Project summary of the funded parent award Huntington disease (HD) is a slowly progressing genetic disorder caused by an expansion of glutamine repeats in the huntingtin protein (wtHTT), leading to mutant HTT (mHTT) that is widely expressed throughout the brain and peripheral tissues. Despite this ubiquitous expression, mHTT shows regional effects by promoting degeneration of medium spiny neurons (MSNs) in the striatum and loss of cortical mass. With aging, the effects spread to other brain areas (1-5). The molecular basis for the regional specificity that encompasses many mHTT processes is unclear; thus, etiology-based therapies for this devastating disease remain elusive. To fill this knowledge gap, we will test the hypothesis that Ras-homolog enriched in the striatum (Rhes) and small ubiquitin-like modifier (SUMO)-1 signaling circuitry orchestrate striatal vulnerability and HD progression. This hypothesis is based on our prior finding that Rhes promotes SUMO-1 modification of mHTT (SUMO1– mHTT) and enhances soluble forms SUMO1–mHTT, leading to toxicity in cell and transgenic animal models of HD (6-14). However, the downstream mechanisms of the Rhes–SUMO1–mHTT pathway in HD remain obscure. Serendipitously, we found that Rhes promotes the formation of actin-containing membrane protrusions known as tunneling nanotubes (TNTs) and that Rhes is transported through TNTs to distant cells (15). Rhes also transports mHTT, but not wtHTT, via the TNTs that form between cultured cells. This intercellular transport requires post-translational modifications (PTMs), such as the farnesylation of Rhes and SUMOylation of mHTT, revealing a new role for the Rhes–SUMO1 pathway in mHTT transmission (15). We now demonstrate that Rhes can move between MSNs and spread mHTT in vivo. We tested using cell-type specific reporter mice, Flex (“Cre- On”) and bicistronic viral vectors, and organotypic brain slices and found that Rhes moves from D1R-MSNs to D2R-MSNs and potentiates mHTT spread from the striatum to the cortex in the brain (16). These results indicate that Rhes is a major driver of mHTT transport, both in vitro and in vivo. We also found that SUMO1 deletion diminishes mHTT protein levels and prevents the HD-like phenotype by upregulating autophagic activity in animal (Q175DN) and cellular HD models (17). Taken together, these new results indicate that the Rhes- SUMO1 pathway alters mHTT levels and promotes mHTT spread in the brain. However, the mechanisms of mHTT spread remain unknown. Therefore, uncovering the mechanisms that enable Rhes to spread mHTT and the in vivo neuropathological role of spread remain essential areas to address. Our preliminary data suggest that SUMO1 regulates striatal mTORC1 signaling, a major regulator of autophagy, in Q175DN mice. Defining whether or how SUMO1 contributes to mHTT spread in vivo and its role autophagy dysregulation is therefore critical both for modeling the disease progression and for drug discovery. Our specific aims...