PROJECT SUMMARY Huntington’s disease is a progressive neurodegenerative disorder caused by a mutation in exon1 of the huntingtin protein (mHttex1), which causes the protein to fibrillize and aggregate. While the exact genetic basis of this disease has been known for decades, there is still no cure or effective clinical treatment. Therapeutic development is complicated by the fact that it is unclear how mHttex1 exerts its toxic effects. Structural studies have revealed mHttex1 monomer aggregates in a stepwise manner, misfolding into several intermediate structures before eventually forming the fibrillar aggregates characteristic of the disease. Previous studies have also shown that some of these intermediate conformers, such as protofibrils, are toxic. To better understand these structures, the Langen lab collaborated with Dr. Richard Roberts to make small peptide binders for protofibrils. Our first set of peptide binders, HD1 and HD8, have shown a high affinity for not only these toxic protofibrils, but also with mHttex1 oligomers, another early mHttex1 conformer posited to be toxic. Even more exciting, our binders appear to prevent a phenomenon known as “seeding,” where introducing a small amount of misfolded mHttex1 will accelerate fibrillization of mHttex1 monomer through template-assisted misfolding. In this proposal, I will leverage our novel peptide binders to learn how HD1 and HD8 prevent seeding of mHttex1 monomer, and what effect these peptide binders will have on mHttex1 aggregation and toxicity. In Aim 1, I will use a combination of biophysical methods such as electron paramagnetic resonance (EPR), solid-state nuclear magnetic resonance (ssNMR), and a new fluorescence microscopy application to monitor fibril growth in real time. These experiments will reveal both the seeding inhibition mechanism of HD1 and HD8 as well as structural information of these peptide binders, which will be important for developing future treatments. Additionally, preliminary evidence shows that co-expressing mHttex1 with HD1 or HD8 in cell culture decreases the amount of overall mHttex1. Using both cell culture and mouse models of Huntington’s disease, Aim 2 will explore how HD1 and HD8 interact with mHttex1 within the cell and whether these peptide binders can rescue the deleterious effects of mHttex1. These experiments will provide a picture of how exactly HD1 and HD8 exert their effects on mHttex1 and evaluate the therapeutic potential of these peptide binders.