Exploring central and enteric nervous system vulnerability in Huntington’s Disease: hints from a knock-in animal model

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an aberrant expansion of CAG repeats within the HTT gene, for which there is currently no cure. The disease is driven by the mutant huntingtin protein, which predominantly leads to the degeneration of the striatum within the brain. Medium-sized spiny neurons (MSNs), the main neuronal components of the striatum, are selectively affected by HD, and they can be further categorized by their surface expression of Dopamine receptors 1 (D1R) and 2 (D2R). These two populations exhibit distinct vulnerabilities, with D2R-expressing MSNs impacted earlier in the disease process, though the reasons for this differential susceptibility remain unclear. Understanding the mechanisms underlying the specific vulnerability of D2R-MSNs, or the resilience of D1R-MSNs, could guide strategies to delay neurodegeneration. In this study, we utilized an HD knock-in mouse model carrying 18 (HttQ20, “control”) or approximately 190 (HttQ175, “HD”) CAG repeats. This model also expresses tdTomato and EGFP fluorescent proteins under the control of Drd1 and Drd2 promoters, respectively, enabling visualization and differentiation of MSNs. Using this model, we conducted a multidimensional analysis to identify features that contribute to MSNs vulnerability. Transcriptomic analysis of small pools of MSNs collected from 8-week-old, presymptomatic mice revealed an overall downregulation of retrotransposable elements in both neuronal classes in the HD group. Notably, in D1R-MSNs, pathways related to oxidative phosphorylation and translation were upregulated in HD mice, suggesting a potential compensatory mechanism that might prevent from mutant huntingtin aggregation. Consistently, we observed an early increase in huntingtin aggregates in D2R-MSNs compared to D1R-MSNs. Furthermore, D2R-MSNs showed greater sensitivity to CAG somatic instability, potentially contributing to their earlier susceptibility to HD. While HD central nervous system symptoms are well-documented, the ubiquitous expression of mutant huntingtin results in a range of peripheral symptoms, including gastrointestinal (GI) disturbances. These symptoms, which appear as early as the prodromal phase, contribute significantly to patient isolation and reduced quality of life. Although some studies have reported alterations in the enteric nervous system (ENS) of HD patients, including mutant huntingtin aggregation, little research has explored this characteristic in-depth. Using the HttQ175 mouse model, we investigated whether ENS and GI alterations observed in patients could be modelled and characterized. To this aim, we successfully established a number of protocols to isolate and visualize ENS ganglia and GI structures, and to culture enteric primary cells. Then, preliminary studies seemed to suggest morphological abnormalities and elevated expression of pro-inflammatory markers such as S100b and IL-6 in HD enteric cultures. In vivo experiments revealed increased expression of specific Bdnf isoforms at presymptomatic stages, and elevated levels of the 55 kDa Gfap protein isoform in symptomatic-stage colonic tissue. While these molecular changes were not conclusively linked to functional or gross structural GI tract alterations, these preliminary findings further support the need to explore GI molecular, cellular and functional alteration in HD, with potential implications for therapeutic development to enhancing patient quality of life. Overall, this thesis offers valuable insights into the cellular and molecular mechanisms of HD, spanning central and peripheral nervous system effects, and contributes to the identification of novel biomarkers, potentially paving the way for improved diagnostic and therapeutic strategies.