Poly-glutamine (Q) expansion mutation in the protein huntingtin (HTT) causes Huntington?s disease (HD). How the mutation imparts an unknown effect on full-length HTT function has yet to be determined. Importantly, postmortem brain ammonia levels are elevated in HD cases, indicating aberrant protein catabolism and seems to occur prior to symptoms of HD. Although HTT expression occurs in all cell types, the metabolic mechanisms of protein catabolism and amino acid-homeostasis in response to nutrient-deprivation controlled by HTT are poorly understood and under-studied. This proposal focuses on defining the conserved cellular pathways regulated by HTT and the impact of polyQ-expansion on these cellular processes. Strategies that use novel model systems represents an innovative approach to understanding both normal and mutant HTT function. Dictyostelium genetics may also identify conserved genetic modifiers as therapeutic targets for the treatment of HD. Data suggests that HTT has an ancestral role in the regulation of protein recycling and clearance in eukaryotic cells, yet it is unclear how HTT controls these cellular processes. HTT deficiency in Dictyostelium results in strong organismal defects indicative of altered protein catabolism that confers hypersensitivity to chemicals that alter autophagic flux. Importantly, both expression of human HTT in Dictyostelium htt- cells or ammonia-detoxification treatments independently rescue htt- phenotypes that suggests the presence of genetic modifiers of conserved HTT-dependent catabolic processes. Undergraduate students will use an array of well- characterized phenotypic assays, the power of Dictyostelium genetics to test protein-clearance mechanisms and perform complementary, non-biased suppressor screens coupled with whole-genome sequencing to define key upstream and downstream effectors that regulate HTT-dependent catabolic pathways in the cell.
In Aim 1, the lab will perform chemical mutagenesis screens to saturation using N-methyl-N? -nitro-N-nitrosoguanidine (NTG) plus restriction enzyme-mediated random insertional mutagenesis (REMI) to mutate genes in htt- cells, and screen for suppressors of autophagic defects and developmental sensitivity to NH3+ and chloroquine.
In Aim 2, students will test the hypothesis that HTT regulates autophagy and/or the ubiquitin proteasome system.
This aim will quantify autophagic and proteasomal machinery in both htt- cells, suppressor mutants and cells expressing normal or mutant human HTT using established molecular, biochemical and microscopic methods. Targeting specific human homologs identified in the screen could be a viable way to suppress aberrant catabolic phenotypes in mutant HTT cells. The sustained impact from this approach, implemented by ethnically and economically diverse populations of undergraduate students will help circumvent a significant barrier to understanding HTT normal function. Moreover, orthologs of identified suppressor genes can be studied in relevant mammalian models and importantly, students at UMass Lowell will contribute to a deeper understanding of the normal role of HTT and the impact of polyQ expansion on regulating protein catabolism within the cell.
Huntingtin (HTT) is a pleiotropic protein found in all mammalian cell types yet when mutated impairs cellular mechanisms of protein catabolism. The normal function of HTT remains unclear which impedes progress in understanding the role it has in regulating protein catabolism and ammonia homeostasis in all cells. Using the power of Dictyostelium genetics, we will identify second-site suppressors that reverse catabolic defects and define the impact of polyQ mutation on HTT normal function.