A15 Investigating the Mutli-Omic Impact of Huntingtin Aggregates on the D. Elanogaster Brain

Huntingtin is a highly conserved and ubiquitously expressed protein of unclear function. Mutations within the Huntingtin gene (HTT) are translated into a pathogenic expansion (>36 glutamines) of the polyglutamine (polyQ) tract, giving rise to an aggressive neurodegenerative disease: Huntington’s disease (HD). Expression of the mutant Huntingtin exon 1 protein (mHTTex1) in Drosophila melanogaster (D. melanogaster) results in reduced lifespan, hindered mobility and formation of intracellular aggregates. Currently, the molecular mechanisms by which mHTTex1 aggregates cause neuronal dysfunction and toxicity remain elusive. To this end, we have generated transgenic D. melanogaster strains co-expressing mHTTex1-mNeongreen (mHTTex1-mNG) and mHTTex1-mScarlet-I (mHTTex1-mSc-I) fusion proteins, which allow us to detect aggregation in situ by measuring Forster Resonance Energy Transfer (FRET). Through neuronal expression of this FRET-based mHTTex1 aggregation sensor, we can track the localisation of mHTTex1 aggregates within the D. melanogaster brain. We have validated that the abundance of mHTTex1 aggregates was increased in flies expressing mHTTex1 with pathogenic polyQ tract (HTTex1Q75-mNG/-mSc-I) compared to a non-pathogenic HTTex1 fragment (HTTex1Q17-mNG-mSc-I). Furthermore, the formation of fluorescently tagged mHTTex1 aggregates in brains was associated with decreased survival. Finally, we have optimised a FRET-based fluorescence-activated cell sorting method (FACS) to isolate single neurons containing mHTTex1 aggregates from whole D. melanogaster brains. Through this in vivo mHTTex1 aggregation model, we hope to reveal the direct transcriptomic and proteomic impact of mHTTex1 aggregate species on D. melanogaster neurons. Furthermore, by examining the transcriptomic profiles at a single cell resolution, we hope to begin dissecting the cell-specific molecular mechanisms of toxicity in HD.