beneficial effects against the inflammatory process (Huntley, 2006; Arévalo-Martín et al., 2003; Eljaschewitsch et al., 2006).

5.6 Use of FAAH and MGL Inhibitors for Inflammatory Pain

Pharmacological inhibition of FAAH and monoacylglycerol lipase has been used as therapeutic target for pain in inflammatory pain models. Inhibition of monoacylglycerol lipase by JZL184 (Fig. 5.10) reduces capsaicin-mediated nocifensive 150 5 Cannabinoids in the Brain: Their Metabolism, Roles…

a N N

O O bdO O PF NH2 C O CH3 O OCH3 H O N O

HN c OH O O f O

NH OH e O NH

Fig. 5.10 Chemical structures of inhibitors of endocannabinoids metabolism used for the treatment of pain in neurological disorders. Fatty acid amide hydrolase inhibitor (OL135) (a); diacylglycerol lipase inhibitor (03841) (b); anandamide membrane transporter (AMT inhibitor) (UCM707) (c); JZL184 (d); and AMT inhibitor (e); and JZL 195 (f) behavior and thermal hyperalgesia. It is suggested that this may be due to increasing accumulation of 2-AG. In contrast, inhibition of fatty acid amide hydrolase by URB597 (Fig. 5.10) blocks capsaicin-mediated mechanical allodynia. This may be caused by the accumulation of anandamide (Spradley et al., 2010). Combined treatment with inihibitors of monoacylglycerol lipase and fatty acid amide hydrolase produces additive effect due to the accumulation of anandamide and 2-AG. Based on these observations, it is proposed that specific inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase may serve as attractive therapeutic targets for the treatment of pain in neurotraumatic and neurodegenerative diseases (Bari et al.,

2006). Recently, JZL 195, a potent inhibitor of both FAAH and MAGL (IC50 = 2 and 4 nM, respectively) has been synthesized. Although it has no effect on neuropathy target esterase, ABHD6 and other brain serine hydrolases (Long et al., 2009), this compound blocks monoacylglycerol lipase and fatty acid amide hydrolase activities in time- and dose-dependent manner in vivo. The in vivo inhibitory actions of JZL 195 against fatty acid amide hydrolase and monoacylglycerol lipase are comparable to those of the selective inhibitors JZL 184 and PF-3845, respectively. Through its inhibitory actions, JZL 195 simultaneously augments brain levels of anandamide and 2-AG, producing antinociceptive, cataleptic, and hypomotility effects like References 151

those produced by direct CB1 agonists. Collective evidence suggests that activating the endogenous cannabinoid system by targeting fatty acid amide hydrolase and monoacylglycerol lipase through fatty acid amide hydrolase and monoacylglycerol lipase inhibitors may be a promising strategy to treat pain and inflammation. This strategy lacks untoward side effects typically associated with Cannabis sativa (Schlosburg et al., 2009).

5.7 Conclusion

Endocannabinoids are lipid mediators that modulate brain function by interacting with cannabinoid receptors as well as the transient receptor potential vanilloid 1 (TRPV1) receptor. Anandamide and 2-AG are well-characterized retrograde messenger at synapses in terms of their production, mode of action, and degradation. These endocannabinoids mimic several pharmacological effects of the exogenous cannabinoid d9-tetrahydrocannabinol. The activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase or by a monoacylglycerol lipase. Although mechanisms for biosynthesis and degradation of anandamide and 2-AG are fairly well established, the manner by which these endocannabinoids accumulate in cells remains controversial. Many studies indicate that endocannabinoids are taken up into neural cells via a facilitated process. Other reports indicate that the uptake of endocannabinoids is facilitated not only by the diffusion, but also through endocytosis. It is proposed that endocannabinoid system modulates a broad range of physiological processes in the CNS and periphery, including pain and inflammation, mood and anxiety disorders, neurodegenerative disorders, cancer, epilepsy, stroke, hypertension, glaucoma, and obesity (Pacher et al., 2006).

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6.1 Introduction

4-Hydroxynonenal (4-HNE) is a major end-product of peroxidation of membrane arachidonic acid (ARA). This nine carbon a, b-unsaturated aldehyde contains three functional groups, which often act in concert and help to explain its high reactivity (Poli and Schaur, 2000). Most importantly, there is a conjugated system of a C=C double bond and a C=O carbonyl group which provide a partial positive charge to carbon 3 due to the presence of mobile pi-electrons. This positive charge is further enhanced by the inductive effect of the hydroxy group at carbon 4. Therefore, 4-HNE is considered to be soft electrophiles and is prone to be attacked by nucleophiles, such as thiol or amino groups. This reaction occurs primarily at carbon 3 and secondarily at the carbonyl carbon 1. Amino acids that react with 4-HNE are cysteine, histidine, and lysine, proteins and peptides leading to the formation of stable Michael adducts with a hemiacetal structure (Schaur, 2003; Poli et al., 2006) (Fig. 6.1). Among the protein residues that react with 4-HNE, Cys exhibits the highest reactivity, followed by His and Lys (Petersen and Doorn, 2004), but Cys–HNE adducts are less stable than His–HNE adducts (Uchida, 2003). These chemical reactions proceed through reactions between the C1/4C double bond with a nucleophile (Cys, glutathione (GSH) and amine) via 1,2- and 1,4-Michael addition (Nadkarni and Sayre, 1995). The 1,2-Michael addition involves the reaction of a primary amine (Lys) with the a,b- unsaturated carbonyl, resulting in the formation of a Schiff base at acidic pH. This step is reversible (Petersen and Doorn, 2004) (Fig. 6.1). The biological effects of 4-HNE are mostly modulated by its lipophilic properties. Although hydrophilic properties play some role in the reactivity of 4-HNE, their effect is less pronounced than lipophilic properties. Because of this reason 4- HNE tends to interact and concentrate in biomembrane rather than in the aqueous space of cells. In neural membranes, concentration of 4-HNE reaches as high as 50 mM. Compared to free radicals, 4-HNE is relatively stable and can diffuse within or even escape from the cell and attack targets far from the site of its generation. It exhibits great reactivity

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 159 DOI 10.1007/978-1-4419-9940-5_6, © Springer Science+Business Media, LLC 2011 160 6 Neurochemical Aspects of 4-Hydroxynonenal

OH OH O His O H C 3 4-HNE H3C Michael adduct His Lys OH OH O O H C H3C 3 Cyclic hemiacetal NH His Lys Lys

OH NH-Lys N-Lys O H3C NH H C Lys 3 NH-Lys Schiff base Michael adduct- crosslink formation

Fig. 6.1 Chemical structures of 4-HNE and its amino acid adducts. Lysine (Lys); and histidine (His)

with proteins, DNA, and phospholipids, generating a variety of intra and intermolecular covalent adducts. At the membrane level, proteins and amino lipids can be covalently modified by 4-HNE. This aldehyde also acts as bioactive lipid mediator that produces rapid cell death not only through the depletion of sulfhydryl groups, but also through alterations in calcium homeostasis (Farooqui, 2009).

6.2 Synthesis of 4-HNE in the Brain

As mentioned above, 4-HNE is a degradation product of 9- and 13-hydroperoxides of n-6 fatty acids (arachidonic and linoleic acids) (Esterbauer et al., 1991). Studies on model system of 9-hydroperoxides of linoleic acid have indicated that b-cleavage of 9(S)-hydroperoxy-octadecadienoic acid (9(S)-HPODE) is decomposed through a three-step process involving Hock cleavage (Fig. 6.2) (Schneider et al., 2001, 2004). The Hock cleavage splits C–C bond as well as O–O bond yielding nonenal and 9-oxo-nonaic acid. Peroxidation of nonenal in the 4-position results in a racemic 4-hydroperoxy-nonenal (4-HPNE), which can be generated either nonenzymically or through enzymic reactions catalyzed by hydroperoxide lyase and alkenal oxygenase. The hydroperoxy group of 4-HPNE is then reduced to produce 4-HNE. Similarly, 13(S)-HPODE is also decomposed first through peroxidation and then 6.3 Metabolism of 4-HNE in the Brain 161

9 9(S)-Hydroperoxy- HOOC octadecadienoic acid HOO 9(S)-HPODE Hock Hyroperoxide lyase cleavage

HOOC nonenal O 9-Oxo-nonanic acid O - H•

O - Alkenal oxygenase O2 , Reduction

OOH O Reduction

OH O 4-HNE

Fig. 6.2 Synthesis of 4-HNE from 9-hydroxy derivative of linoleic acid

by Hock cleavage producing 4-HNE (Schneider et al., 2001, 2004). Ferrous iron (Fe2+)- or vitamin C-mediated decomposition of lipid hydroperoxides also result in generation of 4-HNE (Gu et al., 2007; Lee et al., 2001). Recently, an alternative mechanism involving an intermolecular cross-linking of a peroxyl radical has also been proposed for the formation of 4-HNE. This mechanism involves the styrene– oxygen polymerization and depolymerization (Schneider et al., 2008).

6.3 Metabolism of 4-HNE in the Brain

The primary enzymic pathways of 4-HNE detoxification in brain and liver involves aldehyde dehydrogenase, alcohol dehydrogenase, aldehyde reductase, and glutathione S-transferase (Gallagher and Gardner, 2002; Gardner et al., 2003). Among above enzyme systems, in brain glutathione S-transferase is a predominant mechanism for neuroprotection against 4-HNE-mediated neurotoxicity (Gardner et al., 2003). In vitro and in vivo studies indicate that 4-HNE undergoes many enzymic and nonenzymic reactions (Fig. 6.3), which are closely associated with the modulation of cellular functions. Oxidation of 4-HNE to 4-hydroxynon-2-enoic acid (4-HNA) is catalyzed by the mitochondrial aldehyde dehydrogenase, an NAD-dependent 162 6 Neurochemical Aspects of 4-Hydroxynonenal

C=C bond reduction 4-Hydroxynonenal Michael addition

C=O group C=O group reduction oxidation

Schiff-base Acetal and thio-acetal formation formation Epoxidation

Fig. 6.3 Metabolic fate of 4-HNE

enzyme, which contains three domains; two dinucleotide-binding domains and a small three-stranded b-sheet domain, which is involved in subunit interactions in this tetrameric enzyme (Mitchell and Petersen, 1987). 4-HNE is not only a substrate for mitochondrial aldehyde dehydrogenase, but also a potent inhibitor (Ki = 0.5 mM) of this enzyme, when acetaldehyde was used as a substrate (Mitchell and Petersen, 1991). In addition, reduction of 4-HNE is catalyzed by aldoketo reductase and/or alcohol dehydrogenases in a NADPH-dependent manner yielding 1,4-dihydroxy-2- nonene (DHN) (Burczynski et al., 2001; Srivastava et al., 1999). The conjugation between 4-HNE and glutathione is catalyzed by glutathione-S-transferase resulting in formation of glutathione-4HNE conjugate. This enzyme catalyzes the conjugate formation 600 times faster than the spontaneous nonenzymic reaction (Alin et al., 1985). Glutathione-4-HNE conjugate is also utilized by aldose reductase, an NADPH-dependent enzyme that reduces glutathione-4-HNE conjugate glutathione- 1,4-dihydroxy-2-nonene (glutathione-DHN) (Esterbauer et al., 1991). The reduction of C=C bond in 4-HNE is catalyzed by alkenal/one oxidoreductase yielding 4-hydroxynonenal acid (4-HAA). C=C double bond in 4-HNE undergoes Michael addition reaction with cysteine or glutathione (GSH), resulting in the formation of Michael adduct. This reaction is catalyzed by glutathione-S-transferase. Similarly, amino compounds such as lysine, ethanolamine, guanine, and the imidazole group of histidine also undergo Michael additions with 4- HNE (Zhou and Decker, 1999). Thus, brain tissue contains multiple enzymic pathway for the detoxification of 4-HNE. Although 4-HNE is reduced by aldehyde dehydrogenases and aldolase reductases, the majority of cellular 4-HNE is metabolized by glutathione-S-transferase reaction. Overexpression of glutathione-S-transferase causes marked reduction in 4-HNE levels (Sharma et al., 2004). 6.4 Effect of 4-HNE on Enzyme Activities 163

6.4 Effect of 4-HNE on Enzyme Activities

At low concentrations (0.8–2.8 mM), 4-HNE affects many enzymes (Fig. 6.4) associated with cell growth, gene expression, long-term potentiation, inflammation, apoptosis, and blood–brain barrier permeability (Fig. 6.5). Accumulation of 4-HNE during the initial stages of oxidative injury can exacerbate the cellular oxidative damage cascade by eliciting further protein and oxidative injury not only by altering mitochondrial function and redox status and decreasing ROS clearance (Raza et al., 2008), but also by electrophilically attacking the nucleophilic sites on proteins (Esterbauer et al., 1991). Thus, treatment of PC12 cells with 4-HNE induces oxidative stress by compromising the mitochondrial redox metabolism (Raza et al., 2008). As stated above, 4-HNE treatment reduces glutathione pool and increases ROS, protein carbonylation, and apoptosis. These processes are not only accompanied by marked inhibition in the activities of the mitochondrial respiratory enzymes, cytochrome c oxidase and aconitase, but also by increase in nuclear translocation of NF-kB/p65 protein leading to upregulation of tumor necrosis factor alpha (TNF-a). Furthermore, in PC12 cells, 4-HNE treatment also results in the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA

Glycerophospholipids

COX-2 (↑) Arachidonic acid PLC (↑)

Ca2+ATPase (↓) PLD (↑)

Na+, K+ATPase (↓) PKC (↑)

Adenylate cyclase (↑) 4-HNE MAP-Kinase (↑)

Cysteine Ligase (↑) JNK (↑)

Caspase-3 (↑) Aldolase Reductase (↑)

iNOS (↑) Nrf2 translocation G 6-P dehydrogenase (↓)

Nrf2/ARE

Detoxification

Fig. 6.4 Modulation of enzyme activities by 4-HNE (Upward arrow indicate stimulation and downward arrow indicate inhibition) 164 6 Neurochemical Aspects of 4-Hydroxynonenal

Cell growth

Gene expression

Protein turnover

4-Hydroxynonenal BBB permeability

Long-term potentiation

Apoptosis

Inflammation

Fig. 6.5 Modulation of neurochemical processes by 4-HNE fragmentation, and decreased expression of antiapoptotic Bcl-2 proteins. In addition, 4-HNE modulates MAP kinases, PKC isoforms, cell-cycle regulators, receptor tyrosine kinases, and caspases. Collectively these studies suggest that 4-HNE- induced cytotoxicity may be associated, at least in part, with the altered mitochondrial redox, mitochondrial energy metabolism, membrane dynamics, and respiratory dysfunctions that ultimately leads to apoptotic cell death (Raza et al., 2008).

6.4.1 Modulation of Kinases by 4-HNE

Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that are activated by dual phosphorylation on threonine and tyrosine residues in response to a wide array of extracellular stimuli (Davis, 1999). Three distinct groups of MAP kinases, namely extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 have been identified in mammalian cells and are closely associated with signal transduction processes involved in transfer of signal from the cell surface to the nucleus. ERK plays a major role in cell proliferation and differentiation as well as survival processes mediated by various growth factors (Karin, 1995). Exposure of cultured microglial Ra2 cells with 4-HNE significantly increases expression levels of cPLA2. The activated form of cPLA2 is phosphorylated (p-cPLA2) at amino acid residue S(505) on immunoblots (Shibata et al., 2010a). Pretreatment of Ra2 cells with the antioxidant N-acetylcysteine, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen- activated protein kinase (MAPK) inhibitor SB203580 blocks the 4-HNE-mediated increased expression of cPLA2 and p-cPLA2. Immunocytochemical analysis indicates that staining for p-cPLA2 in Ra2 cells is localized in the cytoplasm and more intense in the 4-HNE-stimulated group than in the vehicle group (Shibata et al., 2010a).

These results support the view that 4-HNE upregulates and phosphorylates cPLA2 in microglia via the ERK and p38 MAPK pathways. In contrast, the stress-activated 6.4 Effect of 4-HNE on Enzyme Activities 165

NMDA A

PtdCho R PtdIns-4,5-P 2 NMDA-R 2 + + PLC Cell cycle arrest Ca2+ cPLA ARA + DAG PKC InsP 4-HNE + 3 Depletion of GSH + Mitochondria MARK signaling Ca2+ pathway Alterations in Ca2+ Oxidative stress homeostasis P38, ERK, JNK Cytochrome c

RNA binding factor (AUF) NUCLEUS Apoptosis COX-2 c-foc, c-jun, AP-1 NF-kB

Fig. 6.6 Modulation of signal transduction processes by 4-HNE. N-Methyl-d-aspartate receptor

(NMDA-R); phosphatidylcholine (PtdCho); cytosolic phospholipase A2 (cPLA2); Receptor (R); phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2); inositol 1,4,5-trisphosphate (InsP3); phospholipase C (PLC); diacylglycerol (DAG); arachidonic acid (ARA); intracellular calcium (Ca2+); and protein kinase C (PKC); 4-hydroxynonenal (4-HNE); glutathione (GSH); mitogen-activated kinase (MARK); extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen- activated Protein Kinase (p38) and nuclear factor-kappaB (NF-kB) protein kinase (SAPK) or JNK (Kyriakis et al., 1994) and p38 mitogen-activated protein kinase (p38 MAP kinase) (Han et al., 1994) are associated with the coordinated activation of various inflammatory cytokines and environmental stressors (Raingeaud et al., 1995). JNK is closely associated with signal transduction pathway that modulates apoptosis (Chen et al., 1996). 4-HNE stimulates tyrosine phosphorylation through the activation of c-Jun N-terminal kinase (JNK) and MAP-kinase (p38), which facilitate transfer of extracellular signals through signal transduction process into intracellular responses through phosphorylation cascades (Fig. 6.6) (Uchida, 2003; Leonarduzzi et al., 2004). 4-HNE-mediated JNK activation promotes its translocation in the nucleus (Fig. 6.6) where JNK-dependent phosphorylation of c-Jun and the transcription factor activator protein (AP-1) binding take place (Camandola et al., 2000; Cheng et al., 2001). This leads to the transcription of a number of genes having AP-1 consensus sequences in their promoter regions. Treatment of PC12 cells with 4-HNE results in maximal activation of JNK within 15–30 min before returning to control level after 1 h post-treatment. In contrast, activities of extracellular signal-regulated kinase (ERK) and p38 MAP kinase remain unchanged from their basal levels (Song et al., 2001). SEK1, an upstream 166 6 Neurochemical Aspects of 4-Hydroxynonenal kinase of JNK, is also activated (phosphorylated) within 5 min after 4-HNE treatment and remains activated for up to 60 min. Pretreatment of PC12 cells with 8-(4-chlorophenylthio)-cAMP, a survival promoting agent blocks both the 4-HNE- mediated JNK activation and apoptotic cell death. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, has no effect on 4-HNE-mediated apoptotic cell death. Collective evidence suggests that 4-HNE mediates early activation of JNK and p38 proteins but downregulate the basal activity of ERK-1/2. In 3 T3 fibroblasts, overexpression of dominant negative c-Jun and JNK1 is blocked by 4-HNE-mediated apoptosis, which indicate a role for JNK-c-Jun/AP-1 pathway during apoptosis (Kutuk and Basaga, 2007).

6.4.2 Modulation of Caspases by 4-HNE

Caspases are a family of at least 14 aspartate-specific cysteine proteases that are involved in the initiation and execution of apoptotic cell death and the proteolytic maturation of inflammatory cytokines such as IL-1b and IL-18 (Creagh et al., 2003; Cohen, 1997). Caspases are normally expressed as inactive proenzymes (zymogens) that become activated during apoptosis. In neural and non-neural cells, 4-HNE induces apoptosis through the release of cytochrome c from mitochondria and caspase-9 and caspase-3 activation (Raza et al., 2008). Detailed investigations on the involvement of 4-HNE in apoptotic cell death in HepG2 cells indicate that 4-HNE-mediated apoptotic signaling involves both the Fas-mediated extrinsic and the p53-mediated intrinsic pathways (Chaudhary et al., 2010). Fas-mediated death inducing signaling complex (DISC)-independent apoptosis pathway is associated with the activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and caspase-3. In contrast, p53-mediated apoptotic pathway in HepG2 cells involves the activation of Bax, p21, JNK, and caspase-3. Treatment of HepG2 cells to 4-HNE not only results in the activation of both Fas and Daxx, but also the export of Daxx from the nucleus to cytoplasm, where it binds to Fas and activates Fas-mediated apoptosis via the ASK1, which in turn activates the JNK pathway and that this pathway is independent of the Fas-FADD-caspase8 pathway operative in various cell types (Yang et al., 1997). Thus, Daxx is primarily a nuclear protein, which translocates from the nucleus to cytoplasm during stress and acts as a death receptor adaptor at the cell surface (Salomoni and Khelifi, 2006). Depletion of Daxx by siRNA blocks apoptotic cell death. 4-HNE-mediated translocation of Daxx is also accompanied by the activation and nuclear accumulation of heat shock factor 1(HSF1) and upregulation of heat shock protein Hsp70 (Sharma et al., 2008; Chaudhary et al., 2010) (Fig. 6.7). Although the molecular mechanism associated with Hsp70-mediated neuroprotection is not fully understood, it is proposed that phosphorylated Hsps interact with Daxx and prevent its interactions with both ASK1 and Fas resulting in prevention of Daxx-mediated apoptosis. These effects of 4-HNE in HepG2 cells can be attenuated by ectopic expression of hGSTA4-4, the isozyme of glutathione S-transferase with high affinity for 4-HNE. In the nucleus, HSF-1 also promotes the expression of hsp-40 6.4 Effect of 4-HNE on Enzyme Activities 167

4-HNE FasL NMDA

PtdCho Fas-R NMDA-R cPLA

+ + 2+ Depletion of GSH 2+ 2 Ca Alterations in Ca homeostasis ARA ASK1 Hsp70 Hsp90 HSF1 Mitochondria - JNK Oxidative stress 4-HNE

Hsp90 Hsp70

MARK signaling HSF1 Caspase-3 Cytochrome c Nrf2 pathway

S-S P Client S-S Nrf 2 P38, ERK, JNK Apoptosis Keap 1 BAG3 Hsp70

Client Gene regulation Daxx Client BAG3 (BclxL, Mcl Hsp70 Degradation) Nrf2 HSF1

ARE AOE h2 c-fos, cjun, AP-1 HSF1 Hsp induction NUCLEUS

Fig. 6.7 Hypothetical representation showing the involvement of 4-HNE in Fas-mediated apoptotic signaling and Nrf2-mediated antioxidant effects. N-Methyl-d-aspartate receptor (NMDA-R); phosphatidylcholine (PtdCho); cytosolic phospholipase A2 (cPLA2); Fas Receptor (Fas-R); Fas ligand (FasL); arachidonic acid (ARA); intracellular calcium (Ca2+); 4-hydroxynonenal (4-HNE); glutathione (GSH); mitogen-activated kinase (MARK); extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated Protein Kinase (p38) heat shock protein 70 (Hsp70); heat shock protein 90 (Hsp90) and heat shock transcription factor 1 (HSF1) and hsp-70, which facilitate the recovery of cells from thermally and chemically induced damage by promoting the stabilization of Bcl-XL. This inhibits Bax translocation to the mitochondrion and the consequent stress-induced apoptosis. In addition, 4-HNE induces phosphorylation of Daxx at Ser668 and Ser671 and promotes its cytoplasmic export. These observations indicate that while 4-HNE induces toxicity through multiple mechanisms, in parallel it evokes signaling for defense mechanisms to self-regulate its toxicity and can simultaneously affect multiple signaling pathways through its interactions with membrane receptors and transcription factors/ repressors (Sharma et al., 2008; Chaudhary et al., 2010).

6.4.3 Modulation of Glutathione S-Transferases by 4-HNE

Glutathione S-transferases or glutathione transferases (GSTs) are a functionally diverse family of soluble multifunctional enzymes that catalyze the conjugation of reduced glutathione to electrophilic centers on a wide variety of substrates including 168 6 Neurochemical Aspects of 4-Hydroxynonenal

HOO HO COOH COOH

GSH

CH3 GSTA1-1 CH3 A2-2 9-Hydroperoxy LNA 9-Hydroxy LNA

O O

O OCROOH O OCROH O GSH O C O C O O PO O PO ROOH GSTA1-1 ROH O A2-2 O (H C) N+ + 3 3 (H3C)3N PtdCho hydroperoxide PtdCho hydrooxide

H C HO CH3 O CH3 O GSH OH GSTA4-4 GS

4-HNE GS-4-HNE

Fig. 6.8 Detoxification of lipid peroxidation products by GST. Glutathione S-transferase (GST); glutathione (GSH); Linoleic acid (LNA); and 4-hydroxynonenal (4-HNE) xenobiotic and endogenous electrophiles including 4-HNE. Several forms of GSTs are known to occur in brain tissue. Although the GSTA1-1 isoform is regarded as a highly promiscuous enzyme, GSTA4-4 is distinguished by a striking specificity toward 4-HNE and related alkenal substrates (Hou et al., 2007). In addition to GST activity, these enzymes also have peroxidase and isomerase activities. They protect neural cells from H2O2-mediated cell death by blocking JNK activity (thus protecting cells against H2O2-induced cell death). GSTs not only reduce lipid hydroperoxides through their Se-independent glutathione peroxidase activity, but also detoxify lipid peroxidation end products, including 4-HNE (Sharma et al., 2004) (Fig. 6.8). Thus, GSTs are a major contributors to the eukaryotic cell’s defenses against oxidative stress. Oxidative stress-mediated elevation in 4-HNE levels is accompanied by transient induction of hGST5.8 and 76 kDa Ral-binding GTPase activating protein (RLIP76). A coordinated action of GSTs (GSTA4-4 and hGST5.8) promotes the conjugation of 4-HNE with GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76) facilitates ATP- dependent transport of GS-HNE. This observation strongly supports the view that both proteins play an important role in the regulation of the intracellular levels of 4-HNE (Cheng et al., 2001; Yang et al., 2003). Cells with induced hGST5.8 and 6.4 Effect of 4-HNE on Enzyme Activities 169

RLIP76 transport GS-HNE at a several fold higher rate as compared to the controls which further confirms the role of these proteins in regulations of cellular concentrations of 4-HNE. Collective evidence suggests that conjugation to 4-HNE with GSH is the primary route of HNE detoxification followed by oxidative/reductive modifications and, ultimately, mercapturic acid formation (Alary et al., 1998). Induction of GSTs involves an antioxidant-responsive response element (ARE) and the transcription factor nuclear factor E2-related factor 2 (Nrf2), which is bound to the Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm (Fig. 6.7). Antioxidants and phytochemicals (isothiocyanates, curcumin, caffeic acid phen- ethyl ester, and polyphenols) disrupt the Keap-Nrf2 complex, allowing Nrf2 to translocate to the nucleus and mediate expression of Phase II genes via interaction with ARE. These genes include genes for glutathione S-transferase (GSTA1 and GSTA4), NAD(P)H:quinone oxidoreductase 1, UDP-glucuronosyltransferase, g-glutamate cysteine ligase, and hemeoxygenase-1. Phosphorylation of Nrf2 by MARK cascade may also facilitate dissociation of Keap-Nrf2 complex, promoting translocation of Nrf2 to the nucleus GSTs. Since 4-HNE stimulates MARK cascade, it is likely 4-HNE may indirectly modulate Nrf2-mediated signaling in the nucleus (Jeong et al., 2006). Silencing Nrf2 by siRNA increases the cytotoxicity of 4-HNE, but not as effectively as silencing of HSF1 (see below). Silencing HSF1 expression promotes the activation of JNK proapoptotic signaling and selectively decreases expression of the antiapoptotic Bcl-2 family member Bcl-XL.

Overexpression of Bcl-XL attenuates 4-HNE-induced apoptosis in HSF1-silenced cells. Based on these results, it is proposed that the activation of HSF1 and stabilization of Bcl-XL mediate a protective response that may control the molecular aspects of lipid peroxidation and oxidative stress-mediated cellular injury.

6.4.4 Modulation of ATPases by 4-HNE

4-HNE also interacts and inhibits a number of membrane-bound enzymes, including Na+, K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase in neural and non-neural tissues. The Ki values for Na+, K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase are 40, 91, and 12 mM, respectively. In synaptosomes, 4-HNE inhibits Na+, K+-ATPase, but has very little effect on synaptosomal Ca2+- and Mg2+-ATPase activities. On the basis of detailed kinetic analysis of the Na+, K+-ATPase activity, it is suggested that 4-HNE- induced effect is caused by the decreased affinity for the substrate. ATP completely protects the ATPase from the 4-HNE attack. Treatment of synaptosomes with 4-HNE reduces membrane lipid fluidity near the lipid/water interface, but has no effect in the interior of lipid bilayer. In addition, 4-HNE-mediated inhibition of the ATPase activity in synaptosomes is closely associated with membrane fluidity and dynamics (Kadoya et al., 2003). In addition, inhibition of Na+, K+-ATPase by 4-HNE results in the depolarization of neuronal membranes leading to the opening of NMDA receptor channels and the influx of additional calcium ions into the cell (Kadoya et al., 2003). This calcium entry can be very harmful for neuronal integrity. 170 6 Neurochemical Aspects of 4-Hydroxynonenal

In the inner mitochondrial membrane, 4-HNE blocks ADP and ATP transport (Picklo et al., 1999). This event may play a substantial role in disruption of the energy-producing capacity of mitochondria. Beside this, 4-HNE also modulates activities of enzymes of lipid, protein, and nucleic acid metabolism, glucose and glutamate transporters (Mark et al., 1997; Blanc et al., 1998).

6.4.5 Modulation of Other Enzymes and Cell Cycle by 4-HNE

In addition to above metabolic changes, 4-HNE stimulates basal and GTP-stimulated phospholipase C and adenylate cyclase activities. It inhibits the activity of ornithine decarboxylase (Rossi et al., 1993). In non-neural cells at low levels (i.e. <1.00 mM), 4-HNE can differentially modulate cell cycle signaling (Barrera et al., 2004). 4-HNE not only orchestrates the expression of the c-myc gene involved in cell proliferation but also decreases cyclin D1, D2, and A in the G0/G1 phase of the cell cycle. In addition, 4-HNE also increases the expression of p21 (the most important CDK inhibitors), but has no effect on the expression of cyclin-dependent kinases (CDK). Cyclins D/CDK2 and cyclin A/CDK2 contribute to the phosphorylation of pRB and 4-HNE produces an increase in hypophosphorylated pRb. In the signal transduction pathway, hypophosphorylated pRb interacts and inactivates E2F transcription factors. Based on band-shift experiments, it is suggested that 4-HNE decreases “free” E2F, as well as increases pRb bound to E2F with consequent repression of the transcription (Barrera et al., 2004). Collectively, these studies suggest that 4-HNE may be an important signaling molecule for the regulation of cell proliferation and differentiation.

6.4.6 Modulation of Heat Shock Response by 4-HNE

Detailed investigations indicate that 4-HNE facilitates the activation of heat shock gene expression by disrupting the inhibitory interaction between Hsp70-1 and HSF1 (Jacobs and Marnett, 2007). The inducible expression of Hsps is mediated by heat shock transcription factor 1 (HSF1), which translocates from cytosol to the nucleus upon activation and enhances the expression of genes from promoters containing heat shock elements (HSE) (Sarge et al., 1993; Baler et al., 1993). A major function of Hsps is to chaperone other proteins by binding to nascent polypeptide chains as well as to unfolded and damaged proteins. Their function as protein chaperones aids in the recovery of cells from thermal and chemical-induced damage (Hahn and Li, 1982; Howard et al., 1993). Silencing of HSF1 expression by siRNA treatment not only results in the reduction in Hsp expression, but increases apoptotic cell death 6.5 Modulation of NMDA Receptor by 4-HNE 171

(Jacobs and Marnett, 2007). Microarray analysis of gene expression in HSF1- silenced RKO cells indicates that Hsp70 co-chaperone, BAG3 (Bcl-2-associated athanogene domain 3), is strongly induced by HNE in control but not in HSF1- silenced colon cancer cells (Jacobs and Marnett, 2009). Silencing BAG3 expression with siRNA results in a dramatic reduction in Bcl-X(L), Mcl-1, and Bcl-2 protein levels in colon cancer cells and increases apoptosis, similar to the effect of silencing HSF1. This observation is supported by immunoprecipitation studies that indicate specific interactions among athanogene 3 (BAG3), Hsp70, and the Bcl-2 family member, Bcl-XL. Collective evidence suggests that BAG3 is not only HSF1-inducible, but also plays a unique role in promoting cancer cell survival during proapoptotic stress by stabilizing the level of Bcl-2 family proteins (Jacobs and Marnett, 2009). Among HSF1-induced genes, BAG3 is notable for its actions in promoting cell survival through stabilization of antiapoptotic Bcl-2 proteins, which appear to have a critical role in mediating cellular protection against 4-HNE-induced death.

6.5 Modulation of NMDA Receptor by 4-HNE

4-HNE also exerts a biphasic effect on the NMDA current. Thus, in hippocampal neurons an early enhancement of NMDA current within the first 30–120 min is followed by a delayed decrease in current that is significantly less than the basal current at 6 h after 4-HNE exposure (Lu et al., 2001). The early enhancement of NMDA current may involve increased phosphorylation of NR1, a NMDA receptor subunit, and the delayed suppression of NMDA current by 4-HNE may be due to the depletion of ATP resulting in impairment of NMDA receptor channel function (Lu et al., 2001). In addition, 4-HNE not only enhances dihydropyridine-sensitive whole-cell Ca2+ currents, but also increases depolarization-mediated increases of intracellular Ca2+ levels in hippocampal neurons (Lu et al., 2002). Prolonged exposure of cultures cells to 4-HNE produces neuronal death, which can be blocked by glutathione treatment and attenuated by nimodipine, a Ca2+ channel blocker. 4-HNE treatment also increases tyrosine phosphorylation of a1 voltage-dependent Ca2+ channels (VDCC) subunits, and inhibitors of tyrosine kinases and phosphatases block the increase in phosphorylation of a1 VDCC subunits, indicating the importance of phosphorylation/dephosphorylation in VDCC-mediated Ca2+ influx. In cortical neurons, 4-HNE also disrupts G-protein-linked muscarinic cholinergic receptors (mAChRs) and metabotropic glutamate receptors (mGluRs). This may alter the activity of phospholipase C, indicating that 4-HNE modulates signal transduction process in brain tissue. Accumulating evidence suggests that modulation of Ca2+ channel activity by lipid peroxidation product may play important roles in altering the response of neurons to oxidative stress in both physiological and pathological settings (Lu et al., 2002). 172 6 Neurochemical Aspects of 4-Hydroxynonenal

6.6 Modulation of Gene Expression by 4-HNE

There is compelling evidence that levels of 4-HNE are markedly increased in neurotraumatic and neurodegenerative diseases (see below), and high levels of 4-HNE can contribute to neurodegeneration (Farooqui et al., 2001, 2008). In contrast, sublethal concentrations of 4-HNE stimulate stress resistance pathways, to facilitate neuroprotection. As stated above, studies on the mechanism of neuroprotection mediated by 4-HNE indicate that 4-HNE mediates the translocation of Nrf2 from cytosol to the nucleus, where this transcription factor in addition to Hsp enhances the expression of g-glutamylcysteine ligase (GCL) and the core subunit of the Xc(–) high-affinity cystine transporter (xCT), thereby increasing the intracellular GSH levels by several folds (Chen et al., 2005). In addition, 4-HNE also modulates the expression of various genes, including PKCII, c-myc, procollagen type I, c-myb, and transforming growth factor 1 (Poli and Schaur, 2000). At low concentrations, 4-HNE induces transient activation of extracellular signal-regulated protein kinase ½, Akt/protein kinase B, cyclooxygenase, and 5-lipoxygenase (Lee et al., 2010). Pharmacological inhibition of both of these kinase pathways effectively attenuates 4-HNE-induced thioredoxin reductase 1 expression and subsequent adaptive protection (Chen et al., 2005). In non-neural systems, silencing of Nrf2 by specific siRNA or the GCL inhibitor l-buthionine sulfoximine (BSO) blocks 4-HNE-mediated gene expression. Collectively, these studies indicate that at low concentrations, 4-HNE activates Nrf2-mediated gene expression and stimulates GSH biosynthesis, thereby conferring neuroprotection against ischemia-reperfusion injury (Chen et al., 2005; Muralikrishna and Hatcher, 2006). In kainic acid (KA) neurotoxicity, marked increase in 4-HNE levels is accompanied by dense labeling to 4-HNE in the nucleus and cytoplasm of injured neurons (Fig. 6.9). Accumulation of 4-HNE is consistent with activation of cytosolic phospholipase A2 and increased release and oxidation of

Fig. 6.9 4-Hydroxynonenal (4-HNE) immunostained sections of field CA3 of the hippocampus, from an untreated rat (a) and a rat which received intracerebroventricular injection of the potent glutamate analog and excitotoxin kainate one day earlier (b). Dense labeling to 4-HNE is observed in the nucleus and cytoplasm of injured neurons (arrows in b), consistent with increased release and oxidation of arachidonic acid after kainate excitotoxicity. Scale = 50 mm (Reproduced with kind permission from Elsevier, Farooqui et al., 2010) 6.8 Modulation of Nucleic Acid Metabolism by 4-HNE 173

ARA after KA-mediated neurodegeneration (Farooqui et al., 2001, 2008). At high concentration, 4-HNE upregulates the synthesis of growth factors, chemokines, and cytokines. By inhibiting adenine nucleotide translocator, 4-HNE suppresses ADP and ATP transport through the inner mitochondrial membrane (Picklo et al., 1999). These events play a substantial role in the disruption of the energy-producing capacity of mitochondria and gene expression in the nucleus. 4-HNE interacts with Fas and promotes proapoptotic signaling via ASK1, JNK, and caspase 3. In parallel, 4-HNE mediates Daxx translocation and promotes its export from the nucleus to the cytosol, where it interacts with Fas to self-limit the extent of apoptosis by inhibiting the downstream proapoptotic signaling (Sharma et al., 2008). Cytoplasmic translocation of Daxx also results in the upregulation of HSF1-associated stress-responsive genes. Collective evidence suggests that 4-HNE modulates gene expression.

6.7 Modulation of Peroxisomes by 4-HNE

Proteasomes are multiprotein complexes that consist of a catalytic core, the 20S proteasome, with additional cap-like structures capable of binding to the 20S to form a 26S proteasome complex. They are responsible for degradation of oxidized and ubiquitinated proteins in both the nucleus and cytoplasm (Coux et al., 1996). 4-HNE modified proteins are poorly degraded by proteasomes (Grune and Davies, 2003). The extensive modification of cellular proteins by 4-HNE also results in the formation of protein aggregates that accumulate in neural cells and are not degraded by the proteasome (Sitte et al., 2000; Grune and Davies, 2003). The decreased degradation of modified protein and their accumulation produces a direct inhibition of proteasome by oxidized and cross-linked proteins (Sitte et al., 2000; Grune and Davies, 2003), and by 4-HNE-modified proteins (Friguet,2 006). At high concentration, 4-HNE directly forms adducts with trypsin, chymotrypsine, and peptidylglu- tamyl peptide hydrolase (Okada et al., 1999; Ferrington and Kapphahn, 2004), which readily inhibits the enzymic activity of proteasome and contributes to the accumulation of modified proteins (Grune and Davies, 2003; Vieira et al., 2000). Accumulation of 4-HNE-modified proteins also interferes with mitochondrial function leading to the release of cytochrome c and caspase -3-mediated apoptotic cell death. The accumulation of ubiquitinated modified proteins resulting from proteasome inhibition in neuronal cells triggers a proinflammatory response characterized by an upregulation of COX-2 and the production of prostaglandin PGE2, which contributes to neurodegeneration (Rockwell et al., 2000).

6.8 Modulation of Nucleic Acid Metabolism by 4-HNE

4-HNE inhibits DNA and RNA synthesis. At low concentrations 4-HNE produces adaptive changes, such as heat shock response and antioxidant response, which protect cell against further damage, but at higher concentrations 4-HNE produces 174 6 Neurochemical Aspects of 4-Hydroxynonenal

N NH dG N NH N 2 OH dR O

H3C 4-HNE

O OH O OH

N N N N

OH OH N N N N N N H H dR dR

4-HNE-dG1 4-HNE-dG2 (6R, 8S, 11S) (6S, 8R, 11R )

CH3 CH3 OH OH O O

N N N N

OH OH N N N N N N H H dR dR

4-HNE-dG3 4-HNE-dG4 (6R, 8S, 11R) (6S, 8R, 11S)

CH3 CH3

Fig. 6.10 4-HNE and guanosine adduct formation. Chemical structures of four diastereoisomers of 4-HNE-deoxyguanosine adducts several covalent adducts (Fig. 6.10), which overwhelm the protective mechanisms and the cells undergo apoptotic cell death. Finally, at extreme concentrations of 4-HNE, cells undergo a necrotic cell death. Two pathways are involved in adduct formation. One pathway is the formation of adduct (4-HNE-dGp-adducts) by direct interaction with the guanosine moiety of DNA (Wacker et al., 2000) and the other 6.10 Modulation of Blood–Brain Barrier by 4-HNE 175 involves the oxidation of 4-HNE to its epoxide (Chen et al., 1998). 4-HNE-dGp-adduct is a bulky product, whereas epoxy products are smaller and are generated after further oxidative metabolism. They react with DNA by forming etheno adducts of adenosine, guanosine, and cytidine (Chung et al., 1996). Furthermore, the reaction of 4-HNE with deoxyguanosine (dG) generates three new chiral centers in the nucleoside (Choudhury et al., 2004), resulting in the formation of four stereoisomers (6R, 8S, 11R), (6S, 8R, 11S), (6R, 8S, 11S), and (6S, 8R, 11R) with 8-hydroxyl and 6-(1-hydroxyhexyl) in the trans configuration (Choudhury et al., 2004). Some of these adducts are mutagenic. These adducts exist in equilibrium with diastereo- meric cyclic hemiacetals, in which the latter predominate at equilibrium. Incorporation of the exocyclic 1,N2-dG structure into duplex DNA precludes Watson-Crick hydrogen bonding and induces structural and thermodynamic perturbations at the lesion and complementary nucleotides (Weisenseel et al., 2002; Plum et al., 1992; Wang et al., 2003).

6.9 Modulation of Phospholipid Metabolism by 4-HNE

Studies on the interactions of 4-HNE with phosphatidylethanolamine (PtdEtn) in an aqueous-organic biphasic system indicate that 4-HNE forms Michael adducts plus a minor Schiff base adducts with amino group of PtdEtn. Other phospholipids such as phosphatidylserine (PtdSer) are not modified by 4-HNE. The Michael adduct of 4-HNE and PtdEtn reaction (PtdEtn/4-HNE) is a poor substrate of secretory phospholipase A2 and is not cleaved by phospholipase D. Ethanolamine plasmalogen (PlsEtn) is also covalently modified by 4-HNE, but is further degraded on its sn-1 position, the alkenyl chain, which may alter the antioxidant potential of ethanolamine plasmalogen (Guichardant et al., 2002). Other aldehydes, which are homologous to 4-HNE e.g. 4-hydroxyhexenal (4-HHE) and 4-hydroxy-2E,6Z- dodecadienal (4-HDDE) also form adducts with amino group containing phospholipids (Bacot et al., 2007) . These three hydroxy-alkenals are highly reactive because of a double bond conjugated with the carbonyl group. The reactivity of 4-HHE, 4-HNE, and 4-HDDE toward PtdEtn depends on their hydrophobicity. Indeed, 4-HDDE is more active in making covalent adducts than 4-HNE, which is also more reactive than 4-HHE (Bacot et al., 2007). Michael adducts of 4-HNE and PtdEtn may alter membrane fluidity and the activity of anchored proteins in the adduct-containing membranes as well as the accessibility of ligands and other functional components, which may result in neural membrane dysfunction.

6.10 Modulation of Blood–Brain Barrier by 4-HNE

The mammalian brain is protected by the blood–brain barrier (BBB), a dynamic and complex interface between blood and the brain that strictly controls the exchanges between the blood and brain compartments. Therefore it plays a key role in brain homeostasis and provides protection against many toxic compounds and pathogens. 176 6 Neurochemical Aspects of 4-Hydroxynonenal

The integrity and stability of BBB is controlled by continuous and tightly linked junctions of endothelial cells with astrocytes, and microglia. This arrangement insu- lates the brain tissue from the blood stream. BBB facilitates supply and disposal of nutrients and metabolites by the expression of transporters and transcytotic receptors at the polarized endothelial cell (EC) surface (Pflanzner et al., 2010). 4-HNE also increases the permeability of the blood–brain barrier (Mertsch et al., 2001) during excitotoxicity. The physiological significance of this observation remains unknown, but elevation in levels of 4-HNE may contribute to increased permeability of BBB that occurs in neurotraumatic and neurodegenerative diseases (Farooqui, 2010).

6.11 4-HNE in Neurological Disorders

Neurodegenerative diseases are characterized by the slow death of neurons in a specific area of brain. Oxidative stress and inflammatory reactions are closely associated with the pathogenesis of neurodegenerative diseases, which include Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), and others. In neurodegenerative diseases, oxidative stress and inflammation overwhelm antioxidant and anti-inflammatory defenses and initiate the process of neurodegeneration (Farooqui, 2010). Among neural cells, neurons are most susceptible to oxidative stress. Oxidative stress may indirectly contribute to brain damage by activating a number of cellular pathways resulting in the expression of stress-sensitive genes and proteins to cause oxidative injury. Moreover, oxidative stress also activates mechanisms that result in a glia cell-mediated inflammation that also causes secondary neuronal damage. Associated neuronal injuries caused by many neurodegenerative diseases are due to activation of glial cells (particularly astrocytes and microglia) at the sites of neurodegeneration. Activated glial cells are thus histopathological hallmarks of neurodegenerative diseases. Even though direct contact of activated glia with neurons per se may not necessarily be toxic, lipid and immune mediators (e.g. nitric oxide, 4-HNE, isoprostane, platelet activating factor, proinflammatory cytokines, and chemokines) released by activated glial cells are major candidate neurotoxins (Farooqui, 2009, 2010). In neurodegenerative diseases, 4-HNE not only reacts with nucleophiles to form Michael adducts, but also forms Schiff bases, resulting in the modification of many enzymes and proteins (particularly cytoskeletal proteins). Protein modification by 4-HNE may be involved in neurodegenerative processes associated with cell death in neurodegenerative diseases (Table 6.1).

6.11.1 4-HNE in Alzheimer Diseases

AD is characterized by accumulation and deposition of aggregated Ab peptide and neurofibrillary tangles composed of hyperphosphorylatedt protein (Farooqui, 2010). 6.11 4-HNE in Neurological Disorders 177

Table 6.1 Levels of 4-HNE in neurological disorders Neurological disorder 4-HNE level Reference Alzheimer disease Increased Bradley et al., 2010; Williams et al., 2006 Parkinson disease Increased Qin et al., 2007; Selley et al., 1998 Amyotrophic lateral sclerosis Increased Zarkovic, 2003; Periluigi et al., 2005 Multiple system atrophy Increased Shibata et al., 2010c Ischemia Increased McKracken et al., 2001 Spinal cord injury Increased Springer et al., 1997 Head injury Increased Zhang et al., 1999

In AD, b-Amyloid may initiate the formation of 4-HNE by stimulating PLA2 activity (Kanfer et al., 1998). 4-HNE colocalizes with intraneuronal neurofibrillary tangles and may contribute to the cytoskeletal derangement found in AD. Detailed investigations on mild cognitive impairment subjects (MCI) and AD patients using proteomics indicate that there is a marked increase in levels of protein-bound 4-HNE to many enzymes (ATP synthase, a-enolase, aconitase, aldolase, glutamine synthetase, Mn-superoxide dismutase) and proteins (peroxiredoxin 6, dihydropyriminidase- related protein-2, and a-tubulin) (Perluigi et al., 2009). These enzymes and proteins are associated with glucose metabolism, maintenance of glutamate levels, antioxidant defense systems, axonal growth, and maintenance of cytoskeleton (Butterfield et al., 2010). In addition, neprilysin (NEP), a major protease which cleaves Ab in vivo, forms adducts with 4-HNE leading to decrease in its activity in the brain of AD patients and cultured cells (Wang et al., 2010). Treatment with N-acetylcysteine results in sparing of NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in AD (Wang et al., 2010). 4-HNE also binds to histones and this binding alters the ability of the histone to bind DNA. It is proposed that alterations in DNA-histone interactions may contribute to the vulnerability of neurons in AD brain (Drake et al., 2004).

6.11.2 4-HNE in Parkinson Disease

PD is a progressive and degenerative disorder caused by the gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta (Beal, 1998; Jenner and Olanow, 2006). Loss of these neurons causes pathological changes in neurotransmission in the basal ganglia motor circuit leading to movement disorders such as tremor, rigidity, and akinesia. There are several parameters, which may contribute to oxidative stress and vulnerability of dopaminergic neurons in the substantia nigra pars compacta. One pathway involves monoamine oxidase-mediated abnormal dopamine metabolism, generation of hydrogen peroxide, formation of superoxide radicals, and auto-oxidation of dopamine producing dopamine-quinone, 178 6 Neurochemical Aspects of 4-Hydroxynonenal a molecule that damages proteins by reacting with cysteine residues (Graham, 1978). Another pathway may involve the release of ARA and generation of 4-HNE in brain and plasma of PD patients (Beal, 1998; Jenner and Olanow, 2006; Selley, 1998; Seet et al., 2009). 4-HNE interacts with a-synuclein, a protein that accumulates and is associated with the pathogenesis of PD (Qin et al., 2007). 4-HNE-mediated modification of a-synuclein not only produces a major conformational change in beta- sheet structure of this protein, but also inhibits the fibrillation in 4-HNE concentration-dependent manner. Addition of HNE-modified oligomers to primary mesencephalic cultures produces marked neurotoxicity. Collective evidence suggests that 4-HNE-mediated modification of alpha-synuclein prevents fibrillation but may generate toxic oligomers, which may contribute to the demise of neurons through oxidative stress (Qin et al., 2007).

6.11.3 4-HNE in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a major motor neuronal disorder that causes progressive loss of motor neurons leading to muscle loss, paralysis, and death from respiratory failure. An important pathological hallmark of ALS is the presence of axonal spheroids and perikaryal accumulations/aggregations comprised of the neuronal intermediate filament proteins, neurofilaments, and peripherin (Beaulieu and Julien, 2003). Although the exact cause of neurodegeneration in ALS is not known, multiple pathophysiological mechanisms have been proposed. These mechanisms include oxidative stress, mitochondrial impairment, protein aggregation, axonal dysfunction, mutant superoxide dismutase expression, cytoskeletal disorganization, glutamate cytotoxicity, inflammation, and apoptotic cell death (Almer et al., 2001; Liu et al., 2002; Farooqui and Horrocks, 2007;

Farooqui, 2010). Stimulation of cPLA2 has been recently reported in motor neurons, reactive astrocytes, and activated microglia of patients with sporadic ALS (Shibata et al., 2010b). The released ARA is metabolized to 4-hydroxynonenal (4-HNE), which reacts with nucleophilic sites of proteins on lysine, cysteine, and histidine residues. In G93A SOD-1 Tg mice, levels of 4-HNE are increased around zinc-accumulating cells and mSOD-1 positive cells, suggesting a link between 4-HNE, SOD-1 mutation, and zinc accumulation. The exposure of G93A SOD-1 Tg mice cultured spinal neurons and astrocytes to 4-HNE increases labile zinc levels and decreases glutamate transporter (Kim et al., 2009; Yao, 2009). Administration of the zinc chelator TPEN increases the survival of G93A SOD-1 Tg mice, suggesting that zinc dyshomeostasis may be involved in the spinal cords of Tg mice, and that this dyshomeostasis may contribute to motoneuron degeneration (Kim et al., 2009; Yao, 2009). Levels of 4-HNE are also elevated in spinal cord motor neurons in the cerebrospinal fluid of patients with ALS (Vigh et al., 2005; Perluigi et al., 2005). 6.11 4-HNE in Neurological Disorders 179

6.11.4 4-HNE in Prion Diseases

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of abnormal isoforms of a host protein known as cellular prion protein (PrPC), motor dysfunctions, dementia, and neuropathological changes such as spongiosis, astroglyosis, and neuronal loss. A significant increase in 4-HNE levels along with other lipid peroxidation product, malondialdehyde has been reported in early preclinical stages of scrapie-infected mice (Yun et al., 2006). Similar increase in 4-HNE and 4-HNE adducts also occurs in Creutzfeldt–Jakob disease (CJD) patients compared to normal control subjects (Andreoletti et al., 2002). Detailed investigation in scrapie mice indicates that massive 4-HNE accumulation occurs in astrocytes, but not in neurons or microglial cells. These findings suggest an important oxidative stress in astrocytes, with possible consequences on their neuronal trophic function (Andreoletti et al., 2002).

6.11.5 4-HNE in Ischemia

Ischemia is a metabolic trauma caused by severe reduction or blockade in cerebral blood flow due to cerebrovascular disease. This blockade not only decreases oxygen and glucose delivery to brain tissue but also leads to the breakdown of blood–brain barrier (BBB) and build-up of potentially toxic products in brain. Earliest event in ischemia is the release of excessive amount of glutamate in the extracellular space and overstimulation of glutamate receptors, which dramatically increases the intracellular calcium resulting in activation of multiple intracellular death pathways (Farooqui and Horrocks, 1994, 2007) (Fig. 6.11). Thus, ischemic injury triggers a complex series of molecular mechanisms, which not only impair the neurologic functions through the breakdown of cellular and subcellular integrity and initiation of Ca2+-influx, but also alterations in ionic balance and redox status, and free-radical generation. These processes are supported through the activation of signaling mechanisms involving phospholipases A2, C, and D (PLA2, PLC and PLD), calcium-dependent kinases, nitric oxide synthases (NOS), calpains, calcinurin, and endonucleases (Farooqui and Horrocks, 2007; Farooqui, 2010). ARA is oxidized to 4-HNE. As stated above, this metabolite at high concentration impairs the activities of Na+, K+-ATPase, glucose 6-phosphate dehydrogenase, and several kinases, including c-jun amino-terminal kinase (JNK) and p38 mitogen- activated protein kinase (Mark et al., 1997; Camandola et al., 2000). The impairment of Na+, K+-ATPase depolarizes neuronal membranes leading to the opening of more NMDA receptor channels and influx of additional Ca2+ into neurons. At low concentration in PC12 cells, 4-HNE exerts adaptive cytoprotective effect primarily through the induction of thioredoxin reductase 1 via transcriptional activation of NF-E2-related factor 2 (Nrf2) (Fig. 6.7), which modulates the expression of important cytoprotective enzymes. 180 6 Neurochemical Aspects of 4-Hydroxynonenal

Ischemia (ATP↓) Ca2+ Glu

Activated NADPH oxidase PtdCho NMDA-R 2 + Xanthine oxidase ↑ 2+ 2+ Ca Alterations in Ca cPLA homeostasis Catecholamine ARA Reperfusion oxidation COX and LOX Mitochondrial dysfunction

Depletion of GSH ROS

Cytochrome c Lipid peroxidation

4-HNE JNK Caspase-3 Oxidative stress

Protein and DNA modifications

Apoptosis

Fig. 6.11 Ischemic injury-mediated generation of 4-HNE and its involvement in apoptotic cell death. N-Methyl-d-aspartate receptor (NMDA-R); phosphatidylcholine (PtdCho); cytosolic phospholipase A2 (cPLA2); cyclooxygenase (COX); lipoxygenase (LOX); reactive oxygen species (ROS); arachidonic acid (ARA); glutathione (GSH); 4-hydroxynonenal (4-HNE); glutathione (GSH); and c-Jun N-terminal kinase (JNK)

6.11.6 4-HNE in Spinal Cord Injury

Spinal cord injury (SCI) is a catastrophic event that leads to the loss of motor and sensory functions of the body innervated by the spinal cord below the injury site. Spinal cord injury induces autodestructive alterations that lead to varying degrees of tissue necrosis and paralysis, depending on the severity of the injury, which consists of two broadly defined events: a primary event, attributable to the mechanical insult itself, and a secondary event, attributable to the series of systemic and local neurochemical and pathophysiological changes that occur in spinal cord after the initial traumatic insult (Klussmann and Martin-Villalba, 2005). In contrast, secondary event that occurs in rostral/caudal spinal levels involves ischemic injury, edema, excitotoxicity, and oxidative stress. In addition, 4-HNE directly and differentially impairs spinal cord mitochondrial function, through the modulation of pyruvate dehydrogenase and complex I-associated proteins (Vaishnav et al., 2010). These processes not only affect neuronal activities, glial cell reaction involving astrocytic activation, and demyelination involving oligodendrocytes, 6.11 4-HNE in Neurological Disorders 181 but also modulate leukocyte infiltration, and activation of macrophages and vascular endothelial cells (Bramlett and Dietrich, 2004). Among non-neural cells following spinal cord injury, macrophages are present at the injury site in large numbers and for the longer duration. Levels of 4-HNE are markedly increased in traumatized spinal cord compared to uninjured control spinal cord (Springer et al., 1997; Gard et al., 2001). 4-HNE not only provokes release of substance P and calcitonin gene-related peptide from central spinal cord and peripheral esophagal nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues, but also induces pain and pain-related behavior (Trevisani et al., 2007). 4-HNE interacts and activates TRPA1 channel though cysteine and lysine residues within its N-terminal domain. These residues are required for activation by 4-HNE. 4-HNE-mediated pain is inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels (Trevisani et al., 2007). These findings suggest that 4-HNE activates and promotes acute pain through the release of neuropeptide, which may also contribute to neurogenic inflammation (Trevisani et al., 2007).

6.11.7 4-HNE in Traumatic Brain Injury

Like spinal cord injury (SCI), traumatic brain injury (TBI) consists of two broadly defined components: a primary component, attributable to the mechanical insult itself, and a secondary component, attributable to the series of systemic and local neurochemical and pathophysiological changes that occur in the brain after the initial insult (Raghupathi, 2004). The primary injury rapidly causes rapid deformation of brain tissue and rupture of neural cell membranes leading to the release of intracellular contents, disruption of blood flow, breakdown of the blood–brain barrier, and intracranial hemorrhage. In contrast, secondary injury to the brain results in neurochemical alterations, activation of microglial cells and astrocytes, and demyelination involving oligodendroglia (Raghupathi, 2004). TBI releases glutamate from intracellular stores (Panter et al., 1990; Sundström and Mo, 2002). Glutamate causes neural cell death through the hyperstimulation of glutamate receptors resulting in the influx of Na+, efflux of +K , and a large Ca2+ influx into neurons (Farooqui et al., 2008). This process is called as excitotoxicity. It results not only in uncoupling of mitochondrial electron transport, but also in stimulation of many calcium-dependent enzymes, including lipases, phospholipases, calpains, nitric oxide synthase, protein phosphatases, and various protein kinases (Pavel et al., 2001; Ray et al., 2003; Arundine and Tymianski, 2004). Like spinal cord injury, levels of 4-HNE are increased in TBI (Zhang et al., 1999; Ansari et al., 2008). In TBI, 4-HNE may not only directly and differentially impair brain mitochondrial function, through the modulation of pyruvate dehydrogenase and complex I-associated proteins (Vaishnav et al., 2010), but also provoke release of substance P and calcitonin gene-related peptide from injured brain resulting in pain and pain-related behavior (Trevisani et al., 2007). 182 6 Neurochemical Aspects of 4-Hydroxynonenal

Fig. 6.12 Increase in cPLA2 (a–c) and 4-HNE (d–f) immunoreactivities in slices from CA1 subfield (hippocampus) following KA-induced neurotoxicity. Slices of CA1 subfield of the rat hippocampai are immunostained with monoclonal antibody of cPLA2 (a–c) and 4-HNE monoclonal antibody (d–f). (a) and (d) are untreated control slices. (b) and (e) are slices have been stained with 6.12 4-Hydroxyhexenal (4-HHE) 183

6.11.8 4-HNE in Kainic Acid Neurotoxicity

Kainic acid (KA) is a cyclic and nondegradable analog of glutamate. It is 30- to 100- fold more potent than glutamate as a neuronal excitant. Intracerebroventricular injections of KA produce dense staining of cPLA2 and 4-HNE at 1 day post injection, before there is any histological evidence of neurodegeneration (Ong et al., 2000). Degenerating neurons in CA1 and CA3 regions of hippocampus of KA-injected rats are observed at 3 days and 1 week after injection. The increased immunoreactivity remains confined to a cluster of neurons at the edge of the degenerating CA1 and CA3 regions at 2 and 3 weeks after KA injections (Ong et al., 2000). No 4-HNE immunoreactivity is observed in reactive astrocytes. Treatment of rat hippocampal slices (CA1 subfield) with 1 mM KA also results in marked increase in cPLA2 and 4-HNE immunoreactivities, which can be blocked by quinacrine, a nonspecific cPLA2 inhibitor (Fig. 6.12). As stated earlier, in degenerating neurons, 4-HNE impairs the activities of Na+, K+-ATPase, glucose 6-phosphate dehydrogenase, and kinases (Camandola et al., 2000; Tamagno et al., 2003). 4-HNE also disrupts transmembrane signaling and the glucose and glutamate transporters in astrocytes (Mark et al., 1997). Collective evidence suggests that KA-mediated increase in 4-HNE may produce neuronal cell death by interacting with many enzyme systems in brain tissue.

6.12 4-Hydroxyhexenal (4-HHE)

Like ARA, DHA is oxidized to 4-hydroxyhexenal (4-HHE). 4-HHE has a conjugated double bond between the a and b carbon, so the g carbon of 4-HHE is electron deficient and reacts readily with nucleophiles such as thiols and amines, whereas the carbonyl group forms a Schiff base with amino groups such as N-termini of proteins and the e-amino group of lysine (Farooqui, 2009). In spite of the structural similarity between 4-HNE and 4-HHE, neurochemical effects and efficacies of these aldehydes vary greatly. For example, 4-HHE inhibits mitochondrial ATP translocator and acts more effectively on the mitochondrial permeability transition than 4-HNE (Kristal et al., 1996; Picklo et al., 1999). In endothelial cells, 4-HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in antiapoptotic Bcl-2. It is shown that 4-HHE promotes the formation of ROS, nitric oxide,

Fig. 6.12 (continued) 1 mM KA, followed by fixation and immunocytochemical staining 7 days after treatment. (c) and (f) are slices that have been treated with 1 mM KA, followed by addition of quinacrine 3 h later and fixation and immune cytochemical staining 7 days later. Arrows indicate hippocampal pyramidal neurons. (a–c) Very little or no staining for cPLA2 is observed in pyramidal neurons in control hippocampal slices (a), while an increase in staining is observed in neurons after KA treatment (b). The increase in cPLA2 immunoreactivity is prevented by treating the slices with quinacrine after the KA application (c). (d–f) Very little or no staining for 4-HNE is observed in pyramidal neurons in the normal hippocampus (d), while an increase in staining is observed in neurons after KA treatment (e). The increase in 4-HNE immunoreactivity is prevented by treating the slices with quinacrine after KA application (f). Scale bar + =160 mm (Reproduced with kind permission from Elsevier, Farooqui et al., 2001) 184 6 Neurochemical Aspects of 4-Hydroxynonenal and ONOO–, leading to imbalance of redox status. The antioxidant N-acetyl cysteine and ONOO– scavenger, penicillamine prevents HHE-induced apoptotic cell death. In addition, 4-HHE modulates endothelial nitric oxide synthase (iNOS) through NF-kB activation (Lee et al., 2004). In contrast, 4-HNE is known to inhibit NF-kB activation, suggesting that peroxidation of ARA and DHA generates end products that have different effects on transcription factor activities of neural and non-neural tissues (Camandola et al., 2000; Lee et al., 2004).

6.13 Conclusion

4-HNE is a by-product of lipid peroxidation. It modulates many neurochemical processes. At low concentrations, 4-HNE significantly increases the expression of antioxidant enzymes, such as glutamate cysteine ligase catalytic subunit, aldo–keto reductase 1C family member 1, and glutathione S-transferase-a4. In addition, 4-HNE not only induces the expression of the transcription factor Nrf2, which coordinates the upregulation of detoxifying enzymes, but also promotes the transcription of c-fos, c-jun, and NFkB, which regulate physiological protective responses against oxidative stress. At high concentration, 4-HNE facilitates oxidative stress and apoptotic cell death through the depletion of glutathione, inactivation of thiol-containing enzymes, and inhibition of calcium sequestration. It inhibits Na+,K+-ATPase, impairs glucose transport in cultured hippocampal neurons, and impairs glutamate transport in cortical astrocytes. 4-HNE disrupts coupling of muscarinic receptors and metabotropic glutamate receptor to PLC in cerebrocortical neurons. The intracellular concentration of 4-HNE not only modulates cell cycle signaling, but also mediates the signaling associated with differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8), which conjugate 4-HNE to GSH to form the conjugate, GS-HNE. Collective evidence suggests that 4-HNE dramatically effects the cell viability due to covalent adducts formation, particularly Michael adducts. Levels of 4-HNE are markedly increased in neurodegenerative diseases (AD, PD, ALS, and prion diseases) and neurotraumatic diseases (ischemia, spinal cord injury, and traumatic head injury). It is not known whether 4-HNE-mediated protein modification is the primary event or a secondary process associated with neuronal injury. Based on the above discussion, it is suggested that determination of 4-HNE can be used as an index of oxidative stress in neurodegenerative and traumatic diseases.

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7.1 Introduction

Isoprostanes (IsoPs) are prostaglandin-like mediators generated nonenzymically by free radical-catalyzed peroxidation of esterified g-linoleic acid (LA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), adrenic acid (AA), and docosahexaenoic acid (DHA) in vivo (Fig. 7.1). The minimum requirement for the synthesis of isoprostanes is a polyunsaturated fatty acid with three contiguous, methylene- interrupted double bonds (Basu, 2004; Nishio et al., 2006). Thus, in neural cells, two classes of F1-isoPs are derived from LA; four classes of F2-isoPs are obtained from ARA, six classes of F3-isoPs are synthesized from EPA, F2-dihomo-isoPs are generated from AA, and eight classes of D4-isoPs and eight classes of E4-isoPs arise from DHA (Janssen, 2001; Musiek et al., 2005; Morrows, 2000; Fam and Morrow, 2003; VanRollins et al., 2008). Each of these classes comprises up to eight racemic isomers, leading to a large number of IsoPs molecular species. It is suggested that IsoPs are reliable biomarkers for oxidative stress. This is because IsoPs can not only be measured accurately at picomolar concentrations with several procedures, including HPLC, gas chromatography–mass spectrometry and radioimmunoassay (Helmersson and Basu, 1999; Wang et al., 1995; Schweer et al., 1997) in samples of body fluids, such as cerebrospinal, pericardial, and bronchoalveolar fluids, plasma, and urine, but also their levels do not exhibit diurnal variations (Awad et al., 1993; Greco et al., 1999; Goil et al., 1998). In addition to reliable biomarkers for oxidative stress, IsoPs have emerged as important lipid mediators that modulate not only excitatory neurotransmission, but several other biochemical processes, including vasoconstriction in brain microvasculature, epithelial cell permeability, and upregulation of cytokine expression at nanomolar concentrations (Roberts et al., 2005;

Scholz et al., 2003) (Fig. 7.2). It is reported that ARA-derived, F2-IsoPs produce their effect on the vascular bed by inducing the generation of thromboxane in the endothelium, which not only facilitate the contraction in the vascular smooth muscle, but also promotes endothelial cell death (Lahaie et al., 1998).

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 193 DOI 10.1007/978-1-4419-9940-5_7, © Springer Science+Business Media, LLC 2011 194 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

O C OH Linoleic acid

O C OH Arachidonic acid

HO HO COOH COOH 12 15 HO HO OH OH

12-F2t - isoprostane 15-F2c - isoprostane HOOC OH OH O HO COOH 5 8

HO O 5-E2t - isoprostane 8-D2t - isoprostane

Fig. 7.1 Chemical structures of polyunsaturated fatty acids and isoprotanes

Isoprostanes

Modulation of Enhanced excitatory Epithelial permeability neurotrans mission

Modulation of endothelial IncreaseIncrease inin vaso- - cell viability constriction Up-rUp- egulation of cytokine expression

Fig. 7.2 Proposed roles of isoprostanes

7.2 Generation of IsoPs in the Brain

Unlike prostaglandins, the synthesis of isoprostanes in situ is initiated at the esterified ARA on the glycerophospholipid molecule (Fam and Morrow, 2003) and is independent of cyclooxygenase activity (Roberts and Milne, 2009). IsoPs are 7.3 Degradation of IsoPs in the Brain 195 produced in vivo primarily by a free radical-catalyzed peroxidation of polyunsaturated fatty acids. There are several important structural differences between prostaglandins (PGs) and IsoPs. In IsoPs, side chains are predominantly oriented cis to the cyclopentane (prostane) ring while in PGs side chains they are exclusively in trans orientation (O’Connor et al., 1984). A second important difference between IsoPs and PGs is that IsoPs are formed primarily in situ esterified to phospholipids and are subsequently released by a phospholipase A2 (Morrow et al., 1992; Stafforini et al., 2006), while PGs are generated only from free ARA, which is oxidized by cyclooxygenase reaction (Fam and Morrow, 2003; Montuschi et al., 2007). Thus, the molecular mechanism by which isoPs are generated is analogous to the synthesis of prostaglandins by cyclooxygenases (Morrow et al., 1999). The biosynthesis of IsoPs involves several steps: (a) an abstraction of a bis-allylic labile hydrogen atom from ARA, (b) addition of an oxygen molecule to ARA forming four positional peroxyl radical, (c) endocyclization, (d) addition of another oxygen and formation of four unstable PGG2-like bicyclic endoperoxide intermediates, and (e) reduction of bicyclic endoperoxide intermediates by glutathione (Fig. 7.3). Depending on the mechanism of formation, four F-ring isoP regioisomers are generated (“F” refers to a diol substitution on the cyclopentane ring) from ARA. These are called as 5-, 12-, 8-, and 15-series regioisomers on the basis of the carbon atom to which the side chain hydroxyl group is attached (Morrow, 2000; Roberts et al., 2005). IsoP regioisomers may also rearrange to form D-, E-, and J-rings. Levels of D2/E2/J2-IsoP esterified in rat tissues are approximately one-third to one-fourth the levels of F2-IsoPs (Fam and Morrow, 2003) (Fig. 7.4). Another mechanism of isoprostane generation starts with a 4-exocyclization of a peroxyl radical leading to an intermediate dioxetane (Durand et al., 2005). Measurement of F2-isoP is considered to be one of the most reliable approaches for assessing oxidative stress status in vivo and is the most reliable index of in vivo lipid peroxidation.

7.3 Degradation of IsoPs in the Brain

Studies on intravenous administration of tritium-labeled 8-iso-PGF2a in rabbits indicate that radioactivity disappears rapidly from circulation. About 80% of the total radioactivity is excreted in urine within 4 h. The plasma half-life of 8-iso-PGF2a is 1 min at the distribution phase. The terminal elimination phase half-life is about 4 min. At 1.5 min after administration 64%, 19%, and 13% of the plasma radioactivity represents 8-iso-PGF2a, 15-keto-8-iso-PGF2a and b-oxidized products, respectively (Basu, 1998). Based on detailed pharmacokinetics in rabbits, it is suggested that 8-iso-PGF2a is metabolized into several degraded polar products through dehy- drogenation at C-15, reduction of d13-double bond, and b-oxidation, which are excreted efficiently into the urine. a-Tetra-15-keto-13, 14-dihydro-8-iso-PGF2a has been identified as a major urinary metabolite in the rabbits. Studies on the degradation of 8-Iso-PGF2a in rabbits indicate that 15-prostaglandin dehydrogenase (15-PGDH) 196 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

COOH

CH3

RH R

COOH

CH3

COOH O

CH3 O

COOH O

O CH3

O2 O2 Exclusion OO COOH COOH O O

O CH3 O CH3

Other products including isofurans

O COOH Isoprostanes CH3 O

Fig. 7.3 Biosynthesis of isoprostanes from arachidonic acid. Biosynthesis of isoprostanes is summarized from Fam and Morrow (2003), Basu (2004), Montuchi et al. ((2007)), and Roberts and Milne (2009) 7.3 Degradation of IsoPs in the Brain 197

Glycerophospholipids Arachidonic acid

G2 IsoP

H2 IsoP

E2-IsoP D2-IsoP IsoP -TXA2 D2-IsoK E2-IsoK

A2-IsoP J2-IsoP IsoP -TXB2

Fig. 7.4 Generation of IsoPs, isothromboxanes, and isoketals from arachidonic acid

HO HO 15-PGDH COOH COOH

HO HO OH O 15-keto-8-iso-PGF 8-iso-PGF2a 2a

∆13-Reductase

HO HO COOH β-Oxidation COOH

HO HO O -tetranor-15-keto-13, 14-dihydydro-8-PGF a 2a 15-keto-13, 14-dihydro-8-iso-PGF2a

Fig. 7.5 Degradation of isoprostanes. 15-prostaglandin dehydrogenase (15-PGDH). Summarized from Basu (1998) and Basu (2004)

converts this metabolite into 15-keto-8-iso-PGF2a, which is reduced to 15-keto-13, 13 14-dihydro-8-iso-PGF2a. This reaction is catalyzed by D -reductase. b-Oxidation of

15-keto-13, 14-dihydro-8-iso-PGF2a results in the formation of a-tetranor-15-ke- to-13, 14-dihydro-8-iso-PGF2 (Basu, 1998) (Fig. 7.5). 198 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

7.4 IsoPs and Signal Transduction Processes

IsoPs are vasoconstrictors not only in brain microvasculature, but also in kidney, lung, heart, retina, and lymphatics. The vasocontractile response to IsoPs depends on extracellular Ca2+ by both L- and T-type Ca2+ channels, and perhaps also involves protein kinase C (Fukunaga et al., 1993). IsoPs also produce platelet aggregation and promote atherogenesis (Tang et al., 2005). In addition, 15-F2-IsoP promotes endothelin release and facilitates proliferation of vascular smooth muscle. Although several reports indicate that discrete receptors for isoP do exist on platelets and vascular tissues (Ting and Khasawneh, 2010; Gong et al., 2010), it is becoming increasingly evident that IsoPs interact with thromboxane (TXA2) receptors (Fig. 7.6). These interactions can be blocked by thromboxane receptor antagonist (Takahashi et al., 1992). IsoP and thromboxane receptors signaling starts by the activation of phospholipase C (PLC), which in turn metabolizes phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) into inositol 1,4,5-trisphosphate (InsP3) and

Ca2+ PGE IsoP 2 Extracellular IsoP-R A1

PtdCho PtdIns-4,5-P EP1 2 PM NMDA-R TBXA2-R G Gs cPLA G 2 + PLC PGs Ca2+

ARA Lyso-PtdCho DAG InsP3

Adenylate kinase ↑ cAMP PKA

Tyrosine kinases Isoprostanes PAF PKC MLCK & CamK-II

Oxidative stress Inflammation Anti-aniogenicSmooth response musclePlatelet cell proliferatio aggregationn

Cellular response Disturbed ion homeostasis Alterations in cellular redox

Neurodegeneration

Fig. 7.6 Involvement of isoprostane in signal transduction processes. N-Methyl-D-aspratate receptor (NMDA-R); phosphatidylcholine (PtdCho); lysophosphatidylcholine (Lyso-PtdCho); cytosolic phospholipase A2 (cPLA2); arachidonic acid (ARA); platelet activating factor (PAF); phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2); inositol 1,4,5-trisphosphate (InsP3,); phospholipase C (PLC); diacylglycerol (DAG); arachidonic acid (ARA); intracellular calcium (Ca2+); and protein kinase C (PKC); prostaglandin E2 (PGE2); PGE2 receptor (EP1); Isoprostane (IsoP); thromboxane receptor (TBXA2-R); isoprostane receptor (IsoP-R); protein kinase A (PKA); cyclic AMP (cAMP); myosin light chain kinase (MLCK); and calmodulin-dependent protein kinases II (CamK-II) 7.4 IsoPs and Signal Transduction Processes 199

2+ diacylglycerol (DAG) . InsP3 then binds to its receptor and raises cytosolic Ca concentrations by inducing Ca2+ release from vesicles into the cytoplasm. DAG serves to stimulate protein kinase C (PKC), which in turn activates phospholipase

A2 (PLA2), an enzyme responsible for the release of ARA from membrane phospholipids (Farooqui and Horrocks, 2007). Signaling through IsoP and TXA2 receptors has been shown to enhance phosphorylation of several tyrosine kinase families.

These enzymes are coupled to isoP, TXA2, and PG receptors through G proteins. These receptors modulate endothelial cell viability, platelet aggregation, vasoconstriction in non-neural tissues and cytokine expression and neurotransmission in brain. TXA2 is highly unstable (a half-life of around 30 s) and functions primarily as an autocrine or local paracrine signal, allowing tight spatial regulation of cellular activation (Ting and Khasawneh, 2010; Gong et al., 2010). In contrast, IsoP is more stable than TXA2. IsoPs exert potent biological actions both via receptor-dependent and independent mechanisms. IsoPs circulate in vivo at concentrations orders of magnitude higher than other ARA metabolites such as TXA2 and remain much more chemically stable (Ting and Khasawneh, 2010). This family of lipid-mediators, particularly 8-iso-PGF2a, is strongly correlated with the oxidative microenvironments found in various disease states.

F2-IsoP exerts its receptor-mediated effect in vascular beds by promoting interactions between endothelial cells and monocytes (Lahaie et al., 1998; Fam and Morrow, 2003). IsoP-mediated monocyte adhesion does not depend on VCAM-1, but involves protein kinases, such as protein kinase A and mitogen-activated protein kinase kinase 1. F2-IsoP also modulates the p38 MAPK pathway during monocyte adhesion (Cracowski, 2004). Thus, F2-IsoP not only affects vascular and bronchial smooth muscles function, but also modulates cellular proliferation (Fam and Morrow, 2003). 8-IsoP increases IL-8 expression in human macrophages involving both ERK 1/2 and p38 MAPK, but not NF-kappaB signaling pathway. These findings further support a link between oxidative stress/lipid peroxidation and inflammation in human macrophages and suggest a role for 8-isoP in this process. This 8-isoP-induced chemokine expression might be involved in the pathogenesis of atherosclerosis as well as other inflammatory disorders. In brain microvasculature, these processes may relate to inflammation and oxidative stress. Receptor independent action of IsoP is due to adduct formation. Some IsoPs (A2/J2-IsoPs) contain unsaturated carbonyl moieties that render them highly reactive and capable of adducting relevant biomolecules such as thiols via Michael addition. For example,

15-A2t-IsoP efficiently conjugates with glutathione in vitro. This reaction is catalyzed by human and rat glutathione transferases (GSTs), with the isozyme GSTA4-4 displaying the highest activity (Milne et al., 2004).

In plasma, HDLs contain significantly higher levels of F2-IsoP than LDL or VLDL (Proudfoot et al., 2009). Furthermore, HDL3 particles contain higher levels of F2-IsoP than HDL2. Platelet activating factor acetylhydrolase (PAF-AH), a secretory calcium-independent Group VII phospholipase A2, which hydrolyzes esterified

F2-isoPs from phospholipids, is predominantly associated with LDL. Low levels of

F2-isoPs in LDL may be related to higher PAF-AH activity in LDL. Paraoxonase 1, a lactonase that requires calcium for activity, is associated with HDL2 and may be 200 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

responsible for lowering F2-isoPs in HDL2 compared with HDL3 (Proudfoot et al., 2009). Low PON-1 activity is correlated with an increased risk of coronary heart disease (Mackness et al., 2003). Physiological significance of these observations is not fully understood. However, HDL is the main carrier of F2-isoPs in the lipoproteins of human plasma. The relevance of F2-isoPs in HDL particles and its relationship to cardiovascular disease has been emphasized in several recent reports. F2-isoPs is associated with atherosclerosis (Morrow, 2005), and angiographic evidence of coronary artery disease (Shishehbor et al., 2006). Plasma 8-isoprostanes in type 2 diabetic subjects are negatively correlated with antioxidative activity associated with the HDL3 subfractions (Nobécourt et al., 2005). Therefore, more studies are required to establish the implications of these findings on HDL function (Proudfoot et al., 2009). Similar to nonenzymic oxidation of arachidonic acid, in vivo and in vitro oxidation of eicosapentaenoic acid (EPA) and DHA generates F3-IsoPs and neuroprostanes, respectively (Gao et al., 2006; Roberts et al., 1998). The amounts F3-IsoP formed are extremely large (up to 8.7 ± 1.0 mg/mg EPA) and greater than levels of F2-IsoPs generated from ARA. EPA supplementation markedly reduces levels of arachidonate-derived F2-IsoPs by up to 64%. This is because EPA competes with ARA for the active site of cyclooxygenases and cytochrome P450 (CYP) enzymes.

These studies provide the evidence that F3-IsoPs and 17,18-epoxyeicosatrienoic (17,18-EEQ) are novel in vivo oxidation product of EPA (Gao et al., 2006; Arnold et al., 2010), which not only induce antiarrhythmic effects, but also suppress Ca2+- induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. Recent studies indicate that oxidized EPA, rather than native EPA, possesses anti-atherosclerotic, anti-inflammatory, and antiproliferative effects (Brooks et al., 2008). Gas chromatography/mass spectrometry (GC/ MS) and liquid chromatography/mass spectrometric (LC/MS) studies indicate that levels of A3/J3-IsoPs increase approximately 200-fold with oxidation of EPA in vitro from a basal level of 0.8 ± 0.4 ng/mg EPA to 196 ± 23 ng/mg EPA after 36 h (Brooks et al., 2008). Supplementation of EPA markedly reduces levels of pro-inflammatory

ARA-derived F2-IsoPs by up to 64% (p < 0.05). These results support the view that dietary n-3 PUFAs may influence the formation of bioactive peroxidation products derived from n-6 PUFAs by channeling the free radical pathway away from the F2- IsoPs. Furthermore, oxidation products of EPA activate the Nrf2 transcription factor that mediates important endogenous antioxidant responses in response to reactive electrophiles via induction of gene transcription (Brooks et al., 2008). These nonenzymically derived products of EPA (F3-IsoP) interact with Keap1, the direct inhibitor of Nrf2, initiating Keap1 dissociation from Cullin3 to facilitate the translocation of Nrf2 to the nucleus, where it binds to AREs as a heterodimer and regulates ARE- directed gene expression (Venugopal and Jaiswal, 1996) (Fig. 7.7). It is proposed that EPA-derived oxidation products containing an a,b-unsaturated carbonyl moiety, specifically J3-IsoPs, generation of resolving E1 and E2, and 3-series prostaglandins and thromboxanes and 5-series leukotrienes may be responsible for eliciting this biological activity and beneficial effects of fish oil on human health (Nourooz- Zadeh et al., 1997; Gao et al., 2007; Farooqui, 2009). This may be responsible for 7.4 IsoPs and Signal Transduction Processes 201

Dietary EPA Thromboxane receptor Glu

PtdChoPtdCho PtdIns-4,5-PtdIns-4,5-P22

NMDA-R G

2 + EPA Ca2+ PLC Inhibition of PG + RvE1 and & ROS generation cPLA RvE PGE3, PGI3, 2 TXA , TXB InsP 3 3 Inhibition of F -IsoP generation ARA DAG 3 F -IsoP 2 3 DAG-lipase + + + F -IsoP ROS PGs PKC Keap1 2 Ca2+ Nrf2 Low levels Protein phosphorylation

Nrf2 NUCLEUS Gene transcription for antioxidative enzymes

Neuroprotection

Fig. 7.7 Effect of EPA-derived isoprostane (F3-IsoP) on Nrf2-mediated gene transcription. N-Methyl-d-aspartate receptor (NMDA-R); phosphatidylcholine (PtdCho); cytosolic phospholipase A2 (cPLA2); Receptor (R); phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2); inositol

1,4,5-trisphosphate (InsP3,); phospholipase C (PLC); diacylglycerol (DAG); arachidonic acid (ARA); intracellular calcium (Ca2+); and protein kinase C (PKC); reactive oxygen species (ROS); prostaglandins (PGs); F2-Isoprostane (F2-IsoP); eicosapentaenoic acid (EPA); resolving E1 (RvE1); resolving E2 (RvE2); F3-Isoprostane (F3-IsoP); 3-series prostaglandins and thromboxanes (PGE3,

PGI3, TXA3 and TXB3); nuclear factor (erythroid-derived 2)-like 2 (NrF2); and kelch-like erythroid Cap‘n’Collar homolog-associated protein 1 (Keap1) the beneficial effects of n-3 fatty acids on human health. Similarly, the nonenzymic oxidation of DHA results in the generation of a series of metabolites known as neuroprostanes. These metabolites are found in rat and pig brain and human spinal fluid and represent a more selective index of brain oxidant injury than isoprostanes (Roberts et al., 1998). In isolated bovine retinae isoprostanes produce dual effects. Low concentrations + of 8-isoPGF2a inhibit, whereas higher concentrations of 8-isoprostane stimulate K - mediated [3H]D-aspartate overflow (Opere et al., 2005). The excitatory effect of

8-isoPGF2a is mimicked by thromboxane receptor agonist, U-46619 and retarded by thromboxane receptor antagonist, SQ 29,548. Based on detailed pharmacological characterization, it is proposed that inhibitory effect of 8-isoPGF2a may be due to the activation of EP1/EP2 receptors while the excitatory effects may be caused by the activation of thromboxane receptors (Opere et al., 2005). In addition, 8-iso-

PGF2a not only augments sensory neuronal function by increasing the release of 202 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived… neurotransmitters from isolated sensory neurons, but also enhances the firing of C-nociceptors in situ. In the anterior uvea of the eye, 8-isoprostanes produce both excitatory and inhibitory effects on sympathetic neurotransmission in isolated mammalian iris ciliary bodies (Opere et al., 2008). This effect is mediated by the thromboxane receptor, which modulates the release of norepinephrine (NE) from sympathetic nerves. IsoPs also attenuate K+-induced [3H]dopamine release in isolated bovine retinae through an indirect action on COX pathway leading to the synthesis of prostaglandins, which in turn activate EP receptors (Liu et al., 2008). Collective evidence suggests that isoPs modulate the release of neurotransmitter in the brain.

7.5 IsoPs in Neurodegenerative Diseases

Levels of IsoPs are increased in neurodegenerative diseases (Alzheimer disease, AD; Huntington disease, HD; Creutzfeldt–Jakob disease, CJD; and multiple sclerosis, MS) and neurotraumatic diseases (stroke, spinal cord injury, SCI, and traumatic brain injury, TBI) (Table 7.1). Although, the molecular mechanism of IsoP-mediated neurodegeneration is not fully understood, Iso-P-mediated inflammation and oxidative stress have been reported to contribute in neurodegenerative process. In AD, the exposure of neurons or synaptosomes to amyloidogenic Ab peptide results in increased synthesis of F2-IsoP (Mark et al., 1999; Brunetti et al., 2004). Furthermore, in a mouse model of AD significant increase in F2-IsoPs precedes amyloid plaque formation (Pratico et al., 2001; Praticò, 2010). Delivery of F2-IsoP into the brains of Tg2576 mice results in elevated levels of Ab in the brain. Increase in Ab levels and plaque-like deposits, can be blocked by a thromboxane (TXA2) receptor antagonist, suggesting that TXA2 receptor activation is associated with the effects of F2-IsoP on Ab (Shineman et al., 2008). It is well known that APP is normally processed by two membrane-associated, catalytic proteases, a- and b-secretase. Such proteolytic cleavage results in the formation of harmless peptide fragments that are readily

Table 7.1 Levels of isoprostanes in neurological disorders Neurological disorder Levels of isoprostanes Reference Alzheimer disease Increased Roberts et al., 1998; Montine et al., 1999a, 2007 Aneurysmal subarachnoid Increased Lin et al., 2006 hemorrhage Huntington disease Increased Montine et al., 1999b Multiple sclerosis Increased Greco et al., 2000 Creutzfeldt–Jakob disease Increased Greco et al., 2000 Scrapie-infected mice Increased Minghetti et al., 2000 KA-induced neurotoxicity Increased Farooqui et al., 2007 Stroke Increased Zeiger et al., 2009 Traumatic brain injury Increased Bayir et al., 2002 Spinal cord injury Increased Oner-Iyidoğan et al., 2004 Schizophrenia Increased Dietrich-Muszalska and Olas, 2009 7.5 IsoPs in Neurodegenerative Diseases 203

Thromboxane receptor S18886 Glu

PtdCho PtdIns-4,5-P2 NMDA-R G

+ 2 + Ca2+ PLC

cPLA +

InsP ARA DAG 3 IsoP DAG-lipase + + PKC + ROS PGE2 Ca2+

NF-kB MAPK

α-secretase

↑ APP ↑ APP Aβ42 protein mRNA

Gene transcription NUCLEU S β-secretase (Proinflammatory enzymes and cytokines)

Amyloid plaques Neurodegeneration

Fig. 7.8 Hypothetical model showing IsoP-mediated generation of Ab. N-Methyl-d-aspartate receptor (NMDA-R); phosphatidylcholine (PtdCho); cytosolic phospholipase A2 (cPLA2); Receptor (R); phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2); inositol 1,4,5-trisphosphate (InsP3,); phospholipase C (PLC); diacylglycerol (DAG); arachidonic acid (ARA); intracellular calcium (Ca2+); and protein kinase C (PKC); mitogen activated kinase (MARK); reactive oxygen species (ROS); prostaglandin E2 (PGE2); Isoprostane (IsoP); amyloid beta (Ab); and amyloid precursor protein (APP) degraded in the brain. In AD, a third membrane associated protease, g-secretase, cleaves APP within the transmembrane portion of the protein (Farooqui, 2010). This cleavage is combined with the extracellular cleavage of APP by b-secretase, which appears to be the rate-limiting enzyme in this degenerative pathway and releases the toxic Ab peptide (40 or 42 amino acids), which is characteristic of AD

(Farooqui, 2010). As stated above, TXA2 receptor activation increases Ab synthesis and secretion of APP ectodomains (Shineman et al., 2008). It is proposed that elevation in APP mRNA may be due to increased stability of mRNA leading to the elevation in APP protein levels. This increased availability of APP provides more substrate for g-secretase-mediated proteolytic cleavages, thereby increasing the levels of Ab40−42 and amyloid plaque deposition (Shineman et al., 2008). Long-term treatment of Tg2576 mice with S18886 leads to reduction in amyloid plaques, insoluble Ab, and decrease in APP levels, supporting the view that blocking of TXA2 receptor can be used for the AD therapy. Furthermore, TXA2 receptor-mediated increase in Ab can directly stimulate cPLA2 resulting in increased ROS generation (Fig. 7.8) directly and through the oxidation of released ARA. Increase in ROS not only induces oxidative stress, but also facilitates NF-kB activation and its nuclear translocation, where it promotes the expression of inflammatory cytokine (TNF-a 204 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

and IL-1b). These cytokine activate both cPLA2 and PLC leading to the production of ARA and DAG, which on hydrolysis by DAG-lipase can provide more ARA (Farooqui and Horrocks, 2007) (Fig. 7.8). The use of urinary iso-P for the detection of AD has been controversial. A series of studies indicated greater free radical damage in DHA-containing compartments than in ARA-containing compartments in regions (hippocampal area) involved in the neuropathology and pathogenesis of AD brain of AD brain (Montine et al., 1999a).

7.6 IsoPs in Neurotraumatic Diseases

Stroke is a metabolic trauma caused by severe reduction or blockade in cerebral blood flow due to the brain. It is accompanied by increase in production of 15-A2t-

IsoP (8-iso-PGA2), one of the most abundant A2-IsoPs generated in vivo in stroke- infarcted human cortical tissue (Zeiger et al., 2009). Exposure of primary neuronal cultures with 15-A2t-IsoP does not alter ATP content, but in combination with oxygen glucose deprivation results in a significant hyperpolarization of the mitochondrial membrane and dramatic increase in neuronal cell death. In the presence of Ca2+,

15-A2t-IsoP treatment leads to a rapid induction of the permeability transition pore and release of cytochrome c, which promotes rapid cell death through apoptosis. Traumatic injury to the brain (TBI) consists of two broadly defined components: a primary component attributable to the mechanical insult itself, and a secondary component attributable to the series of systemic and local neurochemical and pathophysiological changes that occur in the brain after the initial insult (Raghupathi, 2004). The primary injury results in rapid deformation of brain tissue and rupture of neural cell membranes, leading to the disruption and reduction in blood flow, breakdown of the blood brain barrier, and intracranial hemorrhage. In contrast, secondary injury to the brain induces neurochemical alterations, activation of microglial cells and astrocytes, and demyelination involving oligodendroglia (Raghupathi, 2004; Farooqui, 2010). Levels of IsoP are markedly increased in CSF of TBI patients (93.8 ± 30.8 pg/ml) compared to age-matched controls (7.6 ± 5.1 pg/ml, p < 0.05) (Bayir et al., 2002). It is proposed that IsoPs may play a role in the reduction of cerebral blood flow that occurs after brain injury and subsequent oxygen radical production responsible for neurodegeneration in TBI (Farooqui, 2010). Similarly, levels of IsoP are also increased in the spinal cord injury (SCI). It is suggested that isoPs are not only responsible for oxidative stress, but also for neurogenic bladder dysfunction (Oner-Iyidoğan et al., 2004).

7.7 NPs in Neurological Disorders

Neuroprostanes (NPs) are 22 carbons and 4 double bonds containing prostaglandin- like metabolites generated by free radical-mediated peroxidation of DHA, a major unsaturated fatty acid in neural tissues. They are analogous to isoprostanes (Roberts 7.7 NPs in Neurological Disorders 205

OH HOOC(H2C)11

C2H5 11 OH

OH 11-Series F4-NP

OH OH 7 HOOC(H2C)2

OH 7-Series F4-NP

OH HOOC

14 OH OH 14-Series F4-NP OH OH 10 HOOC(H2C)2

OH 10-Series F4-NP

Fig. 7.9 Chemical structures of F4-neuroprostanes

et al., 1998; Nourooz-Zadeh et al., 1999; Roberts and Fessel, 2004; Yin et al., 2005), and are considered to be specific markers for neuronal oxidative stress. During NP synthesis, oxygen-mediated DHA radicalization generates peroxyl radicals, which undergo endocyclization followed by the addition of molecular oxygen and reduction to form the F ring of NP or can isomerize to molecules with E-type and D-type prostane rings (E4/D4-NPs). Like isoPs, NPs is also synthesized in situ esterified to phospholipids and are subsequently released by a phospholipase A2. NPs containing phospholipids and free NPs induce changes in physicochemical properties, such as membrane fluidity and permeability. These changes may lead to impairment in neuronal function and promote oxidative stress and neurodegeneration (Fam and Morrow, 2003; Yin et al., 2005; Greco and Minghetti, 2004). At present, nothing is known about PLA2 activity that releases NP from NP bound phospholipids. F4-NP is the first characterized neuroprostane (Fig. 7.9). It occurs in cerebrospinal fluid

(CSF) from normal individuals. The levels of F4-NP are significantly increased in

CSF from patients with Alzheimer disease (Reich et al., 2001). E4-NP and D4-NP are also detected in normal rat and human brain. Levels of E4/D4-NP in normal brain are found to be one third compared to levels of F4-NP (Roberts and Fessel, 2004). 206 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

Patients with aneurysmal subarachnoid hemorrhage (aSAH), a type of hemorrhagic stroke, also show an increase in levels of F4-NPs in CSF (Hsieh et al., 2009). It is proposed that in aSAH CSF, F4-NP may be a better predictor for outcome of aSAH than F2-IsoPs at early time points. Levels of F4-NPs are also increased in AD patients. (Reich et al., 2001).

7.8 Isoketals (Isolevuglandins) in Neurological Disorders

Isoketals (IsoKs) are g-ketoaldehydes generated via the isoprostane pathway of ARA peroxidation, and are among the most reactive by-products of lipid peroxidation. The formation of isoKs occurs through the rearrangement of H2-IsoP endoperoxides. Isoketals differ from isoprostanes in containing a characteristic aldehydic group in a 1,4-dicarbonyl array, making them extremely reactive toward primary amino groups in proteins (Boutaud et al., 2005). Unlike F2-IsoP, isoKs can modify biologically important proteins rather than activation of specific receptors (Davies et al., 2004). IsoKs are highly reactive g-ketoaldehydes that form pyrrole adducts with the e-amino group of lysine residues on protein (Davies et al., 2004). These pyrrole adducts are unstable in the presence of oxygen and are further transformed to lactam and hydroxylactam adducts, which accumulate as stable end products. IsoKs have remarkable ability to cross-link proteins through oxidation of the pyrrole (Brame et al., 2004). IsoKs also forms adduct with phosphatidylethanolamine (PtdEtn) in vitro. Isoketals also form pyrrole and Schiff base adducts with phosphatidylethanolamine (PtdEtn) in vitro. In addition, the ability of isoketals to covalently modify PtdEtn is greater than that of 4-hydroxynonenal. Whether IsoK-PtdEtn adduct mediates some of the biological effects of IsoKs relevant to physiological and disease processes remains unknown (Sullivan et al., 2010), but it is becoming increasingly evident that their levels are increased in neuodegenerative diseases (Table 7.2). Incubation in heart/brain mitochondria with synthetic IsoKs in the presence or absence of Ca2+ not only results in alterations in mitochondrial respiration and membrane potential (DeltaPsi), but also pyridine nucleotide redox state (Stavrovskaya et al., 2010). IsoKs dose dependently increases liver mitochondria swelling mediated by low concentrations of Ca2+ and Zn2+ or by the prooxidant tert- butylhydroperoxide, and release of cytochrome c. The mitochondrial permeability transition (mPT) inhibitor cyclosporine A delays IsoK-mediated mitochondria dysfunction. The actions of IsoKs are consistent with interactions with cytochrome c, a

Table 7.2 Levels of isoketals in neurological disorders Disease Effect Reference AD Increased Boutaud et al., 2002 MS Increased Greco et al., 2000 HD Increased Montine et al., 1999b Glaucoma Increased Govindarajan et al., 2009 AD Alzheimer disease, MS multiple sclerosis, HD Huntington disease 7.9 Neuroketals in Neurological Disorders 207 protein rich in lysine residues. Accumulating evidence suggests that IsoKs may mediate their cytotoxic effects through induction of the mPT and subsequent activation of downstream cell death cascades involved in apoptosis (Stavrovskaya et al., 2010). IsoKs produce several effects in neural cells. They inhibit the activity of proteasomes in glial cells and induce cell death. Intra-hemispheric injections of 15-E2- IsoK disrupt the blood brain barrier. IsoKs have been detected in tissues as well as biological fluids. These mediators interfere with protein function and are among the most potent neurotoxic products of lipid oxidation. In smooth muscle cells, isoKs interfere with proteosomal degradation of proteins that ultimately lead to oxidative stress-mediated apoptotic death and destabilization of atherosclerotic plaques (Zhang and Salomon, 2005). In blood, IsoK-protein adducts are associated with low-density lipoprotein (LDL). IsoK-protein adducts not only interfere with proteosomal degradation of proteins, but may also contribute to atherogenesis through recognition and endocytosis of the modified LDL by macrophage cells. These processes may ultimately lead to oxidative stress-mediated apoptotic death and destabilization of atherosclerotic plaques (Zhang and Salomon, 2005). It is suggested that levels of IsoK-protein adducts in human blood plasma are more closely correlated with cardiovascular disease than are the classical risk factors LDL or total cholesterol (Salomon et al., 2000).

7.9 Neuroketals in Neurological Disorders

Neuroketals are oxidized products of DHA that contain either a 1,4-pentadiene or 1,4,7-octatriene side chain structure (Bernoud-Hubac et al., 2001) (Fig. 7.10). Synthesis of neuroketal involves the formation of docosahexaenoyl radicals, which are converted to peroxyl radicals following the addition of oxygen. These peroxyl radicals undergo endocyclization followed by further addition of molecular oxygen to form bicyclic endoperoxide intermediate regioisomers, which can then rearrange to form D4-neuroketals and E4-neuroketals regioisomers (Bernoud-Hubac et al., 2001). Due to the presence of 1,4-pentadiene or 1,4,7-octatriene side chain, neuroketals undergo further oxidation to form neuroketals with an additional hydroxyl group. In vitro oxidation of DHA results in the generation of a series of neuroketals that have been identified by mass spectrometric analyses (Bernoud-Hubac and Roberts, 2002). These neuroketals form adducts with lysine through the formation of Schiff base and hydroxylactam. Oxidized hydroxylactam neuroketal-lysyl protein adducts are not detected in nonoxidized rat brain synaptosomes, but are readily detectable following oxidation of synaptosomes (Bernoud-Hubac and Roberts, 2002). It is proposed that because of their capacity to covalently modify proteins, neuroketals may be highly injurious to neurons. The neurotoxic effects of neuroketals is expected to be similar to that of 2,5-hexanedione, a g-diketone that reacts with the e-amine group of lysine with reaction chemistry similar to that of neuroketals. g-Diketone neuropathy is characterized by cross-linking of neurofilaments, via the formation of pyrrole adducts, leading to axonal atrophy and swelling (Lehning et al., 2000). 208 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

O OH O OH OH C O O HO O OH OH Isofuran Isoketal (isolevuglandin) OH

COOH O HO Neurofuran

OH O OH

COOH

O E4-Neuroketal

Fig. 7.10 Chemical structures of isoketal, isofuran, neuroketal, and neurofuran

7.10 Isofurans in Neurological Disorders

Lipid peroxidation under high oxygen tension generates substituted tetrahydrofuran derivatives (Fessel et al., 2002). These ARA-derived mediators are called as isofurans (IsoF) (Fig. 7.10). The molecular mechanism of isofuran generation is not fully understood. However, two mechanisms have been proposed: a cyclic peroxide cleavage pathway and an epoxide hydrolysis pathway. Oxygen concentration modulates the generation of IsoFs. Increased oxygen concentrations favor the formation of isoFs and retard the formation of isoP. Seizures are known to induce the formation of isoFs (Patel et al., 2008). In status epilepticus (SE), synthesis of F2-IsoP overlaps with IsoF formation in hippocampal subregions, but time courses of generation of these mediators are different. IsoF, but not F2-IsoP formation coincides with mitochondrial oxidative stress (Patel et al., 2008). SE induces a transient decrease in hippocampal pO2 measured by in vivo electron paramagnetic resonance oximetry suggesting an early phase of seizure-mediated hypoxia. It is also reported that seizure-induced F2-IsoP formation coincides with the peak hypoxia phase, whereas IsoF formation coincides with the “reoxygenation” phase. These results demonstrate seizure-induced increase in IsoF formation and its correlation with changes in hippocampal pO2 and mitochondrial dysfunction (Patel et al., 2008).

Determination of levels of F2-IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD and age-matched controls as well as 7.12 IsoPs in Kainic Acid Neurotoxicity 209 patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and AD indicate that levels of

IsoFs but not F2-IsoPs are increased significantly in the SN of patients with PD and DLB than those of control subjects (Fessel et al., 2003). Levels of IsoFs and

F2-IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age-matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA (Fessel et al., 2003). Collective evidence suggests that oxygen concentration differentially modulates the formation of isoPs and isoFs. These metabolites are present and readily detectable in normal fluids and tissues, and their levels are dramatically increased in animal model of oxidant injury and chronic neurodegenerative diseases (Fessel et al., 2002).

7.11 Neurofurans in Neurological Disorders

Neurofurans are 22-carbon compounds that are generated by nonenzymic oxidation of DHA. The neurofurans are similar to the isoFs, and are formed under similar conditions of oxidative stress, containing a substituted tetrahydrofuran ring (Song et al., 2008). Levels of neurofurans are elevated in the brain cortex of a mouse model of AD and are depressed in mouse brain cortex by deletion of p47(phox), an essential component of the phagocyte NADPH oxidase. Measurement of the neurofurans may ultimately prove useful in diagnosis, timing, and selection of dose in the treatment and chemoprevention of neurodegenerative disease.

7.12 IsoPs in Kainic Acid Neurotoxicity

Systemic administration of kainic acid (KA), a cyclic and nondegradable analog of glutamate in adult rats induces persistent seizures and seizure-mediated brain damage syndrome (Coyle, 1983; Farooqui et al., 2008). KA-mediated toxicity is accompanied by the selective degeneration of neurons, especially in striatal and hippocampal areas of brain after (Coyle, 1983; Farooqui et al., 2008). Studies on the determination of F2-IsoPs in microdissected hippocampal CA1, CA3, and dentate gyrus regions at various times following subcutaneous KA administration indicate that KA produces large elevations in F2-IsoP levels in the highly vulnerable CA3 region 16 h post injection (Patel et al., 2001). The CA1 region shows small, but statistically insignificant increase in2 F -IsoP levels. The dentate gyrus, a region resistant to KA-induced neuronal death also shows marked (2.5–5-fold) increase in

F2-IsoP levels 8, 16, and 24 h post injection. The increase in F2-IsoP levels in CA3 and dentate gyrus is accompanied by inactivation of mitochondrial aconitase, increase in superoxide production, and neuronal vulnerability in these regions 210 7 Isoprostanes and Other Nonenzymic Polyunsaturated Fatty Acid-Derived…

4 -IsoP (ng/g) 2 F 2

0 C3d1W2W84W W KA treatment time

Fig. 7.11 Levels of F2-isoPs following KA-mediated neurotoxicity. Kainic acid (KA); days (d); and week (W)

(Patel et al., 2001). Although, KA injections into the right lateral ventricle (coordinates: 1.0 mm caudal to bregma, 1.5 mm lateral to the midline, 4.5 mm from the surface of the cortex) produce no changes in F2-isoPs levels at 3 days, 1 week, and 2 weeks after KA administration, there is a significant increase (~134%) in 2F -isoP levels at 4 weeks after kainic acid injection compared to controls. At 8 weeks after injection, the F2-isoP levels are increased (~180%) compared to those in the 4 weeks post-KA injected rats (Fig. 7.11) (Farooqui et al., 2007).

7.13 Conclusion

IsoP and NPs are prostaglandin-like compounds formed in vivo primarily by free radical-catalyzed peroxidation of ARA and DHA. They are not only useful as a specific, sensitive, chemically stable, noninvasive index of free radical generation in vivo, but also mediate several pharmacological effects on neural cells, vascular cells, and smooth muscles cells. Activities of IsoPs are mediated by thromboxane receptors. Levels of these lipid mediators are increased in brain, CSF, and urine of patients with neurological disorders. Nothing is known about NP-mediated receptor stimulation. Like IsoPs and NPs, isoK, and neuroketal are nonenzymic oxidized products of ARA and DHA. IsoKs and neuroketals form pyrrole adducts with the e-amino group of lysine residues on protein. These pyrrole adducts are unstable in the presence of oxygen and are further transformed to lactam and hydroxylactam adducts, which accumulate as stable end products. The ability of IsoK and neuroketals to form protein and phospholipid adducts may be highly injurious to neurons. Oxidation of ARA and DHA under high oxygen tension results in the generation of isofurans and neurofurans. Although, levels of isoPs, NPs, IsoKs, neuroketals, isoFs, References 211 and neurofurans are increased in body fluids of patients with neurological disorders, and are used as reliable biomarkers for oxidative stress: but their (patho) physiological roles in neurological disorders are not fully understood. More studies are needed on relative contribution of ARA and DHA-derived lipid mediators in cell death.

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8.1 Introduction

Ceramide (N-acylsphingosine) forms the backbone of all complex sphingolipids. It is composed of the long-chain sphingoid base, sphingosine, in N-linkage to a variety of acyl groups (varying in length from C14 to C26) (Fig. 8.1). In addition to serving as a precursor to complex sphingolipids, ceramide is a potent signaling molecule capable of regulating vital cellular functions. Thus, ceramide is associated with the regulation of cell growth, viability, differentiation, cell signaling, apoptosis, cytokine biosynthesis, secretion, regulation of enzyme activities, neutrophil adhesion to the vessel wall, vascular tone, and senescence (Fig. 8.2) (Hannun and Obeid, 2002; Kitatani et al., 2008). A chronic increase in intracellular ceramide has been reported to inhibit axonal elongation and receptor-mediated internalization of the nerve growth factor (Costantini et al., 2005). In brain, ceramide also modulates synaptic activity. Thus, a rapid generation of ceramide by sphingomyelin phosphodiesterase-3 (nSMase2) plays a key role in modulating excitatory postsynaptic currents by controlling the insertion and clustering of NMDA receptors (Wheeler et al., 2009). Inhibition of nSMase2 is reported to impair spatial and episodic memory formation in mice. At the molecular level, inhibition of nSMase2 not only reduces ceramide and elevates PSD-95, but also increases the number of AMPA receptors along alterations in the subunit composition of NMDA receptors. It is suggested that nSMase2 is an important component for efficient memory formation. The generation of ceramide through this enzyme emphasizes the importance of ceramide in regulating synaptic events related to learning and memory (Tabatadze et al., 2010). In the outer leaftet of neural membrane, ceramide aggregates into lateral domains in association with sterols and other sphingolipids forming distinct membrane domains called rafts. Lipid rafts contain a given set of proteins, which can alter their size and composition in response to intra- or extracellular stimuli, thereby favoring specific protein–protein interactions. This process not only facilitates ligand receptor binding, but also results in activation of specific signaling cascades. The generation and incorporation of ceramide through the stimulation of many receptors involving

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 217 DOI 10.1007/978-1-4419-9940-5_8, © Springer Science+Business Media, LLC 2011 218 8 Ceramide and Ceramide 1 Phosphate in the Brain

CH2OH Dihydroceramide NH

O OH

CH2OH Ceramide NH

O OH O

CH2O P OH Ceramide 1 phosphate O NH

O HO P PCERA-1 HO OO

Fig. 8.1 Chemical structures of sphingomyelin, dihydroceramide, ceramide, and ceramide 1 phosphate

Modulation of enzyme activity

Modulation of neural cell differentiation

Modulation of immune response

Ceramide 1 Phosphate Modulation of signal transduction

Modulation of neural cell migration

Modulation of mitogenesis

Modulation of apoptosis

Fig. 8.2 Roles of ceramide and ceramide 1 phosphate in the brain sphingomyelinase activation or stress stimuli within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms (Gulbins and Li, 2006; Zhang et al., 2009). These platforms serve to cluster receptor molecules and to organize intracellular signaling molecules to facilitate 8.1 Introduction 219

Extracellular TNF-α A2 A3 o R

TNF-R PM PtdCh

SM PtdIns PtdCho

P75-NT G Intracellular cPLA 2 SMase + SMS PLC + Lyso-PtdCho +

ARA C1P Ceramide DAG + InsP3 + S1P COX-2 SphK PKC + DAG-L Sph MAG + FFA Eicosanoids Caspase cascade

Free radicals & ROS

PAF Apoptosis

CellularResponse

Fig. 8.3 Interactions between glycerophospholipid and sphingolipid-derived lipid mediators. Tumor necrosis factor-a (TNF-a); tumor necrosis factor-a-receptor (TNF-R); agonist A2 (A2); agonist A3 (A3); phosphatidylcholine (PtdCho); lyso-phosphatidylcholine (lyso-PtdCho); cytosolic phospholipase A2 (cPLA2); sphingomyelin (SM); sphingomyelinase (SMase); ceramide 1 phosphate (C1 P); sphingomyelin synthase (SMS); sphingosine (Sph); sphingosine 1 phosphate (S 1 P); diacylglycerol (DAG); phosphatidylinositol (PtdIns); phosphatidycholine-specific phospholipase C

(PLC); inositol 1,4,5-trisphosphate (InsP3); monoacylglycerol (MAG); free fatty acid (FFA); platelet activating factor (PAF); protein kinase C (PKC); arachidonic acid (ARA); cyclooxygenase-2 (COX-2); and reactive oxygen species (ROS) signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also facilitate stress-mediated signaling events (Gulbins and Li, 2006; Zhang et al., 2009) through the recruitment of intracellular signaling molecules. Treatment of neural cells with ceramide produces variety of effects. In general, neurons are less sensitive to ceramide treatment than glial cells, and their response depends upon levels of ceramide (Luberto et al., 2002). Rat hippocampal and spinal cord motor neurons show a biphasic response when treated with ceramide. C6-ceramide produces differentiation, cell survival, and promote neurite outgrowth at a low concentration through its interaction with p75 low-affinity NGF receptor, p75NTR (Barrett, 2000; Arévalo and Wu, 2006), and the TNF-a receptor, p55 (Adam-Klages et al., 1998), but at higher concentrations it promotes apoptosis through the activation of caspase-3 and the protease responsible for the cleavage of polyADP-ribose polymerase (Fig. 8.3). Caspase-3 in turn hydrolyzes a number of proteins related to signal transduction processes, including protein kinase

C, cPLA2, iPLA2, PLC; and cytoskeletal proteins such as a-spectrin, b-spectrin, actin, vimentin; members of the Bcl-2 family of apoptosis-related proteins, preseni- lins, amyloid precursor protein; and DNA modulating enzymes (Farooqui, 2009). 220 8 Ceramide and Ceramide 1 Phosphate in the Brain

Table 8.1 Modulation of enzyme activities by ceramide Enzyme Effect Reference Death-associated protein kinase Increased Herrmann et al., 1996; Ruvolo, 2003; Mao and Obeid, 2008; Jana et al., 2009 Protein kinase B (Akt) Increased Herrmann et al., 1996; Ruvolo, 2003; Mao and Obeid, 2009; Jana et al., 2009 Kinase suppressor of Ras Increased Herrmann et al., 1996; Ruvolo, 2003; Mao and Obeid, 2008; Jana et al., 2009 Protein kinase Cz Increased Herrmann et al., 1996; Ruvolo, 2003; Mao and Obeid, 2008; Jana et al., 2009 Rac Increased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009 Inducible nitric oxide synthase Increased Herrmann et al., 1996; Ruvolo, 2003; Mao and Obeid, 2008; Jana et al., 2009 Ceramide-activated protein kinase Increased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009 c-Jun N-terminal kinase Increased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009 Ceramide protein phosphatase 1 Increased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009 Ceramide protein phosphatase 2A Increased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009; Chalfant et al., 1999 Cathepsin D Increased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009

Phospholipase A2 Increased Liu et al., 1999; Hannun, 1997 Phospholipase C Increased Liu et al., 1999 Phospholipase D Increased Liu et al., 1999 Akt-bcl2 pathway Decreased Herrmann et al., 1996; Mao and Obeid, 2008; Jana et al., 2009

The degradation of these proteins results in abnormal signal transduction and key morphological changes associated with apoptotic cell death. Ceramide is known to promote protein–protein interaction, for example, dimerization of the TrkA receptor (MacPhee and Barker, 1999). Another potential mode of action through which ceramide mediates intracellular signaling is possibly via direct interaction with proteins that have a ceramide binding domain, for example, protein kinase C isoforms (Zhang et al., 1997). Ceramide also binds to cathepsin D in lysosome and translocates to mitochondria upon agonist stimulation resulting in cytochrome c release and activation of downstream caspases. This is another mechanism by which ceramide promotes apoptosis. These observations indicate the importance of ceramide concentration in determining the survival or death of neural cells (Luberto et al., 2002; Hisaki et al., 2004). In neural cells, ceramide modulates signal transduction pathways that are not only involved in apoptotic cell death, but also associated with inhibitory pathways to cell growth through the modulation of stress-activated protein kinase (SAPK) and other pathways (Table 8.1). In contrast, diacylglycerol (DAG), a metabolite derived from neural membrane phospholipids through the action of phospholipase C (PLC), not only activates the classical isoform of PKC that is 8.2 Synthesis of Ceramide and Ceramide 1-phosphate in the Brain 221 associated with cell growth and cell survival, but also stimulates cell proliferation through mitogen-activated protein kinase (MAPK) pathways (Ruvolo, 2001). The ceramide also inhibits the phospholipase D and c-Fos-dependent signaling pathway, retinoblastoma protein dephosphorylation, arrests the serum growth factor- mediated activation of protein kinase C (PKC) and DNA synthesis (Cutler and Mattson, 2001) and reduces the insulin/phosphatidylinositol 3-kinase-mediated Akt/protein kinase B translocation to membranes and its activation (Stratford et al., 2004). Accumulating evidence suggests that ceramide promotes growth arrest and apoptosis while DAG induces cell growth and survival (Ruvolo, 2001). Another important enzyme that regulates the levels of ceramide is sphingomyelin synthase (SMS). This enzyme transfers phosphorylcholine from PtdCho to ceramide, thereby forming SM and DAG and lowering the levels of ceramide (Kitatani et al., 2008).

8.2 Synthesis of Ceramide and Ceramide 1-phosphate in the Brain

Ceramide is not only present in plasma membrane and endoplasmic reticulum, but also in mitochondria (Sidkind, 2005). In neural cells, synthesis of ceramide begins in the endoplasmic reticulum and continues in the Golgi apparatus and plasma membrane. Thus, ceramide is synthesized in the endoplasmic reticulum (ER) and transported to the PM via Golgi apparatus (Riezman and van Meer, 2004). At the Golgi apparatus, ceramide participates in a metabolic flux toward sphingomyelin, diacylglycerol, and glycosphingolipids, which drives lipid raft formation and vesicular transport toward the plasma membrane. At the plasma membrane, receptor clustering in lipid rafts and formation of endosomes is promoted by the transient synthesis of ceramide. The transport of ceramide from the ER to the Golgi apparatus is facilitated by a specific protein called ceramide transfer protein (CERT). This protein has two domains: one that recognizes ceramide and mediates its intermembrane transfer. It is termed as the START domain. The other domain is known as phosphatidylinositol binding domain (PH domain) (Hanada et al., 2003). This domain selectively binds to phosphatidylinositol-4-phosphate, a lipid that is enriched in the Golgi and that promotes the CERT-mediated delivery of ceramide to the Golgi and plasma membranes. The synthesis of ceramide involves three different mechanisms. The de novo synthesis starts with the condensation of serine with palmitoyl- CoA. This reaction is catalyzed by the serine palmitoyltransferase (SPT). It results in the generation of 3-ketodihydrosphingosine. This metabolite is reduced to dihydrosphingosine by 3-ketoreductase. Acylation of dihydrosphingosine to dihydroceramide is catalyzed by dihydroceramide synthase (Fig. 8.4). Introduction of a 4,5 double bond in the sphingoid base in dihydroceramide is catalyzed by a specific desaturase (Smith and Merrill, 1995; Smith and Merrill, 2002; Vaena de Avalos et al., 2004; Gómez-Muñoz, 2006). Several factors modulate the de novo synthesis of ceramide. These factors include tumor necrosis factor-a, hypoxia, and some chemotherapeutic agents (Huwiler et al., 2001). The transport of ceramide from 222 8 Ceramide and Ceramide 1 Phosphate in the Brain

Salvage synthesis pathway De novo synthesis pathway

Complex Palmitoyl CoA + Serine Sphingomyelin sphingolipids

1 10

3-keto-dihydrosphingosine Ceramide 2 7 Dihydrosphingosine Fatty acyl CoA Sphingosine 3

Dihydroceramide 11 UDP-Gal 4 Ceramide

Ceramide Galactosyceramide PAPS 8 10 5 7 8 Ceramide 1 P 9 6

Sphingomyelin Ceramide 1 P Sphingosine Sulfogalactosyceramide (Sulfatide)

Fig. 8.4 De novo synthesis and salvage pathways of ceramide production.Serine palmitoyltransferase (1); ketodihydrosphingosine reductase (2); ceramide synthase (3); dihydroceramide desaturase (4); UDP-Gal transferase (5); cerebroside sulfotransferase (6); ceramidase (7); ceramide kinase (8); sphingomyelinase (9); sphingomyelin synthase (10); ceramide synthase (11) endoplasmic reticulum to Golgi apparatus by ceramide transport protein CERT is the crucial step in the biosynthesis of ceramide (Perry and Ridgway, 2005). Once synthesized, ceramide is utilized for the formation of complex sphingolipids, through intervention of different biosynthetic enzymes, including glucosyl or galac- tosyl ceramide synthases to form cerebrosides or gangliosides, or it can incorporate a phosphocholine head group from phosphatidylcholine (PtdCho) to form sphingomyelin (SM) through the action of SM synthases. The second mechanism of ceramide formation involves activation of sphingomyelinases (SMases) to form phosphorylcholine and ceramide directly. Brain contains three distinct forms of SMases, which can be distinguished in vitro not only by their optima pH: acid, neutral, and alkaline SMases, but also by their subcellular localization, physicochemical, and kinetic properties. Thus, acid SMases are mainly associated with lysosomes, and have optimal enzymic activity at ~pH 4.5–5. However, plasma membranes also contain an acidic SMase. Many factors, such as cytokines, growth factor, cytotoxic drugs, and cellular stress stimulate acid SMase activity. Neutral SMases have optimal activity at a neutral pH and are mainly located in the plasma membrane, cytosol, endoplasmic recticulum, or nuclear membranes. These enzymes are further classified into Mg2+/Mn2+ dependent or independent SMases. ROS and 8.2 Synthesis of Ceramide and Ceramide 1-phosphate in the Brain 223

RNS (reactive oxygen and nitrogen species), GSH depletion, and generation of hydrogen activate neutral SMase, while antioxidants (such as reduced glutathione GSH and coenzyme Q) inhibit its activity. Various cellular mediators, such as anionic phospholipids, protein kinases, PLA2, Bcl-2, and Bcl-xL and proteases, modulate neutral SMase activity (Clarke and Hannun, 2006). Alkaline SMases occur in intestine and are activated by bile salts. Acid SMase and neutral SMase are associated with signal transduction processes (Marchesini and Hannun, 2004). Ceramide is present in the neural cell nucleus, where it is mainly synthesized by the action of SMases on sphingomyelin. In contrast, alkaline SMase is involved in digestion of dietary SM in the intestine. The alkaline SMase isoform has now been renamed NPP7 because of its similarity to the nucleotide-pyrophosphatase/phosphodiesterase (NPP) family of enzymes (Duan, 2006). Ceramide derived from neutral SMase activation is probably associated with the modulation of CAPK and

MAP kinases, PLA2, and CAPP while ceramide synthesized through the activation of acid SMase appears to be primarily involved in NF-kB activation (Ballou et al., 1996). Based on pharmacological studies, it is proposed that there occurs an interplay (cross-talk) between ceramide signaling and phospholipid signaling (Fig. 8.3) (Farooqui and Horrocks, 2007; Farooqui, 2009). Thus, in neural and non-neural cell cultures, ARA, a product of PLA2 catalyzed reaction, stimulates SMase (Robinson et al., 1997), and ceramide, a product derived from the hydrolysis of sphingomyelin by SMase, stimulates PLA2 activity (Hayakawa et al., 1996; Sato et al., 1999; Jayadev et al., 1997; Malaplate-Armand et al., 2006; Huwiler et al., 2001). This interplay between metabolite of glycerophospholipid and sphingolipid metabolism can be blocked with cPLA2 and SMase inhibitors as well as by their antisense oli- gonucleotide (Vanags et al., 1997; Gomez-Muñoz, 1998), supporting the view that lipid mediators of glycerophospholipid metabolism modulate sphingolipid metabolism and vice versa. Targets for ceramide-mediated signaling include many enzymes, such as PKCz, kinase suppressor of Ras, cRaf, cathepsin D, and CAPPs composed of protein phosphatase 1 (PP1) and protein phosphate 2A (PP2A) (Bourbon et al., 2000; Zhang et al., 1997; Huwiler et al., 1996; Heinrich et al., 1999; Ogretmen and Hannun, 2004; Kitatani et al., 2008; Chalfant et al., 1999). CAPPs belong to the family of Ser/Thr protein phosphatases, and ceramide mediates the activation of CAPPs leading to the dephosphorylation of various proteins, such as Bax, Bcl-2, PKCa, Rb protein, SR protein, and Akt, which ultimately modulate diverse cellular processes including apoptosis, cell senescence, the cell cycle, and cellular differentiation (Ruvolo, 2003). In PKC signaling, salvage pathway-derived ceramide leads to attenuation/termination of activation of the p38 MAPK through dephosphorylation. Pharmacologic and siRNA-based studies indicate the involvement of PP1 catalytic isoforms (PP1c-a, PP1c-b, and PP1c-g) in mediating the effects of ceramide on p38 MAPK (Kitatani et al., 2006, 2008). Moreover, the PP1-dependent dephosphorylation of p38 likely depends on LASS5 because knock-down of LASS5 attenuates PP1 dephosphorylation of p38 concomitant with suppression of ceramide synthesis, following stimulation with phorbol ester. This suggests that there is a link between the salvage pathway and PP1, and this link is crucial in control of p38 dephosphorylation (Kitatani et al., 2008). Additional downstream targets for 224 8 Ceramide and Ceramide 1 Phosphate in the Brain

Modulation of protein kinase C

Modulation of protein phosphatase 1 & 2

Modulation of PLA2 & COX-2

Signaling targets for ceramide Modulation of cRaf

Modulation of cathepsin D

Modulation of IL-6 & IL-2

Modulation of cMyc, c-Fos, & c-Jun

Fig. 8.5 Signaling targets for ceramide

ceramide action include COX, IL-6 and IL-2 gene expression, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators (Fig. 8.5) (Ballou et al., 1996). The third mechanism of ceramide synthesis involves the sphingosine salvage pathway (Kitatani et al., 2008). In this pathway, sphingosine (formed from the metabolism of complex sphingolipids) is recycled to ceramide through the action of ceramide synthase. A number of enzymes are associated with the salvage pathway. These enzymes include SMases, cerebrosidases, ceramidases, and ceramide synthases. The salvage pathway not only regulates levels of various sphingolipid- derived lipid mediators, but it also modulates the synthesis of ceramide and subsequent ceramide-dependent cellular signals. Treatment of cultured cell of neural and non-neural origin with stressful stimuli, including proinflammatory and proapoptotic cytokines (TNF-a , IL-1b , FAS ligand, and interferon g, doxorubicin vitamin D and retinoic acid, and serum-deprivation) increases levels of ceramides (Mao and Obeid, 2008). Preventing increase in ceramide levels by inhibitors blocks cell-growth arrest and/or apoptosis in response to these stressful stimuli (Mao and Obeid, 2008). Ceramide can also be transformed into glucosylceramide in the Golgi, but this process does not require CERT (D’Angelo et al., 2007). Importantly, non- vesicular transport of glucosylceramide from its site of synthesis (early Golgi) to distal Golgi compartments is carried out by four-phosphate adaptor protein (FAPP2), four-phosphate adaptor protein, controlling the synthesis of glycosphingolipids, which might essentially play crucial roles in determining the lipid composition of the plasma membrane (D’Angelo et al., 2007). Above described pathways (the de novo pathway, the SMase pathway, and the salvage pathway) individually or coordinately facilitate the generation of ceramide that leads to ceramide-mediated signaling and subsequent modulation of downstream cell responses. The conversion of ceramide to ceramide 1-phosphate is catalyzed by ceramide kinase (CerK) (Fig. 8.6). CerK is found in both the microsomal membrane fraction, and the cytosolic fraction of cells (Mitsutake et al., 2004). CERK is stimulated by calcium, and optimally active at neutral pH. CERK activity is regulated through interactions with lipids (PtdIns-4,5-P2) and cardiolipin. Molecular cloning studies 8.2 Synthesis of Ceramide and Ceramide 1-phosphate in the Brain 225

CH2OH

NH Cer O ATP Pi Cer kinase Cer phosphatase ADP

O of OH c tion s CH2O P OH ni ma ct lation toge du O

NH effe flam Mi in Mo C1P O

Inhibition of apoptosis Modulation of enzymes Cell cycle arrest

Fig. 8.6 Synthesis and roles of cermide 1 phosphate

indicate that CERK contains a pleckstrin homology (PH) domain at the N terminus, which binds with PtdIns-4,5-P2 (Sugiura et al., 2002), and this PH domain is essential for the localization of CERK to specific membranes in cells (Carre et al., 2004). In addition, CERK also interacts with cardiolipin, but the physiological significance of these interactions remains unknown. CERK is upregulated in response to phosphorylation of the cyclic AMP response element-binding protein (CREB) and iden- tifies a CREB binding site on the promoter region (Euskirchen et al., 2004). Thus, elevation in the expression of CERK may be an important mechanism of increasing C1P levels in the cell if ceramide is available. It is postulated that C1P traffics from the Golgi apparatus along the secretory pathway to the plasma membrane, and then released into the extracellular milieu to bind with acceptor proteins such as albumin or lipoproteins (Boath et al., 2008). In contrast to the effects of ceramide, C1P produces opposite effects. C1P produces mitogenic effects and has prosurvival properties (Arana et al., 2010), where as ceramide produces apoptosis. Thus, in murine bone marrow-derived macrophages, C1P not only stimulates DNA synthesis, but also modulates cell division. C1P induces rapid phosphorylation of protein kinase B (Akt), an enzyme that is a key mediator of pro-survival, proliferative, and metabolic effects and downstream target of phosphatidylinositol 3-kinase (PtdIns3-K). Selective inhibition of PtdIns3-K prevents both DNA synthesis and cell growth 226 8 Ceramide and Ceramide 1 Phosphate in the Brain

(Gangoiti et al., 2008). C1P stimulates Akt-mediated phosphorylation of GSK-3b, and this stimulation is also abolished by inhibition of PtdIns3-K. In addition, C1P also increases the expression of cyclin D1 and c-Myc. These are two major targets of GSK-3b that are closely associated with regulation of cell proliferation. Similarly, C1P also modulates phosphorylation of the MAPK, extracellularly regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK) and inhibition of these enzymes is also coupled with macrophage proliferation. C1P stimulates the activity of NF-kB, and inhibitors of this transcription factor not only prevent NF-kB stimulation, but also inhibit macrophage proliferation. Collective evidence suggests that C1P stimulates macrophage proliferation through activation of the PtdIns3-K/PKB, ERK, and JNK pathways, and that GSK-3b, c-Myc, cyclin D1, and NF-kB are important downstream effectors in this process (Gangoiti et al., 2008). In addition, ceramide and C1P are important mediators of inflammatory responses, involving the stimulation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (ARA) and the production of prostaglandin. Thus, C1P produces a dramatic increase (>15-fold) in cPLA2-a activity. This activation is highly specific. With the exception of PtdIns 4,5-bisphosphate, no other lipid has a significant effect on cPLA2-a activity (Subramanian et al., 2005). The effect of C1P on cPLA2 is through its interaction with the CaLB/C2 domain of cPLA2 and facilitates its translocation. These interactions require Ca2+. In the absence of Ca2+, C1P is not able to activate cPLA2 (Subramanian et al., 2005; Nakamura et al., 2006). Lyso- phosphatidylcholine, the other product of cPLA2 catalyzed reaction, is transformed into platelet activating factor, another proinflammatory lipid mediator (Farooqui, 2009; Arana et al., 2010) (Fig. 8.3). All these processes are supported and promoted by intracellularly generated ceramide and C1P. Microglial cells in brain and macrophages in visceral tissues are involved in induction, maintenance, and unregulated chronic inflammation. Exogenous application of C1P to RAW 264.7 macrophage cultures results in modulation of cell migration. The migration process involves specific receptor, which is coupled to

Gi proteins and causes phosphorylation of extracellularly regulated kinases 1 and 2, and Akt upon ligation with C1P. Inhibition of either of these pathways completely abolishes C1P-stimulated macrophage migration (Fig. 8.7). Collective evidence suggests that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration (Grando et al., 2009; Arana et al., 2010). In addition, C1P stimulates the DNA-binding activity of NF-kB, and blockade of this transcription factor results in complete inhibition of macrophage migration. Synthesis of a C1P analog called phosphoceramide analog-1 (PCERA-1) has been recently reported (Goldsmith et al., 2009; Levi et al., 2010). This analog is a potent inhibitor of inflammation. It is proposed that PCERA-1 acts onmac- rophages through its own GPCR receptor (Fig. 8.7). Although very little is known about the properties of C1P and PCERA-1 receptors, based on pharmacological studies, it is proposed that PCERA-1 receptor is different from C1P receptor. More studies are needed on characterization of C1P and PCERA-1 receptors (Goldsmith et al., 2009; Levi et al., 2010). 8.3 Degradation of Ceramide and Ceramide 1-phosphate in the Brain 227

TNF-α PCERA-1 C1P ?

er rt

SM S C1P-R anspo FA Tr PCERA-1-R e Gγ Gα AC

SMas cAMP ATP CER K 3

2 Ceramide C1P PtdIns3K 5-P ERK

JNK

PtdIns-4.5-P NF-kB p38 CREB PtdIns-1,4. Akt

IKK GSK3 iNOS Cell migration Apoptosis Inflammation Modulation of TNF-a and IL-10

Fig. 8.7 Hypothetical model showing activities of ceramide 1 phosphate receptor and phosphoceramide analog 1 (PCERA-1) receptor in macrophages. Ceramide 1 phosphate receptor (C1P-R); ceramide 1 phosphate (C1P); phosphoceramide analog 1 (PCERA-1); phosphoceramide analog 1receptor

(PCERA-1-R); phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2); phosphatidylinositol 1,4,5, triphosphate (PtdInsP3); protein kinase B (Akt); c-Jun terminal kinase (JNK); ceramide kinase (CER K); adenylyl cyclase (AC); extracellular signal regulated kinase (ERK); nuclear factor-kappaB (NF-kB); IkappaB kinase (IKK); glycogen synthase kinase 3 (GSK3); inducible nitric oxide synthase (iNOS); cAMP response element binding protein (CREB); and MAP kinase (p38)

8.3 Degradation of Ceramide and Ceramide 1-phosphate in the Brain

Ceramidases are enzymes that cleave the amide-linked fatty acid in ceramide to generate sphingosine and free fatty acid (Fig. 8.3). Thus, they regulate cellular levels of ceramide, sphingosine, and sphingosine 1-phosphate. Five different ceramidases, namely, one acid ceramidase (ACER1), one neutral ceramidase (ACER2), and three alkaline ceramidases (ACER3) have been reported to occur in mammalian tissues. These enzymes are encoded by five distinct genes. ACER1 (mol. mass ~53– 55 kDa) catalyzes the lysosomal hydrolysis of ceramide to sphingosine and free fatty acid. ACER1 is a heterodimeric protein that has a non-glycosylated a subunit (13 kDa) and a glycosylated b subunit (40 kDa) that is processed into the mature enzyme posttranslationally. Because ceramide degradation is the only catabolic source of intracellular sphingosine, ACER1 activity is postulated to be the rate- limiting step in determining the intracellular levels of sphingosine, and the enzyme plays a central role in the maintenance of cellular ceramide levels. ACER2 is a glycoprotein located in the plasma membrane and has apparent molecular masses of 118–142 kDa (Hwang et al., 2005). Three ACER3 are 264–275 amino acid proteins with molecular masses of ~31 kDa. It contains several putative transmembrane domains, and it is localized to the endoplasmic reticulum (Sun et al., 2007). 228 8 Ceramide and Ceramide 1 Phosphate in the Brain

8.4 Roles of Ceramide and Ceramide 1-phosphate in the Brain

In mitochondria, ceramide forms large protein permeable channels with planar phospholipid and mitochondrial outer membranes and modulates numerous functions (Sidkind, 2005). It enhances generation of ROS, induces alteration of calcium homeostasis of mitochondria, promotes ATP depletion, induces collapse in the inner mitochondrial membrane potential, modulates activities of various components of the mitochondrial electron transport chain, and release of intermembrane space proteins (Sidkind, 2005). Mounting evidence suggests that generation of ceramide through the activation of SMases mediates clustering of lipid rafts to form large ceramide-enriched platforms, in which transmembrane signals are transmitted or amplified in neural and non-neural membranes (Dumitru et al., 2007; Li et al., 2010). ROS-generating enzymes (NADPH oxidases) are localized to membrane rafts, and the integrity of these rafts is required for the release of cellular ROS. Both ceramide and ROS are involved in the modulation of intracellular ion channels, cell proliferation, and apoptotic cell death. Ceramide triggers the generation of ROS and increases oxidative stress in neural and non-neural cells. The inhibition of ROS systems by treatment with antioxidants impairs both SMase activation and ceramide production. These observations support the concept that ceramide-enriched raft platforms are important redox signaling platforms that amplify activation of NADPH oxidases and sphingomyelinases (Dumitru et al., 2007; Li et al., 2010). Involvement of ceramide in formation of redox signaling platforms amplifying oxidative stress may be associated with many neurological disorders (Farooqui, 2009). Ceramide also selectively modulates the phosphorylation state members of the MAPK superfamily, promoting dephosphorylation of ERK1/2 and hyperphosphorylation of p38 MAP kinases, but has no affect on the phosphorylation of JNK or ERK5 (Stoica et al., 2005). Inhibitors of the p38 MAP kinase pathway (SB-202190 or SB-203580) and an inhibitor of the ERK1/2 pathway (U0126) retard ceramide-mediated neuronal death. These p38 and ERK1/2 inhibitors appear to block ceramide-activated apoptotic signaling upstream of the mitochondria, as they not only attenuate mitochondrial release of cytochrome c, Omi, AIF, and SMAC, but also reduce ceramide- mediated caspase-3 activation (Stoica et al., 2005). In the effector phase of apoptotic cell death, the breakdown of sphingomyelin to ceramide in mitochondrial membrane may be a consequence of lipid scrambling, and may regulate apoptotic body formation. Ceramide generation at the plasma membrane exerts distinct and specific functions, including aggregation of the Fas receptor, and effects on protein kinase C (PKC), apoptosis, and cell cycle arrest (Hannun and Obeid, 2002) (Fig. 8.2). Studies on the regulation of PKC activity by ceramides have been controversial and depend upon membranes and cell types used. Another important target of ceramide is phospholipase D (PLD), which is a key regulatory enzyme responsible for the generation of phosphatidic acid (PtdH), a potent mitogenic metabolite, and a precursor of lyso- PtdH and diacylglycerol (DAG) (Gómez-Muñoz, 1998, 2006). Mounting evidence suggests that ceramide generation serves many different functions at distinct locations 8.5 Ceramide in Neurological Disorders 229 in the cell. Since biomembranes have the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of natural ceramide species, the topology and membrane sidedness of ceramide formation may be crucial determinants of its importance in neural membranes. Accumulating evidence suggests that ceramides participate in a variety of cellular functions ranging from proliferation and differentiation to growth arrest, inflammation, and apoptosis (Hannun and Obeid, 2002; Yu et al., 2000; El Alwani et al., 2006). Ceramides also modulate cellular senescence, oxidative stress responses, and nitric oxide signaling (Mathias et al., 1998). The generation of endogenous ceramide is regulated by TNF-a, CD95 (APO-1/Fas), ionizing and ultraviolet radiation, and chemotherapeutic drugs. The targets for ceramide include specific kinases, phosphatases, phospholipases, cyclooxygenases, and various transcription factors including AP1, NF-kB, and IL-6 (Ohanian and Ohanian, 2001; Sawai et al., 2005). Above enzymes and transcription factors closely are associated with regulation of proliferation, differentiation, growth arrest, inflammation, and apoptosis. As stated above, C1P is a mitogenic metabolite. It exerts its effects through the stimulation of the mitogen-activated protein kinase kinase (MEK)/Extracellularly regulated kinases 1–2 (ERK1-2), PtdIns3-K/Akt, and c-Jun terminal kinase (JNK) pathways (Gangoiti et al., 2008). In addition to its mitogenic effect, C1P also modulate apoptotic cell death. Its low concentrations inhibit apoptosis, whereas at high concentrations it promotes apoptosis (Gómez-Muñoz, 1998, 2006). As mentioned above, C1P produces proinflammatory effects by interacting with calcium-binding regions of cPLA2 and leading to its translocation of this enzyme from the cytosol to the perinuclear region in cells. It is interesting to note that cPLA2 and CERK are not only activated by PtdIns-4,5-P2, but translocated from cytosol to perinuclear membranes supporting the view that these enzymes play an important role in the induction of inflammation. In macrophages, C1P modulates macrophage migration in neural and non-neural tissues. In brain, activation of microglial cells and macrophages is involved a number of chronic diseases that are characterized by activation of macrophages and up-regulation of chronic inflammation (Farooqui et al., 2007a, b; Farooqui, 2009). These include neurodegenerative diseases and autoimmune diseases (see below) (Farooqui, 2010), as well as tumor progression and metastasis (Condeelis and Pollard, 2006).

8.5 Ceramide in Neurological Disorders

Alterations in neural membrane sphingolipid metabolism and composition have been reported to occur during neurodegenerative process in neurotraumatic (ischemia, spinal cord trauma, and head injury) and neurodegenerative diseases (Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis). These alterations are accompanied by significant increase in ceramide (Table 8.2) (Cutler et al., 2002, 2004; He et al., 2010; Brugg et al., 1996), which plays important roles in neural cell proliferation, cell cycle arrest, apoptosis, and angiogenesis modulating 230 8 Ceramide and Ceramide 1 Phosphate in the Brain

Table 8.2 Levels of ceramide in neurological disorders Disease Levels of ceramide Reference Ischemia Increased Yu et al., 2000; Feng and Le Blanc, 2006 Alzheimer disease Increased Han et al., 2002; Cutler et al., 2004; He et al., 2010; Haughey et al., 2010 Parkinson disease Increased Brugg et al., 1996 Amyotrophic lateral sclerosis Increased Cutler et al., 2002 Major depressive disorder Increased Kornhuber et al., 2009 KA-induced neurotoxicity Increased Guan et al., 2006; He et al., 2007

Stroke

Alzheimer disease

Multiple sclerosis

Ceramide in neurological disorders Amyotrophic lateral sclerosis

HIV-associated dementia

Batten disease

Kainic acid-induced neurotoxicity

Fig. 8.8 Involvement of ceramide in neurological disorders. Upward arrow indicate increase in ceramide levels cell survival and neurodegeneration. Although, the molecular mechanism associated with ceramide-mediated neurodegeneration is not fully understood, based on many studies it is proposed that increase in intensity of interactions among phospholipid-, sphingolipid-, and cholesterol-derived lipid mediators along with increase in oxidative stress, neuroinflammation, and alterations in energy metabolism may be responsible for neurodegeneration in neurotraumatic and neurodegenerative diseases (Farooqui, 2009, 2010). There is mounting evidence that ceramide-induces oxidative stress, insulin resistance, impairment in energy metabolism, and apoptotic cell death in Alzheimer disease (Tong and de la Monte, 2009). At present, it is not known whether increase in ceramide occurs at the onset of neurological disorder (primary effect), or at the end of neurodegenerative process (secondary effect) (Fig. 8.8). Many neurotraumatic and neurodegenerative diseases are accompanied by the activation of microglia and astrocytes, which contribute to the onset of neuroinflammation through the production of proinflammatory cytokines (TNF-a and IL-b). These cytokines and chemokines stimulate SMases and PLA2 activities leading to the generation of high levels of ceramide and phospholipid-derived proinflammatory lipid mediators, such as eicosanoids, and platelet-activating factors 8.5 Ceramide in Neurological Disorders 231

IGF-1 Glu TNF-α

TNF-R NMDA-R PtdCho SM IGF-1 R

Reacylation 2+

2 Ca SMase IRS-1 Lyso-PtdCho cPL A + + Ceramide PtdIns3K ARA Mitochondrial IκK dysfunction COX-2 p65p50 C1 P PAF Eicosanoids NF-κB ROS Degradation Akt Caspase cascade

IκB-P Growth, glucose and fa t life span, insulin resistance

Apoptosis metabolism, stress response ,

Neuroinflammation and oxidative stress Transcription of genes related to inflammation and oxidative stress NF-kB RE TNF-a FOXO COX-2, sPLA2, SOD iNOS, MMP, VCAM-1and IL-1β IL-6

Neurodegeneration Nucleus

Fig. 8.9 Hypothetical diagram showing involvement of PLA2 and SMase-mediated degradation of membrane phospholipid and sphingolipid degradation and development of insulin resistance. N-Methyl-d-aspartate (NMDA); N-Methyl-d-aspartate receptor (NMDA-R); insulin-like growth factor-1 (IGF-1); insulin-like growth factor-1 receptor (IGF-1R); tumor necrosis factor-a (TNF-a); tumor necrosis factor-a-receptor (TNF-R); phosphatidyl-choline (PtdCho); lyso-phosphatidylcholine (lyso-PtdCho); cytosolic phospholipase A2 (cPLA2); sphingomyelin (SM); sphingomyelinase (SMase); ceramide 1 phosphate (C1 P); nuclear factor-kB (NF-kB); insulin receptor substrate (IRS-1); phosphatidylinositol 3 kinase (PtdIns3K); protein kinaase B (Akt); forkhead transcription factors (FOXOs); platelet activating factor (PAF); arachidonic acid (ARA); cyclooxygenase-2

(COX-2); and reactive oxygen species (ROS); secretory phospholipase A2 (sPLA2); superoxide dismutase (SOD); inducible nitric oxide synthase (iNOS); matrix metalloproteinase (MMP); vascular adhesion molecule-1 (VCAM-1); interleukin-1b (IL-1b); and interleukin-6 (IL-6)

(Fig. 8.9). I propose that increased intensity of interplay among sphingolipid, phospholipid, and cholesterol-derived lipid mediators may contribute to neurodegeneration in neurotraumatic and neurodegenerative diseases (Farooqui, 2010; Farooqui and Farooqui, 2011).

8.5.1 Ceramide in Ischemic Injury

Stroke (ischemia) is a metabolic insult caused by severe reduction or blockade in cerebral blood flow leading to long-term disability and death. The blockade of cerebral blood flow not only decreases oxygen and glucose delivery to brain but also results 232 8 Ceramide and Ceramide 1 Phosphate in the Brain in the breakdown of blood–brain barrier and buildup of potentially toxic products in brain (Farooqui, 2010). Approximately 12% of strokes are hemorrhagic (rupture of a cerebral blood vessel), whereas the remaining 88% are ischemic and result from occlusion of a cerebral artery (either thrombolic or embolic). Depletion of ATP not only results in collapse of ion gradients, and excessive release of neurotransmitters such as dopamine and glutamate, but also leads to neuronal death and development of an infarct. Ischemic injury not only enhances the production of ceramide, but also reduces size of infarct after stroke (Adibhatla and Hatcher, 2010). Ceramide mediates cell-cycle arrest by upregulating of cyclin-dependent kinase (Cdk) inhibitors p21 and p27 through activation of protein phosphatase 2A (PP2A). Treatment with tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of PLC and PLD, increases ceramide levels after transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR) probably due to inhibition of sphingomyelin synthase (SMS) (Adibhatla and Hatcher, 2010). It is also reported that ischemic injury-induced ceramide production is largely restricted to glia cells in the rat hippocampus. N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), an inhibitor of proteolysis, but not ketamine or fumonisin B1, blocks the ceramide pathway and its downstream molecules, JNK and PP2A (Feng and Le blanc, 2006; Tian et al., 2009). At present no information is available on C1P in ischemic brain injury.

8.5.2 Ceramide in Alzheimer Disease

Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by deposition of amyloid beta-peptide (Ab), accumulation of amyloid neuritic plaques, neurofibrillary tangles, inflammation, oxidative stress, loss of synapses, degeneration of neurons in the nucleus basalis, and hippocampus, progressive impairment of memory, and severe dementia. Age is the most important factor that predisposes persons to the nonfamilial form of the disease. The pathogenesis of AD is tightly linked to Ab deposition, oxidative stress, and inflammation, but it remains unclear as to how these factors result in neuronal dysfunction and death. Levels of ceramide are markedly increased in AD. This increase in ceramide is accompanied by elevation in acid sphingomyelinase (ASMase) and acid ceramidase expression in AD (Han et al., 2002; Cutler et al., 2004; He et al., 2010). Importantly, significant correlations are observed between the brain ASMase activity and the levels of Ab peptide, and hyperphosphorylated t protein in AD patients. Treatment of neural cell cultures with Ab oligomers not only activates ASMase, but also increases ceramide and induces apoptotic cell death (He et al., 2010). Studies on the effect of statins, the well-known inhibitors of cholesterol synthesis, indicate that these drugs affect g-secretase activity, increasing the catabolism of APP and reducing the risk of AD (Buxbaum et al., 2002). The proteolytic activity of g-secretase is stimulated by cerebrosides, anionic glycerophospholipids, and cholesterol, pointing to the involvement of lipid rafts in the modulation of secretases (Kalvodova et al., 2005). 8.5 Ceramide in Neurological Disorders 233

These studies are supported by reports on the increase in levels of ceramide in the cerebrospinal fluid (CSF) of AD patients compared to age-matched control subjects (Satoi et al., 2005). Based on the effect of retinoic acid, it is suggested that increase in ceramide level in the CSF from AD patients may be due to astroglial cell activation (Satoi et al., 2005). Type 2 diabetes mellitus increases the risk of AD. Although, the nature of link between type 2 diabetes mellitus and AD risk remains unclear, several mechanisms including insulin and insulin resistance, inflammatory cytokines, and oxidative stress may contribute to this link (Haan, 2006; de la Monte et al., 2010). In brain, IGF-I receptors are found not only in the hippocampus and parahippocampal areas, but also in the amygdala, cerebellum, and cortex. IGF-I enhances excitatory synaptic transmission in the CA1 region of the hippocampus and improves the spatial learning, memory, and cognitive function by inducing neurogenesis and neuroplasticity in the hippocampus (Ramsey et al., 2005; Aberg et al., 2000). These processes involve interactions between insulin or insulin-like peptides with insulin receptors (IGF-R) leading to recruitment of insulin receptor substrate (IRS) proteins, subsequently activating two major signaling pathways: (a) the PtdIns3K-pathway and (b) the MAPK-pathway (White, 2003; de la Monte et al., 2010). PtdIns3K activity mediates activation of Akt, which phosphorylates and thereby deactivates forkhead transcription factors (FOXOs). These transcription factors regulate transcription of many genes involved in glucose and lipid metabolism, growth, stress response, and the aging process. Thus, insulin-like signaling controls many processes through FOXO regulation and other signaling cascades (Huang and Tindall, 2007; Belgardt et al., 2010). Chronic intake of high energy and high fat diet coupled with lack of exercise leads to hyperlipidemia and hyperglycemia, which through several mechanisms (including JNK1 activation) reduce cellular insulin sensitivity, thereby disrupting metabolic homeostasis and inducing obesity (metabolic syndrome), which may influence the onset of AD through their effects on oxidative stress, hypertension, insulin resistance and neuroinflammation (Haan, 2006). Insulin functions not only by controlling neurotransmitter release at the synapses, but also by activating signaling pathways associated with learning and long-term memory. Insulin regulates processes such as neuronal survival, energy metabolism, and plasticity (de la Monte, 2009). It is proposed that AD represents a form of diabetes mellitus that selectively afflicts the brain (de la Monte, 2009). It is called as type 3 diabetes (de la Monte and Wands, 2008). Studies on human postmortem brain tissue link many of the characteristic molecular and pathological features of AD to reduced expression of the insulin and insulin-like growth factor (IGF) genes and their corresponding receptors (Lester-Coll et al., 2006). Furthermore, intracerebral administration Streptozotocin (STZ) produces chemical depletion of insulin and IGF signaling mechanisms combined with oxidative injury. These changes produce neurodegeneration similar to AD (de la Monte et al., 2010). Alterations in insulin sensitivity and signaling may contribute to the instability of synapses in AD (Kroner, 2009). Administration (i.p. injection) of C2Cer:N-acetylsphinganine or its inactive dihydroceramide analog (C2DCer) in Long Evans rat pups results in hyperglycemia, hyperlipidemia, mild insulin resistance, reduce brain lipid content, and increase 234 8 Ceramide and Ceramide 1 Phosphate in the Brain ceramide levels in liver, brain, and serum (de la Monte et al., 2010). It is suggested that ceramide toxicity promotes impairment in neurocognitive function and insulin/ IGF signaling needed for neuronal survival, plasticity, and myelin maintenance in the brain. At present, no information is available on levels of C1P in patients with AD.

8.5.3 Ceramide in Parkinson Disease

Parkinson disease (PD) is a chronic and progressive neurological disorder characterized by uncontrolled muscle tremor, rigidity, and bradykinesia. It affects over 1% of people over the age of 65 years. PD is characterized by degeneration of dopaminergic neurons in substantia nigra due to oxidative stress (Farooqui, 2010). Typical PD cases have intracellular proteinaceous inclusions called Lewy bodies and Lewy neurites in the brainstem and cortical areas. Clinical manifestations of PD include bradykinesia, rigidity, tremor, and postural instability. No information is available on levels and effect of endogenous ceramide and C1P in PD. Endogenous toxins and other stress signals activate the sphingomyelin pathway increasing the levels of ceramide, an important regulator of cell death. Tretment of catecholaminergic cell with C2-ceramide inhibits the phosphorylation of PtdIns3K/AKT and ERK, followed by activation of JNK phosphorylation, and de-phosphorylation (activation) of GSK3b. Although NT3 and IGF-1 increase survival at early time points, only IGF-1 is capable of attenuating C2-ceramide-mediated neuronal death, and this neuroprotection is associated to strong and permanent activation of AKT and inhibition of GSK3b (Arboleda et al., 2009, 2010). Like AD, levels of C1P have not been determined in brains from PD patients.

8.5.4 Ceramide in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative and fatal human disorder characterized by progressive loss of motor neurons. It is characterized by the presence of axonal spheroids and perikaryal accumulations/aggregations comprised of the neuronal intermediate filament proteins, neurofilaments, and peripherin (Beaulieu and Julien, 2003). Although, the molecular mechanism associated with neurodegeneration in ALS is not known, multiple pathophysiological mechanisms, including oxidative stress, mitochondrial impairment, protein aggregation, axonal dysfunction, reactive astrocytosis, and mutant superoxide dismutase expression, inflammation, and apoptotic cell death, have been suggested (Farooqui, 2010). Increase in levels of sphingomyelin and ceramides have been reported in the spinal cords of ALS patients and in a transgenic mouse model (Cu/ZnSOD mutant mice) (Cutler et al., 2002). It is well known that p75NTR-mediated apoptotic pathway in motor neurons is coupled with increased ceramide synthesis and elevation in cytochrome c release (Casaccia-Bonnefil et al., 1996; Bhakar et al., 2003; Pehar et al., 2007). 8.5 Ceramide in Neurological Disorders 235 p75NTR-mediated ceramide synthesis in motor neurons is accompanied by activation of the neutral isoform of SMase and subsequent JNK activation (Brann et al., 2002). However, in p75NTR-expressing NIH-3T3 and PC12 cell lines, the acid isoform of SMase is linked to ceramide synthesis induced by p75NTR signaling (Dobrowsky and Carter, 1998). Accumulating evidence suggests that p75NTR is able to activate different SMases depending on the cell type. Interestingly, the spinal cord of ALS patients and SOD1G93A mice exhibit a remarkable increase in ceramides and cholesterol esters, which have been shown to sensitize motor neurons to different death stimuli, including oxidative and excitotoxic insults (Cutler et al., 2002). Therefore, NGF signaling through p75NTR and the subsequent increase in ceramide production may also modulate the sensitivity of motor neurons to other apoptotic stimuli. It is also suggested that increase in sphingomyelin and ceramide levels along with increase in cholesterol esters and lipid mediators of phospholipid metabolism intensify neuroinflammation and oxidative stress in ALS, resulting in neurodegeneration (Cutler et al., 2002; Pehar et al., 2007; Farooqui, 2010). At present, nothing is known about the level and involvement of C1P in ALS.

8.5.5 Ceramide in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease characterized by loss of oligodendrocytes that maintain the myelin sheath as well as oxidative stress-mediated damage to axons with the loss of neurons. ROS generated by activated macrophages and microglial cells are thought to play a major role in damaging myelin and myelin-producing cells, oligodendrocytes, in MS (Qin et al., 2010). Studies on the determination of ceramide by quantitative liquid chromatography-tandem mass spectrometry technology indicate that levels of ceramide and sphingosine are increased, while C1P levels are decreased in affected white matter and plaques from brains of multiple sclerosis patients (Qin et al., 2010; Hicks et al., 2006). Although, the significance of alterations in ceramide and its metabolite is not fully understood, it is suggested that increase in ceramide levels may be due to activation of N-SMase and ROS-mediated stimulation of death signal through the activation of stress-related kinases (JNK/SAPK), mobilization of Ca2+, stimulation of caspases, and upregulation of a number of death proteins (Bax, Bad) (Ruvolo, 2003).

8.5.6 Ceramide in HIV-1

Human immunodeficiency virus type 1 (HIV-1) infection affect central nervous system and often cause HIV dementia (HIVD). Alterations in sphingolipid metabolism, along with alterations in cytokines and chemokines have been linked to neuronal dysfunction and death in HAD. Brain tissues and CSF from patients with HIVD show increased oxidative stress with abnormal accumulation of sphingomyelin 236 8 Ceramide and Ceramide 1 Phosphate in the Brain and ceramide (Haughey et al., 2004). This observation is supported by studies on HIV-1 coat protein gp120 (glycoprotein 120)-mediated neuronal apoptosis in the HIVD. It is reported that CXCR4-NADPH oxidase-superoxide-NSMase-ceramide pathway is coupled with neuronal apoptosis in HIVD (Jana et al., 2009).

8.5.7 Ceramide in Batten Disease

Batten disease is a rare and fatal inherited disorder of the brain characterized by blindness, seizures, cognitive decline, and early death. The disease begins in childhood and accompanied by changes in personality and behavior, slow learning, and clumsiness, or stumbling. Cognitive and motor decline and seizures in Batten disease may be linked to a buildup of substances called lipopigments, which are made up of fats and proteins. Although, the nature of the defect in Batten disease remains unknown, a common form of Batten disease is caused by mutations in CLN3, a multipass transmembrane protein of unknown function. Significant increase in ceramide levels has been reported to occur in brain tissue from Batten disease patients (Puranam et al., 1997). No information is available on C1P levels in Batten disease.

8.5.8 Ceramide in Major Depressive Disorders

Major depressive disorder (MDD) is a severe mood disorder that is accompanied not only by increase in oxidative stress, but also by increase in proinflammatory cytokines (IL-1b), and increase in serum PLA2. MDD represents a major risk factor for both the development of cardiovascular disease, osteoporosis, and for the development of reversible hippocampal atrophy. Activity of acid SMase is also increased in peripheral blood cells of patients with major depressive disorder (MDD) and several antidepressant drugs have been reported to inhibit acid SMase activity. It is proposed that ASM/ceramide pathway may be associated with the pathogenesis of MDD (Kornhuber et al., 2009).

8.5.9 Ceramide in Kainic Acid Neurotoxicity

Kainate (KA)-mediated neurotoxicity also produces a significant increase in ceramide in the hippocampus, at 1 day and 3 days postinjection. Dense labeling to ceramide is observed in the nucleus and cytoplasm of injured neurons (arrows in B), consistent with increased levels of ceramide in the kainate lesioned hippocampus (Fig. 8.10). Determination of ceramide levels in hippocampus of KA-injected rats indicate significant increases in ceramides with different molecular species including 8.6 Conclusion 237

Fig. 8.10 Ceramide immunostained sections of field CA3 of the hippocampus from an untreated rat (a), and a rat which has received intracerebroventricular injection of kainate 1 day earlier (b). Dense labeling to ceramide is observed in the nucleus and cytoplasm of injured neurons (arrows in b), consistent with increased levels of ceramide in the kainate lesioned hippocampus (Guan et al., 2006). Scale = 50 mm

16:0, 18:0, 20:0, 22:0, and 24:1 fatty acids compared with control rats (Guan et al., 2006). KA-mediated increase in ceramide levels involves increased expression and activity of the first enzyme in the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT) (He et al., 2007). Inhibition of SPT by L-cycloserine or myri- ocin produces a significant neuroprotective effect for a short time after kainate toxicity (He et al., 2007). It is suggested that increase in ceramide may facilitate the opening of the mitochondrial permeability transition pores, which disrupts the transmembrane potential, facilitating release of cytochrome c and activation of caspase-3 in apoptotic cell death (He et al., 2007; Farooqui, 2009). In recent years, investigators have been using lipidomic, proteomic, and genomic procedures with increasing frequency not only to identify and determine levels of ceramide and its metabolites, but also for developing diagnostic test in CSF from patients with neurotraumatic and neurodegenerative diseases (Farooqui, 2010). The use of lipidomic and proteomic strategies for characterizing ceramide-metabolizing proteins in subcellular organelles of human brain and CSF may provide new information on molecular species of ceramide and properties of ceramide-metabolizing enzymes. Combining lipidomics, proteomics, and genomics may greatly enhance the existing knowledge of molecular mechanism associated with neurotraumatic and neurodegenerative diseases and may increase the likelihood of discovering specific biomarkers.

8.6 Conclusion

Ceramide forms the backbone of sphingolipids. It is synthesized by the de novo synthesis or in response to stress or agonists through the action of sphingomyelinases on sphingomyelin. It participates in numerous biochemical processes including 238 8 Ceramide and Ceramide 1 Phosphate in the Brain proliferation and differentiation, growth arrest, inflammation, stress responses, and apoptosis. Ceramides reside and functions within lipid rafts. The targets for ceramide include specific kinases, phosphatases, phospholipases, cyclooxygenases, proteases, and various transcription factors and cytokines including AP1, NF-kB, TNF-a, and IL-b. Activation of microglial cells and astrocytes enhance the synthesis of ceramide through the activation of SMases. Levels of ceramide are increased in neurotraumatic and neurodegenerating diseases, where they contribute to oxidative stress and neuroinflammation. High levels of ceramide facilitate membrane blebbing, DNA laddering, and activation of caspase cascade along with other morphological changes associated with apoptotic cell death in neurotraumatic and neurodegenerative diseases.

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9.1 Introduction

Sphingosine is an 18-carbon amino alcohol with an unsaturated hydrocarbon chain. It is found in sphingolipids (cerbroside, sulfatide, and ganglioside) as well as phospholipid (sphingomyelin) (Fig. 9.1). Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for ceramide and sphingosine. Like ceramide, sphingosine not only regulates activities of phospholipases (PLA2, PLC, and PLD), and protein kinases (PKC and PKA), but also ion channels, CB1 receptors, and SF1 nuclear receptors (Fig. 9.2). These enzymes are involved in signal transduction processes associated with cell survival and neurodegeneration. Phospholipases contribute to the generation of eicosanoids and platelet-activating factors, whereas protein kinases are associated with phosphorylation of protein involved in signal transduction processes (Farooqui, 2009). In addition, Sphingosine strongly inhibits the extracellular signal-regulated kinase (ERK1/2) (Sakakura et al., 1998) and the Akt kinase pathway (Chang et al., 2001), and activates the p38-MAPK (Frasch et al., 1998). Sphingosine also regulates Mg2+ dependent form of phosphatidate phosphohydrolase, and diacylglycerol kinase in a variety of cell types. Modulation of phospholipase and protein kinase activities by sphingosine is closely associated with the induction of apoptosis, cell differentiation, and growth. Sphingosine-mediated apoptotic cell death involves the mitochondrial pathway (Cuvillier et al., 2000). Sphingosine exhibits different domain morphology depending on the surrounding lipid matrix in biomimetic systems such as giant vesicles. Sphingosine acts as a modulator in lipid domain formation, and thus exerts its effect not only through direct interactions with proteins, but also indirectly by influencing their sorting in membranes and modulating the signal transduction processes (Georgieva et al., 2010). Like ceramide, sphingosine forms channels in membranes, but sphingosine channels differ greatly from the large oligomeric barrel-stave channels formed by ceramide (Siskind et al., 2005). Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Based on the effect of sphingosine

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 245 DOI 10.1007/978-1-4419-9940-5_9, © Springer Science+Business Media, LLC 2011 246 9 Sphingosine and Sphingosine 1 Phosphate in the Brain

OH O OP choline Membrane component NH O

Sphingomyelin O

OH CH OH 2 Apoptosis, cell differentiation & NH tumor suppression Ceramide O

OH Inhibition of enzymes CH2OH (PKC and PLA2) Sphingosine NH3

O OH Induction of cell CH2O P OH differentiation, growth & survival O NH Sphingosine 1 phosphate 3

Fig. 9.1 Chemical structures of sphingomyelin, ceramide, sphingosine, and sphingosine 1 phosphate

Phospholipase A2

PtdIns 4 -kinase DAG kinase Modulation of enzyme activities, ion channels and receptors Protein kinase C Protein kinase A by sphingosine Phosphatidylglycerol Phosphate synthase

Ion channels

CB1 and SF1 receptors

Fig. 9.2 Effect of sphingosine on enzyme activity. Upward arrow indicates stimulation and downward arrow indicates inhibition on biomembranes, it is suggested that sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm (Siskind et al., 2005). The degradation of ceramide by ceramidases results in the generation of sphingosine, which in turn is phosphorylated by sphingosine kinases (SphKs) to generate 9.1 Introduction 247

CH2OH

Ceramide O

Ceramidase Fatty acid

CH2OH O Sphingosine NH3 H ATP Trans-2-hexadecenal + S1P SphK phosphataase S1P lyase CH2O-PO3

ADP O OH NH2

CH2O P OH Phosphoethanolamine O NH Sphingosine 1-phosphate 3 PtdEtn

Fig. 9.3 Metabolism of sphingosine in brain. Upward arrow indicates stimulation and downward arrow indicates inhibition sphingosine 1-phosphate (S1P) (Fig. 9.3). S1P is degraded by specific phosphatases, which belong to the family of magnesium-dependent, N-ethylmaleimide-insensitive type 2 lipid phosphate phosphohydrolases that reside in the endoplasmic reticulum. S1P is also hydrolyzed by a pyridoxal phosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine, with the latter subsequently being reused for the biosynthesis of phosphatidylethanolamine (Fig. 9.3). Sphingomyelin is resynthe- sized from sphingosine by the enzymes ceramide synthase (converting sphingosine to ceramide) and sphingomyelin synthase (ceramide to sphingomyelin). SphK, ceramidase, and S1P lyase tightly control levels of S1P, which is widely distributed not only in neural cells, but also in the vascular system throughout the body. Unlike ceramide and Ceramide 1 phosphate, S1P occurs naturally in plasma at relatively high concentrations (Yatomi, 2008). S1P is known to be an integral and functionally important constituent of lipoproteins. The concentration of S1P in plasma is around 200–1000 nM. More than 60% is bound to lipoproteins, with the majority bound to high-density lipoproteins (HDL) (86%). Inside the cell, S1P moves freely between different membranes but requires specific transport mechanisms for translocation to the outer leaflet of the cytoplasmic membrane. One S1P transport mechanism involves ABC-type transporter (Kobayashi et al., 2006). In brain and vasculature, S1P promotes proliferation, cell survival, and angiogenesis. In addition, it is also involved in vasculogenesis, neuritogenesis, and immune function (Spiegel and Milstien, 2003; Alvarez et al., 2007). Accumulating evidence suggests that S1P is a pro-survival and pro-angiogenic mediator, whereas sphingosine and 248 9 Sphingosine and Sphingosine 1 Phosphate in the Brain ceramide are pro-apoptotic sphingolipids. The balance among levels and activities of S1P, ceramide, and sphingosine acts as a cellular rheostat that signals cell survival or death (Ohanian and Ohanian, 2001; Spiegel and Milstien, 2000; Farooqui et al., 2007). Treatment of hippocampal slices with neutral sphingomyelinases (nSMase) results in generation of ceramides and S1P, which can not only be detected neuro- physiologically, through selective enhancement in the population spike amplitude in fEPSP-PS potentiation at the CA3-CA1 schaeffer collateral synapse (Norman et al., 2010), but also by mass spectrometry-based measurements. The ability of nSMase to increase CA1 neuron excitability can be prevented by inhibitors of sphingosine kinase. These findings support the view that in hippocampal slices, S1P may positively regulate the excitability of hippocampal neurons (Norman et al., 2010). In addition, S1P is also an oncogenic lipid that promotes tumor growth and progression (Fyrst and Saba, 2010). S1P is present in nanomolar concentrations from leukocytes, erythrocytes, platelets, and endothelial cells and resides on albumin and lipoproteins, particularly HDL in the circulation (Whetzel et al., 2009).

9.2 Sphingosine Kinases in the Brain

Two sphingosine kinases (SphK) isoenzymes, namely, SphK1 and SphK2, have been reported to occur in mammalian tissues (Spiegel and Milstien, 2003; Takabe et al., 2008). They are encoded by different genes. These enzymes have five conserved domains (C1–C5) with a unique catalytic domain, which is present within C1–C3 (Alemany et al., 2007). The ATP-binding site is present in the C2 domain. SphK1 has no hydrophobic transmembrane domains, whereas SphK2 has four pre- dicted transmembrane domains. SphK1 and SphK2 differ from each other in sequence, catalytic and kinetics properties, subcellular localization, developmental and tissue expression. These enzymes phosphorylate sphingosine and produce S1P, a lipid mediator that has been implicated in a number of agonist-driven cellular responses, including stimulation of cell proliferation, inhibition of apoptosis, and expression of inflammatory molecules (Fig. 9.4). Basal SphK1 activity is involved in housekeeping. SphK1 is responsible for S1P-mitogenic and anti-apoptotic effects. Its exogenous expression induces cell proliferation and pro-survival signals, whereas its downregulation activates proapoptotic events (Taha et al., 2006). In contrast, SphK2 is a putative BH3-only protein that inhibits cell growth and enhances apoptosis (Liu et al., 2003). Stress promotes its translocation from the cytosol to the ER and/or nucleus to stop proliferation and induce, Ca2+ mobilization, leading to apoptosis (Liu et al., 2003; Maceyka et al., 2005). Treatment of neural and non-neural cell cultures with growth factors and cytokines (TNF-a, and IL1-b) or phorbol esters activates SphK1as well as SphK2 and increases S1P levels rapidly and transiently (Pitson et al., 2000). This increase in S1P levels triggers S1P receptor-mediated signaling that modulates cell proliferation, suppression apoptosis, and inhibition of neuroinflammation (Fig. 9.5). Growth factor or cytokine-initiated and ERK2- mediated phosphorylation at Ser225 of SphK1 is essential for enhancing the membrane 9.2 Sphingosine Kinases in the Brain 249

Modulation of enzyme activity

Modulation of cytoskeleton rearrangement

Modulation of cell motility and growth

Sphingosine 1 phosphate Modulation of signal transduction

Modulation of cell migration and adhesion

Modulation of Immediate early genes

Suppression of apoptosis

Fig. 9.4 Roles of sphingosine and sphingosine 1 phosphate in brain

TNF-α S1P

PtdCho SM TNF- α -R ABC transporter cPLA 2 Neutral SMase + ARA ROS Cer Arg SphK1 IκK IκB-P

COX-2 Sphingo S1P PtdIns3K + p65 p50 iNOS + NF-κB NO Caspase • - O 2 cascade Eicosanoids Positive loop Positive loop Akt - Inflammation ONOO

PARP breakdown Proinflammatory cytokines

Apoptosis

Fig. 9.5 Hypothetical diagram showing the involvement of TNF-a and S1P receptors in apoptotic cell death in brain. Tumor necrosis factor-a (TNF-a); tumor necrosis factor-a-receptor (TNF-R); phosphatidyl-choline (PtdCho); cytosolic phospholipase A2 (cPLA2); sphingomyelin (SM); sphingomyelinase (SMase); ceramide (Cer); sphingosine (Sphingo); sphingosine kinase1 (SphK1); sphingosine 1 phosphate (S1P); nuclear factor- kB (NF- kB); phosphatidylinositol 3 kinase (PtdIns3K); protein kinase B (Akt); arachidonic acid (ARA); cyclooxygenase-2 (COX-2); and reactive oxygen species (ROS); secretory phospholipase A2 (sPLA2); superoxide dismutase (SOD); − − inducible nitric oxide synthase (iNOS); peroxynitrite (ONOO ); and superoxide (O2 ) 250 9 Sphingosine and Sphingosine 1 Phosphate in the Brain

Table 9.1 Agonists that stimulate sphingosine kinase1 Agonist Reference Platelet-derived growth factor Hobson et al., 2001 Vascular endothelial growth factor Shu et al., 2002 Epidermal growth factor Sarkar et al., 2005 Hepatocyte growth factor Duan et al., 2004 Tumor necrosis factor-a Pitson et al., 2003 Interleukin-1b Bryan et al., 2008 Steroid hormone Sukocheva et al., 2006 GPCR ligand (acetylcholine) van Koppen et al., 2001 PtdH and Lyso-PtdH Delon et al., 2004; Duan, 2006 S1P Meyer zu Heringdorf et al., 2001

affinity for SphK1 (Table 9.1). This process not only results in its activation, but also its translocation from the cytosol to the plasma membrane, where sphingosine mainly resides and nuclear membrane, where this translocation may lead to the induction of cyclooxygenase 2 (COX2) (Pitson et al., 2003). Both SphK1 and SphK2 contain a CaM-binding site in the region spanned by residues 191–206. SphK1 and SphK2 share overall homology and generate the common product (S1P), but it is becoming increasingly evident that their activities are involved in different cell functions. SphK1 promotes cell growth and proliferation, whereas SphK2 is associated with opposite effects (Maceyka et al., 2005). This suggestion is based on the differential effects of Sphk1 and SphK2 siRNA in animal model of arthritis. Prophylactic i.p. administration of SphK1 siRNA significantly prevents the incidence of arthritis, disease severity, and articular inflammation compared with control siRNA recipients. Treatment of SphK1 siRNA also reduces serum levels of S1P, IL-6, TNF-a, IFN-g, and IgG2a anti-collagen Ab. Ex vivo analysis indicates significant suppression of collagen-specific proinflammatory/Th1 cytokine (IL-6, TNF-a, IFN-g) release in SphK siRNA-treated mice. In contrast, mice receiving SphK2 siRNA develop more aggressive disease; higher serum levels of IL-6, TNF-a, and IFN-g; and proinflammatory cytokine expression to collagen in vitro when compared with control siRNA recipients. These results support the view that SphK1 and SphK2 have distinct immunomodulatory roles in the development of inflammatory arthritis through the regulation and release of proinflammatory cytokines and T cell responses, which can be used for treating inflammation in murine collagen-induced arthritis model (Baker et al., 2010). Although single SphK1 or SphK2 knockout mice develop and reproduce normally, the double-knockout show a deficiency of S1P, which severely disturbs neurogenesis, including neural tube closure and angiogenesis along with embryonic lethality (Mizugishi et al., 2005). This observation suggests that SphK1 or SphK2 may at least partially compensate for the lack of the other, and S1P is critical for neurogenesis and angiogenesis during development.

S1P1 receptor-null mice also show severe defects in neurogenesis, indicating that the mechanism by which S1P promotes neurogenesis is, in part, signaling from the

S1P1 receptor. Accumulating evidence suggests that S1P can be included in a growing 9.3 Sphingosine 1 Phosphate Receptors in the Brain 251 list of signaling molecules, such as vascular endothelial growth factor, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis (Mizugishi et al., 2005).

9.3 Sphingosine 1 Phosphate Receptors in the Brain

Agonist-mediated activation of sphingosine kinases increases intracellular S1P. This sphingolipid either functions as intracellular second messenger or secreted/ transported out of the cell, where it acts extracellularly by binding to S1P receptors in autocrine and/or paracrine manners. To date, five members of S1P family, S1P1

(EDG-1), S1P2 (EDG-5), S1P3 (EDG-3), S1P4 (EDG-6), and S1P5 (EDG-8/NRG-1) have been identified (Spiegel and Kolesnick, 2002). These receptors have seven transmembrane spanning domains and are coupled through different intracellular second messenger systems (adenylate cyclase, PLC and PtdIns3K/protein kinase Akt, as well as Rho- and Ras-dependent signal transduction pathways) through multiple heterotrimeric G proteins (Fig. 9.6) (Waeber et al., 2004; Spiegel and Milstien,

2003; Sanchez and Hla, 2004; Kihara et al., 2007). S1P1 is coupled with Gai, S1P2, and S1P3 are linked preferentially with Gaq (leading to activation of PLC and intra- 2+ cellular Ca release) and Ga12/13 (causing activation of monomeric G-protein RhoA) (Sanchez and Hla, 2004) (Fig. 9.6). Consistently with multiplicity of S1PRs

S1P1 S1P2 S1P3

Gq Gi Gi Gi G12/13 G12/13 Gq

Gi Gq G12/13

Adenylate cyclase Phospholipase C PtdIns 3-kinase ERK cascade Rho cascade

cAMP generation IPtdins 3 Kinase ↑ Ras ↑, Raf ↑ Rho-GEF , Rho ↓ InsP3/DAG ↑ ↑ ↑ PKA activity ↓ PKC↑, Ca2+ ↑ Akt↑, Rac-GE↑, Rac ↑ MEK ↑, ERK ↑ Rac-GAP ↑, Rac ↓ Enzymic pathways and target s

Response

Fig. 9.6 S1P receptors, their G proteins and their target enzymes. Upward arrow indicates increase and downward arrow indicates decrease 252 9 Sphingosine and Sphingosine 1 Phosphate in the Brain and pleiotropic signaling mechanisms. S1PRs modulate numerous cellular processes.

S1P1 and S1P3 receptors modulate angiogenesis by enhancing platelets, endothelial cell and vascular smooth muscle cell proliferation and migration, whereas S1P2 receptors block the migration of above cell types in cardiovascular and cerebrovascular systems because of their unique stimulatory effect on a GTPase-activating protein inhibiting the activity of Rac. S1P receptors can also mediate relaxation and constriction of blood vessels (Waeber et al., 2004). The former effect is promoted by S1P1, which is located on the endothelium. S1P1 enhances PtdIns3K/Akt/ endothelial nitric oxide synthase (eNOS) signaling pathway. The vasoconstricting effects of S1P are induced by S1P2 and/or S1P3 receptors through Rho-Rho-kinase pathway. S1P also protects endothelial cells from apoptotic cell death through activation of S1P1 and S1P3 receptors, which are coupled to PtdIns3-K/Akt/eNOS signaling pathway (Fig. 9.6) (Waeber et al., 2004). S1P receptors (S1PRs) are widely expressed and distributed in the brain (Toman and Spiegel, 2002; Jaillard et al., 2005; Ohuchi et al., 2008; Chun and Hartung, 2010). Thus, S1PRs are detected in the cerebral cortex, molecular layer, amygdala, basal ganglia, periaqueductal gray, hippocampus, and hypothalamus, and lower levels are detected in the thalamus and corpus callosum (Nishimura et al., 2010).

Among neural cells, astrocytes express mRNA for S1P1, S1P2, and S1P3. Astrocytes express mainly S1P1 and S1P3 receptors (Anelli et al., 2005; Mullershausen et al.,

2007; Brinkmann, 2009; Chun and Hartung, 2010), with little expression of S1P2,

S1P4, or S1P5 receptors. S1P5 receptors are expressed in oligodendrocytes (Jaillard et al., 2005). Neurotrophin-3 (NT-3) plays important roles in oligodendrocyte development and myelination. Knockout mice lacking NT-3 or its receptor tyrosine- kinase TrkC contains reduced numbers of oligodendrocyte progenitors as well as attenuated expression of oligodendrocyte specific markers (Kumar et al., 1998). NT-3 not only regulates proliferation and survival, but also differentiation of oligodendrocytes (Rubio et al., 2004). Treatment of oligodendrocyte progenitors with NT-3 produces rapid phosphorylation and activation of the transcription factor CREB (cyclic AMP-response element-binding protein) (Johnson et al., 2000). Activation of CREB facilitates mechanisms by which NT-3 induces the stimulation of DNA synthesis and increased activity of the anti-apoptotic gene Bcl-2 in oligodendrocyte progenitors. These observations support the role of CREB in oligodendroglial cell proliferation and survival (Johnson et al., 2000). It is also shown that in oligodendrocyte progenitors, NT-3-mediated activation of CREB not only requires extracellular regulated kinase 1/2 (ERK1/2) and protein kinase C (PKC) [53], but also translocation of SphK1 to plasma membrane where its substrate (sphingosine) is located (Coelho et al., 2010). In addition, S1P by itself stimulates CREB phosphorylation in the oligodendrocyte progenitors, further supporting the role of SphK1 in NT-3 signaling (Fig. 9.7).

S1P4 has a more restricted expression pattern than S1P2 and S1P5. S1P4 is detectable predominantly within immune compartments and leukocytes. Based on several studies, it is proposed that stimulation of S1PR is a potentially important proliferative signal for astrocytes. Schwann cells express S1P3 > S1P2 > S1P1 > S1P4 = S1P5 in contrast to cells of oligodendroglial lineage where expression of S1P1 and S1P5 9.3 Sphingosine 1 Phosphate Receptors in the Brain 253

NT3 IL-1b S-1-P-R

S1P PtdIns-4,5-P Trk C 2 IL1 b -R ABC transporter G Ca2+ PLC Raf Sph K1 InsP3 DAG P MEK Sph S1P + + P ErK PKC Ca2+

P Creb Internal stores Protein phosphorylation

NUCLEUS Transcription

Synaptic plasticity, cell proliferation, differentiation, and cell survival

Fig. 9.7 Hypothetical diagram showing interactions between neurotrophin 3 and S1P receptors. Neurotrophin3 (NT3); Raf, MEK, and ErK (serine/threonine-selective protein kinases); cAMP Response Element Binding (CREB); S1P receptor (S1P-R); phosphatidylinositol-4,5-bisphosphate

(PtdIns-4,5-P2); Inositol 1,4,5-trisphosphate (InsP3)phospholipase C (PLC); diacylglycerol (DAG); protein kinase C (PKC)

predominates (Jaillard et al., 2005). Although, terminal Schwan cells express S1P1 receptors, these receptors are absent from myelinating Schwann cells (Kobashi et al., 2006). It is proposed that S1P mediates its effect through the activation of Rac1 and RhoA and elevation in Schwann cells migration (Barber et al., 2004). Direct injection of S1P into the striatum of mice induces astrocyte proliferation and astrogliosis (Sorensen et al., 2003). In cultured astrocytes, S1P treatment results in the stimulation of multiple S1P receptors, which results in the activation of PLC and ERK that leads to astrocytic cell proliferation (Wu et al., 2008). All S1PRs have been cloned and accumulating evidence suggests that S1P/S1PR signaling not only plays an important role in neural development, regulation of neural stem cells, and glial migration, but also facilitates astrocyte proliferation, neuroprotection against apoptosis, modulation of neuronal excitability, and glutamatergic neurotransmission (Chun et al., 2000; Toman and Spiegel, 2002; Yamagata et al., 2003; Harada et al., 2004; Kajimoto et al., 2007; Kimura et al., 2007; Milstien et al., 2007; Ohuchi et al., 2008). S1P also produces direct intracellular effects, which are independent of S1PRs (Spiegel and Milstien, 2003). The molecular mechanisms of intracellular action of 254 9 Sphingosine and Sphingosine 1 Phosphate in the Brain

S1P are still elusive. S1P not only enhances Ca2+ mobilization, but also stimulates the ERK/MAPK pathway to promote cell growth and survival (Takabe et al., 2008). It may also activate the PI3K/Akt pathway to modulate the balance between pro- and anti-apoptotic Bcl-2 proteins and activator protein 1 and NF-kB, which promotes survival (Takabe et al., 2008).

9.4 Sphingosylphosphorylcholine in the Brain

Sphingosylphosphorylcholine (SPC) is a metabolic product of sphingomyelin (Fig. 9.8). It occurs in plasma and is a constituent of lipoproteins. It is synthesized by the action of sphingomyelin deacylase on sphingomyelin (Nixon et al., 2008). SPC increases [Ca2+]i in rat brain preparations (Furuya et al., 1996), and stimulates sodium hydrogen antiporter mechanism in a pituitary cell line (Tornquist et al., 1997). However, the functional significance of these observations is not clearly understood. SPC acts as an endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM). Using fluorescence stopped-flow technique, it is shown that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities, supporting the view that SPC is a competitive inhibitor of CaM-target peptide interactions. It not only disrupts the CaM-binding domain of

O OH OP choline

NH O

O Sphingomyelin

Sphingomyelin deacylase

O OH OP choline

Sphingosylphosphorylcholine O NH2 Sphingosylphosphorylcholine

Modulation of Ca2+ homeostasis Modulation of inflammation Modulation of enzyme activities

Fig. 9.8 Generation and roles of sphingosylphosphorylcholine 9.5 FTY720 and Its Neurochemical Effects 255 ryanodine receptor type 1and PtdIns3P receptor type 1, but also the plasma membrane Ca2+ pump. Accumulating evidence suggests that SPC regulates signal transduction processes by inhibiting Ca2+/CaM-dependent enzymes, such as phosphodiesterase and calcineurin. It is also reported that SPC, along with S1P and lyso- PtdH increases nerve growth factor synthesis in astrocytes (Furukawa et al., 2007), suggesting that SPC may play a role in the survival and functional maintenance of neural cells. These effects may be mediated through S1PRs, which are expressed in the CNS (MacLennan et al., 2001). In brain, SPC not only acts as a mitogen, but also as a proinflammatory mediator. In cultured human melanocytes, SPC inhibits melanin synthesis in a dose-dependent manner, and reduces the activity of tyrosinase, the rate-limiting melanogenic enzyme (Kim et al., 2006). In human melanocytes, SPC induces short-thick dendrites, but has no effect on tyrosinase activity in a cell-free system. Furthermore, SPC reduces melanin synthesis by downregulating microphthalmia-associated transcription factor (MITF) and ERK pathway involved in the melanogenic signaling cascade (Kim et al., 2006). Under physiological conditions, levels of SPC are very low in brain tissue, but under pathological conditions, such as subarachnoid hemorrhage (SAH), SPC levels are significantly increased in CSF of SAH patients (Kurokawa et al., 2009; Wirrig et al., 2010). It is suggested that SPC may be involved in the development of cerebral vasospasm. Although, the molecular mechanism of SPC-mediated cerebral vasospasm is not known, in rat cerebral arteries, SPC not only activates p38 mitogen- activated protein kinase (MAPK), but also increases levels of nuclear factor-kappaB (NF-kB) and CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)-enhancer- binding protein. Both transcription factors are activated by SPC in a p38MAPK- dependent manner along with increased expression of MCP-1 in the cerebral arteries (Wirrig et al., 2010). Collective evidence suggests that SPC, but not S1P, acts as a proinflammatory mediator in cerebral arteries. This may contribute to inflammation observed after SAH and may be part of the initiating event in vasospasm (Kurokawa et al., 2009; Wirrig et al., 2010).

9.5 FTY720 and Its Neurochemical Effects

FTY720 is an immunosuppressant and structural analog of sphingosine (Fig. 9.9), which is highly effective in inhibiting autoimmunity and graft rejection in animal models of allotransplantation in an autoimmune disease. It is phosphorylated in vivo by SphK2 (Mandala et al., 2002), which are abundantly expressed in the brain (Blondeau et al., 2007; Bryan et al., 2008). Like endogenous S1P, the phosphorylated form of FTY720 (FTY720-P) activates S1PRs, mainly S1P1, in a dose-dependent manner. Binding of FTY720-P to the S1P1 receptor has been shown to result in its internalization and long-term downregulation of its expression (Oo et al., 2007). FTY720 also inhibits ceramide synthases, decreasing cellular levels of dihydrocer- amides, ceramides, sphingosine, and S1P, but increasing levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P) (Berdyshev et al., 2009). 256 9 Sphingosine and Sphingosine 1 Phosphate in the Brain

Drug Target Effect

Inhibition of cPLA2 Neuroprotection

Cannabinoid receptor 1 Neuroprotection OH NH2 Stimulation of ERK1/2 Neuroprotection OH

Inhibition of ceramide synthase Neuroprotection FTY720 Inhibition of S1P lyase Neuroprotection ATP

Sphingosine Kinase 2 OH ADP NH2

O OH P S1P1 Neuroprotection OH O

FTY720-phosphate

Fig. 9.9 Phosphorylation FTY720 and target enzymes and receptor for FTY720 and FTY720- phosphate. Cytosolic phospholipase A2 (cPLA2); ERK1/2 (serine/threonine-selective protein kinase); and S1P receptor (S1P1)

The FTY720-mediated modulation of sphingolipid de novo biosynthesis is similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differs in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies indicate that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent Ki of 2.15 mM. FTY720-induces upregulation of DHS1P, which is mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA (Berdyshev et al., 2009). FTY720-phosphate is stable in the presence of active sphingosine-1-phosphate lyase, indicating that the lyase does not degrade FTY720. Conversely, FTY720 inhibits sphingosine-1-phosphate lyase activity in vitro (Berdyshev et al., 2009). Administration of FTY720 in mice inhibits tissue sphingosine- 1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression are not significantly affected by this drug (Bandhuvula et al., 2005; Berdyshev et al., 2009). In addition, FTY720 induces numerous effects on the immune system including inhibition of egress of both naive and activated CD4+, CD8+, and B lymphocytes from peripheral lymphoid organs and thymus, cysteinyl leukotriene-dependent T cell chemotaxis to lymph nodes, peripheral blood lymphopenia, egress of lymphocytes from the spleen, displacement of B cells from the marginal zone of the spleen, decreased b1 integrin expression on marginal zone B cells, homing of hematopoietic progenitor cells to the bone marrow, and decreased vascular permeability (Mandala et al., 2002; Sanchez et al., 2003; Kimura et al., 2004). 9.6 Sphingosine 1 Phosphate in Neurological Disorders 257

In addition, FTY720 retards inflammation (Coelho et al., 2007; Payne et al., 2007) by inhibiting cPLA2 and reducing antigen-induced secretion of PGD2 and cysteinyl- leukotriene. Although FTY720 also reduces cPLA2-mediated arachidonic acid release in response to antigen, it has no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcepsilonRI-mediated events, leading to cPLA2 activation (Payne et al., 2007). Thus, FTY720 drastically inhibits recombinant cPLA2a activity, whereas FTY720-phosphate, sphingosine, or S1P have no effect. It is proposed that FTY720 reduces inflammation by blocking the production of inflammatory lipid mediators, like eicosanoids and platelet-activating factor (Ong et al., 2010). Treatment of oligodendrocyte progenitors with FTY720 produces activation of extracellular signal-regulated kinase 1/2 and Akt and protects cells from apoptosis, but arrests their differentiation (Coelho et al., 2007).

9.6 Sphingosine 1 Phosphate in Neurological Disorders

In brain sphingolipid metabolism, sphingolipid-derived lipid mediators are associated with several signaling pathways that control neuronal survival, migration, and differentiation, responsiveness to trophic factors, synaptic stability, synaptic transmission, and neuron–glia interactions. These processes are involved in stabilizing of central and peripheral myelin. Many neurotraumatic and neurodegenerative diseases are accompanied by increase in expression of cytokine and enrichment of sphingolipid metabolisms due to upregulation of SMase activity (Farooqui, 2009). Thus, sphingolipid metabolism is deregulated in neurotraumatic and neurodegenerative diseases, leading not only to the expression of abnormal sphingolipid patterns and abnormal sphingolipid–protein interactions, but also increase in levels of ceramide, ceramide 1 phosphate, decrease in sphingosine, and sphingosine 1 phosphate, alterations in calcium homeostasis, and altered neural membrane organization (Farooqui et al., 2007; Farooqui, 2009). In addition, most neurodegenerative diseases also involve the activation of microglial cells along with neuroinflammation, which is supported by increased expression of proinflammatory cytokines, elevation in eicosanoids, increase in platelet-activating factor levels, and increase in nitric oxide production (Farooqui, 2009, 2010). Collective evidence suggests that abnormal sphingolipid metabolism and above neurochemical processes are closely associated with the pathogenesis of neurotraumatic and neurodegenerative diseases (Piccinini et al., 2010; Farooqui, 2009, 2010).

9.6.1 Sphingosine 1 Phosphate in Ischemia

Stroke is accompanied by the interruption of blood flow to the brain due to the blockage of a cerebral artery. Neurochemically, stroke is characterized by the disruption of glucose and oxygen supply, oxidative stress, and neuroinflammation. 258 9 Sphingosine and Sphingosine 1 Phosphate in the Brain

Sphingosine 1 phosphate and FTY720

Traumatic brain Alzheimer injury disease

Spinal cord Multiple sclerosis injury Ischemic injury

Fig. 9.10 Use of FTY720 for the treatment of neurotraumatic and neurodegenerative diseases

In addition, interactions between the brain and the peripheral immune system results in a biphasic immune response, which consists of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia leading to massive immune cell loss in spleen and thymus. These processes not only lead to the development of infarction, but also apoptotic and necrotic cell death. Cerebral ischemia upregulates the sphingomyelin-ceramide pathway, which involves calcium- independent c-Jun NH2-terminal kinase and serine/threonine phosphatase PP2A activation in hippocampal glia (Hasegawa et al., 2010; Adibhatla et al., 2008). Furthermore, S1P has neuroprotective effect in oxygen-glucose deprivation (OGD) model of ischemia in SH-SY5Y human neuroblastoma cells. Treatment of cell cultures with specific PKC epsilon (V1-2), retards neuroprotective effect of S1P on OGD/recovery-induced necrosis (Agudo-Lopez et al., 2010). FTY720 is an analog of S1P. It is known to induce depletion of circulating lymphocytes by preventing the egress of lymphocytes from the lymph nodes. At the molecular level, the depletion of circulating lymphocyte is due to a downregulation of the S1P1 (Brinkmann et al., 2004). Injections of FTY720 in a mouse model of ischemia decrease lesion size and improve neurological function (Fig. 9.10). FTY720-treated animals not only significantly retain Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression, but show decrease in the expression of caspase-3 and terminal deoxynucleotidyl transferase-mediated uridine 5¢-triphosphate-biotin nick end- labeling-positive neurons at 24 h after middle cerebral artery occlusion along with decrease in the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion, and reduction in immunohistochemical features of apoptotic cell death in the ischemic lesion (Czech et al., 2009). Accumulating evidence 9.6 Sphingosine 1 Phosphate in Neurological Disorders 259 suggests that abnormalities in sphingolipid metabolism may contribute to the pathogenesis of stroke, and FTY720 can be used for the treatment of ischemic injury (Fig. 9.10).

9.6.2 Sphingosine 1 Phosphate in Traumatic Brain and Spinal Cord Injuries

Traumatic injury to brain and spinal cord (TBI and SCI) may lead to permanent disability in young adults. Sphingolipid signaling has been implicated in pathophysiology of TBI and SCI (Singh and Hall, 2008; Farooqui, 2010). Generation of S1P may represent a novel neuroprotective target to counteract the pathophysiology of acute brain and spinal cord injury in regard to apoptotic cell death mechanisms, mitochondrial dysfunction, lipid hydrolysis, and oxidative damage mechanisms (Singh and Hall, 2008). Furthermore, it is suggested that S1P agonist may increase the resistance of the brain tissue to injury by promoting neurotrophic activity, neurogenesis, and angiogenesis. In contrast, treatment with antagonists of S1P activity may cause proregenerative effects via promotion of neurite growth and inhibition of astrogliotic scarring (Singh and Hall, 2008). Many immune inhibitors including tacrolimus (FK506) have been reported to produce beneficial effects by inducing regeneration in brain and spinal cord following TBI and SCI (Zhang et al., 2009). The combination of FTY720 and tacrolimus has been reported to produce synergistic immunosuppresive effects in rat allograft models without causing critical adverse effects (Zhang et al., 2009).

9.6.3 Sphingosine 1 Phosphate in Alzheimer Disease

Alzheimer disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia in the aging population. Loss of synapse, defect in cholinergic system, accumulation of insoluble amyloid b (Ab), and neuronal degeneration are some of the physiological sequelae that are closely associated with the pathogenesis of this devastating disease (Farooqui, 2010). Sphingolipid metabolism is altered in AD (Haughey et al., 2010). Determination of sphingolipid metabolism- derived lipid mediators in brain from AD patients and age-matched normal subjects indicate that increase in expression of acid SMase and ceramidase is accompanied with reduction in levels of sphingomyelin and elevation in ceramide and sphingosine (He et al., 2010). Levels of S1P are decreased. Importantly, there is significant correlation among the brain acid SMase activity, S1P levels, and levels of Ab and hyperphosphorylated t protein. Treatment of neuronal cell cultures with Ab oligomers also leads to the activation of acid SMase activity, elevation in ceramide levels, and induction of apoptotic cell death (He et al., 2010). Pretreatment of the 260 9 Sphingosine and Sphingosine 1 Phosphate in the Brain neuronal culture with purified, recombinant ceramidase blocks the induction of Ab-mediated apoptosis. Based on these observations, it is proposed that acid activation of SMase ceramidase along with elevation in ceramide and sphingosine and reduction in S1P may be important neurochemical events involved in the pathogenesis of AD (He et al., 2010).

9.6.4 Sphingosine 1 Phosphate in Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune, neurological disease of unknown etiology. It is a chronic inflammatory disease, which is characterized by demyelination in young adults. Recently, sphingolipid metabolism has been linked to the pathophysiology of MS and it is reported that oligodendrocyte apoptosis is one of the critical events followed by glial activation and infiltration of lymphocytes and macrophages (Jana and Pahan, 2010). Current therapeutic approaches to treat MS focus on the suppression of the immune system and on blockade of T cell blood– brain barrier transmigration into the brain parenchyma. S1P can be recognized by receptors located on T and B cells. Interaction of FTY720 phosphate with S1P1 not only antagonizes S1P1 on T cells, but also inhibits S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs (Chun and Hartung, 2010).

FTY720 phosphate initially activates lymphocyte S1P1 via high-affinity receptor binding, leading to downregulation of S1P1. This downregulation of S1P1 prevents lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the brain tissue. S1P receptors are also expressed by many CNS cell types. Prophylactic administration of FTY720 to animals with experimental autoimmune encephalitis (EAE), a model of MS, completely prevents development of EAE features, whereas therapeutic administration significantly reduces clinical severity of EAE (Fig. 9.10). MS pathology also involves axonal loss. The extent of axonal transection at the early stages of MS lesion formation correlates with inflammatory infiltration by macrophages and lymphocytes. Inflammatory mediators and toxic products that are secreted by activated macrophages include nitric oxide (Smith et al., 2001), matrix metalloproteinases (Newman et al., 2001), glutamate excitotoxicity (Smith et al., 2000) and direct CD81 T-cell cytotoxic action (Medana et al., 2001). All these factors are capable of damaging axons. It is shown that FTY720 protects against further axon loss when administered in a clinically relevant manner, after the onset of clinical disease. It is likely that the decrease in the expression of iNOS and reduction in numbers of CD31 lymphocytes following therapeutic treatment may at least partly explain the ability of FTY720 significantly preventing further inflammation- associated axon loss. Therapeutic efficacy observed in animal studies has been substantiated in phase 2 and 3 trials involving patients with relapsing or relapsing- remitting MS (Chun and Hartung, 2010). In phase 3 trials, FTY720 has not been proven superior to standard treatments. Thus, more studies are needed on the beneficial effects of FTY720 in MS patients. 9.7 Conclusion 261

9.6.5 Sphingosine 1 Phosphate in Kainic Acid Neurotoxicity

Kainic acid (KA) is a cyclic and nondegradable analog of glutamate. Its systemic administration into adult rats induces excitotoxicity, a common mechanism of neuronal injury after head injury, stroke, and certain neurodegenerative diseases. Astrocytes respond to KA-mediated injury by proliferation and hypertrophy, induction of PLA2 isoforms, elevation in levels of prostaglandins and lipid peroxides, increase in caspase-3 activity, and increase in expression of glial fibrillary acidic protein (GFAP) (Farooqui et al., 2008). In addition, studies on temporal changes in sphingosine kinase 1 (SPHK1)/sphingosine 1 phosphate receptor 1(S1P1) in mouse hippocampus following KA-induced neurotoxicity indicate that lowest level of SPHK1 protein expression is found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P1 proteins steadily increases in the hippocampus. Immunohistochemical analysis indicates that SPHK1 and S1P1 are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P1 signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity (Lee et al., 2010).

9.7 Conclusion

S1P has emerged as a new class of potent bioactive lipid mediator which is associated with a variety of cellular processes, such as cell differentiation, apoptosis, and proliferation. Sphingomyelin and ceramide are precursor for sphingosine, which on phosphorylation by SphK yields S1P. An important feature of the sphingolipid metabolic pathway is the compartmentalization into endoplasmic reticulum, the Golgi apparatus, lysosome and plasma membrane, and this compartmentalization makes the transport of sphingolipids critical for proper neural cell functioning. In neural cells, S1P regulates differentiation, proliferation, inflammation, and cell migration. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in neurochemical actions of S1P is focused recently on two distinct cellular actions of this lipid, namely, its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduces or even blocks cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis,2+ Ca mobilization, cytokine production, and apoptosis. Deregulation of sphingolipid metabolism occurs in neurotraumatic and neurodegenerative diseases. FTY720, a fingolimod, has been used for the treatment of ischemic and traumatic injuries in the brain and spinal cord. Moreover, blocking S1P1 inhibits lymphocyte egress from lymphatic organs. This drug can be used for the treatment MS in patients. 262 9 Sphingosine and Sphingosine 1 Phosphate in the Brain

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10.1 Introduction

The brain has higher concentration of cholesterol compared to other body parts (25% of the body’s free cholesterol) (Dietschy and Turley, 2004). In brain, cholesterol resides in two pools: one major pool associated with the myelin sheaths playing an important role in propagation of the electrical signals along the axons and the other minor pool associated with the plasma membranes of astrocytes and neurons (Dietschy and Turley, 2004). The distribution of cholesterol in neural membranes is asymmetric across the plane of the membrane, with outer and innerleaflets containing 25% and 75% cholesterol, respectively. The asymmetric distribution of cholesterol along with asymmetric distribution of glycerophospholipids (PtdEtn in the exofacial layer and PtdCho in the inner leaflet) is necessary for optimal neural plasticity and synaptic transmission. The half-life of cholesterol in the brain is 170 days (Andersson et al., 1990). Cholesterol not only serves as a precursor for the synthesis of oxysterols, steroid hormones, vitamin D, and lipoproteins, but regulates activities of membrane-bound enzymes, receptors, and ion channels (Simons and Ikonen, 2000). Accumulating evidence suggests that neural membrane cholesterol not only provides neural plasticity and facilitates synaptic transmission, but also modulates endocytosis, and antigen expression (Fig. 10.1). Dynamic clustering of cholesterol along with sphingolipids results in the formation of specialized structures called microdomains or rafts (Farooqui, 2009). In neural membranes, raft formation occurs by self-association of sphingolipids via their long saturated hydrocarbon chains. Cholesterol condenses this packing by positioning between hydrocarbon chains below the large head groups of the sphingolipids (Farooqui, 2009). These interactions lead to the formation of a less fluid, liquid ordered phase, separate from a phosphatidylcholine-rich liquid-disordered phase (Simons and Ikonen, 2000). In neural and non-neural cells, the depletion of cholesterol induces autophagy, a process

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 267 DOI 10.1007/978-1-4419-9940-5_10, © Springer Science+Business Media, LLC 2011 268 10 Cholesterol and Hydroxycholesterol in the Brain

Roles of cholesterol in brain

Raft formation Synaptogenesis

Neuroplasticity Endocytosis

Generation of neurosteroids

Fig. 10.1 Roles of cholesterol in the brain

by which cells digest their own components (Cheng et al., 2006; Clark et al., 2008). Autophagy is a homeostatic process for recycling of proteins and organelles that are increased during times of nutrient deprivation. The structure of cholesterol makes it susceptible to a variety of radical attacks due to the 5,6-double bond and the concomitant vinylic methylene group at C-7 in the B ring. In addition to C-17, cholesterol has isooctyl side chain, which undergoes enzymic oxidation largely by cytochromes P450-dependent oxygenases, but this site of oxidation is typically not a target for ROS relevant to cellular biochemistry (Smith, 1981). Brain contains cytochrome P450-dependent oxygenases, which convert cholesterol into 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol (Fig. 10.2). It is becoming increasingly evident that the major mechanism of brain cholesterol elimination is the conversion of cholesterol into 24(S)-hydroxycholesterol by CYP46A1, a neuron-specific cytochrome P450. Cholesterol is also oxidized to cholesterol oxides and converted into cholesterol ester via acyl-CoA:cholesterol acyltransferase (ACAT) (Björkhem et al., 1998). ACAT is an integral membrane protein, which is found in the endoplasmic reticulum (ER) (Chang et al., 2009). This enzyme modulates the dynamic equilibrium between free and esterified cholesterol in the brain. Two isoforms, namely, ACAT1 and ACAT2 of ACAT have been identified in neural and non-neural tissues (Rudel et al., 2001). ACAT activity not only plays a role in the reesterification process of cholesterol (Zhang et al., 2003), but also acts as a cholesterol sensor protein. Hydroxycholesterols (oxysterols) are agonists for nuclear receptors, such as LXR, which act as cholesterol sensors: when cellular hydroxysterols accumulate as a result of increasing concentrations of cholesterol, LXR induces the transcription of genes that protect cells from cholesterol overload. In addition, LXR are also involved in cholesterol homeostasis (Fig. 10.3). 10.2 Synthesis of Cholesterol and Hydroxycholesterols in the Brain 269

HO Cholesterol 25-hydroxylase CYP27A1

CH2OH b c OH CYP46A1

HO d

Fig. 10.2 Conversion of cholesterol into hydroxycholesterols. Cholesterol (a), 25-hydroxycholesterol (b). 27-hydroxycholesterol (c), and 24-hydroxycholesterol (d). CYP27 (sterol 27-hydroxylase), and CYP46A1 (24-cholesterol hydroxylase)

Raft perturbation, alterations in membrane permeability

Gene expression

Cholesterol homeostasis

Hydroxycholesterols Exocytosis

Agonists for LXR

Apoptosis

Modulation of enzymic activities

Fig. 10.3 Roles of hydroxycholesterols in the brain

10.2 Synthesis of Cholesterol and Hydroxycholesterols in the Brain

As stated above, cholesterol accounts for 20–25% of the total body cholesterol in fresh brain. Most brain cholesterol is found in the myelin sheets (70%) and the remaining 30% of brain cholesterol is divided between membranes of glial cells 270 10 Cholesterol and Hydroxycholesterol in the Brain

(20%) and neurons (10%). Cholesterol contents in brain are independent of dietary uptake or hepatic synthesis because cholesterol does not cross the blood–brain barrier (BBB) (Jurevics and Morell, 1995). Brain synthesizes it own cholesterol through de novo synthesis and total cholesterol levels steadily increase between ages 20–65 years in both men and women. The highest rate of cholesterol synthesis in the first postnatal weeks in humans and rodents may be associated with glial cell proliferation, neurite outgrowth, microtubule stability, synaptogenesis, and myelination. After 65 years, cholesterol levels begin to decrease. Thus, cholesterol is an integral component of neural membranes, where it has been implicated in the assembly and maintenance of lipid rafts. In brain, cholesterol plays a crucial role in membrane organization, dynamics, function, and sorting (Simons and Ikonen, 2000). Cholesterol also plays an important role in modulating the function of many proteins in the brain. This modulation either involves direct binding of cholesterol with proteins or alterations in physical properties of neural membranes, such as fluidity, curvature, and stiffness. At the cellular level, cholesterol is synthesized by oligodendrocytes, astrocytes, and neurons, particularly during early development along with myelin synthesis. Plasma lipoproteins are unable to deliver cholesterol to the brain. The de novo synthesis of cholesterol in the brain is an energy-consuming process comprising multiple intermediates and requiring many enzymes. Although biosynthesis mainly occurs in the endoplasmic reticulum, some cholesterol is also synthesized in peroxisomes (Kovacs et al., 2001). Acetyl-CoA and acetoacetyl-CoA are transformed into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG-CoA reductase converts HMG-CoA into mevalonate, which is phosphorylated to isopentenyl pyro- phosphate (IPP) and other active isoprenoid units. These units condense to form a 10-carbon lipid GPP, 15-carbon lipid FPP, and the 20-carbon lipid GGPP. These reactions are catalyzed by farnesylpyrophosphate, and geranylgeranyl-pyrophos- phate synthases. Formation of FPP is a branch point, where the synthesis of squalene starts. This reaction is catalyzed by squalene synthase. Two alternative pathways exist in the later stages of cholesterol synthesis. Squalene is converted to lanosterol, followed by desmosterol and cholesterol (Figs. 10.4 and 10.5). This is called as Bloch pathway, whilst the synthesis of cholesterol via the conversion of lanosterol to lathosterol, and conversion of lathosterol to 7-dehydrocholesterol is called as Kandutsch–Russell pathway (Lütjohann et al., 2002; Thelen et al., 2006) (Figs. 10.4 and 10.5). In addition to cholesterol, mevalonate is also metabolized to heme, ubiquinones, and vitamin D. Cholesterol contents of neural membranes are tightly regulated by several mechanisms that balance the level of cholesterol synthesis, uptake, and efflux (Farooqui et al., 2007). Accumulating evidence suggests that nuclear receptors, a superfamily of ligand-activated transcription factors provide an indispensable regulatory frame- work in modulating and controlling genes for cholesterol metabolism. The mechanism of transcriptional regulation by nuclear receptors, such as LXRs involves formation of heterodimers with RXRs, which is activated by 9-cis-retinoic acid (a vitamin A derivative). Upon heterodimerization, LXR/RXR binds to specific DNA sequences called LXR-responsive elements (LXREs) in the target genes (Fig. 10.6). 10.2 Synthesis of Cholesterol and Hydroxycholesterols in the Brain 271

Acetyl-CoA + Acetoacetyl-CoA

HMG-CoA

2 Statins

Mevalonate

IPP

7-Dehydrocholesterol GPP Kandutsch-Russell pathway 3 Dolichol CYP51 Farnesylated Cholesterol Lanosterol Squalene FPP NADPH Proteins (Ras) 4 5 oxidase

Bloch pathway Ubiquinone GGPP eNOS Desmosterol 6

Geranylgeranylated proteins (RhoA, RhoB, Rap1)

Fig. 10.4 Biosynthesis of cholesterol in the brain. HMG-CoA synthase (1), HMG-CoA reductase (2), FPP synthase (3), squalene synthase (4), farnesyltransferase (5), GGPP synthase (5), and ger- nylgeranyltransferase (6). Isopentenylpyrophosphate (IPP), geranylpyrophosphate (GPP), farnesylpyrophosphate (FPP), farnesyl protein transferase (FPTase), geranylgeranylpyrophosphate (GGPP), geranylgeranyl protein transferase (GGPTase), and endothelial nitric oxide synthase (eNOS)

Thus, LXR/RXR functions as a sensor of cellular cholesterol concentration and mediates cholesterol efflux by inducing the transcription of key cholesterol shuf- fling vehicles, namely, ATP-binding cassette transporter A1 (ABCA1) and ApoE. ABCA1 is a membrane protein that facilitates the formation of ApoE-cholesterol- phospholipid complex and requires the hydrolysis of ATP for its activity, whereas ApoE is a cholesterol transport protein that plays a central role in the brain response to injury and neurodegeneration in mammalian species (Fig. 10.7). In addition, biosynthesis of cholesterol is regulated by insulin-induced genes (INSIGs) and sterol regulatory element binding proteins (SREBP), in particular SREBP-2 and controlled through feedback regulation by sterol, including cholesterol itself. SREBP2 controls the expression of enzymes involved in cholesterol biosynthesis, including the rate-limiting enzyme HMG-CoA reductase (HMGR) (Brown and Goldstein, 1997; DeBose-Boyd, 2008). In mammals, most growth and differentiation of the brain occurs in the first few weeks or years after birth, and the cholesterol required for such growth apparently comes exclusively from de novo synthesis. The highest rate of synthesis occurs during first postnatal weeks in humans and rodents (Jurevics and Morell, 1995; Jurevics et al., 1997). As the brain matures and cholesterol pools in the brain become constant, the rate of de novo synthesis of cholesterol in the brain 272 10 Cholesterol and Hydroxycholesterol in the Brain

HO HO cd

HO HO

Fig. 10.5 Chemical structures of various intermediates of cholesterol biosynthesis. Lanosterol (a), lthosterol (b), 7-dehydrocholesterol (c), zymosterol (d), and desmosterol (e) markedly declines. The Bloch pathway contributes more to cholesterol biosynthesis in young animals, whereas the Kandutsch–Russell pathway is more involved in aged animals (Thelen et al., 2006; Ong et al., 2010). The majority of brain cholesterol is present in myelin sheets and whilst there is a clear role of oligodendrocyte cholesterol in myelin synthesis, the respective roles of neurons and glial cells in cholesterol biosynthesis in the adult brain are less well understood (Pfrieger, 2003). As stated above, the accepted view is that in the adult nervous system, astrocytes are responsible for brain cholesterol, and synaptic plasticity is modulated by cholesterol homeostasis, which is controlled by cell export proteins and lipoproteins such as apolipoprotein E (apoE) (Fig. 10.7). The latter plays an important role in the translocation of cholesterol from astrocytes to neurons (Levi et al., 2005). Compared to glial cells, neurons show different profiles of cholesterol biosynthetic enzymes, post-squalene sterol precursors and cholesterol metabolites, and produce cholesterol less efficiently possibly due to low levels of lanosterol-converting enzymes (Pfrieger, 2003; Nieweg et al., 2009). Nevertheless, there have been suggestions that besides glial cells, adult neurons are also capable of cholesterol synthesis (Saito et al., 1987; Suzuki et al., 2007). 10.2 Synthesis of Cholesterol and Hydroxycholesterols in the Brain 273

A1 A2

Cholesterol R1 R1

cPLA PlsEtn-PLA2 2 Hydroxysterols ARA (-) DHA 15-LOX (-) Docosanoids ER - ROS 5-LOX

PPAR RXR Eicosanoids LXR RXR

Receptor dimerization NF-kB

Mature SREBP

LXR RXR PPAR RXR NF-kB-RE LXRE SRE PPRE mRNA for cytokines, Posttranslational mRNA for cholesterol Fatty acid desaturase proinflammatory modification synthesizing enzymes, & elongase mRNA & Enzymes , & Transcription & cholesterol transcription transcription efflux NUCLEUS

Fig. 10.6 Transcriptional regulation of cholesterol homeostasis regulating enzymes, fatty acid desaturases and elongases, and proinflammatory enzymes and cytokines in the brain. Agonists (A1 and A2), receptors (R1 and R2), plasmalogen (PlsEtn), phosphatidylcholine (PtdCho), plasmalogen-selective phospholipase A2 (PlsEtn-PLA2), cytosolic phospholipase A2 (cPLA2), 15-lipoxygenase (15-LOX), 5-lipoxygenase (5-LOX), arachidonic acid (ARA), liver X receptor (LXR), liver X receptor response element; retinoid receptor (RXR), sterol regulatory element binding protein (SREBP), peroxisome proliferator alpha (PPARa), sterol response element (SRE), nuclear factor- kB (NF-kB), nuclear factor-kB response element (NF-kB-RE), endoplasmic reticulum (ER), and negative sign (−) indicate inhibition

Astrocytes synthesize and secrete two- to threefold more cholesterol than neurons, and secrete it as lipoprotein particles, which serve as cholesterol carriers (DeMattos et al., 2001; Vance et al., 2005). Although neurons synthesize enough cholesterol to survive and grow, the formation of numerous mature synapses during synaptogenesis demands additional amounts of cholesterol, which is provided by astrocytes (Fig. 10.7). Thus, the availability of cholesterol appears to limit synaptogenesis and development of mature synapse. This may explain the delayed onset of synaptogenesis after glia differentiation and neurobehavioral manifestations of defects in cholesterol or lipoprotein homeostasis (Mauch et al., 2001; Pfrieger, 2003). Inhibition of cholesterol synthesis by inhibitor in hippocampal slices reduces synaptic plasticity. This observation supports the view that one of the major roles of cholesterol is to modulate neural membrane plasticity and that it can cause either a positive or negative effect depending upon membrane constituents. The enrichment of cholesterol in lipid rafts may be responsible for reduction in lateral mobility of raft components. Collective evidence 274 10 Cholesterol and Hydroxycholesterol in the Brain

+ Acetate LXR 24-Hydroxycholesterol Astrocyte +

NADP + ABCA1

O + Neuron 2 ABCG1 de novo synthesi s Memory consolidation CYP46 Nucleus NADPH + H

+ ApoE ApoE

Cholesterol 27-Hydroxy- cholesterol Cholesterol

27-Hydroxycholesterol

Brain

X BBB Cholesterol 24-Hydroxycholesterol 27-Hydroxycholesterol

Blood

Metabolism

Fig. 10.7 Interactions between cholesterol metabolism of neurons and astrocytes. CYP46 (24-cholesterol hydroxylase), liver X receptor (LXR), ATP-binding cassette A1 (ABCA1), ATP- binding cassette G1 (ABCG1), and blood–brain barrier (BBB) suggests that supply of astrocyte-derived cholesterol is necessary not only for fine-tuning neural cholesterol dynamics for basic synapse function, but also for optimal neuroplasticity and behavior. In addition, cholinergic function, ionotropic and metabotropic receptor machinery, and neural oxidative stress reactions are also modulated by brain cholesterol homeostasis (Nelson and Alkon, 2005). 10.3 Degradation of Cholesterol in the Brain 275

10.3 Degradation of Cholesterol in the Brain

Four cytochrome P450-dependent hydroxylases, namely, CYPs 7A1, 27A1, 11A1, and 46A1 are known to occur in neural and non-neural tissues. These enzymes play key roles in cholesterol homeostasis through the elimination of excess cholesterol through blood–brain barrier into blood from where it is taken to liver by plasma lipoproteins. CYP46A1 enzyme is a microsomal enzyme, which is selectively expressed in hippocampal and cortical neurons, and is responsible for the majority of cholesterol turnover in the central nervous system (Ramirez et al., 2008). Substrate specificity of P450 46A1 is not limited to cholesterol. A number of other structurally related steroids are also hydroxylated by this enzyme. Hydroxysterols bind two different targets, liver X receptors (LXRs) and oxysterol-binding proteins (ORPs). Among three major naturally occurring hydroxysterols (27-hydroxycholesterol, 24(S)- hydroxycholesterol, and 25-hydroxycholesterol), only 25-hydroxycholesterol is known to bind ORPs. The brain-specific 24(S)-hydroxycholesterol and the peripher- ally synthesized 27-hydroxycholesterol have been reported to be primarily ligands for LXR ligands (Russell, 2000; Fu et al., 2001). LXR ligands increase cholesterol efflux by inducing the expression of cholesterol-binding (ApoE and ApoD) proteins and cholesterol transporters belonging to the ATP-binding cassette (ABC) family (Zelcer and Tontonoz, 2006). Furthermore, LXRs produce their anti-inflammatory effects by antagonizing NF-kB-induced gene expression (Joseph et al., 2003) (Fig. 10.8). The mechanism associated with the anti-NF-kB activity of LXRs involves the recruitment of sumoylated LXR to the NF-kB motif, which then retards the degradation of associated nuclear receptor corepressor (N-CoR) complexes (Ghisletti et al., 2007). In addition, LXR signaling also represses TLR4-induced expression of iNOS, COX-2, and IL-6 in murine macrophages. Disruption of the cholesterol 24-hydroxylase gene in the mouse reduces both cholesterol turnover and synthesis in the brain, but does not alter steady-state levels of cholesterol in the tissue. Mice deficient in 24-hydroxylase exhibit impaired learning and defective hippocampal long-term potentiation, suggesting that the metabolism of cholesterol by this enzyme is required for learning and memory formation (Ramirez et al., 2008). Accumulating evidence suggests that neurons meet the majority of their cholesterol supply from astrocytes not only by ApoE-dependent transport, but also through the sterol transporters ATP-binding cassette A1 (ABCA1) and G1 (ABCG1), which are present in glial cells and under control of LXR. The other target of hydroxycholesterol is linked to APP processing. Oxysterol Binding Protein 1 (OSBP1) overexpression or silencing reduces or increases APP processing through the regulation of APP trafficking (Zerbinatti et al., 2008). Studies on the expression pattern of Cyp27A1 and other related genes in primary glial and neuronal cultures of rat brain indicate that primary astrocytes express different sterol hydroxylases and are able to uptake exogenous 27-hydroxycholesterol (Gilardi et al., 2009). Since astrocytes are associated with maintenance of homeostasis in the brain, it is hypothesized that impairment of CYP27 activity in astrocytes may alter their role from providing delivery of cholesterol to neurons to the release of signaling molecules (Gilardi et al., 2009). 276 10 Cholesterol and Hydroxycholesterol in the Brain

Cholesterol efflux Glu

1(-) Cholesterol PtdCho NMDAR ABCA1

cPLA + 2 2+ Ca Cholesterol sPLA2 hydroxylases ApoE Lyso-PtdCho + ARA -

Hydroxycholesterols ROS IkB/NFkB (O -, OH., H O ) Eicosanoids 2 2 2 PAF + + + + - Oxidative stress - Neuroinflammation IkB Positive loop (+)

NF-kB-RE LXR Apoptosis COX-2 RXR sPLA2 iNOS Genes related to inflammation TNF-α Cholesterol IL-1β efflux IL-6 Suppression COX-2, iNOS PLTP1, ABCA1. IL-1β,& IL-6 of Arc ApoE, & LPL

Fig. 10.8 Interactions between hydroxycholesterols and PLA2-mediated inflammatory signaling.

Phosphatidylcholine (PtdCho), cytosolic phospholipase A2 (PLA2), secretory phospholipase A2

(sPLA2), lysophosphatidylcholine (lyso-PtdCho) arachidonic acid (ARA), platelet-activating factor (PAF), reactive oxygen species (ROS), N-Methyl-d-aspartate receptor (NMDA-R), nuclear factor- kB (NF-kB), nuclear factor-kB response element (NF-kB-RE), retinoic acid receptor (RXR), liver X receptor (LXR), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-a (TNF-a), and interleukin1b (IL-1b) upward arrows indicate increase and downward arrows indicate decrease

In liver, CYP27A1 is a microsomal enzyme, which transforms cholesterol into 7-a hydroxycholesterol. This enzyme is also found in mitochondria, where it metabolizes cholesterol to 27-hydroxycholesterol. CYP27A1 is a polyfunctional enzyme that in addition to generating 27-hydroxycholesterol and 3b-hydroxy-5- cholestenoic acid in extra-hepatic tissues also oxygenates bile acid intermediates in the liver and vitamin D3 in the kidney (Masumoto et al., 1988). 27-Hydroxycholesterol and 3b-hydroxy-5-cholestenoic acid are ligands for the nuclear receptors, LXRa and LXRb. These receptors activate the transcription of several genes involved in lipid metabolism (Song and Liao, 2000). Catalytic efficiencies of cholesterol- metabolizing P450-dependent hydroxylases vary significantly in brain, liver, lungs, and probably reflect physiological requirements of different organs for the rate of cholesterol turnover. Collective evidence suggests that cytochrome P450-dependent hydroxylases (CYP7A1, 27A1, 11A1, and 46A1) represent a unique system that 10.4 Roles of Hydroxycholesterols in the Brain 277 facilitates elimination of cholesterol from different tissues to fit their needs (Pikuleva, 2006). 24(S)-hydroxycholesterol plays a pivotal role in promoting altered inflammatory signaling, apoptotic responses, and Alzheimer disease (AD)-type changes in brain (Schippling et al., 2000; Michikawa, 2004; Vaya and Schipper, 2007) (See below). In addition, 24(S)-hydroxycholesterol stimulates the synthesis and secretion of ApoE, an effect which may be modulated by the ApoE allele (Abildayeva et al., 2006). ATP-Binding Cassette (ABC) transporters, particularly the A and G subfamilies play important roles in cholesterol efflux (Borst et al., 2000). ABCA1 is known to play a critical role in peripheral lipid transport by regulating cholesterol efflux from the plasma membrane to the lipid acceptor protein-AI (apoA-I). High expression of ABCA1 occurs in neurons of hypothalamus, thalamus, amygdala, cholinergic basal forebrain, and hippocampus in the adult rat brain (Koldamova et al., 2003). ABCA1 expression is known to be regulated by LXRs (Venkateswaran et al., 2000). In liver, cholesterol metabolite, 24(S)-hydroxycholesterol is efficiently converted into bile acids and excreted in bile in its sulfated and glucuronidated form (Pikuleva, 2006).

10.4 Roles of Hydroxycholesterols in the Brain

As stated above, cholesterol homeostasis is maintained through the oxidation of cholesterol to several hydroxycholesterols and cholesterol oxidation products (COPs). Among hydroxyl-cholesterols and COPs, 24(S)-hydroxycholesterol is a major metabolite of cholesterol metabolism (Russell, 2000; He et al., 2006). The conversion of cholesterol to 24(S)-hydroxycholesterol is catalyzed by the cholesterol- 24-hydroxylase (CYP46), an enzyme, which is enriched in the striatum and the cortex. Knocking out cholesterol 24-hydroxylase in mice produces 40% reduction in de novo cholesterol synthesis in the brain (Lund et al., 1999). In the human adult, brain cholesterol turnover gives rise to a daily flux of about 7 mg of 24(S)- hydroxycholesterol from the brain into the circulation, so that 24-hydroxycholesterol levels in plasma are taken as an index of brain cholesterol elimination (Lütjohann et al., 1996). Although downregulation of cholesterol synthesis via the SREBP/ SCAP regulatory pathway is common for 24(S)-hydroxycholesterol synthesis, more variations exist with respect to other hydroxycholesterols, which may also act as ligands for the nuclear receptor LXRa. Because this receptor regulates the expression of cholesterol 7a-hydroxylase and ABC transporter proteins, hydroxycholesterols and their intermediate steroid metabolites modulate a number of biological processes, including differentiation, exocytosis, enzyme activities, and immune function (Schroepfer, 2000) (Figs. 10.3 and 10.8). In addition, hydroxycholesterols also modulate cholesterol trafficking, gene transcription, PtdCho synthesis, inflammation, and cognitive function. In addition to having a direct physical effect on membranes and modulation of Ca2+ signals, hydroxysterols also inhibit the phosphorylation of endothelial nitric oxide synthase (eNOS) and cPLA2 through LXR- mediated mechanism (Fig. 10.8). 278 10 Cholesterol and Hydroxycholesterol in the Brain

10.4.1 Hydroxycholesterols in Neural Cell Differentiation

Cellular differentiation is defined as a process by which a generic cell transforms into a specific type of cell in response to specific triggers from the body or the cell itself. This process results in the maturation of a less specialized cell into a more specialized cell type in order to perform a specific function without any alterations in the genetic material. In multicellular organisms differentiation occurs many times not only during the development, but also in adulthood (Sue O’Shea, 2002). Hydroxycholesterols modulate neural cell differentiation and cell death in various types of neural cell cultures. Thus, 22 (R)-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, stimulates neurite outgrowth formation in rat pheochromocytoma cells (PC12 cells) and differentiated human Ntera2/D1 teratocarcinoma neurons. Although the mechanism associated with neuronal differentiation is not fully understood, it is proposed that 22 (R)- hydroxycholesterols interact with LXRs through NF-kB-dependent mechanism and modulate neuronal differentiation in rat PC12 cells. No neurite outgrowth formation occurs without the participation of LXRs (Schmidt et al., 1999). In human Ntera2/ D1 teratocarcinoma precursor cells (NT2), 22(R)-hydroxycholesterol inhibits the proliferation, but induces differentiation in neurons and astrocytes to produce “neuron-like” or “astrocyte-like” cells (Yao et al., 2007). 22 (R)-Hydroxycholesterol- mediated differentiation of NT2 cells is accompanied by elevations in the expression of neurofilament protein NF200, the cytoskeletal proteins microtubule-associated protein type II (MAP2a and MAP2b), glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor receptor-a 2 (GFRa 2). These effects are produced only by 22(R)-hydroxycholesterol. 22(S)-Hydroxycholesterol, an enantiomer of 22(R)-hydroxycholesterol, does not produce differentiation and has no effect on the expression of above parameters indicating that these effects are stereospecific. Furthermore, other steroids have also failed to induce differentiation in NT2 cells (Yao et al., 2007). Similarly, 22 (R)-hydroxycholesterol also modulates neurosteroid formation in SH-SY5Y cells. This process is inhibited by the cytP450scc inhibitor, aminoglutethimide. Treatments of differentiated SH-SY5Y cells with the microtubule-depolymerizing drug colchicine and the actin microfilament- altering agent cytochalasin D reduces pregnenolone synthesis without affecting cell viability. Since the addition of the cell-permeant cholesterol analog, 22(R)- hydroxycholesterol promotes pregnenolone synthesis, it is proposed that modulation of progesterone synthesis by 22(R)-hydroxycholesterol may be associated with neuronal plasticity, cognitive function, and also neurodegenerative disorders (Guarneri et al., 2000). 22-Hydroxycholesterol also regulates cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuronal development (Sacchetti et al., 2009). The deletion of LXRa and LXRb slows cell cycle progression and VM neurogenesis, causing a decrease in DA neurons at birth. Activation of LXRa and LXRb with hydroxycholesterols increases the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type, but not in LXRa and LXRb(−/−) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) 10.4 Roles of Hydroxycholesterols in the Brain 279 during DA differentiation upregulate neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors (Sacchetti et al., 2009). In contrast, 25-hydroxycholesterol produces neurotoxicity in NGF-differentiated PC12 cells in dose- and time-dependent manner (Chang et al., 1998a). 25-Hydroxycholesterol- mediated cell death can be prevented by treatment with vitamin E and methyl-beta- cyclodextrin (Chang et al., 1998b). This suggests that 25-hydroxycholesterol-mediated cell death may involve oxidative stress.

10.4.2 Hydroxycholesterols in Exocytosis

Exocytosis is defined as a process by which intracellular vesicles containing soluble proteins, lipids, and neurotransmitters are released into the extracellular compartment. This process involves binding of vesicles with plasma membrane and dis- charge of vesicles out of the cell. Hydroxycholesterols (7-ketocholesterol, 24(S)-hydroxycholesterol, and cholesterol 5, 6 b-epoxide) have reported to induce exocytosis in PC12 cells (Ma et al., 2010). 7-Ketocholesterol-mediated exocytosis can be attenuated by pretreatment with methyl-beta-cyclodextrin (MbCD). Moreover, treatment of PC12 cells with thapsigargin, an agent that depletes intracellular calcium, or addition of lanthanum chloride, an agent that blocks calcium channels also attenuates 7-ketocholesterol-mediated exocytosis in PC12 cells. Fura-2 imaging studies indicate that treatment with 7- ketocholesterol results in rapid and sustained increases in intracellular calcium levels, and this effect can also be attenuated by MbCD, thapsigargin, or lanthanum chloride (Ma et al., 2010). Collectively, these results indicate that by 7-ketocholesterol-mediated neurotransmitter release depends on the integrity of cholesterol-rich lipid domains on PC12 membranes and a rise in intracellular calcium, either through release from internal stores or influx via calcium channels may be closely associated with exocytosis.

10.4.3 Hydroxycholesterols in Apoptosis

Apoptosis is characterized by nuclear chromatin condensation, DNA fragmentation, cell shrinkage, and bleb and apoptotic body formation (Farooqui et al., 2004). Plasma membrane and other subcellular organelles such as mitochondria and endoplasmic reticulum remain active during apoptosis. Neurochemically, apoptosis is characterized by the externalization of PtdSer from inner leaflet to the outer leaflet, cytochrome c release, caspase-3 activation, and DNA ladder formation. Apoptosis is a characteristic feature of the normal developmental process as well as a response of cells to stress or other environmental insults (Oppenheim, 1991). While the mechanisms leading to apoptotic cell death are not yet understood, several factors including expression of bcl-2 or bcl-xL genes and neurotrophic survival factors have been reported to prevent apoptotic cell death (Farooqui, 2009). Hydroxycholesterols are 280 10 Cholesterol and Hydroxycholesterol in the Brain

Table 10.1 Neurochemical effects of various hydroxycholesterols in brain Mediator Effect Reference 24-Hydroxycholesterol Modulation of cholesterol homeostasis, Velázquez et al., 2006; apoptosis, oxidative stress, and Ong et al., 2003 inflammation 25-Hydroxycholesterol Modulation of apoptosis, oxidative Kim et al., 2006; Ong stress, and inflammation et al., 2003 27-Hydroxycholesterol Elevation in Ab, suppression of Arc, Velázquez et al., 2006; apoptosis, oxidative stress Lizard et al., 2000; Mateos et al., 2009 22-Hydroxycholesterol Modulation of neurogenesis Sacchetti et al., 2009 7-Ketocholesterol Modulation of apoptosis, oxidative Chang et al., 1998a stress, and inflammation Ab beta amyloid, Arc activity-regulated cytoskeleton-associated protein cytotoxic to human neuroblastoma cell line, SH-SY5Y, oligodendrocyte cell line (158N), and endothelial cells. In SH-SYSY and 158N cells, 24(S)-hydroxycholesterol and 25-hydroxycholesterol induce apoptotic cell death through the stimulation of caspase-3 (Kolsch et al., 2001; Trousson et al., 2009; Rantham Prabhakara et al., 2008) (Table 10.1). This enzyme causes proteolytic cleavage of a variety of enzymes (protein kinase C, cytosolic phospholipase A2, calcium-independent phospholipase A2, phospholipase C), cytoskeletal proteins (a-spectrin, b-spectrin, actin, vimentin, Bcl-2 family of apoptosis-related proteins), and DNA modulating enzymes (poly [ADP- ribose]polymerase) (Kolsch et al., 2001; Garcia et al., 1992). Other oxysterols, such as 25-hydroxycholesterol, 7b-hydroxycholesterol, and 7-ketocholesterol, also produce toxic effects on neural cell cultures (Chang et al., 1998a; Ong et al., 2003). 7b-Hydroxycholesterol, and 7-ketocholesterol modulate secretion of IL-1b, which is synthesized as an inactive 33 kDa propeptide. Proteolytic cleavage of this propeptide produces active IL-1b. The release of this cytokine facilitates apoptotic cell death. Studies on the effect of 7-ketocholesterol and 1-methyl-4-phenylpyridinium (MPP+) in differentiated PC12 cells indicate that 7-ketocholesterol significantly increases the MPP+-mediated nuclear damage, reduces the mitochondrial transmembrane potential, increases cytosolic cytochrome c, activates caspase-3, enhances reactive oxygen species production, and depletes reduced glutathione (Kim et al., 2006). N-Acetylcysteine, ascorbate, trolox, and calmodulin antagonists block the cytotoxic effect of MPP+ in the presence of 7-ketocholesterol indicating that 7-ketocholesterol produces a synergistic effect with MPP+ (Kim et al., 2006; Han et al., 2007). The molecular mechanism associated with hydroxyl- and ketocholesterol- mediated toxic effect is not fully understood. However, 7-ketocholesterol triggers the stimulation of NADPH oxidase, generation of superoxide anions, loss of mitochondrial transmembrane potential (∆Ym), release of cytochrome c, and activation of caspase-3. These processes are closely associated with apoptotic cell death (Lizard et al., 2000). The presence of 24-hydroxycholesterol and 25-hydroxycholesterol in plasma is an indication of neurodegeneration in brain. Other cholesterol-derived metabolite, such as 7-oxocholesterol not only modulates Ca2+ signals, but also inhibits the phosphorylation of endothelial nitric oxide synthase and cPLA2 10.5 Cholesterol and Hydroxycholesterols in Neurological Disorders 281

(Millanvoye-Van Brussel et al., 2004). Collective evidence suggests that hydroxysterols have neurotoxic effects on neural cell cultures, and among them 24-hydroxycholesterol can be used as a marker for neurodegeneration (Rojo et al., 2006).

10.5 Cholesterol and Hydroxycholesterols in Neurological Disorders

Intake of cholesterol enriched high fat diet not only causes BBB dysfunction in New Zealand white rabbits (Ghribi et al., 2006), but also results in cerebral amyloidosis in wild-type rabbits (Sparks et al., 1994), suggesting that high fat cholesterol- enriched diet may be a risk factor for neurodegenerative diseases, such as AD. Epidemiological and biochemical investigations also indicate that there is a link between cholesterol turnover and neurological diseases, and hypercholesterolemia per-se is an important risk factor for AD, PD, and HD (Wolozin, 2004; Huang et al., 2007; Wolozin et al., 2007). These diseases involve the accumulation of intracellular or extracellular cerebral deposits of misfolded protein called amyloidogenic proteins. Thus, accumulation of b-amyloid (Ab), a-synuclein, and huntingtin occurs in AD, PD, and HD respectively (Farooqui, 2010). Although molecular mechanisms by which amyloidogenic proteins produce neurodegeneration are not fully understood, marked increase in oxidative stress and neuroinflammation along with elevation in hydroxycholesterol levels and lipid mediators of phospholipid and sphingolipid metabolism have been reported to occur in AD, PD, and HD (Farooqui, 2010). In addition, increase in hydroxycholesterol levels also occurs in multiple sclerosis (MS), ischemic injury, and traumatic brain injury (TBI), Niemann-Pick disease (NPD), and cerebrotendinous xanthomatosis (CTX) (Fig. 10.9).

Traumatic Hydroxycholesterols Cerebrotendinous brain injury xanthomatosis

Ischemia Niemann-Pick type C

Multiple sclerosis Alzheimer disease Huntington disease

Fig. 10.9 Alterations in hydroxycholesterol in neurological disorders upward arrows indicate increase in hydroxycholesterol 282 10 Cholesterol and Hydroxycholesterol in the Brain

10.5.1 Cholesterol and Hydroxycholesterol in Alzheimer Disease

It is well known that AD is characterized by progressive neuronal degeneration, gliosis, and accumulation of plaques and tangles, which are aggregates of amyloid- b (Ab) peptides derived from proteolytic cleavages of amyloid precursor protein (APP), and hyperphosphorylated tau protein, respectively (Farooqui, 2010). APP is processed via a- or the b-secretase pathways (Thinakaran and Koo, 2008), resulting in the shedding of nearly the entire ectodomain and generation of membrane- tethered b-or a-C-terminal fragments, respectively. The b- and a-C-terminal fragments are subsequently cleaved within the transmembrane domain by g-secretase. Cholesterol directly interacts with APP and stimulates its insertion into phospholipid monolayers (Fantini and Yahi, 2010). It also interacts with Ab protofibrils. However, it remains controversial whether cholesterol enhances or reduces Ab polymerization. Since, the generation of Ab peptides through APP proteolysis occurs within lipid rafts and is sensitive to inhibitors of cholesterol biosynthesis, its association with cholesterol homeostasis in AD may not be simply through the regulation of Ab fibrillogenesis (Wolozin, 2004; Fantini and Yahi, 2010), but other parameters may also play an important role in the above process. Association of sterol homeostasis, particularly cholesterol metabolism, is currently the subject of intense interest in the pathogenesis of AD. In AD, increase in lipid peroxidation not only intensifies neuronal death, but also induces the synthesis of oxidized cholesterol (hydroxysterols) (Arca et al., 2007). These events directly or indirectly may promote APP processing in favor of insoluble b-amyloid deposition (Nelson and Alkon, 2005), tau hyperphosphorylation and NFT formation, mitochondrial insuf- ficiency, and neuronal cell death (Melov et al., 2007). Thus, Ab and APP are known to oxidize cholesterol to form 7b-hydroxycholesterol that is a pro-apoptotic oxysterol with neurotoxicity at nanomolar concentrations. 7b-Hydroxycholesterol also blocks the secretion of soluble APP from cultured rat hippocampal H19-7/IGF-IR neuronal cells (Nelson and Alkon, 2005). Levels of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid (CSF) are significantly higher in AD and vascular demented patients at early stages of the disease compared to healthy subjects (Lutjohann and von Bergmann, 2003). Statins, the inhibitors of cholesterol biosynthesis, modulate APP processing and neuronal secretion of Ab along with de novo cholesterol synthesis. High-doses of simvastatin (80 mg/day) given to AD patients with hypercholesterolemia not only lead to a significant decrease in CSF concentrations of 24(S)-hydroxycholesterol, but also a significant decrease inb A -levels in cerebrospinal fluid supports the view that 24-hydroxycholesterol may be associated with the pathogenesis of AD (Lutjohann and von Bergmann, 2003). 24(S)-Hydroxycholesterol exerts its effect on APP processing by increasing the a-secretase activity as well as the a/b-secretase activity ratio (Famer et al., 2007). In CSF from AD patients, levels of ApoE, tau, p-tau (hyperphosphorylated tau) are significantly increased along with 24(S)- hydroxycholesterol compared to CSF from control subjects. It is stated that during neurodegenerative process cholesterol is converted into 24(S)-hydroxycholesterol. The release of 24(S)-hydroxycholesterol from neurons induces ApoE secretion by 10.5 Cholesterol and Hydroxycholesterols in Neurological Disorders 283 astrocytes, and both processes are related to the intensity of the neurodegenerative process and neuronal injury (Leoni et al., 2010). ApoE is also involved in the scav- enging of tau from neurons. The direct correlations between ApoE, 24(S)- hydroxycholesterol, and tau suggest that cholesterol metabolism may be involved in the generation of both tau and Ab, and ApoE is released by astrocytes in order to counteract this ongoing process (Leoni et al., 2010). Levels of 22(R)-hydroxycholesterol, a steroid intermediate in pregnenolone synthesis pathway are decreased in hippocampal and frontal cortex samples from AD patients compared to age-matched controls (Yao et al., 2002). 22(R)-Hydroxycholesterol has been shown to protect PC12 and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neurons from Ab toxicity and death. The effect of 22(R)-hydroxycholesterol seems to be stereospecific because its enantiomer 22(S)-hydroxycholesterol fails to protect cultured cells from Ab-induced cell death. Moreover, the effect of 22(R)-hydroxycholesterol is specific forb A - induced cell death because it does not protect against glutamate-induced neurotoxicity. It is proposed that 22(R)-hydroxycholesterol offers a new means of neuroprotection against Ab toxicity. There is a significant flux of another oxysterol, 27-hydroxycholesterol from the circulation into the brain. Oxysterols also inhibit HMG-CoA reductase activity in astrocytes. 25-Hydroxycholesterol has a dual effect on cell proliferation: at higher concentrations it produces cell proliferation in a dose-dependent manner, while at lower concentrations it promotes neural cell survival (Velázquez et al., 2006). Cholesterol-enriched diets and hydroxycholesterol (27-hydroxycholesterol) increase Ab and phosphorylated tau levels in rodents. Although the mechanisms by which cholesterol and 27-hydroxycholesterol modulate the production of Ab and tau protein phosphorylation is not fully understood, but in transgenic mice for AD, leptin, an adipocytokine, has been reported to control and modulate Ab production and tau protein hyperphosphorylation. Recent studies indicate that feeding rabbits with a 2% cholesterol-enriched diet for 12 weeks decreases the levels of leptin by approximately 80%. Incubation of hippocampal organotypic slices with 27-hydroxycholesterol not only reduces leptin levels by approximately 30%, but also increases Ab(1–40) and Ab(1–42) levels to 1.5-fold and 3-fold, respectively along with elevation in phosphorylated tau protein levels. Treatment of hippocampal organotypic slices with leptin retards the 27-hydroxycholesterol-mediated increase in Ab and phosphorylated tau protein by decreasing the levels of BACE-1 and GSK-3b, respectively. Accumulating evidence suggests that cholesterol- enriched diets and 27-hydroxycholesterol induce AD-like pathology by altering leptin signaling (Marwarha et al., 2010). Although the molecular mechanism associated with 27-hydroxycholesterol-mediated effect is not known, this metabolite is not only known to increase Ab production, caspase 12 activity, and gadd153 (also called CHOP) expression, but also decreases mitochondrial membrane potential, increases levels of the nuclear factor-kB (NF-kB) and heme-oxygenase 1 (HO-1) along with alterations in calcium homeostasis. Additionally, 27-hydroxycholesterol induces glutathione depletion, ROS generation, inflammation, and apoptotic-mediated cell death (Dasari et al., 2010). In addition, cholesterol-enriched diet turns on 284 10 Cholesterol and Hydroxycholesterol in the Brain expression of genes associated with AD-like pathology in hippocampal and cortical area of mice brain (Mateos et al., 2009). This gene expression includes the expression of Activity-regulated cytoskeleton-associated protein (Arc). High cholesterol diet suppresses Arc in mice. This gene plays an important role in memory consolidation in rodents and its expression is reduced in autopsy brains obtained from AD patients (Ginsberg et al., 2000; Guzowski et al., 2000). The expression of Arc is regulated by NMDA receptor (Fig. 10.8). This glutamate receptor modulates excitotoxicity and alterations in its activities are closely associated with pathophysiology of AD (Farooqui, 2010). Treatment of hippocampal neuronal cultures with 27-hydroxycholesterol produces changes, which are similar to the in vivo effects of cholesterol on the expression of Arc and NMDA receptor. It is proposed that this may be one of the mechanism by which hypercholesterolemia may contribute to the pathogenesis of neurodegenerative diseases (Björkhem et al., 2009). In addition, abnormalities in cholesterol metabolism correlate well with reports indicating that the possession of the apoE allele e4 is a strong risk factor for AD development, because there is a stepwise increase, as a function of alleles (e2 to e3 to e4), in serum total and low-density lipoprotein (LDL) cholesterol levels (Frikke-Schmidt et al., 2000). The finding that statin treatment decreases the prevalence of AD (Wolozin et al., 2000) supports the suggestion that reduction in serum total cholesterol level and LDL-cholesterol may result in decrease in cellular cholesterol level in the CNS, which in turn may retard the synthesis of amyloid b-protein (Ab) in neurons (Simons et al., 1998) and secretion of Ab from cells into the CSF (Fassbender et al., 2002). Collective evidence suggests that alterations in cholesterol and hydroxycholesterols levels may be associated with pathophysiology of AD.

10.5.2 Cholesterol and Hydroxycholesterol in Parkinson Disease

Parkinson disease (PD) is a neurodegenerative disorders that is characterized by the gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta (Farooqui, 2010). Oxidative stress, inflammation, and a-synuclein overexpression are major risk factors for the development of PD and Lewy body dementia. Recent studies implicate cholesterol in the pathogenesis of PD, and it is shown that statins may lower the risk of PD (Johnson et al., 1999; Huang et al., 2007; Wolozin et al., 2007; Hu et al., 2008). Cholesterol and a-synuclein interact with each other in lipid rafts (Fortin et al., 2004) and that consumption of diet enriched in cholesterol increases the risk factor of developing PD. Studies on cell culture model and transgenic mice model of PD indicate that the cholesterol-depleting agent methyl-b- cyclodextrin reduces the level of a-synuclein in membrane fractions (Bar-On et al., 2006). Moreover, metabolic inhibition of cholesterol biosynthesis with statins decreases the levels of a-synuclein accumulation in neuronal membranes, whereas cholesterol supplementation of cultured neurons enhances a-synuclein aggregation (Bar-On et al., 2008). These studies are consistent with recent reports indicating that lovastatin treatment of a-synuclein transgenic mice is accompanied by a marked 10.5 Cholesterol and Hydroxycholesterols in Neurological Disorders 285 reduction of a-synuclein aggregation and abrogation of neuronal pathology (Koob et al., 2010), supporting the view that treatment with statins may be beneficial for PD patients. It is also shown that levels of oxidized cholesterol metabolites are markedly increased in the brains of PD patients and oxidized cholesterol metabolites can enhance a-synuclein aggregation providing a mechanistic link between oxidative stress and development of PD (Bosco et al., 2006). Although the role of a-synuclein in brain remains elusive, recent studies indicate that a-synuclein may be associated with synaptic vesicle trafficking probably via lipid binding (Bar-On et al., 2006, 2008) and in neurotransmitter release. 27-Hydroxycholesterol increases the levels of a-synuclein and induces apoptosis, whereas 24-hydroxycholesterol increases the levels of tyrosine hydroxylase, the enzyme that catalyzes the synthesis of dopamine, reflective specificity of the actions of 27- versus 24-hydroxycholesterol (Rantham Prabhakara et al., 2008). The differential effects of 24- and 27-hydroxycholesterols on a-synuclein may be similar to the role of hydroxycholesterols in APP processing (Prasanthi et al., 2009; Marwarha et al., 2010) and on cholesterol storage in Niemann-Pick C1 disease (Frolov et al., 2003).

10.5.3 Cholesterol and Hydroxycholesterol in Huntington Disease

Huntington Disease (HD) is a hereditary and progressive neurodegenerative disorder that is characterized by mid-life onset causing involuntary movements, cognitive, physical, and emotional deterioration, personality changes, dementia, and premature death. HD is characterized by neurodegeneration in the basal ganglia and cerebral cortex (Cepeda et al., 2001). The genetic defect in HD involves the presence of unstable CAG trinucleotide repeat in exon 1 of the HD gene, which encodes for a polyglutamine expansion near the N-terminal end of a large protein called huntingtin (a 348 kDa protein essential for embryogenesis). Insoluble aggregates containing huntingtin occur in cytosol and nuclei of HD patients, transgenic animal, and cell culture models of HD. Accumulation of mutant huntingtin along with transcriptional repression, proteasome impairment, oxidative injury, and mitochondrial dysfunction may lead to neurodegeneration in HD (Farooqui, 2010). Alterations in cholesterol metabolism occur in murine HD models and HD patients (Valenza et al., 2010; Leoni et al., 2008). The synthesis of cholesterol is reduced in multiple transgenic and knock-in HD rodent models. In animal model of HD, the cholesterol-dependent activities of neurons mainly rely on the transport of cholesterol from astrocytes on ApoE-containing particles. mRNA levels of cholesterol synthesizing enzymes and mRNA for cholesterol efflux transporters are severely reduced in primary HD astrocytes, along with impaired cellular production and secretion of ApoE (Valenza et al., 2010). Plasma 24(S)-hydroxycholesterol levels are significantly higher in controls than in HD patients at all disease stages (Leoni et al., 2008). In pre-HD, subject’s plasma levels of 24(S)-hydroxycholesterol have been reported to be similar to control subjects, and thus significantly higher than 286 10 Cholesterol and Hydroxycholesterol in the Brain those of HD patients at any disease stage. Changes in 24(S)-hydroxycholesterol levels parallel the large decrease in caudate volumes, a process that occurs in HD stage 1 and is associated with neuronal loss (Leoni et al., 2008). It is likely that dysregulation in cholesterol metabolism is linked to specific actions of the mutant huntingtin on sterol regulatory element-binding proteins leading to lower cholesterol levels in caudate areas of the brain. Neural cells expressing mutant huntingtin show increase in NMDA receptor density in cholesterol-enriched domains (del Toro et al., 2010). This increase in NMDA receptors contribute to increase in susceptibility to excitotoxic insults. Treatment of neural cells with simvastatin or b-cyclodextrin protects neural cells against NMDA-mediated excitotoxicity (del Toro et al., 2010). It is proposed that mutant huntingtin-mediated accumulation of cholesterol contributes to NMDA-mediated excitotoxicity. Emerging evidence suggests that dysregulation of cholesterol and sphingolipid metabolism may contribute to the pathogenesis of HD through increased sensitivity to excitotoxicity (del Toro et al., 2010), and changes in 24(S)-hydroxycholesterol levels complement findings of MRI morphom- etry, which has become a valuable tool to follow neurodegenerative changes in the early stages of HD (Leoni et al., 2008).

10.5.4 Cholesterol and Hydroxycholesterols in Niemann-Pick Type C

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder. At the subcellular level, NPC is characterized by improper trafficking of cholesterol, bis(monoacyl-glycerol)phosphate (BMP), and sphingolipids in lysosome-like storage organelles (LSOs), which become engorged with these lipids. Progressive neuronal loss, especially of cerebellar Purkinje cells, is a hallmark of NPC. Ballooned neurons, axonal abnormalities, and astroglyosis along with severe demyelination are pathological characteristics of NPC. It is suggested that the initial defect in cholesterol or sphingolipid trafficking causes eventual traffic jam in these LSOs resulting in not only retention of these lipids, but also transmembrane proteins, which transiently associate with the late endosomes in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN) and plasma membrane (PM) (Mukherjee and Maxfield, 2004). Two cholesterol-binding proteins, NPC1 (a large transmembrane protein with 1278 amino acid residues) and NPC2 (an intra-lysosomal soluble protein comprising 132 amino acid residues) are known to occur in mammalian tissues. They act in concert to export lipoprotein- derived cholesterol and sphingolipids from lysosomes to TGN and PM (Wiegand et al., 2003; Friedland et al., 2003). Thus, NPC1 plays a major role in the sorting of glycolipids as well as cholesterol within the late endosomes, whereas NPC2 primarily plays a role in the egress of cholesterol and, potentially, glycolipids from lysosomes. Mutations in either NPC1 or NPC2 are the cause of the NPC. The most common form of the disease (95% cases) is caused by mutation in NPC1gene and remaining cases result from mutation in the NPC2 gene. Mutation in NPC1 protein causes 10.5 Cholesterol and Hydroxycholesterols in Neurological Disorders 287 impairment of intracellular cholesterol trafficking and dysregulation of cholesterol biosynthesis. Disruption of cholesterol homeostasis also contributes to deregulation of autophagic activity and early-onset neuroinflammation, which may contribute to the pathogenesis of NPC disease. Cholesterol trafficking defects result in diminished production of cholesterol derivatives including neurosteroids, which normally help mediating brain development, growth, and differentiation (Mellon, 2007). NPC1 knockout mice show mitochondrial cholesterol accumulation, mitochondrial glutathione depletion, and release of apoptogenic proteins (Fernandez et al., 2009). NPC1 binds cholesterol and 25-hydroxycholesterol with higher affinity than 24(S)- hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol (Infante et al., 2008). NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5-a-cholestan-3-one) (Liou et al., 2006). Immunoelectron microscopic study of the primate brain showed that NPC1 is localized primarily in astrocytic processes at the sides of synapses (Patel et al., 1999), whilst NPC2 is observed mainly in small diameter dendrites or dendritic spines (Ong et al., 2004). Collectively, these studies suggest that alterations in hydroxycholesterol levels may contribute to the pathogenesis of NPC.

10.5.5 Cholesterol and Hydroxycholesterol in Multiple Sclerosis

Multiple sclerosis is an inflammatory neurodegenerative condition that is characterized by the demyelination of axons in discrete regions of the brain and spinal cord and infiltration of activated macrophages into the brain parenchyma. The most accepted component of MS etiology is crossing of activated T-cells through the blood–brain barrier and initiation of an inflammatory response to myelin (Hartung and Rieckman, 1997). This is followed by scars or plaques formation in the damaged areas leading into faulty nerve conduction. Initial demyelination of CNS axons triggers catastrophic events, resulting in further immune activation and inflammatory injury to axons (Craner et al., 2004). Thus, activated macrophages have been shown to play an important role in the propagation of the disease (Trapp et al., 1998). MS starts as a relapsing-remitting disease (RRMS), and is followed by a progressive phase (SPMS). The progressive phase causes the greatest disability and has no effective therapy, but the processes that drive SPMS are mostly unknown. Determination of 25(S)-hydroxycholesterol levels in MS patients at different stages of the disease indicate that in the oldest groups of patients, the levels of 24(S)-hydroxycholesterol are significantly lower than in the controls, possibly reflecting loss of neuronal cells responsible for the synthesis and this decrease correlates with the disease burden evaluated by magnetic resonance imaging (MRI). Furthemore, there is a significant inverse relation between the expanded disability status scale-grade of the disease and the plasma cholesterol-related levels of 24(S)- hydroxycholesterol. There is a tendency to increased plasma levels of 24(S)- hydroxycholesterol in the younger patients. High levels of 24(S)-hydroxycholesterol 288 10 Cholesterol and Hydroxycholesterol in the Brain have been reported to occur in MS patients in the third and fourth decades of life (Leoni et al., 2002; Teunissen et al., 2003, 2007; Leoni et al., 2003). More studies are needed not only in brain tissue from MS patients, but also in serum and CSF levels in various phases of MS.

10.5.6 Hydroxycholesterol in Traumatic Brain Injury

Secondary phase of traumatic brain injury (TBI) is accompanied not only by alterations in cholesterol homeostasis, but also activation of microglial cells and astrocytes, and oligodendroglia leading to microgliosis, astrocytosis, and demyelination (Raghupathi, 2004; Farooqui, 2010). Inflammatory reactions, oxidative stress, and nitrosative stress are major components of secondary injury. Following TBI, activity of cholesterol 24S-hydroxylase (Cyp46) and levels of 24(S)-hydroxycholesterol are significantly increased in brain tissue (Cartagena et al., 2010). Alterations in levels of this cholesterol metabolite increase SREBP-1 mRNA and full-length protein but have no effect on levels of cleaved SREBP-1. In contrast, 24(S)-hydroxycholesterol decreases levels of LXR- independent SREBP-2 mRNA, full-length protein, and SREBP-2 active cleavage product. In neuroblastoma cells, 24(S)-hydroxycholesterol decreases HMG CoA reductase, squalene synthase, and FPP synthase activities as well as their mRNA levels but has no effect on mRNA of fatty acid synthesis genes acetyl CoA carboxylase or fatty acid synthase. Levels of fatty acid synthase mRNA are not altered by 24(S)-hydroxycholesterol treatment, but acetyl CoA carboxylase mRNA levels are significantly decreased, indicating that changes to transcription of cholesterol synthesis genes after TBI are related with the increase in Cyp-46 activity (Cartagena et al., 2010), but not with alterations in fatty acid synthesis genes, which are modulated by other mechanisms.

10.5.7 Cholesterol and Hydroxycholesterols in Cerebrotendinous Xanthomatosis

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder caused by mutations in the CYP27A1 gene coding for the enzyme sterol 27-hydroxylase, a mitochondrial P-450 enzyme with broad substrate specificity. The rates of hydroxylation of the sterols are: 7a-hydroxy-4-cholesten-3-one > 4-cholesten-3- one > 7a-hydroxycholesterol > 24-hydroxy-4-cholesten-3-one > cholesterol > 25-hydroxy- 4-cholesten-3-one > 24-hydroxycholesterol > or = 25-hydroxycholesterol (Norlin et al., 2003). CTX is characterized by tendon xanthomas, diarrhea, cataracts, and progressive neurological dysfunction, including dementia, psychiatric disturbances, pyramidal and/or cerebellar signs, and seizures (Gallus et al., 2006). CTX patients develop severe premature atherosclerosis and elevated levels of cholestanol (a by-product of abnormal bile acid synthesis) despite normal serum levels of 10.6 Cholesterol and Hydroxycholesterols in Kainic Acid Neurotoxicity 289 cholesterol. Based on this observation, it is suggested that sterol 27-hydroxylase is an anti-atherosclerotic enzyme. Sterol 27-Hydroxylase not only catalyzes bile acid hydroxylation (Russell, 2003) and activation of vitamin D (Usui et al., 1990), but also stimulates cholesterol efflux in cultured cells (Escher et al., 2003) through the formation of 27-hydroxycholesterol. This metabolite is a ligand for LXR receptor. CYP27 mediates LXR activation as a response to cholesterol loading, and this may be the dominant mechanism for induction of LXR-responsive genes in human macrophages. Sterol 27-hydroxylase may accordingly protect these cells from cholesterol overload by two concurrent but separate mechanisms. One mechanism (Björkhem et al., 1994) utilizes the increased solubility of 27-hydroxycholesterol and cholestenoic acid to excrete these compounds from the cell. The second mechanism operates via activation of LXR, stimulating ABCA1-mediated reverse cholesterol transport. Both of these pathways are impaired in CTX patients, lacking sterol 27-hydroxylase activity.

10.6 Cholesterol and Hydroxycholesterols in Kainic Acid Neurotoxicity

Kainic acid (KA) is a cyclic and nondegradable analog of glutamate. Its injections produce loss of neurons in specific striatal and hippocampal areas of the brain (Farooqui et al., 2008). Neuronal axons and nerve terminals are more resistant to the destructive effects of KA than the cell soma. KA induces its effects by interacting with specific receptors, the KA receptors (KAR). KA administration in rodents has been used as an animal model to study molecular mechanism of neurodegeneration (Farooqui et al., 2001). Intraventricular injections of KA are known to produce increase in cholesterol immunostaining with filipin in the CA1 region of rat hippocampus (Ong et al., 2003). Studies on cholesterol biosynthetic pathway in KA-mediated lesions indicate that mRNA level of SREBP-2 is significantly reduced at both 1 day and 2 weeks post-kainate injection. Immunohistochemical analyses show significant reduction in SREBP-2 immunoreactivity in the KA-induced lesions. These studies show that there is high level of SREBP-2 expression in the normal hippocampus, and that KA-mediated neuronal injury results in a significant reduction of SREBP-2 expression in the damaged areas (Kim and Ong, 2009; Ong et al., 2010). KA injections also produce a significant decrease in HMG-CoA reductase. Lanosterol synthase and CYP51, which are responsible for conversion of squalene to lanosterol, and lanosterol to cholesterol, show significant reduction in mRNA expression at 1 week and 2 week post-KA injection, respectively. KA injections also produce increase in ABC1 mRNA expression in the hippocampus after 1 week and 2 weeks. The increase in ABCA1 mRNA is paralleled by an increase in ABCA1 protein expression, in reactive astrocytes in the degenerating hippocampus (Ong et al., 2010). The expression of ABCA1 regulated by LXRs (Venkateswaran et al., 2000) and their major ligands are oxysterols (Repa et al., 2007). Thus, the increase of ABCA1 after 290 10 Cholesterol and Hydroxycholesterol in the Brain

KA-mediated neurodegeneration may be due to increased oxysterols since oxysterols can activate liver X receptors which modulate ABCA1 expression (Cao et al., 2007). In parallel with the increase in ABCA1 and ABCG1, increase in expression of another enzyme involved in cholesterol export into the bloodstream, cholesterol 24 hydroxylase, and its product 24-hydroxycholesterol is found in the degenerating hippocampus after KA-induced lesions (He et al., 2006). Analysis of brain tissue by lipidomics indicates that there is a significant increase in cholesterol, 24-hydroxycholesterol, and 7-ketocholesterol in hippocampal region at 2 weeks after injury (Ong et al., 2003). As stated above, cholesterol oxidation products produce toxic effect on neuronal cultures through decrease in staining of the AMPA receptor subunit GluR1 in the CA field. The addition of glutathione to hippocampal cultures and slices blocks the decrease in GluR1 immunoreactivity. In organotypic slices from the hippocampus of rats injected with KA plus lovastatin show significantly lower levels of cholesterol, 24-hydroxycholesterol, and 7-ketocholesterol, compared to those that have been treated with KA alone, suggesting that lovastatin modulates the loss of hippocampal neurons after KA treatment. This observation suggests that KA-induced toxicity is mediated through cholesterol oxidation products (Park et al., 2000; Ong et al., 2010).

10.7 Conclusion

The brain is the richest source of cholesterol. Cholesterol contents of brain are independent of dietary cholesterol contents. BBB prevents the entry of cholesterol from the circulation into the brain. So that de novo synthesis of cholesterol is responsible for almost all cholesterol present in the brain. Based on the above discussion, it is obvious that alterations in cholesterol composition of lipid rafts can significantly affect cell function. It is anticipated that as the neurochemical importance of cholesterol metabolism in neurodegeneration increases considerably, the methodology to detect subtle changes in lipid raft composition will become perfect. Cholesterol is oxidized to hydroxycholesterols by cytochrome P450-dependent hydroxylases. These enzymes play key roles in cholesterol homeostasis through the elimination of excess cholesterol from brain through blood–brain barrier into blood from where it is taken to liver by plasma lipoproteins. Among hydroxycholesterols, 24-hydroxycholesterol is the major metabolite. High levels of 24-hydroxycholesterol in serum are index for neurodegeneration. Thus levels of 24-hydroxycholesterol are significantly increased in AD, PD, NPC, and TBI. Marked alterations in cholesterol metabolism and increases in hydroxycholesterol occur during KA-mediated neurotoxicity. Alterations in hydroxycholesterols may play important roles in neural differentiation, cell cycle arrest, and apoptosis. At present, it is not known whether the generation of hydroxycholesterols is the initiating point in neurodegeneration (primary effect), or it is the end result of neurodegenerative process itself (secondary effect). References 291

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11.1 Introduction

Neural membranes are composed of phospholipids, sphingolipids, cholesterol, and proteins. The distribution of lipids in two leaflets of lipid bilayer is asymmetric (Ikeda et al., 2006; Yamaji-Hasegawa and Tsujimoto, 2006). Asymmetric distribution of lipids is needed for structural integrity necessary for protein function. Sphingolipids and cholesterol interact with each other via hydrogen bonds, hydrophobic forces, and van der Waal interactions (Simons and Ikonen, 1997). In addition, the head groups of sphingolipids bind to each other through hydrophilic interactions. These interactions result in lateral association of sphingolipids and cholesterol. Phospholipid composition of outer and inner monolayer halves is quite different. Thus, ethanolamine and serine containing phospholipids are located in the cytofacial site of inner monolayer, whereas choline containing phospholipids are localized on the exofacial side of outer monolayer. Among phospholipids phosphatidylethanolamine (PtdEtn), plasmenylethanolamine (PlsEtn), and phosphatidylserine (PtdSer) are enriched in docosahexaenoic acid (DHA, 22:6n-3) at the sn-2 position of the glycerol moiety, whereas phosphatidylcholine (PtdCho), phosphatidylinositol (PtdIns), and phosphatidic acid (PtdH) contain arachidonic acid (ARA, 20:4n-6) (Farooqui et al., 2000; Tillman and Cascio, 2003). In neural membranes, the fate of each lipid class is closely linked to neural cell function. For example, cholesterol and sphingolipids are involved in formation of lipid microdomains or lipid rafts, which float within the membrane and acts as molecular sorting machines and platforms for signal transduction pathways (Zajchowski and Robbins, 2002; Fantini et al., 2002; Lucero and Robbins, 2004). Thus, lipid rafts act as a unique compartment of the plasma membrane, which not only ensures correct intracellular trafficking of proteins and lipids, such as protein–protein interactions by concentrating certain proteins in these microdomains, while excluding others, but also modulate signal transduction processes associated with neural cell functions. In addition, sphingolipids play an important role in inhibiting or stimulating the oligomerization of amyloidogenic proteins, which are closely associated with pathogenesis of neurodegenerative

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 299 DOI 10.1007/978-1-4419-9940-5_11, © Springer Science+Business Media, LLC 2011 300 11 Perspective and Direction for Future Studies on Lipid Mediators diseases (Fantini and Yahi, 2010; Farooqui, 2010). Cholesterol has a dual role: regulation of protein–sphingolipid interactions through a fine tuning of sphingolipid conformation (indirect effect), and facilitation of pore (or channel) formation through direct binding to amyloidogenic proteins (Fantini and Yahi, 2010). Phospholipids, sphingolipids, and cholesterol provide neural membranes with integrity and are storage depots for lipid mediators. Interactions between phospholipids and sphingolipids are involved in maintaining lipid asymmetry, a dynamic process, which is necessary in maintaining normal neural membrane functions such as neuroplasticity and vesicular transport (Farooqui, 2009a). At the same time, local or global changes in lipid asymmetry are essential for cell cycle progression, apoptosis, and platelet coagulation. The disruption of asymmetry may also lead to apoptotic cell death. In neural membranes, the binding of transmembrane and peripheral proteins link lipid metabolism with phospholipid that is necessary for vertical positioning and tight integration of proteins in the lipid bilayer (Palsdottir and Hunte, 2004). The binding between phospholipids and proteins is not only stabilized by multiple noncovalent interactions between protein residues and phospholipid head groups, but also by hydrophobic tails of phospholipids (Palsdottir and Hunte, 2004; Farooqui and Horrocks, 2007). As stated above, neural membrane also contain sphingolipids (sphingomyelin, ceramide, cerebroside, sulfatide, and sphingosine) and cholesterol, which not only provide neural membrane with physicochemical characteristics (organization, fluidity, permeability, and structural integrity), but also modulate many neural cell functions, such as cell activation, differentiation, proliferation, survival, growth arrest, migration, and apoptosis. Collective evidence suggests that lipid signaling not only involves generation of lipid mediators, but also an orches- trated coupling among lipid metabolism, lipid organization, and the action of protein machines that execute appropriate downstream reactions. Remarkable enhancement of phospholipid, sphingolipid, and cholesterol metabolism has been reported to occur at plasma membrane and nuclear levels. Phospholipid, sphingolipid, and cholesterol metabolism in the nucleus may be as complex as that exists in the plasma membrane. However, an important feature of nuclear lipid metabolism pathways is their operational independence from metabolic pathways that are associated with plasma membrane and other subcellular organelles (Albi et al., 2006, 2008; Albi and Viola Magni, 2007; Ledeen and Wu, 2008; Martelli et al., 2005; Farooqui and Horrocks, 2006; Farooqui, 2009a; Farooqui et al., 2010).

11.2 Lipid Mediators in the Brain

Receptor-mediated catabolism of phospholipid-, sphingolipid-, and cholesterol by phospholipases, cyclooxygenases, lipoxygenases, acyltransferases, sphingomyelinases, and cytochrome P450 hydroxylases results in the generation of lipid mediators, which are lipophilic molecules that facilitate signal transduction processes, regulate cell–cell communication, neural cell proliferation, and differentiation along with control of molecular and cellular events associated with oxidative stress, 11.2 Lipid Mediators in the Brain 301

Lipid mediator Synthesizing enzyme

Neural cell Lipid Neural cell survival mediators differentiation

Neural cell Neural cell proliferation migration

Immune Oxidative stress Inflammation Gene expression Mitogenesis responses

Apoptosis

Neural dysfunction

Fig. 11.1 Hypothetical diagram showing interactions between Agonist (A) and Receptor (R) and modulation of signal neural cell proliferation and differentiation, oxidative stress, inflammation, and gene expression by lipid mediators (Modified from Farooqui, 2009a) inflammation, and gene expression in the brain (Fig. 11.1) (Farooqui, 2009a; Farooqui et al., 2010). Phospholipid-derived lipid mediators are diacylglycerols

(DAG), phosphatidylinositol 1,4,5-trisphosphate (Ins(1,4,5-P3), eicosanoids (prostaglandins, leukotrienes, thromboxanes, and lipoxins), docosanoids (D and E series resolvins, protectins, and neuroprotectins), endocannabinoids (2-arachidonylglycerol and anandamide), lysophospholipids, fatty acids, platelet-activating factor (PAF) and oxidized phospholipids. The sphingolipid-derived lipid mediators include ceramide, ceramide 1-phosphate, sphingosine, sphingosine 1-phosphate, and cholesterol-derived lipid mediators include 22-, 24-, 25-, 27-hydroxycholesterols (Fig. 11.2). The nonenzymic lipid mediators of glycerophospholipid metabolism are isoprostanes (isoP) and neuroprostanes (NeuroP), 4-hydroxynonenal (4-HNE), and 4-hydroxyhexenal (4-HHE) (Fam and Morrow, 2003; Phillis et al., 2006; Farooqui and Horrocks, 2007; Bazan, 2005a, b; Serhan, 2005a, b, c). These lipid mediators are involved in controlling the duration and magnitude of acute inflammation, oxidative stress, as well as the return of the injury site to homeostasis in the process of catabasis (the decline of the disease state). Another function of lipid mediators and their signal transduction network is to convey the message of extracellular signals from the cell surface to the nucleus to induce a biological response at the gene level. The conveying process involves the transfer of signal from lipid mediator to nuclear pores (large proteinaceous assemblies) that provide the sole 302 11 Perspective and Direction for Future Studies on Lipid Mediators

Lipid mediators Precursors

24-Hydroxycholesterol

Cholesterol 7-Ketocholesterol

25Hydroxyocholesterol Ceramide-1-phosphate

Sphingosine- Lipids Sphingolipids Ceramide Sphingosine 1-phosphate DAG Eicosanoids

Glycero- Arachidonic acid 4-HNE phospholipids

ROS Lysophosphatidic Docosatrienes acid

Docosahexaenoic acid Resolvins

4-HHE Lysophospholipids PAF

Fig. 11.2 Glycerophospholipid, sphingolipid, and cholesterol-derived lipid mediators in brain tissue. Diacylglycerol (DAG); prostaglandins (PG); leucotrienes (LT); thromboxane (TX); 4-hydroxynonenal (4-HNE); reactive oxygen species (ROS); 4-hydroxyhexanal (4-HHE); platelet- activating factor (PAF); 2-arachidonylglycerol (2-AG); N-arachidonoylethanolamine (AEA); N-arachidonyl-dopamine (NAD); and virodhamine (VDA). isoprostane (IsoP); neuroprostane (NeuroP); isoprostane (IsoP); lysophosphatidic acid (Lyso-PtdH) (Modified from Farooqui et al., 2010)

gateway for the exchange of material between cytoplasm and nucleus at the inter- phase (Fahrenkrog, 2006). Intracellular lipid mediator trafficking is a highly organized and dynamic process involving interactions between plasma membrane and nucleus-derived lipid mediators and proteins associated with intracellular signal transduction processes. Nuclear pore complexes support two modes of transport: (a) passive diffusion of ions, metabolites, and (b) intermediate-sized macromolecules. These processes facilitate receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. Faithful, continuous nuclear pore complex assembly is the key for maintaining normal physiological function and is closely tied to proper cell division. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. As such, the nuclear pore complex and nuclear transport play central roles in translocating signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes related to a particular function (Fahrenkrog, 2006; Naim et al., 2007). 11.3 Interactions Among Phospholipid-, Sphingolipid-, and Cholesterol-Derived… 303

11.3 Interactions Among Phospholipid-, Sphingolipid-, and Cholesterol-Derived Lipid Mediators in the Brain

In neural cells, metabolism of phospholipid, sphingolipid, and cholesterol is interrelated and interconnected. Many cellular stimuli (neurotransmitters, cytokines, and growth factors) modulate more than one enzyme of phospholipid, sphingolipid, and cholesterol metabolism at the same time. This phenomenon produces complexity in regulatory processes associated with phospholipid, sphingolipid, and cholesterol metabolism. Under normal conditions, homeostasis among enzymes of phospholipid, sphingolipid, cholesterol metabolism is based not only on optimal levels of lipid mediators and organization of signaling network, but also on the complexity and interconnectedness of their metabolism. Under pathological conditions, marked increases in levels of lipid mediators disturb the signaling networks, and result in loss of communication among phospholipid, sphingolipid, and cholesterol metabolism. This process not only threatens the integrity of neural cell lipid homeostasis, but also facilitates neural cell death (Farooqui et al., 2007a, b, c; Farooqui and Horrocks, 2007). In addition to above processes, the organization and compartmentalization of phospholipids, sphingolipids, and cholesterol in neural membranes of various subcellular fractions provides structural and functional integrity that facilitates the appropriate interactions with integral membrane proteins. An organized compartmentalization is needed for modulating regular cellular functions such as cell proliferation, differentiation, communication, and controlled adaptive responses through interactions among lipid mediators (Ivanova et al., 2004). However, under pathological conditions, high levels and intense interactions among phospholipid, sphingolipid, and cholesterol-derived lipid mediators produce harmful effects that facilitate apoptotic cell death (Farooqui, 2009a). At the molecular level, close interactions between the cPLA2-generated second messengers (ARA and its metabolites), and the SMase-generated second messenger (ceramide and ceramide 1-phosphate) occurs (Farooqui, 2009a; Vanags et al., 1997; Robinson et al., 1997; Malaplate-Armand et al., 2006; Farooqui et al., 2010) (Fig. 11.3). This process is closely associated with oxidative stress, inflammation, and apoptosis. PAF, a phospholipid-derived lipid mediator, also promote the activation of sphingomyelinase and generation of ceramide leading to the activation of scramblase with subsequent phosphatidylserine exposure, a process associated with apoptotic cell death (Lang et al., 2005). Interestingly, sphingolipid-derived lipid mediators, ceramide, ceramide 1 phosphate (C1P), sphingosine 1 phosphate (S1P) modulates activities of several enzymes associated with the synthesis of phospholipid-derived lipid mediators, such as arachidonic acid and its oxidative metabolites. Thus, ceramide upregulates cPLA2 and COX-2 expression and stimulates PGE2 synthesis (Farooqui, 2009a), a lipid mediator that produces neuroinflammation. Similarly, sphingosine 1-phosphate (S1P) upregulates COX-2 in a concentration- and time-dependent manner, but has no effect on COX-1 expression (Nodai et al., 2007). Suramin, an antagonist of S1P3 receptor, completely prevents S1P-mediated COX-2 expression. Thus, ceramide, ceramide 1-phosphate, sphingosine, and S1P stimulate enzymes associated 304 11 Perspective and Direction for Future Studies on Lipid Mediators

Glu Glu

SM Cholesterol PM PtdCho

2 ROS + + SMase cPLA

Lyso- ARA + Ceramide PtdCho + Cholesterol 24-hydroxylase + 12 24-Hydroxy-

COX-2 cholesterol ROS Ceramide-1-P Sphingosine 7-ketocholesterol PAF PGE2 3

Sphingosine-1-P Inflammation

+++ Mitochondrial dysfunction

Gene transcription

Nucleus Cytochrome c COX-2 TNF-α sPLA2 IL-1β Proinflammatory enzymes Proinflammatory cytokines Oxidative iNOS IL-6 Cathep- stress sin D Apoptosis Caspase cascade

Fig. 11.3 Hypothetical diagram showing interactions among phospholipid-, sphingolipid-, and cholesterol-derived lipid mediators. Phosphatidylcholine (PtdCho); sphingomyelin (SM); cytosolic phospholipase A2 (cPLA2); sphingomyelinase (SMase); arachidonic acid (ARA); reactive oxygen species (ROS); cyclooxygenase-2 (COX-2); reactive oxygen species (ROS); ceramide -1-kinase (1); ceramidase (2); sphingosine-1-kinase (3); ceramide-1-phosophate (ceramide-1-P); sphingosine-1-phosphate (sphingosine-1-P); and plasma membrane (PM). Proinflammatory genes include TNF-a; IL-1b; nitric oxide synthase; cyclooxygenase-2 (COX-2); sPLA2. Positive sign (+) indicates stimulation, upward arrow indicates increase, and downward arrow indicates decrease in levels of precursors and lipid mediators. Upward arrows indicate increase in levels, where as downward arrows indicate decrease with the synthesis of lipid mediators-derived from neural membrane phospholipids (Pyne, 2004; Pettus et al., 2004; Nodai et al., 2007). In addition, in rat brain slices sphingomyelinase and ceramide decrease the levels of plasmalogens through the activation of PlsEtn-PLA2 (Latorre et al., 2003). The decrease in plasmalogens by sphingomyelinase or ceramide is inhibited by quinacrine, ganglioside, and bro- moenol lactone, which are inhibitors of plasmalogen-selective PLA2 activity (Latorre et al., 2003; Farooqui and Horrocks, 2001). Treatment with caspase-3 inhibitor, acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-chloromethylketone (Ac-DEVD- CMK), partially blocks the ceramide-induced stimulation of plasmalogen-selective

PLA2 without altering sphingomyelinase-elicited ceramide accumulation (Latorre et al., 2003; Farooqui and Horrocks, 2004). In addition, S1P is metabolized to phosphoethanolamine and hexadecanol, which are prerequisite for the synthesis of phospholipids (Park et al., 2006). Collective evidence suggests that interactions between sphingolipid- and phospholipid-derived lipid mediators are closely associated with cell proliferation, differentiation, apoptosis, migration, and cell survival 11.3 Interactions Among Phospholipid-, Sphingolipid-, and Cholesterol-Derived… 305

PLA2,PLC, & PLD

COX, LOX, & EPOX

Sphingomyelinases

Lipid mediator synthesizing Ceramide & sphingosine kinases enzymes

Acetyltransferases

N-Acyltransferases

Cytochrome P450-dependent hydroxylases

Fig. 11.4 Enzymes associated with the synthesis of lipid mediators. Phospholipase A2 (PLA2); phospholipase C (PLC); phospholipase D (PLD); Cyclooxygenase (COX); lipoxygenase (LOX); and epoxygenase (EPOX)

(Wymann and Schneiter, 2008; Hannun and Obeid, 2008). Thus, lipid mediator synthesizing enzymes (phospholipases A2, C, D, cyclooxygenases, 5-lipoxygenase, phosphoinositide 3-kinase, sphingomyelinases, ceramide kinases, sphingosine kinase and cholesterol hydroxylases) (Fig. 11.4) and their downstream targets constitute a complex lipid signaling network with multiple nodes of interactions and cross-talk among various lipid mediators. Alterations and imbalances in this network due to abnormalities in lipid mediator production may contribute to the pathogenesis of neural trauma, neurodegenerative, and neuropsychiatric diseases (Farooqui, 2009a; Wymann and Schneiter, 2008; Hannun and Obeid, 2008). Ceramide, the central core in sphingolipid metabolism is not only a precursor of complex sphingolipids, but is also involved in the regulation of signal transduction processes associated with apoptosis, cell growth, differentiation, senescence, inflammation, and neurodegenerative disorders (Farooqui et al., 2007a; Farooqui, 2009a). The presence of cholesterol in neural membrane enhances or inhibits the interactions between ceramide and amyloidogenic proteins (Ab, a-synuclein, and huntingtin). It is suggested that presence of cholesterol in neural membranes has a major impact on interaction between ceramide and amyloidogenic protein. In the case on non-hydroxyfatty acid (NFA) containing ceramide, cholesterol usually accelerates protein binding to sphingolipid due to hydrogen bonding (Fantini et al., 2002) and this also applies for amyloidogenic proteins (Yahi et al., 2010). In contrast, ceramide containing a hydroxylated fatty acid (HFA), the OH group of cholesterol is excluded from the H-bond network and it cannot exert its conformational effect on the ceramide. Thus, cholesterol does not enhance but rather perturbs the organization of ceramide and can even retard ceramide binding to amyloidogenic proteins (Yahi et al., 2010). Thus, according to the distribution of HFAs versus NFAs in ceramide, an increase of membrane cholesterol may produce opposite effects on sphingolipid-mediated amyloidogenic protein binding and aggregation. These effects of 306 11 Perspective and Direction for Future Studies on Lipid Mediators cholesterol are associated with fine tuning of ceramide conformation for interaction between amyloidogenic proteins and ceramide (Fantini and Yahi, 2010). In wild-type and sterol-resistant Chinese-hamster ovary (CHO) cells, oxysterols promote the translocation and activation of CTP: phosphocholine cytidylyltransferase a to nuclear membrane where it promote the synthesis of PtdCho (Gehrig et al., 2009). Increasing evidence suggests that oxysterols act as ligands of liver X receptors, transcription factors with key roles in lipid metabolism. Oxysterols not only bind to transcription factors, but also interact with the INSIG (insulin-induced gene) proteins. This suggests that oxysterols regulate the transport and maturation of sterol-regulatory element binding proteins (Olkkonen and Hynynen, 2009). Nothing is known about the interactions between hydroxycholesterols and sphingolipids in neural plasma membranes. However, in Chinese hamster ovary (CHO)-K1 cells, 25-hydroxycholesterol stimulates the synthesis of sphingomyelin (Ridgway, 1995; Lagace et al., 1995). The role of oxysterol-binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol, in activation of sphingomyelin synthesis has also been assessed by overexpression in CHO-K1 cells. When compared to mock transfected controls, three CHO-K1 clones overexpressing OSBP by 10- to 15-fold show a two- to threefold enhancement of [3H]serine incorporation into sphingomyelin when treated with 25-hydroxycholesterol. The closer examination of one of these clones (CHO-OSBP cells) produces a >8.5-fold stimulation of sphingomyelin synthesis with 25-hydroxycholesterol compared to 3.5-fold in controls, slightly higher basal levels of sphingomyelin synthesis, and a more rapid response to 25-hydroxycholesterol. [3H]Serine incorporation into phosphatidylserine, phosphatidylethanolamine, ceramide, or glucosylceramide is affected only by <15%. Based on the determination of sphinganine N-acyltransferase, sphingomyelin synthase, and serine palmitoyltransferase activities, it is suggested that overexpression of OSBP or 25-hydroxycholesterol has no affect on ceramide content of Golgi-enriched fractions from control or overexpressing cells. However, diglyceride mass is reduced in overexpressing cells and by treatment with 25-hydroxycholesterol. Collective evidence suggest that OSBP potentiates the stimulatory effects of 25-hydroxycholesterol on sphingomyelin synthesis. 25-Hydroxycholesterol promotes the translocation of OSBP to the Golgi apparatus where it appears to stimulate conversion of ceramide to sphingomyelin (Ridgway, 1995; Lagace et al., 1995).

11.4 Detection and Levels of Lipid Mediators in Neurological Disorders by Lipidomics

The assessment of biomarkers of neurodegenerative diseases is complicated by diagnostic imprecision, the variability in clinical features and rates of progression, complex disease genetics, and multiple molecular etiologies. At the same time, most neurodegenerative diseases are accompanied by oxidative stress and neuroinflammation (Farooqui, 2009a, 2010), processes supported by not only lipid mediators, but also by oxidatively modified proteins and nucleic acids in various stages of 11.4 Detection and Levels of Lipid Mediators in Neurological Disorders… 307 the diseases. These days, we have been empowered with technological advances in lipidomics, proteomics, and genomics. Using these techniques, investigators are performing analysis of hundreds to thousands of mRNA and proteins simultaneously, dissecting signaling pathways, determining levels of lipid mediators, characterizing enzyme proteins, and cloning genes from cells in biological fluids, such as CSF and brain extracellular fluid (ECF), mostly obtained by cerebral microdialysis (Maurer, 2010). Both materials are of high diagnostic value in clinical neurology, where CSF and ECF samples from stroke, AD, PD, amyotrophic lateral sclerosis (ALS), traumatic brain injury, and multiple sclerosis can be analyzed for lipid mediators and biomarkers. Lipidomics can detect minute amounts of lipid mediators in biological fluids (German et al., 2007; Bowers-Gentry et al., 2006). This procedure has been used to determine levels of lipoxins, hydroxyeicosatetraenoic acids, F2- isoprostanes, prostaglandins, leukotrienes, nitrotyrosine, carbonyls in proteins, oxidized DNA bases, and 4-HNE in CSF (Serhan et al., 2006; Adibhatla et al., 2006; Milne et al., 2006; Lu et al., 2006; Perluigi et al., 2005). Lipidomics has also been used to determine levels of lipid mediators in small tissue samples from neurotoxin injected rat brain and brain samples from patients with neurodegenerative diseases (Yoshikawa et al., 2006; Butterfield et al., 2006). Proteomics has been used to char- acterize phospholipid, sphingolipid, and cholesterol metabolizing enzymes in biopsy and autopsy samples of brain tissue not only from animal models of neurodegenerative diseases, but from patients with acute neural trauma and neurodegenerative diseases. Phospholipid, sphingolipid, and cholesterol-metabolizing enzymes may be potential therapeutic targets because these enzymes synthesize and degrade lipid mediators required for neuronal growth (Fonteh et al., 2006). Combining lipidomics and proteomics will not only enhance existing knowledge of disease pathology, but will also increase the likelihood of discovering specific markers associated with neurological diseases. Deleterious alterations in lipid homeostasis and levels of lipid mediators may be key factor in the onset and progression of neurodegenerative diseases (Farooqui and Horrocks, 2007). At present, it is unclear whether the generation of lipid mediators is the initiating point in neurodegenerative process (primary event), or it is the end result of neurodegenerative process itself (secondary event). So, temporal determination of levels of lipid mediators and enzymes associated with their synthesis by lipidomics and proteomics will be helpful in understanding the relationship between lipid mediator generation and neurodegenerative process. Establishment of automatic systems including databases and accurate analyses of lipid mediators derived from enzymic and nonenzymic metabolism of neuronal membrane phospholipids, sphingolipids, and cholesterol will facilitate the identification of key biomarkers associated with neurodegenerative diseases (Lu et al., 2006). Genomics analysis of brain samples from affected regions of AD, PD, HD, ALS, MS, and schizophrenia patients may provide information on candidate genes and enzymes that modulate levels of lipid mediators (Farooqui, 2009a). Gene expression profiles of susceptible neuronal populations by genomics may reveal mechanistic clues to the molecular mechanism underlying various neurological diseases. This would not only help in understanding the molecular mechanisms associated 308 11 Perspective and Direction for Future Studies on Lipid Mediators with the development of neurodegenerative diseases, but would also facilitate molecular diagnostics and targets for drug therapy based on gene expression in body fluids such as CSF and blood (Facheris et al., 2004). Levels of lipid mediators can then be used to monitor responses to drug therapy.

11.5 Modulation of Lipid Mediators by Diet

Levels of prostaglandins, leukotrienes, thromboxanes, and lipoxins, resolvins, protectins, and neuroprotectins, 2-arachidonylglycerol, anandamide, and platelet-activating factor (PAF) in neural and non-neural tissues are partly regulated by diet (Farooqui, 2009b). The high intake of food enriched in vegetable oils (ARA containing fats) elevates levels of prostaglandins, leukotrienes, thromboxanes, and PAF. These lipid mediators, by acting through their receptors, not only modulate vasodilation and vasoconstriction in the cardiovascular and cerebrovascular systems, but also regulate the expression of proinflammatory cytokines and chemokines, which induce and support inflammation and oxidative stress in the brain. ARA-containing diet can also increases levels of lipid peroxides and isoprostanes, which produce vasoconstrictive effects in pulmonary artery, coronary arteries, cerebral arterioles, retinal vessels, and portal vein (Montuschi et al., 2007). Accumulating evidence suggests that ARA-derived lipid mediators produce prothrombotic, proaggregatory, and proinflammatory effects in the body. In contrast, diet enriched in EPA and DHA generates docosanoids (E-series resolvins, D-series resolvins, protectins, neuroprotectins, and maresins). The molecular mechanisms of EPA and DHA action are only partially understood. These fatty acids not only induce changes in membrane structures, but their direct interactions with ion channels and transcription factors produce alterations in eicosanoid biosynthesis (Calder, 2006; Jump, 2004). EPA also competes with ARA at all steps of eicosanoid biosynthesis resulting in specific alterations in lipid mediator production and action (Farooqui, 2009b). The enzymic oxidation of EPA generates the 3-series of prostaglandins and thromboxanes and the 5-series of leukotrienes. These eicosanoids have different biological properties than the corresponding analogs produced by the metabolism of ARA. For example, TXA3 is less active than TXA2 in aggregating platelets and constricting blood vessels (Farooqui, 2009b). EPA- and DHA-derived lipid mediators not only downregulate proinflammatory cytokines, but also produce anti-inflammatory, antithrombotic, antiarrhythmic, hypolipidemic, vasodilatory, and anti-exitotoxic effects (Farooqui et al., 2007a, b, c, 2008; Simopoulos, 2002; Hogyes et al., 2003; Bannenberg and Serhan, 2010). EPA- and DHA-derived lipid mediators act as agonists at specific receptors (CMKLR1, BLT1, ALX/FPR2, and GPR32) (Farooqui, 2009a, b). Although occurrence and partial characterization of the above- mentioned receptors have been reported in non-neural tissues, no information on occurrence and characterization is available in brain. Future studies should be directed on isolation and characterization of CMKLR1, BLT1, ALX/FPR2, and GPR32 in neural membranes. In addition to receptor mediated effects, the presence of EPA and DHA in neural membranes not only alters the physical characteristics of the 11.6 Conclusion 309 membrane, but also improves their biological functions such as signal transduction, ion channeling, and ligand binding to nuclear receptors. EPA and DHA also inhibit or quench gene expression of cyclooxygenase-2 and other enzymes, thereby diminishing the formation of proinflammatory molecules. During resolution phase of neuroinflammation, EPA- and DHA-derived resolvins can signal for potent counter-regulatory effects on leukocyte functions, including preventing uncontrolled neutrophil swarming, decreasing the generation of proinflammatory cytokines and chemokines, blocking production of reactive oxygen species, and promoting clearance of apoptotic neutrophils from inflamed brain (Farooqui, 2009a, b). ARA is an essential fatty acid and its presence in diet is not inherently unhealthy. The problem is that these days diet contains very high amounts of saturated fat, with ARA to DHA ratio of about 20:1. The Paleolithic diet on which human beings have evolved and lived for most of their existence had a ratio of 2-1:1, and was high in fiber, rich in fruits, vegetables, lean meat, and fish. (Simopoulos,2 002, 2006, 2008; Farooqui, 2009b). Changes in eating habits, natural versus processed food, which is enriched in corn-based livestock, and increase in the consumption of vegetable oil in past 20 years along with decrease in consumption of sea food has altered ARA to DHA ratio in favor of ARA (Simopoulos, 2000, 2006; Kris-Etherton et al., 2000; Weylandt and Kang, 2005; Kang and Weylandt, 2008). In contrast, consumption of DHA-enriched diet has anti-inflammatory effects that are partly mediated by repression of genes that code for proinflammatory cytokines. DHA-enriched diet also elevates the levels of docosanoids that have antioxidant and antiapoptotic effects in neural and non-neural tissues (Bazan, 2005a, b). ARA and DHA also interact with several transcription factors (PPAR, LXR, HNF-4, NFkB, and SREBP) differently to modulate lipid metabolism for maintaining cellular homeostasis (Jump, 2004). Fatty acids or their metabolites bind directly to specific transcription factors to regulate gene transcription. These fatty acids also modulate gene expression indirectly through their interactions with COX, LOX, PKC, or sphingomyelinase signal transduction pathways as well as pathways that involve changes in membrane lipid/lipid raft composition that affect G-protein receptor or tyrosine kinase-linked receptor signaling (Horrocks and Farooqui, 2004; Farooqui, 2009a, b). Collectively, these studies support the view that levels of ARA- and DHA-derived lipid mediators are partly modulated by the diet (Horrocks and Farooqui, 2004; Farooqui, 2009a, b) and ratio between ARA and DHA must be properly maintained in human diet. The primary goal of future research on neurochemistry of lipid mediators should be how to increase the levels of DHA-derived lipid mediators in the brain tissue through diet, and avoid neurotraumatic, neurodegenerative, and neuropsychiatric diseases.

11.6 Conclusion

Presence of various combinations of polar headgroups, fatty acyl chains, and backbone structures makes neural membrane phospholipids and sphingolipids complex molecules, which perform diverse functions. Phospholipid-derived polyunsaturated 310 11 Perspective and Direction for Future Studies on Lipid Mediators fatty acids (DHA and ARA) are essential fatty acids. They act as precursors for lipid mediators, such as docosanoids (resolvins, protectins, neuroprotectine, and maresins) and eicosanoids (prostaglandins, leukotrienes, thromboxanes, and lipoxins). The incorporation and proportions of DHA and ARA markedly influence neural membrane properties such as fluidity, flexibility, elasticity, permeability, and gene expression. The metabolism of DHA differs from ARA metabolism. Oxygenation of ARA generates proinflammatory mediators (prostaglandins, leukotrienes, and thromboxanes), whereas DHA generates anti-inflammatory lipid mediators, such as resolvins, protectins, neuroprotectins, and maresins, which directly or indirectly suppress the activity of NF-kB. Increased intake of DHA results in a decrease in the level of ARA in neural membrane phospholipids, thereby decreasing the level of substrate for the production of proinflammatory eicosanoids and cytokines. Based on the above observations, it is suggested that DHA is anti-inflammatory while ARA is proinflammatory. Inappropriate amounts of dietary n-6 and n-3 fatty acids may lead to abnormal inflammatory and immune responses because of their abundance in the brain. Thus, an adequate ratio of n-6 and n-3 fatty acids may promote a healthier balance between DHA and ARA-derived lipid mediators, which is necessary to maintain optimal neural membrane function and primary goal of future research on lipid mediators should be how to increase the levels of DHA in human tissue, including the brain, through diet. DHA and ARA-derived lipid mediators have been characterized by lipidomics and can be used as biomarkers for neurological disorders in biological fluids. Identification of biomarkers for neurodegenerative diseases may not only lead to early diagnosis and follow-up of the progression of neurodegenerative diseases, but also monitoring of therapeutic responses.

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A 12-HETE-EA and 15-HETE-EA, 142 Acyl-CoA lysocardiolipin acyltransferase NAT and PLD, 142

1 (ALCAT1), 75–77 PGE2-EA, 142 Acyl-CoA synthetases (ACS) REM and TRPV1, 142 ARA and DHA activation, 75–76 Antiinflammatory intracellular concentrations, 76–77 DHA and EPA, 50 isoforms, 76 DHA-enriched diet consumption, 309 lipid metabolism and homeostasis, 75, 76 hypothetical diagram, 67 AD. See Alzheimer disease IL-1b-mediated suppression, 52 2-AG. See 2-Arachidonylglycerol ARA. See Arachidonic acid Ajulemic acid (AJA), 135 Arachidonic acid (ARA) ALCAT1. See Acyl-CoA lysocardiolipin ARA-derived lipid mediators, 308, 310 acyltransferase 1 bisoxygenation and cyclization, 6 ALS. See Amyotrophic lateral sclerosis CoA, 80 Alzheimer disease (AD) deacylation/reacylation cycle, 75–76 age, 232 description, 309 C2Cer:N-acetylsphinganine, 233–234 DHA ratio, 309, 310 cerebrospinal fluid (CSF), 233 enzymes release, 22–23 dementia, 33, 120 enzymic and non-enzymic products, 4-HNE, 176–177 DHA, 1, 4 22(R)-hydroxycholesterol, 282–283 EPA, 308 24(S)-hydroxycholesterol, 282 generation, 197 27-hydroxycholesterol, 283 4-HNE, 167 lysophospholipids, 95–96 and isoprostanes biosynthesis, 196 microglia, 121 LOXs, 11–13 PD and HD, 281 metabolism, 14, 308, 310 g-secretase activity, 34 nonenzymic oxidation, 200 sphingosine 1 phosphate, 259–260 oxidation, 172 streptozotocin (STZ), 233 peroxidation, 159 type 2 diabetes mellitus, 233 radical-catalyzed peroxidation, 193 Amyotrophic lateral sclerosis (ALS) 2-Arachidonylglycerol (2-AG) ceramide, 234–235 Ca2+ mobilization and COX-2, 140 pathophysiological mechanisms, 35 cytochrome P450 enzymes, 141 sporadic form, 35 degradation and role, 140 Anandamide 12-HETE-GE and 15-HETE-GE, 141 degradation, role, 141 hydrolysis, 1,2-arachidonyl-PtdCho, 139

A.A. Farooqui, Lipid Mediators and Their Metabolism in the Brain, 315 DOI 10.1007/978-1-4419-9940-5, © Springer Science+Business Media, LLC 2011 316 Index

2-Arachidonylglycerol (cont.) Ceramide membrane-associated N-acyltransferase, 139 cathepsin D, 220 monoacylglycerol lipase inhibition, 140 C6-ceramide, 219

PGE2-G levels, 141 chemical structure, 218 and C1P (see also Ceramide 1 phosphate) brain, 228–229 B degradation, 227 Batten disease, 236 description, 217 BDNF. See Brain-derived neurotrophic factor diacylglycerol (DAG), 220–221 BLT receptors, 18, 56–57 enzyme activities, 220 Brain-derived neurotrophic factor (BDNF), 148 glycerophospholipid and sphingolipid derived mediators, 219–220 neural cell treatment, 219 C neurological disorders, 230–236 Cannabinoid receptor. See Cannabinoids nSMase2 inhibition, 217 Cannabinoids rafts, 217–219 AJA, 135 synthesis chemical structures, receptor agonists, 134 conversion to C1P, CerK, 224–227 endocannabinoids de novo, 221–222 2-AG and anandamide, 138 neural cells, 221 anandamide metabolism, 142 sphingomyelinases (SMase) 2-arachidonylglycerol metabolism, activation, 222–224 139–141 sphingosine salvage pathway, 224

CB1 and CB2 receptors, 133–135, Ceramide kinase (CerK) 137–138 ceramide conversion to C1P in neurodegenerative disorders, 147–149 cardiolipin, 225 in neurotraumatic diseases, 145–147 molecular cloning, 224–225 retrograde signalling, 139 stimulation, 224 FAAH and MGL, 149–150 de novo synthesis, 222 glutamate and dopamine receptors, Ceramide 1 phosphate (C1P) basal ganglia and ceramide, inflammatory coordinated interplay (cross-talk), 143 responses, 226 DARPP-32, 144 description, 229

dopamine transmission, 143–144 effect, cPLA2, 226 electrophysiological and neurochemical macrophage proliferation, 226 analysis, 143 protein kinase B phosphorylation, 225–226 GABA modulation, 143 synthase nuclear level and cAMP/PKA, 136 cell division, 225 PPARg and NF-AT, 136 CerK, 224–225 receptor-mediated signaling, 136–137 Ceramide synthase seven-transmembrane-domain, 133–134 conversion to C1P, CerK, 224–227 structures, receptor antagonists, 135 de novo, 221–222 system-mediated signaling, 133 neural cells, 221 CCAAT. See Cytidine-cytidine-adenosine- sphingomyelinases (SMase) adenosine-thymidine activation, 222–224 Cell differentiation sphingosine salvage pathway, 224 defined, 278 Cerebrospinal fluid (CSF), 66, 307 22 (R)-hydroxycholesterols, 278 Cerebrotendinous xanthomatosis (CTX) LXR a and b, 278 activation, 289 multicellular organisms, 278 characteristics, 288–289 Ceramidase description, 288 acid ceramidase expression, AD, 232 hydroxycholesterol levels, 281 sphingosine salvage pathway, 224 CerK. See Ceramide kinase types, 227 Chinese-hamster ovary (CHO), 306 Index 317

Cholesterol epilepsy, 32 brain, 267 gene, 7, 9 conversion, 268, 269 “housekeeping” enzyme, 7 degradation immunoreactivities, 9 CYP27A1, 275, 276 inflammation and upregulation, 28 cytochrome P450-dependent inhibitors, 29 hydroxylases, 276–277 ischemic injury, 30–31 24-hydroxylase, 275 LX and PG biosynthesis, 19 distribution, 267 mRNA, 9, 11 KA, 289–290 traumatic brain injury, 31–32 neurological disorders, 280–289 eicosanoids, LOX and EPOX role, 267 ALS, 35–36 structure, 268 CJD, 36 synthesis PD, 35

astrocytes, 270, 273 LOX, and EPOX, PLA2 isoforms BBB, 270 endogenous activities, 24 chemical structures, 272 enzymic activity, 23–24 HMG-CoA reductase isoforms coupling, 23 (HMGR), 271 reaction rates, 23 homeostasis, 272, 273 regulation, 24–25 LXR/RXR functions, 271 Cytidine-cytidine-adenosine-adenosine- metabolism interactions, 274 thymidine (CCAAT), 255 neurons, 272 Cytochrome P450 oxygenases structures, 270, 272 cholesterol conversion, 268, 269 transcriptional regulation, 273 hydroxylases, 276–277 CJD. See Creutzfeldt–Jakob disease Cytokines COX-2. See Cyclooxygenase-2 AD, 233 COX, LOX, EPOX and eicosanoids and chemokines, 57 in AD, 33–35 COX isoforms, 24 in ALS, 35–36 expression, 109 in CJD, 36 glial cells, 60, 63 in PD, 35 HAD, 235 COXs. See Cyclooxygenases 4-HNE, 173 C1P. See Ceramide 1 phosphate induction, 119–120 Creutzfeldt–Jakob disease (CJD) inflammatory expression, 203 COX-2 immunoreactivity, 36 interleukin-1b, 27 RT-PCR and western blotting MDD, 236 studies, 36 neurotransmission, brain, 197 CTX. See Cerebrotendinous production, monocytes and epithelial xanthomatosis cells, 75 Cyclooxygenase-2 (COX-2), 140–141 profibrotic, 20 Cyclooxygenases (COXs) proinflammatory AD, eicosanoids, LOX and EPOX genes, 66 activity and expression, 34–35 production, 25 ARA metabolism, 36 secretion, 26 dementia characterized, 33 proteolytic maturation, 166 low-fibrillar A deposits, 33–34 roles, isoprostanes, 194

g-secretase activity, 34 SMases and PLA2 activities, catalytic centers, 6 230–231 COX-1, COX-2 and COX-3 tumor necrosis factor, 54

amino acid sequences, 7, 9 Cytosolic phospholipase A2 (cPLA2) ARA concentration, 7 ARA release, 1 cytosolic glycoprotein, 11 interactions, 2

degradation, 6–7 and sPLA2, 26–27 318 Index

D Eicosanoids

Deacylation/reacylation cycle action and isoforms, PLA2, 2 ACS, 75–77 biosynthesis, 308 acyl-CoA: lysophospholipid acyltransferase multiplicity ALCAT1 roles, 77–78 ARA, 4–6 LPEAT2 activity, 78 COXs, 6–11 purification, 77 EPOXs, 13–14 CoA-independent reacylation, 81 LOXs, 11–13 enzymes, 74–75 neural membrane phospholipids, 1 long-chain acyl-CoA hydrolase/ in neurodegeneration, 27–28 thioesterases, 80 neurodegenerative diseases

PLA2, 78–79 AD, 33–35 DHA. See Docosahexaenoic acid ALS, 35–36 Dihydrosphingosine 1-phosphate (DHS1P), 255 CJD, 36 Dihydroxyeicosatrienoic acids (DHETs) COX, LOX and EPOX, activities, 33 and EETs, 21 PD, 35 metabolism, 14 in neuroinflammation, 25–27 Docosahexaenoic acid (DHA) neurotraumatic diseases ARA, 309, 310 (see Neurotraumatic diseases) deacylation/reacylation cycle, 75–76 in nociception (pain state), 28 derived lipid mediators, brain non-enzymic peroxidation, ARA and aspirin-triggered forms, 59 DHA, 3–4 D-series resolvins, 58 numerous eicosanoids, 1 enzymic and non-enzymic, 58–59 PGs, LTs and LXs structures, 2 extracellular matrix (ECM) phospholipid-derived lipid mediators, 301 components, 60 receptors, 15–22 leukocyte trafficking, 60 ROS, 2–3 neuroprotection, 60, 61 in synaptic plasticity, 28–29

non-neural cells, 58–59 upstream PLA2 isoforms, relationship, protectins and neuroprotectin 22–25 (see Neuroprotectin; Protectins) Eicosapentaenoic acid (EPA) 17S D series resolvins, 60 derived lipid mediators, brain and EPA, 308–309 anti-inflammation/pro-resolution intake, 310 effects, 54–55 lysophospholipid, 85 CYP metabolite profiles, 58 metabolize, 1–2 cytochrome P450 isoforms, 57 nonenzymic oxidation, arachidonic acid, 200 neural and non-neural tissues, 53, 55–56 and non-enzymic peroxidation, ARA, 3–4 non-enzymic oxidation, 57 radical-catalyzed peroxidation, 193 oxo product, 56–57 synthesis, 26–27 pain hypersensitivity, 55 Docosanoids neurochemistry polymorphonuclear (PMN) cells, 54 description, 49 prostaglandins and thromboxanes, 52 DHA-derived lipid mediators, brain, 58–67 resolvins, 52–53, 54

EPA-derived lipid mediators, brain, 52–58 RvE1 and RvE2, 54–55 neuronal stimulation, 49–50 and DHA, 308–309 n-3 polyunsaturated fatty acids, brain, 50–52 enzymic oxidation, 308 polyunsaturated fatty acid (PUFA) Endocannabinoids composition, 49 anandamide metabolism, 142 2-arachidonylglycerol metabolism, 139–141 cannabis sativa preparations, 137–138 E chemical structures, 138 EAE. See Experimental autoimmune GABAergic and glutamatergic encephalomyelitis synapses, 139 EETs. See Epoxyeicosatetraenoic acids generation, pathways, 139 Index 319

lipid-like substances, 137 CJD (see Creutzfeldt–Jakob disease) neurodegenerative disorders PD (see Parkinson disease) anti-excitotoxic action, 149 Experimental autoimmune encephalomyelitis antioxidant action and neuroinflam- (EAE), 96, 148 mation, 148 Extracellular fluid (ECF), 307 BDNF, EAE, FAAH and G93A-SOD1, 148 cannabinoid-based drugs, 148–149 F CNS inflammatory process and MS Fatty acid amide hydrolase (FAAH) patients, 149 ABHD6, 146 hSOD1G93A, 148 antiinflammatory and neuroprotective neuroprotective effects, effects, 148 endocannabinoids, 147 chemical structures, inhibitors

non-CB1/non-CB2 receptors, 147 endocannabinoids, 150 PD and HD, 147 extracellular to intracellular space, 138 neurotraumatic diseases postsynaptic membrane bound enzyme, 142 administration, 2-AG, 146 spinal cord lesion, 146 brain damage and dysfunction, 146 time-and dose-dependent manner, 149 FAAH and TBI, 146 URB597, 149 ischemic injury and microglial cell FTY720 and neurochemical effects

migration, 145 cPLA2-mediated arachidonic acid, 257 JWH-133 and focal cerebral DHS1P, 255 ischemia, 145 immunosuppressant and structural proinflammatory/microglial-related analog, sphingosine, 255, 256 responses, 146–147 phosphate, 256 spinal cord trauma, 146 short-chain vs. long-chain ceramides, 256 VR1 vs. anadamide, 146–147 peripheral and neural tissues, 138 post-synaptic neuron and pre-synaptic G terminals, 138–139 Genomics Endothelial nitric oxide synthase (eNOS), 14 analysis, 307 eNOS. See Endothelial nitric oxide synthase technological advances, 307 Epilepsy, 32 Glutathione EPOXs. See Epoxygenases 4-HNE treatment, 163 Epoxyeicosatetraenoic acids (EETs) isozyme, 166 CYP epoxygenase, 13–14 modulation, 167–168 functional link, 21 peroxidase activity, 168 mediated effects, 14 S-transferase, 161 physiologic processes, 24 G-protein coupled receptors (GPCRs) secretion and action, insulin, 14 neurochemical effects sEH increases, 13–14 eicosanoid receptors, 25 Epoxygenases (EPOXs) LTs, 18

COX and LOX, PLA2 isoforms PGs, 17 enzymic activity, 23–24 seven-transmembrane, 20–21 isoforms coupling, 23 cytochrome P450 (CYP), EETs cell-cell communication, CNS, 14 H CYP2C11, 14 HD. See Huntington disease PPARa, 14 HDL. See High-density lipoproteins secretion and action, insulin, 14 Heat shock protein, 166–167 soluble epoxide hydrolase (sEH), 13–14 12-HETE ethamolamide (12-HETE-EA), 142 eicosanoids, LOX and EPOX 15-HETE ethamolamide (15-HETE-EA), 142 AD (see Cyclooxygenases) HETEs. See Hydroxyeicosatetraenoic acids ALS, 35–36 High-density lipoproteins (HDL), 247 320 Index

HPETE. See Hydroperoxyeicosatetraenoic acid glutathione s-transferases, 167–169 Human immunodeficiency virus type 1 heat shock response, 170–171 (HIV-1) infection, 235–236 kinases, 164–166 Human neuronal-glial (HNG) cells, 66 gene expression modulation Huntington disease (HD) intracerebroventricular injection, 173 AD and PD, 281 ischemia-reperfusion injury, 172 description, 285–286 KA-mediated neurodegeneration, models, 285 172–173 stages, 286 neurotraumatic and neurodegenerative Hydroperoxyeicosatetraenoic acid (HPETE) diseases, 172 FLAP, 13 intra and intermolecular covalent lipoxygenases action, 19 adducts, 160 oxygen position, ARA, 12 metabolism Hydroxycholesterol detoxification, 162 apoptosis, 279–280 enzymic pathways, 161 exocytosis, 277 metabolic fate, 161–162 KA, 289–290 neurological disorders, 176–183 neural cell differentiation, 278–279 NMDA receptor modulation, 171 neurological disorders, 280–289 nucleic acid metabolism modulation synthesis, 269–274 diastereoisomers, 173–174 22-Hydroxycholesterol, 278 DNA and RNA synthesis, 173–174 22(R)-hydroxycholesterol, 278, 282–283 etheno adducts, 175 24(S)-hydroxycholesterol guanosine moiety DNA, 174–175 changes, 286 peroxisomes modulation, 173 conversion, 268 phospholipid metabolism modulation, 175 levels, 286–288 synthesis role, 277 Hock cleavage, 161 25-Hydroxycholesterol linoleic acid, 160–161 cell proliferation, 283 and 24-hydroxycholesterol, 280 neurotoxicity production, 279 I ORPs binding, 275 Infantile-onset neuronal ceroid lipofuscinosis 27-Hydroxycholesterol, 268, 269 (INCL), 97 Hydroxyeicosatetraenoic acids (HETEs) Ischemia, sphingosine 1 phosphate action, 19 neurological disorders 15(R)-HETE, 19 biphasic immune response, 258 5-HETE and 12-HETE, 12 interruption, blood flow, 257 4-Hydroxyhexenal (4-HHE) PKC epsilon (V1-2), 258 ATP translocator, 183 sphingomyelin-ceramide pathway, 258 a and b carbon, 183 stroke, neurochemically, 257–258 NF-kB activation, 184 treatment, neurotraumatic and redox status, 183–184 neurodegenerative diseases, 258 4-Hydroxynonenal (4-HNE) Ischemic injury, 30, 231–232 amino acids, 159 Isofurans blood–brain barrier modulation chemical structure, 208 endothelial cell (EC) surface, 176 dopaminergic neurodegeneration, PD and homeostasis, 175–176 DLB, 209

integrity and stability, 176 F2-IsoPs and IsoFs levels, 208–209 chemical structures, 159–160 mechanisms, 208 description, 159 “reoxygenation” phase, 208 enzyme activities modulation Isoketals (IsoKs) ATPases, 169–170 chemical structure, 208 caspases, 166–167 1,4-pentadiene/1,4,7-octatriene side chain enzymes and cell cycle, 170 structure, 207 Index 321

Isolevuglandins. See Isoketals (IsoKs) LOXs convert, 11

Isoprostanes (IsoPs) LTA 4, 12

biomarkers, oxidative stress, 193 LTC4 synthesis, 13 degradation and LXs biosynthesis, 4, 5 biosynthesis, arachidonic acid, 196 neurochemical effects, 18

pharmacokinetics, 195 PGE2 and LTB4 generation, 26 15-prostaglandin dehydrogenase PGs, LXs and TXs (15-PGDH), 197 biosynthesis, 24 radioactivity, 195 brain tissue, 25 and fatty acid, chemical structures, 194 coupling mechanisms, 24 generation proinflammatory effects, 26 biosynthesis, 195 receptors, 24–25 cyclooxygenase activity, 194 synthesis, 23

F2-isop measurement, 195 primary, 13 KA neurotoxicity release, 32 cyclic and nondegradable analog, 209 Leukotrienes

F2-isoPs levels, 210 EPA, enzymic oxidation, 308 neurodegenerative diseases levels, 308 amyloid plaque formation, 202 Lipid mediators APP cleavage, 203 brain hypothetical model, Ab generation, 203 agonist and receptor, modulation neurological disorders (see Neurological interactions, 300–301 disorders) nuclear pore complexes transport neurotraumatic diseases, 204 modes, 302 radical-catalyzed peroxidation, 193 receptor-mediated catabolism, 300–301 roles, 194 sphingolipid-derived, 301, 302 signal transduction processes cholesterol role, 300 EPA and DHA generation, 200 fatty acids, 309–310 excitatory and inhibitory effect, 201 modulation, diet

F2-IsoP receptor-mediated effect, 199 ARA, 308, 309 LDL/VLDL, 199 EPA and DHA, 308–309 Nrf2-mediated gene transcription, 201 paleolithic, 309 plasma 8-isoprostanes, 200 neural

TXA2, 199 cell function, 299, 300 vasoconstrictors, 198 membranes, 299 IsoPs. See Isoprostanes neurological disorders, lipidomics, 306–308 phospholipid, sphingolipid and cholesterol-derived J CHO cells, 306 Juvenile rheumatoid arthritis (JRA), 123, 126 C1P and S1P, 303–304 enzymes, synthesis, 305 K interactions, 303, 304 Kainate (KA)-mediated neurotoxicity, 236–237 neural cells, 303 Kainic acid (KA) NFA, 305 description, 124 OSBP role, 306 neurotoxicity, 183, 209–210, 289–290 rat brain slices sphingomyelinase and ceramide, 304 Lipidomics L analysis, brain tissue, 290 Land’s cycle. See Deacylation/reacylation cycle characterization, ceramide-metabolizing Leukotriene (LTs) protein, 237 converts, 13 lipid mediators detection, 306–308 ischemic injury, 30–31 neurological disorders (see Neurological 5-LOX activation, 34 disorders) 322 Index

Lipoxins (LXs) enzymes, 74–75 activation, 20 long-chain acyl-CoA hydrolase/ ATLs, 20 thioesterases, 80

biosynthesis pathways, 19 PLA2, 78–79 coupling mechanisms, 24 INCL, 97 generation, 26 ischemic injury

levels, 307, 308 Lp-PLA2 activity, 94–95

LXA4 receptor, 20 Lyso-PtdH level, 95 PGs, LTs, and EETs neuroprotective effects, brain tissue, 25 lyso-PtdCho, 94 physiologic processes, 24 Lyso-PtdCho, 82–85 trihydroxytetraene eicosanoids, 19–20 Lyso-PtdEtn, 85–86 Lipoxygenases (LOXs) Lyso-PtdH, 90–93 catalytic pathways, 12 Lyso-PtdIns, 87–88 COX, and EPOX, PLA2 isoforms Lyso-PtdSer, 86–87 (see Cyclooxygenases) MS and EAE, 96 and COX-2 immunoreactivities, 30 oxidative stress, 93 eicosanoids, COX and EPOX plasmalogens, 89–90 AD (see Cyclooxygenases) quantification and seperation, 73 ALS, 35–36 Lysohosphatidylserine (Lyso-PtdSer) CJD (see Creutzfeldt–Jakob disease) deacylation and degranulation, 86 PD (see Parkinson disease) intracellular calcium mobilization, 87 5-LOX, 12-LOX, and 15-LOX gene pertussis toxin, 86–87 EPOX activities, 30–31 Lysophosphatidic acid (Lyso-PtdH) isoform, 12 B103 neuroblastoma cell cultures, 92 LX biosynthesis, 19 brain neuronal immunostaining, 30 mediated signaling, 91, 93 p38 MAPK role, 31 metabolism and roles, 91–92 receptor-mediated stimulation, 13 neural cells, 91 Src homology 3 (SH3), 13 synthesis, 90–91 nomenclature, 12 Lysophosphatidylcholine (Lyso-PtdCho) Liver X receptor (LXR) DHA, 85 activation and deletion, 278 intracerebroventricular CYP27, 289 injections, 84 24(S)-hydroxycholesterol, 275 modulation, receptors, 82–83 hydroxycholesterols, 268 morphological transformation, ligands, 275, 289 microglia, 84 RXR, 270, 271 roles, brain, 82–83 LPEAT2. See Lysophosphatidylethanolamine Lysophosphatidylethanolamine acyltransferase 2 (Lyso-PtdEtn), 85–86 LTs. See Leukotriene Lysophosphatidylethanolamine acyltransferase LXs. See Lipoxins 2 (LPEAT2), 78 Lyso-cardiolipin (Lyso-Ptd2Gro) Lysophosphatidylinositol (Lyso-PtdIns) apoptosis, 88–89 mediated exocytosis, 87–88 described, 88 mitogenic activity, 88 mitochondrial membranes, 89 neural and synaptic membranes, 87 Lyso-glycerophospholipids metabolism Lysoplasmalogens, 89 AD, 95–96 Lyso-PtdCho. See Lysophosphatidylcholine chemical structures, 73, 74 Lyso-PtdEtn. See Lysophosphatidyletha- deacylation/reacylation cycle nolamine ACS, 75–77 Lyso-Ptd2Gro. See Lyso-cardiolipin acyl-CoA: lysophospholipid Lyso-PtdH. See Lysophosphatidic acid acyltransferase, 75–77 Lyso-PtdIns. See Lysophosphatidylinositol CoA-independent reacylation, 81 Lyso-PtdSer. See Lysohosphatidylserine Index 323

M PD, 284–285 Major depressive disorders (MDD), 236 TBI, 281 MAPK. See Mitogen-activated protein kinases endocannabinoids Microphthalmia-associated transcription factor description, 144–145 (MITF), 255 neurodegenerative, 147–149 MITF. See Microphthalmia-associated neurotraumatic diseases, 145–147 transcription factor 4-HNE Mitogen-activated protein kinases Alzheimer Diseases, 176–177 (MAPK), 17, 27, 31, 136 amyotrophic lateral sclerosis (ALS), 178 Multiple sclerosis (MS), 96, 235, 260 definition, 176 glial cells, 176 ischemia, 179–180 N KA neurotoxicity, 183 N-acyl-transferase (NAT), 142 neurodegenerative processes, 176 Neurodegeneration Parkinson disease, 177–178 apoptotic cell death, 27 Prion diseases, 179 hydroperoxides, 27 spinal cord injury, 180–181 neural cell dysfunction and death, 27 traumatic brain injury, 181–182 pathological conditions, 27 in vitro and in vivo models, 145

PGD2 dehydration, 28 IsoPs role, 27 aSAH, 205–206 Neurodegenerative processes, 176 E-and D-type prostane rings, 205 Neurofurans, 208, 209 F4-neuroprostanes, structure, 205 Neuroinflammation isofurans, 208–209 converging mechanisms, 26 IsoKs, 206–207 expression and stimulation, neurofurans, 209

iPLA2, 26–27 neuroketals, 207–208 inflammasome mediators, expression, 26 radical-mediated peroxidation, polymorphonuclear leukocytes (PMN), 26 DHA, 204 protective process, 25–26 levels, endocannabinoids, 144 Neuroketals lipidomics chemical structure, 208 CSF and ECF, 307 docosahexaenoyl radicals formation, 207 genomic analysis, 307 Neurological disorders lipid mediators, 307 ceramide neurodegenerative diseases, AD, 232–234 306–307 ALS (see Amyotrophic lateral sclerosis) lyso-glycerophospholipids metabolism Batten disease, 236 (see Lyso-glycerophospholipids HIV-1, 235–236 metabolism) involvement, 230 PAF ischemic injury, 231–232 AD (see Alzheimer disease) KA neurotoxicity, 236–237 microglia, 121 levels, 229–230 PAF involvement, 120–122 MDD (see Major depressive disorders) receptor-mediated alterations, MS, 235 PAF, 118, 119 PD, 234 subunits types, 120

SMases and PLA2 activities, 230–231 synapse degeneration, 122 cholesterol and hydroxycholesterols TBI and SCI, 118–119 AD, 281–284 sphingosine 1 phosphate CTX, 281 AD, 259–260 HD, 285–286 ischemia, 257–259 KA neurotoxicity, 289–290 KA neurotoxicity, 261 multiple sclerosis, 287–288 MS, 260 NPC, 286–287 TBI and SCI, 259 324 Index

Neuroprostanes (NPs) P chemical structure, 205 PAF acetylhydrolase radical-mediated peroxidation, DHA, 204 plasma, 116, 123 Neuroprotectin role, 120 animal models, effects, 64 short chain phospholipids, 114 anti-inflammatory actions, 67 structure, 115 apoptotic cell death, 62 Type I and II, 115 ARA enriched food, 66 PAF-receptor (PAF-Rs) ARPE-19 cells, 62 enzymes, 118 cerebral cortex, 63 PAF-mediated stimulation, enzymic cerebrospinal fluid (CSF), 66 activities, 116–117 chemical structures, 65 Parkinson disease (PD) epithelium-derived factor, 63–64 AD and HD, 281 glial cells, 63 cell culture and transgenic mice inflammatory signaling, 67 model, 284 macrophages, 63 ceramide, 234 non-amyloidogenic pathway, 66–67 character, 284

NPD1-mediated regulation, 65 eicosanoids, COX, LOX and EPOX total organic synthesis, 62 COX-2 patients, 35 Neurotraumatic diseases neurons loss to causes, 35 COX, LOX and EPOX Activities, 30 4-HNE, 177–178 epilepsy, 32 PD. See Parkinson disease ischemic injury, 30–31 Peroxisome proliferator-activated receptor primary and secondary injury, 29–30 gamma (PPARg), 136 secondary injury, 29 Peroxisome-proliferator-activated spinal cord trauma, 31 receptors (PPAR) TBI, 31–32 coactivators, 24

Neurotrophin-3 (NT-3), 252 15d-PGJ2, 28 NF-AT. See Nuclear factor-activated T cells PPARa, 14 Niemann-Pick type C (NPC) PPARg, 17

NPC1 or NPC2, 286–287 PGE2-G. See Prostaglandin glyceryl esters pathogenesis, 287 PGs. See Prostaglandins N-methyl-D-aspartic acid (NMDA) Phosphatidylethanolamine (PtdEtn), 206

receptor, 55, 84, 139, 165, 171 Phospholipase A1 (PLA1) Nociception (pain state) deacylation hot-plate, 28 PtdIns, 87 peripheral and central mechanisms, 28 PtdSer, 86 Non-hydroxyfatty acid (NFA), 305 Lyso-PtdCho synthesis, 82 NPC. See Niemann-Pick type C neural cells, 73 n-3 polyunsaturated fatty acids, brain Phospholipase A2 (PLA2) cyclooxygenase and lipooxygenase, 52 calcium-independent, 78 nerve growth factor expression in, 52 classification, 78 neuronal function and regulation, 50 paralogs, splice variants and multiple peroxisomal disorders, 50–51 forms, 79 PUFAs, neural membranes, 51 PKA. See Protein kinaseA

NPs. See Neuroprostanes PLA1. See Phospholipase A1

NT-3. See Neurotrophin-3 PLA2. See Phospholipase A2 Nuclear factor-activated T cells (NF-AT), 136 Plasmalogen-selective phospholipase A2, 113 Platelet-activating factor (PAF) catabolism O hydrolysis and 1-alkyl-2-acetyl- Oxysterol-binding protein (OSBP) sn-glycerol, 114, 115 role, 306 SDS-PAGE, 116 translocation, 306 Type I and II PAF-acetylhydrolase, 115 Index 325

de novo synthesis LTs, LXs and EETs CDP-choline phosphotransferase, 110 brain tissue, 25 enzymes, 109–110 physiologic processes, 24

1-O-alkyl-2-acetyl-sn-glycero-3- PGD2, PGF2a and PGE2 modulation, 28–29

phosphate, 110 PGE2 and 8-epi-PGF2a, 36

description, 107 PGH2, 4

KA (see Kainic acid) PGI2 and PGE2 levels, 28 molecular mechanism, PAF-mediated physiological production, 7 neural injury production, 35 neural membrane oxidized Protectins phospholipids, 125 anti-inflammatory actions, 67 PAF-Rs, 124, 125 ARA enriched food, 66 neural and non-neural cells, 118 ARPE-19 cells, 62 neurological disorders, 118–123 cerebral cortex, 63 oxidative fragmentation pathway, chemical structures, 65 synthesis, 114 CSF, 66 PAF-Rs (see PAF-receptor) docosanoids, 301, 308, 310 pathways description, 108–109 epithelium-derived factor, 63–64 remodeling pathway, synthesis glial cells, 63 acetyl CoA, 111, 112 inflammatory signaling, 67 brain and inflammatory cells, 111 levels, 308 cross-talk, 113 macrophages, 63 plasmalogen conversion, 112 non-amyloidogenic pathway, 66–67

receptor-mediated modulation, 113–114 NPD1-mediated regulation, 65 roles, 107, 108 total organic synthesis, 62 synthesis, 108 Protein kinase A (PKA), 5, 89, 90, 136, 137 visceral disorders Proteomics levels, 123 and lipidomics, 307 sepsis, 124 use, 307 SLE and JRA, 123 PtdEtn. See Phosphatidylethanolamine Polymorphonuclear leukocytes (PMN), 26 PPAR. See Peroxisome-proliferator-activated receptors R PPARg. See Peroxisome proliferator-activated Rapid-eye-movement (REM), 142 receptor gamma Reactive oxygen species (ROS) Prion diseases, 179 function, 3 Proinflammatory generation, 28 effects, 26 production, 26, 27 precursors, 26 and proteins reaction, 3 temporal “switch”, 20 Receptors, eicosanoids

Prostaglandin glyceryl esters (PGE2-G) levels, acute inflammatory response, 20 141 BLT, 18 Prostaglandins (PGs) “braking signals”, 20 cyclopentenone cannabinoid (CB), 21 activities, 17 cells and tissues express, 15 covalent adducts, 17 cyclopentenone prostaglandins, 17

dehydration, 17 15-d-PGJ2, 17

15d-PGJ2 EETs, 21

inflammatory response, 17 EP2 activation, 15

mediated cell death, 28 EP1, EP2, EP3 and EP4, 15 signaling pathway, 18 formyl peptide, 20 elevations, 31–32 LXs, 19–20 EPA enzymic oxidation, 308 PGE, 17 levels, 31–32, 308 PGs, 15 326 Index

Receptors, eicosanoids (cont.) signal transduction processes, 245 synthesis, 22 SM, 245 TP isoforms, 21 sphingolipids, 245 Resolvins Sphingosine kinase (SphK) activities, 53 CaM-binding, 250 animal models, effects, 64 five conserved domains (C1-C5), 248 apoptotic cell death, 62 growth factor/cytokine-initiated, 248, 250 DHA, 58 hypothetical diagram, TNF-a and S1P DPA-derived metabolites, 60 receptors, 249 EPA and DHA, 308, 309 SphK1, 248 levels, 308 SphK2, 248, 249 neural and non-neural tissues, 56 treatment, siRNA, 250 17S D series, 60 vascular endothelial growth factor, 250–251 RhoA/Rho kinase (ROCK), 14 Sphingosine 1 phosphate (S1P) ROS. See Reactive oxygen species COX-2 upregulation, 303 enzymes stimulation, 303–304 fEPSP-PS, 248 S HDL, 247 SCI. See Spinal cord injury lipoproteins, 248 SM. See Sphingomyelin neurological disorders SMases. See Sphingomyelinase AD, 259–260 SMS. See Sphingomyelin synthase ischemia, 257–259 S1P. See Sphingosine 1 phosphate KA neurotoxicity, 261 SPC. See Sphingosylphosphorylcholine MS, 260 Sphingomyelin (SM). See also Sphingosine TBI and SCI, 259 apoptotic cell death, 228 pro-survival and pro-angiogenic and ceramides mediator, 247–248 ALS patients, 234–235 transport mechanism, ABC-type HIV dementia (HIVD), 235–236 transporter, 247 chemical structure, 218 Sphingosine 1 phosphate receptor, brain SMases action, 223 Ca2+ mobilization, 254 SMS, 232 eNOS signaling pathway, 252 Sphingomyelinase (SMases) five members, family, 251 activation, rafts, 218 G proteins and target enzymes, 251 ceramide formation, activation hypothetical diagram interactions, 253 acid SMases, 222 in autocrine/paracrine manners, 251 alkaline SMases, 223 intracellular effects, 253–254 CAPK and MAP kinases, 223–224 NT-3, 252 neutral SMases, 222–223 PLC and ERK, 253

signaling targets, 224 S1P4 restricted expression pattern, Sphingomyelin synthase (SMS), 232 252–253 Sphingosine Sphingosylphosphorylcholine (SPC) Akt kinase pathway, 245 CNS, 255 18-carbon amino alcohol, 245 CSF, SAH patients, 255 cell survival and neurodegeneration, 245 fluorescence stopped-flow ceramide degradation, 246–247 technique, 254 channel types, 245–246 generation and roles, 254 chemical structures, 246 MITF and ERK pathway, 255 effect, enzyme activity, 246 SphK. See Sphingosine kinase ERK1/2 and p38-MAPK, 245 Spinal cord injury (SCI) giant vesicles, 245 4-HNE, 180–181 metabolism, 247 PAF, 119 N-ethylmaleimide-insensitive type 2, 247 sphingosine 1 phosphate, 259 pro-apoptotic, 247–248 and TBI, 118, 119 Index 327

Spinal cord trauma microglial cells, 9

eicosanoids PGH2, 6 anti-CD11d mAb treatment, 31 PGs, LTs and LXs APE/Ref-1 expression, 31 receptors, 24 5-LOX, FLAP, and CysLT1 mRNAs, 31 synthases, 23

neurotraumatic diseases, 29 TXA2, 20–21 Stroke. See Ischemic injury Transient receptor potential vanilloid receptor Synaptic plasticity, eicosanoids (TRPV1), 142 COX-1 and COX-2 inhibitors, 29 Traumatic brain injury (TBI) neuronal, 28 4-HNE, 181–182

PGE2-mediated synaptic modulation, 29 hydroxycholesterol, 281, 288 Systemic lupus erythematosus (SLE) and ischemia, 118 PAF levels, 123 and SCI, 119 patients, 123 sphingosine 1 phosphate, 259 and spinal cord trauma accumulation, 32 T components define, 29 TBI. See Traumatic brain injury COX and LOX activities, 31–32 Thromboxanes (TXs) neural cells, 29 ARA oxygenation, 310 persistent accumulation, 32 coupling mechanisms, 24 TRPV1. See Transient receptor potential levels, 308 vanilloid receptor