Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig’s disease, is characterized by progressive and selective loss of the upper motor neurons in the motor cortex and lower motor neurons in the brainstem and spinal cord. ALS is one of the most common motor neuron disorders worldwide, and death occurs on average within three to five years of the disease onset; unfortunately, there is no effective cure available. Over 1000 mutations in about 40 genes have been associated with ALS pathology, including superoxide dismutase 1 (SOD1), transactive response (TAR)-DNA binding protein (TARDBP), fused in sarcoma (FUS), and chromosome 9 open reading frame 72 (C9orf72). FUS mutations induce an early onset (juvenile) form of ALS and are associated with rapid disease progression. A molecular mechanism of pathogenesis common to many forms of ALS is the disruption of the synthesis, folding, trafficking, and degradation of proteins (proteostasis). Therefore, enhancing protein quality control, for example, via autophagy, might be able to protect motor neurons against ALS pathogenesis. One strategy for enhancing protein homeostasis and protecting neurons is the induction of autophagy. BECN1 is a master regulator of autophagy, but it is repressed by direct interaction with BCL2 via a BH3 domain. In this work, we identify a small molecule BH3 mimetic that disrupts the BECN1-BCL2 interaction using an induced pluripotent stem cell model of ALS with mutant P525L FUS. We identified obatoclax, a brain-penetrant drug candidate that rescued neurons at nanomolar concentrations by reducing cytoplasmic FUS levels, restoring protein homeostasis, and reducing degeneration. Proteomics data suggest that obatoclax protects neurons via multiple mechanisms. Thus, obatoclax is a candidate for repurposing as a possible ALS therapeutic and, potentially, for other disorders linked to defects in protein homeostasis.:TABLE OF CONTENTS 1 Introduction 11 1.1 Amyotrophic lateral sclerosis 12 1.1.1 Clinical manifestations 12 1.1.2 ALS therapeutics 12 1.2 Introduction to ALS genetics 14 1.2.1 SOD1 14 1.2.2 C9orf72 17 1.2.3 TARDBP 19 1.3 FUS 21 1.3.1 Introduction to FUS 21 1.3.2 FUS domains 21 1.3.3 FUS protein and its physiological function 23 1.3.4 ALS-associated FUS mutations 26 1.3.5 Pathomechanisms of FUS-ALS 29 1.4 ALS models 31 1.4.1 In vivo ALS models using mice 31 1.4.2 In vitro models using human iPSCs 33 1.5 Protein homeostasis and ALS 36 1.5.1 Ubiquitin-proteasome system 37 1.6 Autophagy 39 1.6.1 Molecular mechanisms regulating autophagy 39 1.6.2 Defects in autophagy in ALS 41 1.6.3 Autophagy as a possible therapeutic target against ALS-RBP pathogenesis 42 1.6.4 BECN1/BCL2 complex as a possible therapeutic target 43 1.7 Project rationale and aims 48 2 Material 49 2.1 Chemicals 49 2.1.1 Table list of chemicals 49 2.2 Purchased kits 50 2.2.1 Table list of kits 50 2.3 Antibodies 50 2.3.1 Table list of primary antibodies 50 2.3.2 Table list of secondary antibodies for immunostaining 51 2.3.3 Table list of secondary antibodies for western blotting 51 2.4 Cell culture 52 2.4.1 Table list of cell culture media and reagents 52 2.4.2 Table list of small molecules 53 2.4.3 Table list of cell culture media in the small molecules-based neuronal differentiation protocol 54 2.5 BH3 mimetics 56 2.5.1 Table list of BH3 mimetics compounds 56 2.6 Proteomics 57 2.6.1 Table list SDS gel electrophoresis 57 2.6.2 Table list gel digestion 58 2.6.3 Table list instrumentation: Q-EXACTIVE HF - DIA 58 2.6.4 Table list instrumentation: THERMO DIONEX3000 RSLC 59 3 Methods 60 3.1 Licenses 60 3.2 Cell culture 60 3.2.1 Induced pluripotent stem cells (iPSCs) 60 3.2.2 Small molecules-based neuronal differentiation 60 3.3 BH3 mimetics screening and analysis 61 3.3.1 BH3 reconstitution 61 3.3.2 Cell viability 61 3.3.3 Stress granules amelioration 61 3.4 Obatoclax evaluation 62 3.4.1 Autophagic activity and efflux 62 3.4.2 Immunostaining 62 3.4.3 Proximity ligation assay 62 3.4.4 Protein isolation and quantification 62 3.4.5 Capillary electrophoresis 63 3.4.6 SDS-PAGE and Western blot 63 3.4.7 Proteomics 63 3.5 Senolytic activity evaluation 64 3.6 Statistical analysis 64 4 Results 65 4.1 Selection of BH3 mimetics for testing 65 4.2 Identifying BH3 mimetics that are non-toxic to iPSC-derived neurons 65 4.3 ABT-737, obatoclax and gambogic acid reduce aberrant P525L FUS-eGFP stress granules 66 4.4 Obatoclax reduces cytoplasmic P525L FUS-eGFP levels 68 4.5 Obatoclax promotes protein homeostasis in iPSC-derived neurons 69 4.6 Obatoclax ameliorates the degeneration of P525L-FUS iPSC-derived neurons 70 4.7 Obatoclax induces autophagy in iPSC-derived neurons 71 4.8 Long-term effects of obatoclax on LC3 are not due to compound instability 75 4.9 Obatoclax disrupts BECN1-BCL2 complex 77 4.10 Obatoclax contributes to neuroprotection via multiple mechanisms suggested by proteomics analysis 79 4.11 Obatoclax has senolytic activity in vitro 84 5 Discussion 88 5.1 The potential of autophagy as a therapeutic target in ALS and its present limitation 88 5.2 Identification of bh3 mimetics protecting mutant fus neurons against aberrant stress granules 88 5.2.1 ABT-737 89 5.2.2 Gambogic acid 89 5.3 Obatoclax is a candidate for drug repurposing for FUS-ALS 90 5.3.1 Obatoclax, a drug that induces autophagy 90 5.3.2 Obatoclax reduces neuronal cell death 91 5.3.3 Obatoclax as a candidate for clinical trials 91 5.4 Molecular mechanisms by which obatoclax protects neurons 92 5.4.1 Proteasome is affected in P525L-FUS but not in WT-FUS iPSC-derived neurons 92 5.4.2 Obatoclax may rescue protein homeostasis 92 5.4.3 Obatoclax may increase gangliosides biosynthesis 93 5.4.4 Obatoclax may rescue endosomal trafficking and the Golgi network 93 5.4.5 Obatoclax may reduce DNA damage 94 5.4.6 Obatoclax may increase chaperone activity 94 5.5 Beyond ALS: obatoclax as a possible anti-aging drug 95 5.6 Outlook and future experiments 95 5.6.1 Enhancing the neuroprotective effect of obatoclax 95 5.6.2 Obatoclax has an effect in mixed culture of neurons 95 5.6.3 Obatoclax interaction with other BCL2 proteins 96 5.6.4 Obatoclax could have an effect on other subtypes of ALS and other neurogenerative diseases. 96 5.6.5 Obatoclax could have potential as anti-aging drug 97 6 Bibliography 98 7 Appendix 121 8 Acknowledgments 126