Investigating the Crosstalk between Small Extracellular vesicles and RNA granules in Huntington’s Disease

Huntington’s disease (HD) is a neurodegenerative disorder marked by progressive neuronal degeneration, with no current cure. Recent research suggests that RNA granules (such as stress granules and p-bodies) and small extracellular vesicles (sEVs) play critical roles in cellular dysfunction in HD. Both compartments share features like liquid-liquid phase separation (LLPS) and RNA-binding proteins, but the relationship between mutant huntingtin (mHTT) and their shared content remains unexplored. In this study, we analyze the transcriptomic and proteomic profiles of sEVs and RNA granules in a model expressing mHTT to understand their molecular interactions in HD. Our results show significant changes in gene expression in both sEVs and RNA granules, with a notable decrease in sEVs. Long non-coding RNAs (lncRNAs) were abundant in both compartments, and their expression shifted in HD, suggesting their involvement in disease progression. Additionally, 139 genes in our marker list are regulated by the Repressor Element 1 Silencing Transcription Factor (REST), which is disrupted in HD. Three marker genes (SNHG7, LHR1 LNC1610-1, and lnc-DUXA-1) were validated in RNA granules using RNA-FISH, showing partial co-localization with YB1-positive stress granules. qRT-PCR confirmed increased expression of all five marker genes (lnc-SLC30A5-6, SNHG7, SNHG12, LHR1-LNC1610-1, and lnc-DUXA-1) in HD RNA granules, with three markers showing increased expression in sEVs, though two exhibited high variability. To validate the relevance of our findings, we compared the expression patterns of HD sEV marker genes with a recently published RNA sequencing dataset of plasma EVs from HD patients. Our results show a stronger correlation between the pre-HD group in the patient dataset and our cell model, suggesting that our model better reflects the early stages of HD progression. We also identified distinct protein profiles in HD sEVs and RNA granules, with 13 shared proteins, highlighting a unique molecular signature for HD. STRING and KEGG pathway analyses revealed enriched pathways related to neurodegenerative diseases, suggesting broader impacts on neurodegenerative processes. Overlapping GO terms between RNA granules and sEVs point to functional interactions, particularly in RNA transport and metabolism. Notably, WDR1, a protein associated with mHTT-RNA complexes, was identified in both HD sEVs and RNA granules, suggesting its role in HD pathogenesis by influencing RNA granule formation and facilitating intercellular communication via sEVs. Our findings demonstrate that mHTT alters the composition of sEVs and RNA granules in HD. The detection of miRNAs, zinc finger proteins (ZNFs), and lncRNAs in sEVs suggests that HD cells may attempt to manage stress and intercellular signaling. The identification of overlapping proteins like WDR1, RANBP6, and ITGAV offers potential biomarkers and therapeutic targets. This study enhances our understanding of HD pathology by revealing the differential sorting of RNA and proteins in HD, with implications for early diagnosis and targeted therapies.