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We have previously shown that the incomplete splicing of exon 1 to exon 2 of the HTT gene results in the production of a small polyadenylated transcript ( Httexon1 ) that encodes the highly pathogenic exon 1 HTT protein. There is evidence to suggest that the splicing factor SRSF6 is involved in the mechanism that underlies this aberrant splicing event. Therefore, we set out to test this hypothesis, by manipulating SRSF6 levels in Huntington’s disease models in which an expanded CAG repeat had been knocked in to the endogenous Htt gene. We began by generating mice that were knocked out for Srsf6 , and demonstrated that reduction of SRSF6 to 50% of wild type levels had no effect on incomplete splicing in zQ175 knockin mice. We found that nullizygosity for Srsf6 was embryonic lethal, and therefore, to decrease SRSF6 levels further, we established mouse embryonic fibroblasts (MEFs) from wild type, zQ175, and zQ175:: Srsf6 +/− mice and transfected them with an Srsf6 siRNA. The incomplete splicing of Htt was recapitulated in the MEFs and we demonstrated that ablation of SRSF6 did not modulate the levels of the Httexon1 transcript. We conclude that SRSF6 is not required for the incomplete splicing of HTT in Huntington’s disease.

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin ( HTT ) gene. HTT mRNA contains 67 exons and does not always splice between exon 1 and exon 2 leading to the production of a small polyadenylated HTTexon 1 transcript, and the full-length HTT mRNA has three 3′UTR isoforms. We have developed a QuantiGene multiplex panel for the simultaneous detection of all of these mouse Htt transcripts directly from tissue lysates and demonstrate that this can replace the more work-intensive Taqman qPCR assays. We have applied this to the analysis of brain regions from the zQ175 HD mouse model and wild type littermates at two months of age. We show that the incomplete splicing of Htt occurs throughout the brain and confirm that this originates from the mutant and not endogenous Htt allele. Given that HTTexon 1 encodes the highly pathogenic exon 1 HTT protein, it is essential that the levels of all Htt transcripts can be monitored when evaluating HTT lowering approaches. Our QuantiGene panel will allow the rapid comparative assessment of all Htt transcripts in cell lysates and mouse tissues without the need to first extract RNA.

Huntington’s disease (HD) is a devastating neurodegenerative disorder, caused by a CAG/polyglutamine repeat expansion, that results in the aggregation of the huntingtin protein, culminating in the deposition of inclusion bodies in HD patient brains. We have previously shown that the heat shock response becomes impaired with disease progression in mouse models of HD. The disruption of this inducible arm of the proteostasis network is likely to exacerbate the pathogenesis of this protein-folding disease. To allow a rapid and more comprehensive analysis of the heat shock response, we have developed, and validated, a 16-plex QuantiGene assay that allows the expression of Hsf1 and nine heat shock genes, to be measured directly, and simultaneously, from mouse tissue. We used this QuantiGene assay to show that, following pharmacological activation in vivo, the heat shock response impairment in tibialis anterior, brain hemispheres and striatum was comparable between zQ175 and R6/2 mice. In contrast, although a heat shock impairment could be detected in R6/2 cortex, this was not apparent in the cortex from zQ175 mice. Whilst the mechanism underlying this impairment remains unknown, our data indicated that it is not caused by a reduction in HSF1 levels, as had been reported.

BackgroundHuntington-lowering approaches are a major focus for therapeutic intervention for Huntington’s disease. In evaluating these treatments, it will be important to understand how the targeting strategy affects levels of the HTT1a and full-length HTT transcripts and the soluble exon 1 HTT and full-length HTT proteins that they encode. It will also be important to determine how a given strategy impacts on the subcellular location of HTT aggregation, dependent on the stage of disease at which the intervention was administered.AimHere, we set out to map the earliest ages at which the appearance of HTT aggregation could be detected throughout the brain in heterozygous zQ175 mice.ResultsAggregation analyses were performed at monthly intervals from 1-6 months of age. Immunohistochemistry with the S830 antibody detected aggregated HTT in the form of a diffuse nuclear immunostain in neuronal nuclei in the striatum, hippocampus and cortex by 2 months. By 6 months, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. We also applied two HTRF assays to track the accumulation aggregated HTT in eight brain regions; one of which could detect aggregation in the striatum and cortex at one month of age. HTRF showed that soluble exon 1 HTT levels decreased over the 6-month period, whilst levels of soluble full-length mutant HTT remained unchanged.ConclusionsThese data support the hypothesis, that exon 1 HTT initiates the aggregation process in knock-in mouse models and paves the way for a detailed analysis of HTT aggregation in response to HTT-lowering treatments.

BackgroundYAC128 transgenic mice carry human HTT with an expanded CAG repeat. This model is particularly useful for evaluating therapies targeting the human HTT gene and/or protein.AimsTo better understand the molecular phenotype of YAC128 mice at the RNA and protein level.MethodsA QuantiGene assay was designed to gain insights into incomplete splicing in the context of human HTT, and to evaluate the lowering efficiency of therapeutic compounds. RNAscope was implemented to visualize HTT transcripts and immunohistochemistry to characterise HTT aggregation spatiotemporally. HTRF measured the levels of the total HTT protein and the pathogenic exon 1 HTT. Mouse embryonic fibroblasts (MEFs) from YAC128 mice were used for screening agents targeting human HTT transcript.ResultsMicroscopic 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.ConclusionsHuman HTTundergoes incomplete splicing in brains of YAC128 mice. The RNA clusters detected may have direct therapeutic implications, with pathogenic or protective consequences

BackgroundHuntington’s disease (HD) is caused by an expanded CAG repeat in exon 1 of the HTT gene. In models of HD, an expanded CAG repeat in HTT causes premature termination of HTT RNA during transcription; this occurs by a process called incomplete splicing. Incompletely spliced HTT (HTTexon1) includes exon 1 of the coding region of HTT, as well as a 5’ region of intron 1, which is non-coding. HTTexon1 encodes a truncated exon 1 HTT protein, which is implicated in HD pathogenesis. Although the precise RNA processing mechanism of Httexon1 is unknown, splicing factor SRSF6 has been shown to co-precipitate with transcripts containing Htt intron 1 in HD mice.AimTo elucidate the role of splicing factor SRSF6 in incomplete splicing of Htt in HD mice.MethodsHeterozygous Srsf6 knock-out (KO) mice (Srsf6±) were generated by CRISPR/Cas9. Characterisation of Srsf6± mice was undertaken by quantitative RT-PCR and western blotting. Viability of homozygous Srsf6 KO (Srsf6-/-) mice was examined by inbreeding of Srsf6± mice. To assess the modulation of incomplete splicing by decreasing SRSF6, Srsf6± mice were bred to HD knock in mice (zQ175) and tissues were analysed. Levels of Httexon1 were measured by Quantigene, a gene expression assay.ResultsSrsf6-/- homozygotes were embryonic lethal, limiting us to the use of Srsf6± mice only. In Srsf6± heterozygotes, Srsf6 mRNA was decreased by 50% in brain and peripheral regions, and SRSF6 protein was decreased by 70% in brain compared to wild type mice. However, heterozygosity for Srsf6 knock out did not modulate the level on incomplete splicing in zQ175 mice.ConclusionAblation of a single Srsf6 allele did not reduce levels of incomplete splicing in HD mice and therefore, further Srsf6 knock down may be required. Accordingly, mouse embryonic fibroblasts (MEFs) have been generated and will be used to measure Httexon1 levels after further Srsf6 knockdown by RNA interference.This work is supported by the CHDI foundation.

BackgroundThe heat shock response (HSR) is responsible for the maintenance of proteome integrity within cells. HSR is induced under conditions of proteotoxic stress such as the presence of aggregates or aberrantly-folded proteins, as observed in Huntington´s disease (HD). The master regulator of the HSR is HSF1, which regulates the transcriptional activation of the heat shock genes. The pharmacological induction of HSR is a promising therapeutic strategy in HD. Previously our group has shown that HSR pharmacological induction reduces aggregation and improves survival in HD mouse models. However, these effects were transient as the ability to induce HSR became impaired with disease progression.AimsTo study the mechanisms involved in HSR impairment in HD and how this progressive impairment could be improved or restored by increasing HSF1 activity.MethodsC57BL/6J mice were dosed with the HSP90 inhibitor NVP-HSP990, which releases HSF1 from its HSP90 inhibitory complex. To quantify heat shock protein (HSP) gene expression, we have established a novel Quantigene multiplex assay (ThermoFisher Scientific) to measure the concomitant expression of 11 HSPs and their regulators. Quantitative RT-PCR (qPCR) was used to validate the expression of a subset of these genes.ResultsOur results have identified a set of HSP genes that are induced by NVP-HSP990 in mouse brain and muscle. The multiplex HSP Quantigene assay produces comparable data to qPCR, whilst its simple workflow on tissues homogenates allows the analysis of many more HSP genes that is practical by qPCR. We have used these tools to define the kinetics of the pharmacological HSR induction.ConclusionsThe establishment of the multiplex HSP Quantigene assay, together with NVP-HSP990 provides the tools with which to investigate approaches to restore the HSR impairment in HD.This work is supported by the CHDI Foundation.

BackgroundHuntington’s Disease (HD) is neurodegenerative disorder caused by the expansion of a CAG repeat in the HTT gene. We have recently shown that incomplete splicing of exon 1 to exon 2 of the HTT (human) and Htt (mouse) mRNAs produces a polyadenylated transcript that contains exon 1 and a short sequence of intron 1, that is translated to an exon 1 HTT protein. Measurement of the abundance of these transcripts has relied on quantitative RT-PCR assays (qPCR), which are time consuming and involve many steps (i.e. RNA isolation, reverse transcription and amplification). Assays employing branched DNA technology to detect nucleic acids involve less steps, can be multiplexed and are commercially available.AimsThe aim of this study was to develop and optimize a Quantigene assay to measure concomitantly all Htt or HTT transcripts in a timely and efficient manner.Methods/techniquesWe designed probes to develop multiplex assays for mouse Htt and human HTT. In each case, the location of the probe sets within exons, introns and the 3’UTR were designed to identify all possible Htt or HTT transcripts. The zQ175 knock-in HD mouse model at 2 months of age was used to validate the mouse multiplex assay and fibroblasts from HD patients and control samples were used for the human assay.Results/outcomeWe optimized our mouse Quantigene assay and show that it generates comparable data to the much more time-consuming qPCRs. We show that transcription of Httexon1 stops after the second polyA site within intron 1. We also measured differences in the Htt 3’UTR transcripts. The human multiplex Quantigene assay needs to be further optimized as to date, only the full length HTT transcript has been detected, in contrast to our qPCR data.ConclusionsWe have developed and optimized a multiplex Quantigene assay to rapidly measure all mouse Htt transcripts directly in tissue lysates. This assay can be used for investigating levels of the Htt transcripts in vivo after administration of therapeutic agents.This work is supported by the CHDI foundation.

Lowering the levels of HTT transcripts has been a major focus of therapeutic development for Huntington’s disease (HD), but which transcript should be lowered? HD is caused by a CAG repeat expansion in exon 1 of the HTT gene, and the rate of somatic expansion of this CAG repeat throughout life is now known to drive the age of onset and rate of disease progression. As the CAG repeat expands, the extent to which the HTT mRNA is alternatively processed to generate the HTT1a transcript and highly aggregation-prone and pathogenic HTT1a protein increases. Several HTT-lowering modalities have entered clinical trials that either target both HTT and HTT1a together, or full-length HTT alone. We have developed siRNAs that target the Htt1a mouse transcript (634/486) and used these, together with a potent Htt-targeting siRNA (10150) to compare the efficacy of lowering either full-length Htt or Htt1a. zQ175 and wild-type mice were treated with 10150 or 634/486 alongside control groups at 2 months of age with treatment to 6 or 10 months, or at 6 months with treatment to 10 months. The siRNA potency and durability were most effective in the hippocampus. Whilst both strategies showed benefits, despite the greater potency of 10150, targeting Htt1a was more effective at delaying HTT aggregation and transcriptional dysregulation than targeting full-length Htt. These data support HTT-lowering strategies that are designed to target the HTT1a transcript, either alone, or together with lowering full-length HTT. Lowering HTT1a transcript levels delays the onset of molecular and neuropathological phenotypes in a knock-in mouse model of Huntington’s disease.