Probes for INS

RESULTS Microscopic analysis revealed that the full-length _HTT_ mRNA (_FL-HTT_) was retained in RNA nuclear clusters together with the incompletely spliced _HTT1a_ transcript. These clusters were not observed in zQ175 HD mouse model where, instead, _FL-Htt_ and _Htt1a_ mRNAs were detected as mostly cytoplasmic molecules. Immunohistochemistry showed a progressive appearance of aggregated HTT in nuclei in the cortex, striatum, hippocampus and cerebellum. HTRF indicated that the level of exon 1 HTT was highest in the cerebellum. Soluble mutant exon 1 HTT decreased with age, with concomitant increase in aggregated HTT. In YAC128 MEFs, _HTT1a_ was detected and ASOs targeting _HTT_ were efficient in lowering _HTT_ levels in this model system.

Epigenetic mechanisms are altered in the striatum of HD patients and mouse models, but how they might contribute to pathogenesis, including cognitive deficits, is unclear. Epigenetic regulation is critical to learning and memory processes, through transcriptional control of gene program promoting neural plasticity. We asked whether memory-associated epigenetic and transcriptional responses were impaired in HD R6/1 mice. To this end, we trained R6/1 mice (and control mice) in an aquatic navigation task, the double H maze, which allows assessing striatum-dependent memory (e.g. egocentric spatial memory). We then generated ChIP-seq, 4C-seq and RNA-seq datasets on striatal tissue of HD and control mice during egocentric memory processing, including memory acquisition and consolidation/recall. Egocentric memory was altered since early symptomatic stage in R6/1 mice, which correlated with dramatic reduction of striatal epigenetic and transcriptional changes induced by memory process. More specifically, multi-omic analysis showed that, during memory acquisition, 3D chromatin re-organization and transcriptional induction at BDNF-related genes were diminished in R6/1 striatum. Moreover, we found that changes in H3K9 acetylation (H3K9ac), which accompanied memory process in normal striatum, were attenuated in R6/1 striatum. Functional enrichment analyses further indicated that altered H3K9ac regulation during late phase of egocentric memory process (e.g. consolidation/recall) contributed to impaired TGFβ-dependent cellular plasticity. Together, this study provides support to the hypothesis that epigenetic dysregulation in HD contributes to cognitive deficits, and shed light on new targets of striatal plasticity, particularly H3K9ac and TFGβ signaling.

We aimed to determine the possible role of CAG somatic expansions on the clinical expression of intermediate allele (IA) carriers of the HTT gene, responsible for Huntington disease (HD). We performed exon one sequencing analysis of the HTT gene on peripheral blood DNA in a Spanish cohort of asymptomatic IA carriers (n=55), symptomatic IA carriers (n=86) and HD subjects (n=124). Additionally, we investigated different brain regions of an individual carrying an HTT allele with 33 CAGs, with neurocognitive symptoms. Linear regression models were used to analyse the association between CAG length and age with somatic mosaicism. Symptomatic IA carriers presented with motor (80%), cognitive (20%) and/or behavioural (22%) signs, and an average age of onset of 58.7 years±18.6. Somatic mosaicism is CAG- and age-dependent in alleles of CAG≥27 CAGs, with b=0.04 (95% CI: 0.035-0.046), and b=0.001 (95% CI: 0.001-0.002), respectively, for all IAs. There was no statistical difference between HTT somatic mosaicism in symptomatic vs asymptomatic IA carriers (p=0.066). Somatic expansions of +1 and +2 CAGs were detected in the brain of the individual with 33 CAGs, with the highest expansion ratio observed in the putamen, where up to 10% of the DNA molecules underwent somatic expansion. In conclusion, somatic CAG expansions observed in blood cannot explain, overall, the neurocognitive signs of IA carriers. However, somatic instability occurs in IAs, which changes with CAG number and age; therefore, the presence of cells in the brain that express up to +2 CAGs may be important when considering the phenotypes of those alleles close to the pathological threshold.

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualise HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role, or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood.