The impact of polyQ-expansion and suppression of huntingtin-deficiency phenotypes in the model organism Dictyostelium - PROJECT SUMMARY/ABSTRACT
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.
