Developing and validating a high-throughput drug screen in a zebrafish model of amyotrophic lateral sclerosis Alexander McGown Department of Neuroscience (SITraN) Supervisors: Dr Tennore Ramesh and Prof Pamela Shaw PhD Thesis Submitted February 2014 1 Abstract Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder that rapidly leads to paralysis and death. Currently the treatment options for ALS are limited and there is a desperate need for new therapeutic agents to combat the disease. Zebrafish were highlighted as having the potential of being both an excellent model for ALS and as a drug screening system due to their rapid development, large numbers, optical clarity and susceptibility to genetic manipulation. This work describes the use of a zebrafish model of ALS that overexpresses the mutant G93R SOD1 gene alongside a hsp70-DsRed stress readout. This has been used to demonstrate that the hsp70- DsRed readout is activated by sod1 toxicity and that in the embryo stages it is characterised by expression in the glycine positive inhibitory interneurons. Further investigation of these neurons identified defective glycinergic transmissions from the stressed neurons. In the adults, the motor neurons of the spinal cord are primarily affected, and also show defective neuromuscular junctions (NMJ’s). This suggests that ALS is a disease which does not start at symptom onset but is a progressive disorder, where specific neuronal subtypes are initially dysregulated, followed by the dysregulation within other neuronal subtypes. Based upon this fluorescence readout, a high-throughput drug screen using the zebrafish was designed, optimised and validated which has the capability to screen 100’s of compounds with a sensitivity and specificity of over 90%. This assay has been utilized to screen a small molecule library of 2000 compounds in order to identify potential therapeutics for ALS. This screen highlighted novel therapeutics that can potentially ameliorate sod1 toxicity and some that upregulate the heat shock response, a known cellular repair pathway. Selamectin, a macrocyclic lactone with known neuroprotective properties, was identified as the most promising hit from the screen. By combining Selamectin and Riluzole, a reduction in neuronal stress was seen, which offered the potential for a therapy with reduced side effects e.g. sedation. In conclusion, this work has highlighted the potential of a novel therapeutic for the treatment of ALS that should be taken towards mouse trials. 2 Statement of contribution I state that all work in this thesis is my own. The only exception to this is the electrophysiology experiments outlined in chapter 3.4 which were performed in a collaboration with Jonathan R. McDearmid and his lab from the University of Leicester and has been published in (McGown et al 2013). 3 Acknowledgments This work is dedicated to my Grandad and Uncle whose words of wisdom, laughs and guidance have given me the drive to succeed I would like to thank Dr Tennore Ramesh and Professor Dame Pamela Shaw for all their guidance and support throughout the years, without their insight and knowledge this would not have been possible. Dr Niki Panagiotaki deserves a special mention for her help and patience with me and for making my time in the lab enjoyable and rewarding. I would also like to thank Danielle for putting up with my moaning, working weekends and endless science chatter! Finally a special mention for my parents who have been so supportive over the years, they are amazing people who have always gone the extra mile and further to help me. 4 Contents Abstract .......................................................................................................... 2 Statement of contribution ............................................................................... 3 Contents ........................................................................................................ 5 List of abbreviations ....................................................................................... 9 List of tables and figures ...............................................................................12 1. Introduction ...............................................................................................16 1.1 Sporadic ALS .......................................................................................17 1.2 Familial ALS ........................................................................................18 1.2.1 SOD1 .............................................................................................20 1.2.2 ALS2 ..............................................................................................21 1.2.3 TDP-43 ..........................................................................................22 1.2.4 FUS ...............................................................................................24 1.2.5 VCP ...............................................................................................25 1.2.6 OPTN ............................................................................................25 1.2.7 C9orf72 ..........................................................................................26 1.3 What causes ALS? ..............................................................................27 1.3.1 Oxidative stress .............................................................................28 1.3.2 Axonal transport defects ................................................................29 1.3.3 Mitochondrial defects .....................................................................30 1.3.4 Protein aggregation .......................................................................31 1.3.5 Excitotoxicity ..................................................................................32 1.3.6 Activation of non-neuronal cells .....................................................33 1.3.7 Dysregulated transcription and RNA processing ...........................34 1.3.8 Endoplasmic Reticulum (ER) Stress..............................................34 1.3.9 Neuro-inflammation .......................................................................35 1.3.10 Deregulated endosomal trafficking ..............................................36 5 1.4 Current treatments ...............................................................................36 1.5 Current drug screen studies ................................................................40 1.6 Models of ALS .....................................................................................41 1.7 Zebrafish model of Neurodegeneration ...............................................45 1.7.1 Zebrafish models of ALS ...............................................................46 1.8 Background to the project ....................................................................47 1.9 Heat shock proteins .............................................................................49 Methods ........................................................................................................54 2.1 Animals ................................................................................................54 2.2 Zebrafish crossing ...............................................................................55 2.3 Dechorinating method ..........................................................................55 2.4 Adult tissue collection, fixing and sectioning ........................................55 2.5 Immunostaining of muscle samples .....................................................56 2.6 Spinal cord staining .............................................................................57 2.7 Zebrafish drug screening – initial screen .............................................58 2.8 Genotyping embryos at 2dpf using the InCell microscopy system .......59 2.9 Genotyping embryos at 6dpf using the InCell microscopy system .......60 2.10 Printing of the spectrum library ..........................................................61 2.11 Loading of drugs into plates using the Echo550 liquid handling system ..................................................................................................................61 2.12 High-throughput drug screening readout ...........................................63 2.13 Pherastar ...........................................................................................63 2.14 Spectrum library .................................................................................63 2.15 Zebrafish behavioural analysis ..........................................................64 2.16 Statistics used in high throughput screening plate screening ............64 2.17 Quality control statistical analysis ......................................................66 2.18 Electrophysiology ...............................................................................67 6 Chapter 3: Cellular changes in a zebrafish model of ALS and the identification of drug effect in this model .......................................................68 3.1 Investigating the Hsp70-DsRed response in G93Ros10 zebrafish embryos .....................................................................................................69 3.2 Investigating the Hsp70-DsRed stress response in sod1 mutant zebrafish ....................................................................................................70 3.3 Inhibitory interneurons show the stress response prior to motor neurons in