circRNAs are covalently closed RNA molecules recently re-discovered thanks to the advances in RNA-seq technology. They are produced by the canonical spliceosome in a non-canonical splicing process, named back-splicing. Heterogeneous in internal composition and highly stable, circRNAs regained the attention of neuronal biologists because of their enrichment in brain and neuronal compartments. Moreover, several pioneering studies revealed a fine orchestration of circRNA expression in crucial stages of neuronal development, such as synaptogenesis. The growing evidence of circRNA enrichment in synapses raises the intriguing question as to the yet unknown molecular mechanisms leading to this unique neuronal sub-compartmentalization. In addition, in which of the compartments composing the synapse – dendrites and axon – circRNAs preferentially localize, is still largely elusive. Here, I have focused specifically on the pre-synaptic compartment – the axon – during specific stages of neuronal development. The proper development of the axon is crucial to guarantee the correct synapse formation. Impairments in this process can lead to severe neurodevelopment diseases. I explored circRNA expression in the axonal compartment, shedding light on circRNA distribution in and trafficking to the neuronal distal compartment. To reach these goals, I used Xenopus laevis retinal ganglion cell (RGC) developing axons as a model. The results presented in this thesis highlight an abundant expression of circRNAs in the axonal compartment, where they are enriched compared to the somatic one. The enrichment in axons led me to deeper explore their preferential axonal sub localization, by investigating how they reach the neuronal distal compartment. circDDX17 was selected as reference model of axonal circRNAs. Its tracking within the axon revealed an heterogenous distribution and shape. circDDX17 trafficking along the axon displayed an anterograde preferential directionality and slow speed giving hints to uncover the molecular mechanisms of circRNA translocation to the axonal compartment. Bioinformatic analysis revealed that the RNA-RBP complex formation, the most common and described mechanism of axonal transport, could underlie circRNA intracellular translocation. Taken together, my data uncover the axonal circRNA population and characterize their localization in the neuronal distal compartment.

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.

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