Citation: Transl Psychiatry (2011) 1, e31, doi:10.1038/tp.2011.27 & 2011 Macmillan Publishers Limited All rights reserved 2158-3188/11 www.nature.com/tp Altered neuroinflammatory, arachidonic acid cascade and synaptic markers in postmortem Alzheimer’s disease brain JS Rao1,2, SI Rapoport1 and H-W Kim1,2 Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is the leading cause of dementia in the elderly. A recent positron emission tomography imaging study demonstrated upregulated brain arachidonic acid (AA) metabolism in AD patients. Further, a mouse model of AD shows an increase in AA-releasing cytosolic phospholipase A2 (cPLA2) in brain, and a reduction in cPLA2 activity ameliorated cognitive deficits. These observations led us to hypothesize that there is an upregulation of AA cascade and neuroinflammatory markers in the brain of AD patients. To test this hypothesis, we measured protein and mRNA levels of AA cascade, neuroinflammatory and synaptic markers in postmortem frontal cortex from 10 AD patients and 10 age-matched controls. Consistent with our hypothesis, AD frontal cortex showed significant increases in protein and mRNA levels of cPLA2-IVA, secretory sPLA2-IIA, cyclooxygenase-1 and -2, membrane prostaglandin (PG) synthase-1 and lipoxygenase- 12 and -15. Calcium-independent iPLA2-VIA and cytosolic PGE2 synthase were decreased. In addition, interleukin-1b, tumor necrosis factor-a, glial fibrillary acidic protein and CD11b were increased. AD postmortem brain also showed signs of cellular injury, including decreased synaptophysin and drebrin, pre- and postsynaptic markers. These results indicate that increased AA cascade and inflammatory markers could contribute to AD pathology. Altered brain AA cascade enzymes could be considered therapeutic targets for future drug development. Translational Psychiatry (2011) 1, e31; doi:10.1038/tp.2011.27; published online 16 August 2011 Introduction gene transcription of these enzymes. The released nitric oxide can also promote AA hydrolysis from the membrane by The postmortem Alzheimer’s disease (AD) brain is character- cPLA2, by stimulating glutamate release from nerve terminals ized by intracellular neurofibrillary tangles with paired helical and thereby increasing intracellular Ca2 þ concentrations via filaments consisting of phosphorylated t-protein, and extra- postsynaptic ionotropic N-methyl-D-aspartate and other glu- cellular senile (neuritic) plaques containing b-amyloid fibrils. tamatergic receptors.10,11 b-Amyloid peptide can also pro- 12 In the United States, nearly 5.3 million people are affected voke glutamate-induced excitotoxicity and PLA2 activation. by AD.1 Common behavioral disturbances, aside from AA is a conditionally essential polyunsaturated fatty acid memory loss, are apathy, depression, agitation and general found mainly in the stereospecifically numbered-2 position of withdrawal. Apathy is the most prevalent disturbance, affect- membrane phospholipids, from which it can be hydrolyzed by ing about 70% of AD patients; depression ranks second, cPLA2 or sPLA2. A portion of the released AA is metabolized occurring in about 54% of patients; and agitation ranks third, to bioactive prostaglandin H2 (PGH2) by COX-1 or COX-2, to appearing in about 50% of patients.2 cytoprotective epoxyeicosatrienoic acid by cytochrome p450 The senile plaques often are infiltrated by activated epoxygenase, or to cytotoxic leukotrienes by lipoxygenase 13 microglia that secrete inflammatory cytokines, release nitric (LOX) subtypes-5, -12 and -15. Bioactive PGH2 is converted 3–6 oxide and express peripheral benzodiazepine receptors. to PGE2 (PGE) by membrane PG synthase-1 (mPGES-1) or Inflammatory cytokines can activate many brain signaling cytosolic PG synthase (cPGES). PGH2 also can be converted RETRACTED 14 pathways, including the arachidonic acid (AA, 20:4n-6) to thromboxane A2 by thromboxane synthase (TXS). Of the metabolic cascade.7 For example, activation of the cytokine two COX isoenzymes, COX-1 is constitutively expressed, interleukin (IL)-1 receptor cascade can increase expression of whereas COX-2 is inducible. cPGES uses PGH2 produced by AA-metabolizing enzymes, including AA-selective cytosolic COX-1, whereas mPGES-1 uses COX-2-derived endo- 8 15 phospholipase A2 (cPLA2), secretory sPLA2 and cyclooxy- peroxide. AA and its metabolites can modulate signal trans- genase (COX)-2,9 and of the transcription factors activator duction, transcription, neuronal activity, apoptosis and many protein (AP)-2 and/or nuclear factor kappa B that regulate other processes within the brain.16–18 1Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA Correspondence: Dr JS Rao, Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, 9000 Rockville Pike, Building 9, Room 1S-126, Bethesda, MD 20892, USA. E-mail: [email protected] 2These authors contributed equally to this work. Keywords: Alzheimer’s disease; arachidonic acid cascade; inflammation; synaptophysin; drebrin Received 28 April 2011; revised 7 June 2011; accepted 16 June 2011 Altered AA cascade enzymes in Alzheimer disease JS Rao et al 2 Recent human imaging and postmortem studies have 100 000 g for 60 min at 4 1C. The resulting supernatant-1 was demonstrated an elevated AA incorporation coefficient (k*) the cytosolic fraction, and the pellet was resuspended in the as well as altered phospholipid fatty acid concentrations19 in homogenizing buffer containing 0.2% (w/v) Triton X-100. The brains of AD patients, particularly in regions reported to have suspension was kept at 4 1C for 60 min with occasional stirring high densities of senile (neuritic) plaques with activated and then centrifuged at 100 000 g for 60 min at 4 1C. The microglia.20 An increase in k* is suggestive of increased AA resulting supernatant-2 was the membrane fraction. Protein incorporation and utilization by the brain. AA utilization by the concentrations in membrane and cytosolic fractions were brain can be initiated by the release of AA from the determined with Protein Reagent (Bio-Rad, Hercules, CA, phospholipid membrane by cPLA2. Genetic deletion of cPLA2 USA). The membrane and cytosolic fractions were confirmed has been shown to improve cognitive performance in a using specific markers cadherin and tubulin, respectively. transgenic animal model of AD.21 Moreover, drugs that decrease turnover and metabolites of the AA cascade, such Western blot analysis. Proteins (50 mg) were separated on as lithium and valproate,22 have been reported to have 4–20% SDS-polyacrylamide gels (Bio-Rad). Following beneficial effects in patients with HIV-associated demen- electrophoresis, the proteins were transferred to a poly- tia.23,24 Further studies have shown beneficial effects vinylidene fluoride membrane (Bio-Rad). Protein blots were of lithium in AD patients.25–27 These findings suggest that incubated overnight in Tris-buffered-saline, containing the AA cascade might have a role in the cognitive deficits 5% non-fat dried milk and 0.1% Tween-20, with specific associated with dementia. primary antibodies (1:200 dilution) for cPLA2-IVA, sPLA2-IIA, In view of evidence of excitotoxicity and neuroinflammation iPLA2-VIA, COX-1 (1:1000), COX-2 (1:500), cytochrome in AD,28,29 and of elevated AA metabolism in AD patients,20 P450 epoxygenase, TXS, 5-, 12- and 15-LOX (Abcam, we hypothesized that the AA cascade is upregulated in the AD Cambridge, MA, USA) and neuron-specific enolase brain and is accompanied by elevated neuroinflammatory (1:10 000) mPGES-1(1:200), cPGES, IL-1beta (IL-1b), markers as well as by synaptic marker loss. To test this tumor necrosis factor-alpha (TNF-a) CD11b, and glial hypothesis in this study, protein and mRNA levels of AA fibrillary acidic protein (GFAP). Synaptophysin, drebrin and cascade enzymes, neuroinflammatory markers and synaptic mPGES-1 were determined using a specific (1:200) primary markers were compared between postmortem frontal cortex antibody (Abcam, Cambridge, MA, USA). Cytoplasmic from 10 AD patients and 10 age-matched controls. We also protein blots were incubated with appropriate horseradish 2 þ compared expression of Ca -independent iPLA2, which is peroxidase-conjugated secondary antibodies (Bio-Rad) and selective for docosahexaenoic acid (22:6n-3) in membrane were visualized using a chemiluminescence reaction (Kodak, phospholipid.30 The frontal cortex (Brodmann area 9) was Rochester, NY, USA). Optical densities of immunoblot bands chosen for study because functional and structural abnorm- were measured using Alpha Innotech Software (Alpha alities have been reported in this region in AD patients.31–35 Innotech, San Leandro, CA, USA) and were normalized to b-actin (Sigma-Aldrich, St Louis, MO, USA) to correct for unequal loading. All experiments were carried out twice with Materials and methods 10 control and 10 postmortem brain samples from AD patients. Values were expressed as percent of control. Postmortem brain samples. The protocol was approved by the Institutional Review Board of McLean Hospital, and by Total RNA isolation and real time RT-PCR. Total RNA the Office of Human Subjects Research of the National was isolated from the frontal cortex using an RNeasy mini kit Institutes of Health (no. 4380). Frozen postmortem human (Qiagen, Valencia, CA, USA). RNA integrity number was frontal cortex (Brodmann area 9) from 10 AD patients (diag- measured using a Bioanalyzer (Agilent, Santa Clara, CA, nosed by history and the presence of senile neuritic