!"!#$ CHEMICAL GENETIC INTERROGATION OF NEURAL STEM CELLS: PHENOTYPE AND FUNCTION OF NEUROTRANSMITTER PATHWAYS IN NORMAL AND BRAIN TUMOUR INITIATING NEURAL PRECUSOR CELLS by Phedias Diamandis A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy. Department of Molecular Genetics University of Toronto © Copyright by Phedias Diamandis 2010 Phenotype and Function of Neurotransmitter Pathways in Normal and Brain Tumor Initiating Neural Precursor Cells Phedias Diamandis Doctor of Philosophy Department of Molecular Genetics University of Toronto 2010 &'(!)&*!% The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer. ! ""% "!+&,&% % When you set out on your journey to Ithaca, pray that the road is long, full of adventure, full of knowledge. The Lestrygonians and the Cyclops, the angry Poseidon -- do not fear them: You will never find such as these on your path, if your thoughts remain lofty, if a fine emotion touches your spirit and your body. The Lestrygonians and the Cyclops, the fierce Poseidon you will never encounter, if you do not carry them within your soul, if your soul does not set them up before you. Pray that the road is long. That the summer mornings are many, when, with such pleasure, with such joy you will enter ports seen for the first time; stop at Phoenician markets, and purchase fine merchandise, mother-of-pearl and coral, amber and ebony, and sensual perfumes of all kinds, as many sensual perfumes as you can; visit many Egyptian cities, to learn and learn from scholars. Always keep Ithaca in your mind. To arrive there is your ultimate goal. But do not hurry the voyage at all. It is better to let it last for many years; and to anchor at the island when you are old, rich with all you have gained on the way, not expecting that Ithaca will offer you riches. Ithaca has given you the beautiful voyage. Without her you would have never set out on the road. She has nothing more to give you. And if you find her poor, Ithaca has not deceived you. Wise as you have become, with so much experience, you must already have understood what Ithacas mean. - Constantine P. Cavafy (1911) ! """% &*,-./#$01$2$-!( I thank God for guiding me down the long and laborious journey of graduate studies. For what started off as a simple pursuit of a diploma and future career, soon became a voyage full of adventure, knowledge and perhaps the most significant part of my personal development. I would like to thank my supervisors Dr. Mike Tyers and Dr. Peter Dirks for the years of quality co-supervision. I truly feel you have both shaped the way I think not only about science, but also the way I see the world around me. Thank you for giving me freedom, independence and guidance when I needed it. I am more creative, efficient and a better self-critic because of you two. I hope that in the spirit of mentoring, I too can one day pass these skills down to my own students and that our collaborations and friendship continue to flourish. To my committee members Dr. Peter Roy and Dr. James Ellis, thank you for your valuable time and thoughts. Although our time together was short, your ideas and constructive criticism have left a forever-lasting improvement to this document. To Ian, you have been a model citizen, taking the time to share your endless wealth of patience and knowledge with us graduates students. You’ve been like a father figure in the lab to so many of us and have always led by good example. Thank you for consistently being there for both the good and bad times. You are a “true” team player and role model. To Kevin and Jenny, I also thank you for your constant support in my science and personal life. I consider you both true friends. To Caroline, Erick and Ryan, I have enjoyed embarking on this long PhD journey with you. I hope your graduate training has also brought you more riches that you could ever imagine. And to the remainder of the Dirks Lab, thank you all for your contributions to this work and for your friendship. You have all made coming to work enjoyable the last few years. Lastly, thank you to my mother Anastasia, my father Eleftherios and my sister Maria. I may have encountered many marvels and wonderful people during this long and difficult voyage, but perhaps the most wonderful realization it has brought me is the magnitude of love you have in your hearts. You have all proven that people can unselfishly provide unlimited belief, support and love to help another succeed. You have ! "#% taken a once troubled adolescent and helped him already surpass the expectations of his given abilities. There are many problems in this world, but if every child had the love you have given me, the earth would be a much better place. I hope that the completion of this journey has given me the insight, skills and experience needed to one day find a more sufficient way to honor the sacrifices you have made for my life. Until then, I dedicate this thesis, the most significant accomplishment of my life so far, to you. Thank you. Phedias Diamandis (2010) ! #% #"(!%.3%&'')$4"&!".-(% (±)-PPHT (±)-2-(N-Phenethyl-N- EGR Early growth response propyl)amino-5-hydroxytetralin 5-HT 5-Hydroxytryptamine (serotonin) Emx2 empty spiracles homeobox 2 5-HTR1A 5-Hydroxytryptamine (serotonin) ENU N-ethyl-N-nitrosourea receptor, 1A AC Adenylate cyclase ER endoplasmic reticulum Ach Acetylcholine ES Embryonic stem ACSF Artificial cerebral spinal fluid FACS fluorescence activated cell sorting AML Acute myeloid leukemia FGF basic fibroblast growth factor (bFGF) AMP adenosine monophosphate FITC fluorescein isothiocyanate B2M beta-2-microglobulin GABA !-aminobutyric acid BDNF Brain-derived neurotrophic factor GABRB1 !-aminobutyric acid receptor, beta 1 BMPs Bone Morphogenetic Proteins GABRB2 !-aminobutyric acid receptor, beta 2 BSA Bovine serum albumin GAD67 Glutamate decarboxylase, 67-kD BTSC brain tumor stem cell GBM glioblastoma multiforme BTX "-bungarotoxin GFAP glial fibrillary acidic protein 2+ CaMK Ca /calmodulin-dependent protein Gi G protein, inhibitory kinases cAMP cyclic adenosine monophosphate GluR Glutamate receptor, metabotropic (mGlu) CD cluster of differentiation GPCR Guanine nucleotide binding protein (G protein), coupled receptor CHRM3 Cholinergic receptor, muscarinic 3 Gq/11 G protein, q polypeptide (M3) CHRNA7 Cholinergic receptor, neuronal GRIN2B glutamate receptor, ionotropic, N- ("7nAChR) nicotinic, "-polypeptide 7 Methyl-D-Aspartate, subunit 2B CNTF ciliary neurotrophic factor GRIA1 glutamate receptor, ionotropic, AMPA-1 CpG cytosine and guanine separated by a Gs G Protein, stimulatory phosphate CREB cAMP response element binding GSK-3 Glycogen synthase kinase 3 CSC cancer stem cell h Human CNS central nervous system HDACS Histone deacetylases DAG Diacylglycerol HEPES N-2-Hydroxyethelypiperazine-N’-2- ethanesulfonic acid DAPI 4’,6’-diamidino-2-phenylindole Hh Hedgehog hydrochloride Dlk6 Delta-like 1 homolog HRP horseradish peroxidase Dlx5 Distal-less homeobox 5 HSCs Hematopoietic stem cells DMEM Dulbecco’s Modified Essential HTS high throughput screening Medium DMSO Dimethyl sulfoxide IP3 inositol 1,4,5-trisphosphate dNTPs Deoxynucleotide triphosphates JAK Janus kinase 3 DPAT (dipropylamino)tetralin Ki-67 Antigen identified by monoclonal antibody Ki-67 DRD2 Dopamine receptor D2 LIF Leukemia inhibitory factor E Embryonic day Lin Lineage EC# effective concentration needed to LOPAC library of pharmacologically active decrease proliferation by #% compounds ECT electroconvulsive therapy m Mouse