INTERACTIONS BETWEEN EXClTOTOXlClTY AND LYSOSOMAL INHIBITION: IMPLICATIONS FOR ALZHEIMER'S DlSEASE PATHOGENESIS Michael Paul Murphy A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Psychology University of Toronto @Copyrightby Michael Paul Murphy, 1998 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibtographic Services services bibliographiques 395 Wellington Street 395, nre Wellington OttawaON K1AON4 Ottawa ON KIA ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence aüowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or selI reproduire, prêter, distribuer ou copies ofthis thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nim, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thése. thesis nor substantial extracts Gom it Ni la thèse ni des extraits substantiels may be printed or othenirise de celle-ci ne doivent être imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. INTERACTIONS BETWEEN EXCITOTOXICITY AND LYSOSOMAL INHIBITION: IMPLICATIONS FOR ALZHEIMER'S DlSEASE PATHOGENESIS. BY Michael Paul Murphy Doctor of Philosophy, 1998 Department of Psychology, University of Toronto ABSTRACT The peptide p-amyloid (AB) is believed to be the crucial elernent in Alzheimer's disease (AD) pathogenesis, being terminally responsible for neuronal death and ensuing dementia. Lysosomal inhibition alters the processing of the p-amyloid precursor protein (PAPP), from which AP is derived, such that potentially amyloidogenic fragments (fpAPP) accumulate intracellularly, largely within lysosomes. Although it has been hypothesized that the lysosornal dysfunction accompanying normal aging (and modeled with lysosomal inhibitors) may be connected to AD, the consequences of this manipulation on the CNS are not known, nor is the potential importance of intracellular Ap (iAP) understood. Hence, rnonkeys, rats and PAPP transgenic mice received direct brain infusions (i.c.v.) of the lysosornal inhibitors leupeptin or chloroquine (CHL) for up to 2 months. Both chernicals caused an increase in iAP, but did not cause significant neuronal death. Both also caused a strong astrocytic response, and CHL caused blood- brain barrier (BBB) dysfunction. These changes do not resemble major aspects of AD pathology. It has been shown that AD is associated with neurotrauma, such as head injury, and that excitotoxic processes cause similar degenerative changes. Also, AP renders cultured neurons vulnerable to excitotoxic damage. Therefore, if dysfunctional lysosomes and iAp represent a risk factor for AD, excitotoxins such as kainic acid (KA) may combine synergistically with lysosomal inhibitors. Hence, CHL (over a range of systemic doses and i.c.v.; 1 week, mice and rats) was combined wi-th KA foilowed by a 1 week survival period; multiple behavioral and histological analyses were conducted at several time points. Accumulation of iAB was evident at high doses. as was astrocytic activation. No deficits in sensorimotor ability (balance, muscle strength, vision, exploratory behavior) were detected. However, animais given KA and doses of CHL sufhkient to cause iAp accumulation were markedly impaired on the Moms watermaze test of spatial learning and exhibited increased lirnbic system damage, particularly in the amygdala. These results indicate that although iAP may not be irnmediately hanful. it may nevertheless be coupied to a pathogenic process relevant to the development of AD and should be further exarnined in the context of the development of appropriate treatment strategies. ACKNOWLEDGMENTS Next to the torture of constnicting a coherent introduction out of the roughly 6000+ AD related articles in the past 5 or 6 years. I could make a case for this being one of the toughest sections to Wte. Ifs difficult to find the tight words to do justice to the contributions that everyone has made to this work (ordeal?), and at times it seems Iike I'm selling them short. On the other hand, I also feel like I'rn giving an acceptance speech at the Academy Awards, and that I'm going to be dragged off the stage at any minute. So ....... be patient. it's a long one. My loving wife Sharon has to appear at the top of the list. For her, the words don't exist to describe how good she's been throughout this whole experience. I've been told that if your mamage can survive a thesis, there just 'ain't much more to worry about. You've stuck with me through the whole anluous journey - the long hours, the sacrifices. and my (sometimes very) short temper. Kitten, I love you al! the more for your patience, and look to the future with the cornfort that you'll always be there for me (and that we can spend more time together. after I do al1 those dishes). Aithough they may not know it, over the years my friends have provided indispensable support and encouragement. Thanks to Bruce, Doug. Faiz. Jared, Jason and Steve for getting me through those critical formative years and helping me to develop my creative edge. as well as fostering my skills of global domination. I owe a special debt to Steve for allowing me ample practice at those special abilities that al1 lrishmen need. as well as much of my Pythonesque sense of humor and affinity for lampposts. Well done. Brother Fat Man! Thanks are also in order for al1 of the regular Euchre and weekend crowd. for providing much needed distraction and ail round good times. Thanks to Brian and Crystal Leila for all those dusk-til-dam card sessions (in spite of Crystal's frightening dominance). and Marc and Leona Nicholas for some of the most mernorable pub crawl moments (the best of which, unfortunately. cannot be mentioned in print). You guys are great, and I would've went totally mental without you. I would never have made it this far if it weren't for the mutual love and support of my parents, Michael and Sylvia Murphy, and their patience with their son (the perpetual student) in letting me find my own path. My other set of parents, Jennie and Jirn Sword, have been terrific in lending that little bit of extra encouragement when needed. Over the years, you've al1 shown genuine interest and enthusiasm for my sometimes cryptic studies, and let me know that 1 wasn't working in a vacuum. My sister, Joanne McCluskey, has always ken there for me when I needed her. From the .... aggressive interactions of our youth (and all those times that I forœd her to endure Dr. Who), to all those long hours at the zoo, introducing me to bingo, and being one of rny closest fnends and confidants - love 'ya, Acha! In the same breath, I couldn't have asked for a better brother in law in Paul. For my etemal comrade in numerous video game extravaganzas (thanks to JO for permitting al1 those hours at the TV, with only occasional psychotic explosions - wocka-wocka-wocka), the many mernorable laughs (Mr. Snuffalopagos, ah, cornes to mind - better get a iife preserver!), and al1 the late night conversations, I have only these words of wisdom: Always gain groond, show no mercy, and remember - you ... are rny number one .... *guy?l I probably would never have made it as a grad student if it weren't for rny close friend and nearly constant cornpanion, Liz (now Dr.) Head. It still amazes me that after al1 these years we can still get together and talk (more like free association. as you say) for 12+ hours. Notice how the campus pub collapsed affer we stopped going - coincidence? I think not. Magic cards, cribbage, bingo, Washington, Miami, hurricanes (real and the bar), pumpkin carving, Washington (again, but better), and a legendary pub crawl through the streets of Toronto.. ....shall 1 go on? Hey! Take mre, MFB. While still on the lab front, thanks to al1 those who've been my cornrades and cornpanions over the years. To those that are long gone, Dr. Mona Sazgar, Mark Hlousek, Andrew Kohn, Janina Ferbinteanu and Chris Reid...... thanks for the memories? Thanks to Mike Michael for al1 the enlightened philosophical banter, and Dr. Moshe Khurgei for showing me the ropes and for some unforgettable Neuroscience Meeting moments (particularly in wind swept Miami). For John Rick, thanks for the many years of good conversation in lounge number 2. the months of gaming (no doubt delaying both our dissertations), and, uh, teaching me the compulsive side of science. And where would I be without my fellow gossip manger and sometimes roornmate. Candace Douglas? Insane, perhaps? Good luck - 1 already miss you. This work would not have been possible without the friendship and lengthy technical assistance of John Mielke (to the Milgram lab!) and Suzi Barsoum (friend-who- is-a-girl), whose loyalty to the project was never questioned. Thanks for patiently listening to al[ my manic ranting and giving me years of more fun than one penon should have in a lab O' rats (roll Conan soundtrack, please). Many others have contributed to vaflous components of these studies: Aliœ Koo, Monika Kaila, Jonathan Maritz, Karen Bengualid, Asheer Sharma, Rahim Hiji, Rishi Narine, Chnstina Beckett, Amin Ladak. Deva Thinichelvam, and Hance Clarke. Thanks also to latecorners Lara Chebaro and Luke Shannon for fiiling in the detaiis, the interesting textures, and the indispensable humor. Thanks to the Wild Strawberrïes. Bj6rk, Depeche Mode. New Order and Erasure for providing the atmosphere. Much thanks to Dr. Bill Milgram, who's been a good friend and colleague, and has yielded many items of gmd advice over an equal number of good beers.