I I CYTOPROTECTIVE ACTIONS PF NICOTINE: THE INCREASED EXPRESSION OF a.7 NICOTINIC RECEPTORS AND NGF/TrkA RECEPTORS ' . ' f !'b y' Ramamohima R. Jonnala ' ' ' I' Submitted to the Faculty o~the School of Graduate Studies of Medical College of Georgia in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy Juiy, 2001 I' l Cytoprotective Actions of Nicotine: The Increased Expression of a.7 Nicotinic Receptors a-!ld NGF/TrkA Receptors This dissertation is submitted by Ramamohana R. Jonnala and has been examined and approved by an appointed committee of the faculty of the school of Graduate Studies of the Medical College of Georgia. The signatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, form and accuracy of presentation. ' This dissertation is therefore in partial fulfillment of the requirements of the degree of Doctor of Philosophy. ACKNOWLEDGMENTS I would like to extend my appreciation to my major advisor, Dr. Jerry J. Buccafusco, for his guidance, encouragement and support. I would also like to thank my committee members, Dr. Alvin V. Terry Jr., Dr. William D. Hill, Dr. Nevin A. Lambert and Dr. Clare M. Bergson and also my thesis readers Dr. Debra Gearhart and Dr. Dale W. Sickles for their time and valuable suggestions. My sincere thanks to Dr. Deborah L. Lewis, Dr. Robert W. Caldwell and Dr. Gary C. Bond for encouraging me to enter the graduate program in the Department of Pharmacology & Toxicology, Medical College of Georgia. My sincere thanks to members of my laboratory Laura, Vanessa, Daniel, Cat, Mark, Nandu, Lu, and Shyamala for their assistance and suggestions. TABLE OF; CONTENTS Page ABSTACT ............................................................. : ............ .. lll LIST OF FIGURES ................................................................ .. vii LIST OF TABLES ................................................................. .. ix LIST OF ABBREVATIONS ..................................................... .. X I. INTRODUCTION A. Significance of study ............... ,..................................... 1 B. Literature review ........................................................ .. 3 I. Alzheierm's disease pathophysiology .......................... 3 2. Genetics of AD ...................................................... 6 3. The selective vulnerability ofBFC neurons .................... 7 4. NGF and NGF receptors ........................................ .. 9 5. Oxidative damage .................................................. 10 6. Cholinomimetic therapy ........................................ .. 12 7. N1cotmic receptors .............................................. .. 14 8. The PC12 cell model system .................................... .. 18 II. MATERIALS AND METHODS Cell culture ................................................................. .. 21 Compounds ................................................................ .. 21 Strucutres of choline and choline analogs .............................. .. 22 Cell vi;;tbility assay ......................................................... 23 125 [ I]a-BGT binding assay ...................................... : ........ .. 23 Elisa assay ..................................................................... 24 Animals ....................................................................... 25 Surgical procedure .......................................................... 25 Westemblot.................................................................. 25 Exposure of PC12 cells peroxynitrite ................................... .. 26 Cytosensor Microphysiometer ............................................ 27 Cell lysate preparation ..................................................... 27 Immunoprec1p1 . ·1at1· on s tudi es ............................................... 28 MAPKassay............................................................... .. 29 Page I.RESULTS I. Stimulation of cx7nAChR protects differentiated PC12 cells from trophic factor withdrawal toxicity. .. 3 0 2. Neuroprotective actions of cholii1e·and choline analogs............ 31 3. The abilities of different nAChR agonists to indµce neuroprotection.. .. .. .. .. .. .. 32 4. The abilities of different nAChR agonists to induce cx7nAChR upregulation............................................................... 33 5. MLA induced increase in cx7nAChR expression and neuroprotection... .. .. .. .. .. .. .. .. .. 34 6. Nicotine up-regulates TrkA receptors................................. 35 7. Relationship between nicotine's neuroprotection and its ability to increase TrkA receptors ............... :................................ 37 8. Metabolic response of PC12 cells to NGF............................ 38 9. Effect of SIN-I on cell viability........................................ 39 10. Inhibition ofNGF mediated metabolic responses by SIN-1...... .. 40 11. Effects of SIN-I on TrkA receptor autophosphorylation.. ..... .. .. 40 12. Effects of SIN-1 on MAPK activity.................................... 41 IV. DISCUSSION I. Neuroprotective actions of nicotine..................................... 42 2. Choline and Choline analogs............................................. 45 3. Relationship between the ability to produce neuroprotection and the upregulation of cx7nAChRs. ... .. .. .. .... 47 4. Relationship between increased TrkA receptors and neuroprotection.. 51 5. Importance ofNGF signaling............................................. 54 V. SUMMARY.......................................................................... 61 VI. REFERENCES....................................................................... 89 Abstract: Certain epidemiological studies have reported a negative correlation between smoking and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease, reflecting perhaps the neurotrophic actions of nicotine. In recent years there has been intense interest in the development° of new nicotinic acetylcholine receptor (nAChR) agonists. These agents have the potential to be used in the treatment of patients with AD. However, the mechanism for the neuroprotective action of the nicotine is not yet known, indeed, it is not yet clear as to which subtype of nAChR mediates the response. In neuronal cell lines, the induction of cytoprotection often requires exposure to nicotine for up to 24 hr to produce a full neuroprotective effect and this chronic exposure of nicotine is also known to increase nAChR receptors and cell surface nerve growth factor (NGF) receptors. The purpose of this study is to determine which subtype of nAChRs are involved in nicotine's neuroprotective actions and also to determine whether nicotine's neuroprotective actions are related to its ability to increase cell surface nAChRs and NGF receptors. Preincubation of differentiated PC 12 _cells with nicotine for 24 hr protected the cells from growth factor withdrawal-induced toxicity in a time and concentration-dependent manner. Nicotine's cytoprotective actions were completely blocked oy non-selective nAChR antagonist mecarnylarnine, and the a7nAChR preferring antagonist methyllcaconitine (MLA) indicating that the response was primarily mediated by the subtype of a7 nAChR receptors. The acetylcholine precursor, choline is a very selective 111 and full agonist at a.7 receptors. Among five choline analogs tested for neuroprotection potential, acetylpyrrolidinecholine and pyrrolidinecholine were found to be more potent and more efficacious than their parent co_mpound, choline. The rank order of the six compounds tested for their cytoprotective ability is as follows: acetylpyrrolidinecholine = pyrrolidinecholine · > choline = monoethylcholine = diethylcholine = triethylcholine. Further, to confirm the above structure activity relationships with respect to their binding affinities at u7nAChR, [1 25I]u-bungarotoxin (BGT) displacement binding studies were performed using differentiated PC12 cells. Choline was only 50 fold less potent than nicotine in displacing [125I]u-BGT binding. Pyrrolidinecholine, the most active analog, fully displaced [125I]u-BGT binding and it exhibited a slightly greater affinity for the site than did choline. Next we compared the ability of seven different nAChR agonists with varying activities at a.7 receptors for their ability to produce cytoprotection. Among the seven compounds tested, nicotine was the most effective and the most potent followed in order of potency by 4OH-GTS21, epibatidine, methylcarbamylcholine, 1, 1-Dimethyl-4-phenyl-piperazinum, cytisine and tetraethylarnmonium. Since, epibatidine and cytisine were less efficacious than nicotine despite their greater affinity for a.7 receptors and because short-term exposure of cells to nicotine did not produce cytoprotective actions, we next compared the ability of these compounds to upregulate cell surface a.7 receptors. After, incubation of cells for 2 hr with either nicotine or cytisine, the number of [1 25I]u-BGT binding sites on differentiated PC12 cells were measured. Nicotine, the most efficacious compound increased the [125I]u­ BGT binding sites by ~40% over the untreated control cells. In contrast, cytisine, the least effective compound failed to do so, indicating that the ability to upregulate a.7 receptors iv may provide one possible mechanism for neuroprotective actions of nicotine. Further, we confirmed that these additional populations of receptors were