Project Overview
The Huntington disease (HD) mutation produces a pathogenic mutant HTT (mHTT) protein with an elongated polyglutamine tract. Presence of mHTT disrupts diverse cellular pathways, leading to adverse molecular phenotypes including inflammation, impaired synaptic transmission, and eventually neurodegeneration. Although HD has a singular genetic cause, there have been recent advances in our understanding of HD gene modifiers (PMID: 26232222). Dr. Hayden’s team previously identified a key cis-acting modifier associated with hastened HD onset – on average 13 years earlier (PMID: 31104771). This variation lacks two interrupting regions: one from within the CAG repeat (CAA-CAG), the other from the adjacent CCG repeat (CCA-CCG), collectively termed the CAG-CCG loss of interruption (LOI). The effect of the CAG-CCG LOI variant on disease progression is greater than for any other known variant in HD, however, mechanisms underlying its effect remain unclear (PMID: 39140258). By increasing our understanding of the neurobiology of the LOI variant, we hope to uncover novel therapeutic targets, with the ultimate goal of identifying new avenues for the development of HD genetic therapies.
Astrocytes form an intricate network throughout the brain through their associations with both the vasculature and nervous systems. This allows these specialized glial cells to support a variety of essential functions, including maintenance of blood flow, metabolic homeostasis, and synaptictransmission. The canonical HD brain is marked by astrogliosis, where astrocytes from these tissues express higher levels of the protein GFAP and have altered morphologies, such as larger cell bodies and reduced branch complexity. Early data from our lab suggests that striatal tissue samples from LOI variant HD donors display increased astrocyte reactivity compared to canonical HD patients matched for CAG repeat length.
Objective:
Validate preliminary findings from our group and determine whether astrocyte pathogenesis plays a role in the exacerbated disease phenotype of LOI variant-expressing cells.
Methodology:
The current study will use primary astrocyte-neuron corticostriatal (CS) cultures isolated from wildtype mouse pups. At DIV 2, CS cultures will be transfected with custom AAV plasmids containing the canonical or LOI variant N-terminal sequence of the mHTT gene. These AAVs will also contain a fluorescent reporter sequence to assess transfection rates. At DIV 16, CS cultures will undergo fixation and immunocytochemical staining with antibodies against GFAP, complement component 3 (C3, marker of astrocyte reactivity), and DAPI (nuclear marker). Astrocytes from these cultures will then be imaged using confocal microscopy. Our analysis will quantify the density and morphology (soma size and branch complexity), as well as C3 intensity levels, of LOI-expressing astrocytes in comparison to CAG length-matched and control cells.
Significance:
This project will improve our understanding of mHTT expression on astrocyte function, an important consideration for the development of HD therapeutics. This study will also help determine whether reactive astrogliosis plays a role in the hastened disease progression seen in carriers of the CAG-CCG LOI variant, a major HD genetic modifier.
Partners & Donors
Huntington Society of Canada
