Circular RNA (circRNA) molecules have critical functions during brain development and in brain-related disorders. Here, we identified and validated a circRNA, circHTT(2,3,4,5,6), stemming from the Huntington's disease (HD) gene locus that is most abundant in the central nervous system (CNS). We uncovered its evolutionary conservation in diverse mammalian species, and a correlation between circHTT(2,3,4,5,6) levels and the length of the CAG-repeat tract in exon-1 of HTT in human and mouse HD model systems. The mouse orthologue, circHtt(2,3,4,5,6), is expressed during embryogenesis, increases during nervous system development, and is aberrantly upregulated in the presence of the expanded CAG tract. While an IRES-like motif was predicted in circH TT (2,3,4,5,6), the circRNA does not appear to be translated in adult mouse brain tissue. Nonetheless, a modest, but consistent fraction of circHtt(2,3,4,5,6) associates with the 40S ribosomal subunit, suggesting a possible role in the regulation of protein translation. Finally, circHtt(2,3,4,5,6) overexpression experiments in HD-relevant STHdh striatal cells revealed its ability to modulate CAG expansion-driven cellular defects in cell-to-substrate adhesion, thus uncovering an unconventional modifier of HD pathology.

Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.

Circular RNAs (circRNAs), single-stranded, circularized RNA molecules, are particularly enriched in neurons and their functional relevance for brain development and neurological disorders has become evident in recent years. Here, we identified and validated the first brain enriched RNA circle originating from the human HTT locus (CircHTT: 484nt, Ex 2-6), conserved also in mouse (circHtt) and minipig. We validated the circularity of the identified molecule by divergent primer amplification, sequencing and RNase R treatment. Then, we analyzed the expression pattern of circHTT/circHtt in human and mouse tissues. This analysis revealed ubiquitous expression in different tissues, including blood. Importantly, and in line with circRNA characteristics, circHTT/circHtt is expressed at very high levels in the brain. To characterize possible implications for Huntington’s Disease (HD), we studied its expression pattern in induced pluripotent stem cell (iPSC)-derived neuronal cells and HD mouse models. We found that circHTT/circHtt expression increases significantly with increasing number of CAG repeats in terminally differentiated cortical neurons and in brain districts of HD mouse models. These findings suggest possible implications for HD pathology. Analysis of circHTT sequence revealed week miRNA recognition elements, binding sites for RNA binding proteins, and the presence of a putative IRES sequence. Follow-up in vivo knock-down and in vitro over-expression studies are currently ongoing to elucidate the biological function(s) of circHTT/circHtt. In conclusion, we uncovered a circRNA sensitive to the HD mutation, which may be relevant for HD pathophysiology or to modulate huntingtin expression.

Alternative Splicing (AS) is crucial for generating protein-coding isoforms and circular RNAs (circRNAs), stable non-coding RNA’s produced by circularization of exons through the back-splicing process. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a strong correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, expanded Htt CAG repeats reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Though the mechanisms of this dysregulation remain uncertain, our study unveils transcriptionally altered micro-RNAs, possibly impacting the expression of RNA-binding proteins, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might result in altered neural fitness and contribute to disease. With the development of new therapies entering clinical trials for HD, there is an increasing need to develop and validate biomarkers in accessible biofluids (such as blood) to follow disease progression and predict treatment’s outcome. Here, we detect and characterize circRNAs in peripheral blood of a cohort of gender and age matched healthy and HD individuals at different stages of the disease. We investigate their potential use as biomarkers by evaluating their correlation with disease progression and the length of the CAG repeat. Interestingly, 35 circRNAs, whose expression significantly increases in HD, possibly identify new disease biomarkers to assess the pathogenic process and the pharmacologic responses to therapeutic intervention.

Background Circular RNAs (circRNAs) are a special group of non-coding RNAs formed by back-splicing. Mostly cytosolic in eukaryotic cells, circRNAs are particularly enriched and conserved in neurons and originate from protein-coding genes to function as global regulators of gene expression. Recent studies showed that circRNAs partake in brain physiology and pathology, contributing to neurological disorders, such as myotonic dystrophy type 1, Parkinson’s and Alzheimer’s Diseases. Here, we identified the first ever known brain enriched RNA circle originating from the Huntington’s Disease gene HTT (CircHTT: 484 nt, Ex 2-6, chr4:3088665-3109150) which is conserved in mouse and minipig. Aims and Methods To functionally characterize circHTT and its implication for HD pathogenesis, we first studied its expression pattern in human and mouse body districts and in iPS-derived neuronal cell lines with different CAG repeats. Then, overexpression and down-regulation of the circle were used to determine the effects on transcription of the HD gene and translation of wild-type and mutant protein. Results CircHTT expression increases significantly with increasing CAG repeats in terminally differentiated cortical neurons. Furthermore, circHtt is significantly more expressed in brain districts of Q111 and zQ175 knock-in mice. These findings indicate that the expression of circHTT/Htt occurs in a CAG repeat dependent manner in neuronal cells, typical hallmark of HD pathology. Strikingly, overexpression of the circular RNA in human HEK293T, PC3, mouse STHdh Q7/7, Q7/111 and Q111/111 cell lines consistently increase wild-type huntingtin, while decreasing mutant huntingtin protein, with no alteration of the HTT/Htt transcript level. Conclusions In conclusion, we identified a brain enriched RNA circle originating from the HD gene locus that is sensitive to the HD mutation and may modulate huntingtin expression. Our observations might pave the way to new trials of therapeutic intervention.

ABSTRACTAlternative splicing (AS) appears to be altered in Huntington’s disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected.Here, taking advantage ofHttCAG knock-in mousein vitroandin vivomodels, we demonstrate a strong correlation betweenHttCAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and,in vivo,the striatum prior to overt behavioral phenotypes stages. Remarkably, expandedHttCAG repeats reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors.Though the mechanisms of this dysregulation remain uncertain, our study unveils network of transcriptionally altered micro-RNAs and RNA-binding proteins (CELF, hnRNPS, PTBP, SRSF) which, in turn, might influence the AS machinery, primarily in neural cells.We suggest that this unbalanced expression of linear and circular RNAs might result in altered neural fitness, contributing to HD striatal vulnerability.

Background With the development of new therapies entering clinical trials for HD, there is an increasing need to develop and validate biomarkers in accessible biofluids, such as blood, to follow disease progression and predict treatment outcomes. Some biomarkers do exist; however, reliable and readily available additional ones will be crucial in assessing the pathogenic process and pharmacologic responses to therapeutic interventions. Aims For the first time in HD, we detect and characterize circRNAs in peripheral blood of a cohort of gender and age-matched controls and HD patients at various stages of the disease. We studied their potential as biomarkers by evaluating their correlation with disease progression and the size of the CAG repeat. Methods 50 blood samples were collected from both HD patients and a healthy cohort of individuals. Total RNA was used for RNA sequencing, and the obtained data were aligned with STAR. DCC program was used to quantify circRNAs and CircTest (R package) to identify differentially expressed circRNA between different disease stages and controls. CAG repeat size correlation was also tested. To validate our findings, candidate circRNAs were quantified via RT-qPCR in our samples. Results Our study led to the discovery of 7, stringently defined circRNAs that possess 3 characteristics: differentially expressed in HD patients (all up-regulated, p-value < 0.05), correlated to CAG repeat size (|Pearson’s R| > 0.3 and p-value < 0.05) and have higher expression as the disease progresses. Using divergent primers, we quantified and validated via RT-qPCR the expression of subsets of circRNA candidates. Conclusions We identified for the first time circRNAs whose expression increases in the blood of HD patients. Currently, we aim to validate these circular molecules in independent blood cohorts as well as in cerebrospinal fluid and plasma samples to fully elucidate whether these highly stable RNA molecules could be used as disease biomarkers.

Huntington’s disease (HD) is a devastating neurodegenerative disorder caused by an aberrant expansion of the CAG tract within the exon 1 of the HD gene, HTT. HD progressively impairs motor and cognitive capabilities, leading to a total loss of autonomy and ultimate death. Currently, no cure or effective treatment is available to halt the disease. Although the HTT gene is ubiquitously expressed, the striatum appears to be the most susceptible district to the HD mutation with Medium-sized Spiny Neurons (MSNs) (D1R and D2R) representing 95% of the striatal neuronal population. Why are striatal MSNs so vulnerable to the HD mutation? Particularly, why do D1R- and D2R-MSNs display different susceptibility to HD? Here, we highlight significant differences between D1R- and D2R-MSNs subpopulations, such as morphology, electrophysiology, transcriptomic, functionality, and localization in the striatum. We discuss possible reasons for their selective degeneration in the context of HD. Our review suggests that a better understanding of cell type-specific gene expression dysregulation within the striatum might reveal new paths to therapeutic intervention or prevention to ameliorate HD patients’ life expectancy.