Characterization of novel pharmacological Caspase-6 inhibitors provides insight on developing treatments against Alzheimer disease by Prateep Pakavathkumar Department of the Integrated Program in Neuroscience, McGill University, Montreal June 2017 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Prateep Pakavathkumar, 2017 "If I have seen further than others, it is by standing upon the shoulders of Giants” - Sir Isaac Newton Table of contents Table of contents ..................................................................................................... i Abstract ................................................................................................................ vii Résumé ............................................................................................................... viii Preface .................................................................................................................... x First-authored manuscripts ................................................................................................. x Author contributions ........................................................................................................... xi Collaborative manuscripts ................................................................................................. xii Acknowledgements ............................................................................................ xiii Abbreviations list ................................................................................................ xiv List of Tables ....................................................................................................... xix List of Figures ...................................................................................................... xx Introduction ............................................................................................................ 1 1. Literature Review ............................................................................................... 4 1.1 Apoptosis .......................................................................................................................... 4 1.1.1 The discovery of apoptosis ........................................................................................ 4 1.2 Discovering caspases and link to cell death proteases ................................................. 4 1.2.1 The evolution of caspases .......................................................................................... 4 1.2.2 The first mammalian caspase ..................................................................................... 5 1.2.3 Apoptosis in Caenorhabditis elegans ......................................................................... 5 1.2.4 The mammalian caspases ........................................................................................... 6 1.3 Caspases are classified based on sequence similarities, substrate preferences, or function ................................................................................................................................ 13 1.3.1 Phylogenetic classification of human caspases ........................................................ 14 1.3.2 Substrate preference-based classification of human caspases ................................. 14 1.3.3 Functional classification of human caspases ........................................................... 15 1.4 Molecular platforms for apoptotic and inflammatory pathways ............................. 16 1.4.1 The death-inducing signaling complex and the extrinsic pathway .......................... 17 1.4.2 The apoptosome and the intrinsic apoptotic pathway .............................................. 19 1.4.3 The inflammasomes and pyroptosis......................................................................... 20 1.4.4 The PIDDosome and mitotic catastrophe ................................................................ 21 1.4.5 The Ripoptosome at the crossroads between apoptosis and necroptosis ................. 22 1.5 Caspase functions in apoptosis and beyond................................................................ 22 1.5.1 Caspase functions in systematic dismantling of dying cells .................................... 23 1.5.2 Non-apoptotic caspase functions in immune cells ................................................... 24 1.5.3 Non-apoptotic caspase functions in the nervous system.......................................... 25 1.5.4 Other non-apoptotic functions ................................................................................. 27 1.6 Caspase-6 transcriptional control, alternative splicing, and tissue expression ....... 29 1.6.1 Little is known about CASP6 gene transcriptional regulation ................................. 29 1.6.2 The alternatively spliced Caspase-6 isoform ........................................................... 31 1.6.3 Caspase-6 is expressed at low levels in the healthy brain ....................................... 32 1.7 Caspase-6 enzyme structural analysis ......................................................................... 33 1.7.1 The 3D structure of Caspase-6 and its active site .................................................... 33 1.7.2 Unprocessed inactive Caspase-6 .............................................................................. 34 1.7.3 Caspase-6 processing by inter- and intramolecular cleavage .................................. 34 1.7.4 Changes in the Caspase-6 active site upon substrate binding and pH ..................... 35 1.8 Caspase-6 post-translation modifications (PTMs) ..................................................... 36 1.8.1 Caspase-6 phosphorylation at serine 257 ................................................................. 37 1.8.2 Caspase-6 palmitoylation at cysteine residues ......................................................... 37 1.9 Caspase-6 activation follows canonical activation cascades and can self-activate .. 38 1.9.1 Caspase-6 is activated both downstream and upstream of other caspases ............... 39 1.9.2 Caspase-6 self-activates in mammalian cells ........................................................... 40 1.10 Caspase-6 substrates and protein interaction analysis ............................................ 40 1.10.1 Caspase-6 cleaves many cytoskeletal proteins in human neurons ......................... 40 1.10.2 Caspase-6 signalling pathways in human T lymphocytes ..................................... 41 1.10.3 Caspase-6 protein-protein interaction network ...................................................... 42 ii 1.11 Caspase-6 mediates axonal degeneration.................................................................. 43 1.11.1 Caspase-6 is activated in peripheral neurons leading to axonal degeneration ....... 44 1.11.2 Caspase-6-mediated axonal degeneration in the CNS ........................................... 45 1.12 Caspase-6 is activated early in Alzheimer disease ................................................... 47 1.12.1 Caspase-6 is activated in sporadic and familial AD brains.................................... 48 1.12.2 Caspase-6 is activated early during AD pathogenesis ........................................... 48 1.12.3 Caspase-6 activity can increase A production, disrupt the cytoskeleton, and impair the ubiquitin-proteasome system ...................................................................................... 49 1.12.4 Caspase-6 is part of a pro-inflammatory cycle in AD brains ................................. 50 1.13 Caspase-6 activation in other neurodegenerative diseases...................................... 51 1.13.1 Caspase-6 cleaves mutant huntingtin protein in mouse models ............................ 51 1.13.2 Caspase-6 cleaves DJ-1 in PD ............................................................................... 52 1.13.3 Caspase-6 is activated following ischemic stroke ................................................. 53 1.14 Caspase-6 inhibitors: proteins, peptides, and small molecules ............................... 55 1.14.1 Endogenous protein Caspase-6 inhibitors .............................................................. 56 1.14.2 Zinc-mediated Caspase-6 inhibition ...................................................................... 58 1.14.3 Pharmacological Caspase-6 inhibitors ................................................................... 58 1.15 Hypothesis and Thesis objectives .............................................................................. 61 1.16 Figures and legends..................................................................................................... 62 1.17 Tables ........................................................................................................................... 72 2. Methylene blue inhibits caspases by oxidation of the catalytic cysteine ......... 77 2.1 Preface ............................................................................................................................ 77 2.2 Abstract .......................................................................................................................... 77 2.3 Introduction ................................................................................................................... 78 2.4 Results ............................................................................................................................ 80