Pulse-shape FACS identifies novel hits upregulated by Huntington’s disease induced ER stress

Huntington’s disease (HD) is a neurodegenerative condition caused by mutant huntingtin (mHtt), containing CAG repeats in the gene, coding for an expanded polyglutamine stretch, which leads to the synthesis of a protein that is unable to fold appropriately. The accumulation and aggregation of this misfolded protein leads, among other cytotoxic effects, to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). In HD, the UPR pathways fail to properly reduce the stress created in the ER, leading neuronal cells expressing mHtt to apoptosis, triggering neurodegeneration, motor and cognitive decline and dyskinesia. Using a cutting-edge technique, Pulse-shape FACS, we separated mHtt (Htt96Q) – expressing HEK293 cells into populations with or without large mHtt aggregates and showing high or low ER stress levels. These cell populations were subjected to RNA seq. The top hits in cells showing ER stress before the appearance of large mHtt aggregates, include known UPR-induced genes, such as Herp and BiP. Among these top hits was mesencephalic astrocyte-derived neurotrophic factor (MANF), a protein known to have pro-survival effects on models of Parkinson’s and other diseases. We are investigating the effects of MANF on mHtt expressing cells, with cause to believe that intervention into cellular pathways involving MANF could lead to a novel therapeutic approach for HD.