UC Irvine

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat mutation in the Huntingtin (HTT) gene. The mutation impacts neuronal protein homeostasis and cortical/striatal circuitry. SUMOylation, a post-translational modification with broad cellular effects, directly modifies the Huntingtin protein (HTT), along with other key neuronal and synaptic proteins. Furthermore, disruptions in protein homeostasis lead to the accumulation of mutant Huntingtin (mHTT) protein and SUMOylated proteins, accompanied by dysregulated synaptic-related proteins and pathways. We investigated proteome-wide changes in striatal protein SUMOylation and SUMO-associated proteins in the context of HD using the R6/2 transgenic mouse model. We performed SUMO protein-enrichment from HD and non-transgenic (NT) tissue, followed by mass spectrometry, to assess changes within the SUMO-associated protein network in HD. I identified both known and potentially novel SUMOylated and/or SUMO-associated proteins, including those necessary for presynaptic function and cytomatrix at the active zone scaffolding, cytoskeleton organization, and glutamatergic signaling. A network-based analysis identified altered pathways in HD striata to include clathrin-mediated endocytosis signaling, synaptogenesis signaling, synaptic long-term potentiation, and SNARE signaling. A novel protein target of SUMOylation, protein interacting with C kinase (PICK1) was validated by an in vitro SUMOylation assay. Furthermore, the metabotropic glutamate receptor 7 (mGluR7) – a Group III mGluR and modulator of glutamatergic signaling (a core neurotransmission pathway disrupted in HD) – was SUMO-enriched. To evaluate functional measures of neuronal activity in HD cells in vitro, and to evaluate how modulation of SUMOylation via reduction of the E3 SUMO ligase, protein inhibitor of activated STAT1 (PIAS1) may alter mGluR localization, we conducted a receptor internalization assay in primary neuronal cultures. We found decreased mGluR7 internalization in R6/2 primary striatal neurons compared to NT cells, and siRNA-mediated knockdown of PIAS1 prevented this HD phenotype. In addition, microelectrode array analysis on primary neuronal cultures indicated early timepoint hyperactivity in several measurements of neuronal activity within HD cortical neurons, while later timepoints demonstrated deficits. HD phenotypes were rescued at select timepoints following knockdown of PIAS1. Taken together, my findings provide a mouse brain SUMO-ome resource, and demonstrate one of the first assessments of SUMO proteomics in the context of neurodegeneration. We find that significant alterations occur within the post-translational landscape and SUMO-protein interactions for synaptic proteins in HD.