Modulating Proteostatic Stress in Human Motor Neurons to Identify Neuroprotective Targets
Watts, Michelle Elizabeth
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CitationWatts, Michelle Elizabeth. 2020. Modulating Proteostatic Stress in Human Motor Neurons to Identify Neuroprotective Targets. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractAmyotrophic lateral sclerosis (ALS) is a paralyzing and fatal motor neuron (MN) disease with few therapeutic options. The ability to produce MNs from ALS patients using induced pluripotent stem cell (iPSC) technologies and neuronal differentiation protocols has improved therapeutic identification by allowing, for the first time, mechanistic studies and drug screening within the correct human cellular context. In this thesis, we investigate how modulating protein homeostasis (proteostasis), a core process dysregulated in ALS, affects both healthy and ALS patient MNs. We demonstrate that proteostatic stressors targeting the endoplasmic reticulum (ER) are preferentially more toxic to MNs than non-MNs, in healthy and ALS patient cultures, without inducing insoluble proteins. In contrast, proteostatic stressors targeting the ubiquitin-proteasome machinery induce insoluble proteins associated with ALS, yet do not uniformly trigger cell-type-specific degeneration. These experiments demonstrate, for the first time, a fundamental vulnerability of iPSC-derived MNs to ER stress, with distinctly differential effects to other proteostatic stressors. These studies additionally define assay conditions necessary to induce ALS MN degenerative phenotypes in a synchronous and scalable manner amenable for drug screening.
We demonstrate that these assays can accurately predict and identify drugs with neuroprotective potential by showing that kenpaullone, a well-established MN protective agent, is able to preserve ALS patient MN viability in proteostatic stressor conditions. We further demonstrate, for the first time, that a small molecule inhibitor to one of kenpaullone’s targets, MAP4K4, is also able to maintain ALS patient MN viability in all conditions of proteostatic stress. As with kenpaullone, this preserved viability includes the maintenance of healthy neurites and is independent of a direct reduction in UPR induction or insoluble protein buildup. Finally, we utilize global phosphoproteomic remodeling analyses to discover a core proteome that is changed with kenpaullone and MAP4K4 inhibition and identify several new compounds that preserve MN viability. These studies cumulatively outline a network of vulnerability points and protective pathways in human MNs, highlighting the value of modulating proteostatic stress in patient MNs to gain insights into degenerative disease mechanisms and to identify new neuroprotective targets.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37369444
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