TraP-MEDD — Translational Platform for MSA: Elucidation of Disease-mechanisms and Drug discovery.

BACKGROUND: Multiple system atrophy (MSA) is a rare neurodegenerative synucleinopathy, which predominantly shows alpha-synuclein (a-syn) aggregation in oligodendrocytes (OL). There is no effective treatment and etiopathogenesis is vastly unknown. There are two theories explaining the aberrant localization of a-syn in OL. One theory first postulates a pathogenic overexpression of a-syn in OL; however, gene expression studies in brain have failed to show a-syn overexpression. A second theory, considers there is an abnormal up-take of neuronal a-syn by OL, mainly supported by mouse models. These studies have demonstrated that a-syn, can redistribute from neuron to OL and spread by prion-like mechanisms; however, this has not been replicated in human OL. Using patient-derived induced pluripotent stem cells (iPSC), and their subsequent differentiation into specific cell types (such as OL) enables generating scalable quantities of disease-relevant tissue (iPSC-OL). OBJECTIVES: (i) To generate MSA-specific iPSC from MSA fibroblasts collected at FCRB, and subsequent differentiation into OL. (ii) To investigate alterations in fibroblasts and iPSC-OL from MSA subjects vs. controls at a transcriptomic, epigenomic, and proteomic level. (iii) To assess the uptake and seeding propensity of structurally distinct a-syn fibrillar strains (amplified from MSA brains) by MSA-derived iPSC-OL. (iv) To identify the interactome of a-syn in iPSC-OL. (v) To create an a-syn/OL molecular network model by computational analysis and predict therapeutic candidates (vi) To create a complimentary humanized mouse model parallel to the iPSC-OL model (vii) To test therapeutic candidates in both in vitro and in vivo models. METHODS: Fibroblasts (MSA=6) will be reprogrammed to iPSC using the non-integrative Sendai virus strategy. OL differentiation will be obtained following Douvaras and Fossatti 2015 Nature protocol. A fully functional ‘OMIC’ iPSC-OL characterization will be performed by RNA sequencing and mass spectrometry. Structurally distinct a-syn fibrillar strains will be amplified from MSA using PMCA. iPSC-OL cell lines will be exposed to a-syn fibrillar strains. The binding of the strains to the plasma membranes, uptake, seeding, and accumulation propensities will be assessed quantitatively using STORM imaging and biochemical assays. The plasma membrane and cytosolic interactomes will be determined by mass spectrometry after pull-down experiments. In coordination with Dr.Douglas Armstrong's lab we will create a molecular network model by clustering algorithms. Network target predictions will be validated in the iPSC-OL and the novel humanized rodent model for MSA. IMPACT: We expect our work to significantly impact MSA's field shedding light on disease mechanisms and creating a warranted cell and animal model for drug discovery. By assessing distinct a-syn fibrillar strains in oligodendrocytes, identifying pathogenic interactions within these cells, and the epigenomic/transcriptomic/proteomic changes that affect them, we will understand the pathological events leading to MSA to an unprecedented level. This should allow modulating disease progression through proteins that interact with pathogenic a-syn fibrillar strains, yielding novel therapeutic tools and strategies.