Huntington?s disease (HD) is a neurodegenerative disorder caused by an expansion of a CAG repeat tract within the huntingtin (HTT) gene, leading to neuronal death primarily in the striatum and the cortex. The CAG repeat is highly unstable, both intergenerationally and in somatic cells. HD patients display a high degree of age- dependent somatic expansion of the CAG repeat in the striatum, although such expansions also occur in other tissues. Recent genome-wide association studies of HD patients have revealed the existence of genetic modifiers of the age of onset of the disease; these include several genes involved in DNA repair, and in particular DNA mismatch repair (MSH3, MLH1, PMS2, PMS1). In addition, studies in mouse models of HD have revealed that genetic knockout of the DNA mismatch repair genes, Msh2, Msh3, Mlh1, or Mlh3 reduces somatic instability of CAG repeats in the striatum. These data suggest that somatic expansion of CAG repeats is likely linked to striatal neuronal loss, and onset of disease symptoms in HD patients. Therefore, in Aim 1, we propose to characterize and compare protein assemblies in mouse striatum and other mouse brain regions that recognize and process CAG extrusions. We anticipate that these protein complexes may shed light on the mechanism of CAG expansions, while also potentially uncovering novel pathological processes unrelated to repeat instability.
In Aim 2, we will develop proximity biotinylation (TurboID)-based assay in human HEK293 cells to evaluate transient and stable protein assemblies on DNA containing CAG extrusions, with a view to eventually applying this approach to cells of neuronal origin. The effects of knockdown of critical mismatch repair genes like MSH3 or FAN1 on the processing of such DNA lesions will also be evaluated. The objective of these approaches is to identify proteins that determine whether CAG extrusions are processed by FAN1 or the mismatch repair pathway.
Huntington's disease, a slowly progressing neurodegenerative disease caused by a CAG repeat tract expansion within the huntingtin gene, belongs to a large group of neurodegenerative diseases characterized by protein misfolding and accumulation. The studies proposed here will provide mechanistic insights into somatic CAG expansions, and could yield novel protein targets for therapeutic intervention.