SUMMARY In our original grant, we proposed to investigate the likely common mechanisms by which the proSAAS chaperone protects neurons from neurotoxic aggregating proteins and peptides, such as alpha synuclein and Abeta. We hypothesized that secreted proSAAS sequesters cytotoxic oligomers and fibrils extracellularly, reducing their concentrations at the synapse, and that endocytosed proSAAS might act intracellularly to similarly sequester cytotoxic species. Using cultured primary hippocampal and nigral neurons, we are currently investigating whether secretory proSAAS is involved in intracellular and extracellular Abeta and alpha synuclein sequestration, and whether secretory proSAAS also accelerates the intracellular degradation of Abeta and alpha synuclein. However, three major findings were made recently in carrying out the above project which take the work in an unexpected but important new direction. The first is the discovery that delivery of proSAAS to the cytoplasm, by expression of signal-less proSAAS (“cyto-proSAAS”), results in the liquid-liquid phase separation and formation of large (2 - 4 µm) symmetric proSAAS spheres, formed by dynamic fusion of smaller spheres. The second major finding is that these cyto-proSAAS spheres specifically interact with TDP-43216-414 aggregates, and efficiently sequester these aggregates within the sphere cores. Thirdly, and most importantly, a collaboration with the Shorter laboratory provided important information that the interaction between proSAAS and TDP-43 is cytoprotective in a yeast model cell system. The proposed supplement to our existing “Common Mechanisms” grant is designed to determine whether cytoplasmic proSAAS should also be studied, not only in proteostatic mechanisms in Alzheimer's disease, but also in the context of TDP-43 aggregation in another neurodegenerative disease, fronto-temporal dementia. Obtaining a one-year supplement to investigate the functional properties of cyto-proSAAS will provide us with the opportunity to exploit our exciting findings regarding the highly unusual physical properties of cyto-proSAAS in forming “aggregate sequestration” spheres. This supplement will also permit us to determine whether cyto-proSAAS expression is relevant to blocking Abeta and TDP-43 cytotoxicity in human cells (indeed, cyto-proSAAS expression may represent an improved avenue to achieve our original specific aim of ameliorating Abeta cytotoxicity -original Proposal Aims 1 and 3). Ultimately, this research will provide insight into whether proSAAS-mediated cytoplasmic aggregate sequestration should be further explored as a possible therapeutic approach in neurodegenerative disease. Lastly, it should be mentioned that given the unpredicted costs of the Covid-19 research shutdown, we clearly require additional funding to work on cyto-proSAAS, as we will otherwise be solely dedicated to recovering lost time in completing our original Aims. 1