Contrasting pathomechanisms of membrane versus cytosol alpha-synuclein excess

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Dysregulation of α-synuclein (αS) results in Parkinson’s Disease (PD). Dementia with Lewy bodies (DLB), PD Dementia (PDD), Multi-System Atrophy (MSA), and also Alzheimer’s disease are likewise characterized by αS imbalance. While most cases are sporadic, several familial αS missense mutations are linked to synucleinopathy and can inform us about pathologic mechanisms. Among them, E46K causes PD/DLB while G51D causes PD/MSA. αS transiently binds to vesicle membranes and, paradoxically, E46K increases αS membrane association whereas G51D decreases it. We seek to test the intriguing possibility that ‘early intervention’ strategies upstream of αS aggregation must take into account the exact nature of the underlying disequilibrium: does αS accumulate at membranes (E46K-like) or in the cytosol (G51D-like)? We will test the hypotheses that: (1) Membrane αS accumulation and cytosol αS accumulation are two distinct pathways to αS toxicity. (2) Pharmacological strategies to correct the two pathways differ. The Dettmer lab has pioneered the strategy of triggering cellular αS stress by familial PD/DLB mutant ‘amplification’ strategies. We took advantage of the αS repeat structure to create the E46K amplification ‘3K’ (E35K+E46K+E61K), which extends fPD-linked E46K to the two neighboring repeats. We have demonstrated that 3K indeed accentuates key aspects of E46K, including increased membranes binding, thereby causing pronounced cellular stress. Our new key innovation is the G51D amplification ‘3D’ (V40D+G51D+V65D), which – opposite to E46K – renders αS largely cytosolic, thereby also causing pronounced cellular stress. We will characterize 3D alongside 3K to compare membrane- (3K) vs. cytosol-associated (3D) αS stress. We consider a strategy of discovery in amplified systems followed by confirmation in patient iPSC neurons ideal for our 2 Aims: Aim 1: Contrasting effects of αS membrane vs. cytosol accumulation on cellular pathways 3K and 3D will bring out shared and mutant-specific stress signatures that are too subtle in the single mutants. a) Focus on 3K, wt, and 3D transduction of human iPSC neurons to perform TMT proteomics and RNAseq b) Confirm compelling changes by Western blot in hiPSC neurons of E46K vs. G51D plus isogenic control lines Aim 2: Finding compounds specific (or not) to lessening cytosolic vs. membrane αS-related stress We will build upon innovative neuronal reporter cell lines of 3K and 3D αS imbalance. a) Compare in detail candidate compounds b) Perform comparative medium-throughput small-molecule screens c) Confirm findings biochemically in hiPSC E46K and G51D neurons. This work pursues research priorities outlined in “Recommendations of the Alzheimer's disease-related dementias conference”. It especially focuses on priorities that address Alzheimer's disease-related dementias (ADRD), specifically dementia with Lewy bodies (DLB) and Parkinson’s disease (PD) dementia (PDD).