PROJECT SUMMARY Poly ADP-ribose (PAR) is an RNA-like protein modification whose dysregulation is linked to age- dependent neurodegenerative diseases including ALS, Parkinson’s and Alzheimer’s disease. PAR is a core component of stress granules, a type of membraneless ribonucleoprotein (RNP) granules that can seed aberrant protein aggregation leading to neuropathology. Unlike RNA, PAR can form into both linear and branched structures. Strikingly, inhibitors of PAR polymerase, which is a class of FDA-approved anticancer drugs, were shown to mitigate neurotoxicity in cell models of neurodegeneration, reflecting a potential role of PAR in neurotoxicity. Two laboratories at Johns Hopkins University led by Sua Myong (Biophysics department) and Anthony K. L. Leung (Department of Biochemistry and Molecular Biology ) bring together orthogonal expertise in molecular imaging, chemical and proteomic methods to investigate the molecular basis of PAR- driven protein condensation and aggregation mechanism responsible for neurodegenerative diseases, which may pave new ways of developing therapy. Our recent discovery of a PAR modifying method (Leung et al, Mol Cell, 2019) and mechanism of FUS liquid-liquid phase separation in ALS/FTLD-linked cases (Myong et al, Mol Cell, 2019) places us in an ideal position for tackling the poorly understood role of PAR in biomolecular condensation implicated in neurodegenerative diseases. In Preliminary Studies, we discovered that (i) PAR is extremely potent in condensing FUS (fused in sarcoma), an RNA binding protein localized in stress granules and implicated in ALS/FTLD and (ii) PAR targeted proteome is enriched in stress granule components. Building on these exciting results, we propose to uncover the role of PAR in driving biomolecular condensation by employing single molecule, biochemical, meso-scale, biophysical and cellular platforms.