PROJECT SUMMARY Protein aggregates accumulate in several neurodegenerative diseases. For example, the proteins FUS and TDP-43 aggregate in frontotemporal dementia, while α-synuclein (αSyn) aggregates in synucleinopathies such as dementia with Lewy bodies and Parkinson’s disease. Protein disaggregases, such as the yeast AAA+ protein Hsp104, directly disassemble aggregated protein structures and soluble oligomers and therefore hold great therapeutic potential for protein misfolding diseases. We have previously potentiated Hsp104 to disaggregate αSyn, FUS, and TDP-43 in vitro and in yeast models. Our potentiated Hsp104 variants exhibit some toxicity when expressed in higher metazoan model systems, perhaps due to a lack of substrate specificity. Thus, I became interested in studying Hsp78 (the yeast mitochondrial homologue of Hsp104) because its divergent sequence might provide clues to mitigating Hsp104 toxicity and it has a putative human mitochondrial orthologue, Skd3. Little is known about the cell biology or biochemistry of Skd3. Three key pieces of data underlie my aims: (1) I targeted mitochondrial (mt) Hsp78 (mtHsp78) to the cytoplasm (cHsp78) and potentiated its activity via homologous mutations that nonselectively potentiate Hsp104. I found that three cHsp78 variants selectively rescue α-synuclein, FUS, or TDP-43 toxicity in yeast. (2) Surprisingly, I discovered that three different mtHsp78 variants rescue αSyn toxicity in yeast without affecting cytoplasmic αSyn aggregation. (3) I have purified Skd3 and shown that Skd3 has robust protein disaggregase activity, in vitro. Three unaddressed problems impede harnessing these protein disaggregases therapeutically. On the basis of my preliminary data, I hypothesize that: (1) the differences in rescue by the cHsp78 variants for αSyn, FUS, and TDP-43 is due to differences in substrate selectivity for disaggregation; (2) the mtHsp78 variants rescue αSyn toxicity by supplementing mitochondrial proteostasis thereby preventing mitochondrial dysfunction and promoting cell survival; (3) the human mitochondrial protein disaggregase Skd3 can be potentiated to increase activity and that potentiated Skd3 variants will rescue toxicity in metazoan model systems of synucleinopathies. Therefore, I will address three aims: (1) I will test the selective disaggregase activity of the cHsp78 variants in vitro using biochemical techniques innovated in our lab; (2) I will determine the mechanism of mtHsp78 rescue of αSyn toxicity using yeast genetics and cell biology; (3) I will characterize and potentiate Skd3 against αSyn toxicity in metazoan model systems using biochemical, cell biology, and genetic engineering techniques. These studies are an important step towards understanding the mitochondrial protein disaggregase machinery and their role as a potential therapeutic for synucleinopathies such as dementia with Lewy bodies and Parkinson’s disease.