Brain CYP2D6 and its role in neuroprotection against Parkinson’s disease by Amandeep Mann A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Amandeep Mann 2011 Brain CYP2D6 and its role in neuroprotection against Parkinson’s disease Amandeep Mann Doctor of Philosophy, 2011 Graduate Department of Pharmacology and Toxicology University of Toronto Abstract The enzyme CYP2D6 can metabolize many centrally acting drugs and endogenous neural compounds (e.g. catecholamines); it can also inactivate neurotoxins such as 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and β- carbolines that have been associated with Parkinson’s disease (PD). CYP2D6 is ideally situated in the brain to inactivate these neurotoxins. The CYP2D6 gene is also highly polymorphic, which leads to large variation in substrate metabolism. Furthermore, CYP2D6 genetically poor metabolizers are known to be at higher risk for developing PD, a risk that increases with exposure to pesticides. Conversely, smokers have a reduced risk for PD and smokers are suggested to have higher brain CYP2D6 levels. Our studies furthered the characterization and involvement of CYP2D6 in neuroprotection against PD. METHODS: We investigated the effects of CYP2D6 inhibition on MPP+-induced cell death in SH-SHY5Y human neuroblastoma cells. We compared levels of brain CYP2D6, measured by western blotting, between human smokers and non-smokers, between African Green monkeys treated with saline or nicotine, and between PD cases and controls. In addition, we assessed changes in human brain CYP2D6 expression with age. RESULTS: Blocking CYP2D6 activity in SH- SY5Y cells with four diverse inhibitors significantly increased MPP+-induced neurotoxicity. Smokers have higher brain CYP2D6 compared to non-smokers. In monkeys, basal expression of CYP2D6 varied across brain regions and was increased by chronic nicotine treatment in II select regions (notably the basal ganglia) and specific cell types. Expression of human brain CYP2D6 increased from fetal to 80 years of age in the frontal cortex; the influence of age on CYP2D6 expression was brain region specific. Compared to age-matched controls, PD cases had ~40% lower CYP2D6 levels in the frontal cortex, cerebellum and hippocampus consistent with lower CYP2D6 increasing the risk for PD. In the caudate and substantia nigra, CYP2D6 levels were similar between PD case and controls using Western blotting. This is likely due to the increase in CYP2D6-expressing astrocytes and much higher cellular CYP2D6 in PD affected areas as observed with immunocytochemical staining. CONCLUSIONS: Brain CYP2D6 can meaningfully inactivate neurotoxins, and it can be increased by nicotine in brain regions of interest to PD. These findings support the contention that higher brain CYP2D6 is protective and lower levels may contribute to increased risk for PD. III Acknowledgments First and foremost, I would like to thank my supervisor Dr. Rachel F. Tyndale for her support, guidance and mentorship during my Ph.D. I am grateful for all the opportunities she provided that allowed me to grow as a student and as a professional. She inspired me to always be learning, thinking, applying and questioning science. Her hard work, dedication and continuous encouragement shaped me to become a better student and scientist. I am very grateful to the excellent Ph.D. Committee Members Dr. Jose Nobrega and Dr. James Wells, who contributed their time, knowledge and expertise. Their guidance and constructive criticism was very helpful in bringing the research within this thesis together. I would also like to thank my all of my friends and colleagues, who provided their support, time, knowledge, advice and technical expertise during my Ph.D. This especially includes Ewa Hoffmann, Charmaine Ferguson, Jibran Khokhar, Sharon Miksys, Daniel Hanna and Andy Zhu. This accomplishment was that much more achievable and enjoyable with the love from my dearest friends Helena Mistry, Nael Al Koudsi, Vaneeta Verma, Jassi Lachhar-Dugha and my best friend Arun Dhanota. Last but not least, I would like to thank my family for their love, support and continuous encouragement. I am sincerely thankful to my parents' hard work, patience and understanding, without which I would not have had the opportunities I did in my life. I am appreciative of my Grandfather's wisdom and advice that helped me gain perspective and make sense of things during challenging times. The constant encouragement provided by my sister-in-law Jasmine and brother Kamal definitely made this goal much easier to achieve. I am also very thankful to my niece Nayha for brightening my days with her smiles, hugs, kisses and paintings when I needed it the most. Lastly, I would like to thank my Grandmother for her love and support. As she had been very excited to see me complete my Ph.D., I dedicate this thesis in her loving memory. IV Table of Contents Abstract ............................................................................................................................................... II Acknowledgments........................................................................................................................... IV Table of Contents...............................................................................................................................V List of Table......................................................................................................................................VII List of figures ................................................................................................................................. VIII Summary of abbreviation............................................................................................................. IX Section I: Introduction.....................................................................................................................1 Statement of Research Problem............................................................................................................. 1 Review of the literature............................................................................................................................ 3 1. Cytochromes P450 2D6 (CYP2D6).................................................................................................... 3 1.1 Cytochromes P450 ..............................................................................................................................................3 1.2 CYP2D6 substrates..............................................................................................................................................4 1.3 CYP2D6 inhibitors...............................................................................................................................................7 1.4 CYP2D6 regulation........................................................................................................................................... 10 1.5 CYP2D6 genetic variation.............................................................................................................................. 11 1.5.A CYP2D pseudogenes..................................................................................................................................................14 1.5.B Interethnic variability CYP2D6.............................................................................................................................14 1.5.C CYP2D6 genetic associations to health risk.....................................................................................................15 1.6 CYP2D expression in different species.................................................................................................... 16 1.6.A CYP2D in rodents .......................................................................................................................................................16 1.6.B CYP2D in non-human primates............................................................................................................................18 2. Brain Cytochromes P450 ...................................................................................................................19 2.1 Brain CYP expression...................................................................................................................................... 19 2.1.A Brain CYP activity.......................................................................................................................................................20 2.1.B Brain CYP regulation.................................................................................................................................................21 2.2 Brain CYP2D6..................................................................................................................................................... 22 2.2.A CYP2D6 CNS expression..........................................................................................................................................22 2.2.B CYP2D6 CNS function ...............................................................................................................................................23 2.2.C Brain CYP2D6 regulation ........................................................................................................................................26