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MicroPubl Biol,
2020]
Huntingtons disease (HD) is an autosomal dominant monogenic neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the gene encoding the protein huntingtin (Htt) (MacDonald et al., 1993). The resultant disease-associated Htt protein harbors a polyglutamine (polyQ) repeat that renders it metastable with respect to folding (Carrell and Lomas, 1997). Htt protein misfolding, characterized by the accumulation of misfolded protein aggregates and neurotoxicity, is first observed in mid- to late-life for most HD patients (Becher et al., 1998). The age-of-onset for HD is inversely proportional to CAG repeat length (Becher et al., 1998). Nonetheless, genetic variation between HD patients is attributed to slight differences in age-of-onset, even when repeat length is the same (Gusella and MacDonald, 2000). Thus, genetic background seems to be an important modifier of Htt protein aggregation and toxicity. We are interested in identifying genes/proteins that enhance or suppress the folding defect of human Htt.To model Htt toxic-gain-of-function in the genetically tractable Caenorhabditis elegans, we previously characterized transgenic animals expressing a YFP-tagged polyQ-expanded disease-associated fragment of human Htt in C. elegans body wall muscle cells (Lee et al., 2017). More specifically, the first 513 amino acids of the human Htt protein were fused to YFP for visualization. Two different polyQ tract lengths (Q15 and Q128) were utilized, resulting in the proteins Htt513(Q15)::YFP and Htt513(Q128)::YFP, corresponding to the strains EAK102 and EAK103, respectively (Lee et al., 2017). For simplicity, these proteins are referred to herein as Htt513(Q15) or Htt513(Q128). As reported, only Htt513(Q128), not Htt513(Q15), formed protein aggregates in body wall muscle cells (Lee et al., 2017), consistent with only longer polyQ tracts being associated with disease.Here, we describe the identification and characterization of genetic modifiers of Htt aggregation (mha). To this end, EAK103 animals expressing Htt513(Q128) were grown to the L4 larval stage and exposed to the alkylating agent ethyl methanesulfonate (EMS) at a final concentration of 50mM for 4hrs, according to established protocols (Brenner, 1974). In short, F1 individuals derived from the mutagenized parents were allowed to self-fertilize for one generation, yielding an F2 population, for the purpose of homozygosing recessive alleles and thereby uncovering mutant phenotypes. Screening of the F2 animals for those with increased or decreased aggregation was performed by eye with a fluorescent stereomicroscope.