Elucidating the Impact of Mutant Huntingtin Aggregates on Neuronal Dysfunction in Drosophila Melanogaster

Abstract

Huntingtin (HTT) is a highly conserved and ubiquitously expressed, multi-functional protein. Mutations within exon 1 of 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). Aggregates are well established as a key hallmark which are linked to the pathogenesis of HD. A small, truncated mutant fragment of HTT (mHTTex1), has been identified as a component of neuronal aggregates in HD patient brains, and its presence recapitulates HD-like symptoms in model systems. However, the molecular mechanisms dictating mHTTex1 aggregation and toxicity remain elusive.

To this end, a novel Drosophila Melanogaster (D. melanogaster) model was developed to investigate mHTTex1 aggregates. Transgenic fly strains were generated that pan-neuronally co-express HTTex1 mNeongreen (HTTex1-mNG) and HTTex1 mScarlet-I (HTTex1-mSc-I) fusion proteins with 17, 52, or 75 glutamine repeats (HTTex1Q17-mNG/-mSc-I, HTTex1Q52- mNG/-mSc-I, and HTTex1Q75-mNG/-mSc-I) which enabled detection of mHTTex1 aggregation in situ by measuring Forster Resonance Energy Transfer (FRET). Through the neuronal expression of this FRET-based HTTex1 aggregation biosensor, the localisation of HTTex1 within the fly brain was tracked. Co-expression of pathogenic mHTTex1 fusion proteins (HTTex1Q52-mNG/-mSc-I and HTTex1Q75-mNG/-mSc-I) resulted in the formation of SDS stable mHTTex1 aggregates , which correlated with reduced lifespan and mobility in flies. A novel FRET-based fluorescence-activated cell sorting method (FACS) was established which enabled quantitative readout of mHTTex1 aggregates within live cells derived from fly brains.

To interrogate the impact of mHTTex1 aggregates on the proteome, label free quantitative proteomics (LC-MS) analysis of both immunoprecipitated (IP) mHTTex1 aggregates from fly head lysates and whole fly brain samples was performed. Immunoprecipitates and whole brain lysates from flies expressing mHTTex1 with both pathogenic and non-pathogenic polyQ tracts were compared. The results obtained revealed that mHTTex1 aggregates significantly associate with proteins involved in intercellular transport and lead to a global increase of proteins associated with the endomembrane system.

The findings of this work establish mHTTex1 aggregates as profound disrupters of the neuronal proteome. RNAi knockdown of the key upregulated protein sff resulted in significant toxicity, highlighting a potential protective role increased sff protein levels may play against HTTex1-induced toxicity.