Speaker: Camille Mitchell (Boyden Lab)

Title: Principles, Development and Neurobiology Applications of In Situ Single-Molecule Protein Sequencing

Abstract: The ability to map protein identity, with resolution sufficient to infer interactions, would support analysis of how proteins work together, or malfunction, in biological processes and diseases. Although several emerging technologies aim towards single-molecule protein sequencing, they require proteins to be removed from the nanoscale spatial context of cells and tissues. Expansion microscopy (ExM) has facilitated a diversity of chemical analyses by isotropically separating molecules throughout a specimen after permeation via a charged hydrogel, followed by gel swelling. Here, we adapt key protein sequencing steps - Edman degradation and amino acid recognition - to the ExM gel context. Using testbed peptides in ExM gels, we show that N-terminal amino acids can be recognized over multiple cycles of in-gel Edman degradation. These results establish principles of in situ protein sequencing and provide a framework for future in situ protein sequencing developments, including the development of higher specificity and affinity amino acid binders. Future work, such as single amino acid recognition reagents, resolving peptides from proteins and early applications of the technology, will also be discussed.

Speaker: Hyeseung Lee (Heiman Lab)

Title: Innate Immune Contributions to Striatal Vulnerability in Huntington’s Disease

Abstract: Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by CAG repeat expansions in the huntingtin (HTT) gene. Although mutant huntingtin (mHTT) is known to disrupt diverse cellular processes, the mechanisms underlying the enhanced vulnerability of striatal neurons remain elusive. Our previous cell type-specific transcriptomic profiling studies using both Translating Ribosome Affinity Purification followed by sequencing (TRAP-seq) and single-nucleus RNA sequencing (snRNA-seq) across human HD and mouse models identified mitochondrial RNA (mtRNA) release and activation of innate immune signaling in the most vulnerable cell type of HD, the indirect pathway striatal projection neurons (iSPNs). These findings suggested that activation of the innate immune sensor PKR may contribute to neurotoxicity in HD. In this talk, I will present follow-up studies examining whether early inhibition of neuronal innate immune activation has therapeutic potential in HD models.