Biology subjects

Bates, G. P.

Publications and source records attributed to Bates, G. P..

8 recordsLinked to original sources

bioRxiv2025

Nuclear RNA Clusters Are Dynamic Structural Entities in Huntington's Disease.

Huntingtons disease research has focused on a toxic protein gain-of-function as the main driver of pathology. The role of mutant HTT mRNA in vivo has been only partially studied and remains largely unexplored. Recently, we discovered that fully processed human HTT mRNA is retained, together with the alternatively processed HTT1a transcript, in RNA nuclear clusters in YAC128 mouse brains. Here, we demonstrate that these clusters were already present in the prenatal stage at day 14.5, indicating early developmental effects. Moreover, these clusters were confined to neurons, implying a neuron-specific mechanism of accumulation, and were colocalised with Prpf8, a core spliceosomal protein, suggesting potential impact on nuclear homeostasis. HTT nuclear RNA clusters showed remarkable dynamics, rapidly dissolving when ionic interactions were disrupted or transcription and splicing were inhibited. This malleability underscores the accessibility of HTT mRNA to therapeutic interventions and may guide the future design of HTT-targeting therapies.

neuroscience↗

bioRxiv2025

Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntingtons disease pathogenesis in mice

Huntingtons disease (HD) is a progressive neurodegenerative disorder with no approved therapies. Two major molecular drivers--somatic expansion of inherited CAG repeats and toxic mutant HTT (mHTT) variants--lead to neuronal dysfunction. Despite multiple trials, HTT-lowering strategies have not shown meaningful clinical benefit. Using therapeutic divalent siRNAs, we assessed the long-term impact of silencing MSH3 (a key regulator of somatic expansion), HTT, or both. In Q111 HD mice (>110 CAGs), which exhibit robust expansion, mHTT inclusions, and transcriptional dysregulation by 12 months, long-term MSH3 silencing blocked expansion, reduced inclusions, and reversed gene expression changes. HTT silencing alone had limited effect, but combined MSH3/HTT targeting synergistically eliminated inclusions and restored transcriptomic profiles. Parallel treatment in wild-type mice showed no toxicity, supporting the safety of long-term intervention. These findings position somatic expansion as a promising therapeutic target and demonstrate the potential of RNAi-based co-silencing of MSH3 and HTT as a disease-modifying strategy for HD.

neuroscience↗

bioRxiv2025

Lowering the HTT1a transcript as an effective therapy for Huntingtons disease

Lowering the levels of HTT transcripts has been a major focus of therapeutic development for Huntingtons 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. One Sentence SummaryLowering HTT1a transcript levels delays the onset of molecular and neuropathological phenotypes in a knock-in mouse model of Huntingtons disease.

neuroscience↗

bioRxiv2024

Mutant HTT protein decreases with CAG repeat expansion: implications for therapeutics and bioassays.

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in the HTT protein. The mutant CAG repeat is unstable and expands in specific brain cells and peripheral tissues throughout life. Genes involved in the DNA mismatch repair pathways, known to act on expansion, have been identified as genetics modifiers, therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of disease progression. In the context of an expanded CAG repeat, the HTT pre-mRNA can be alternatively processed to generate the HTT1a transcript, that encodes the aggregation prone and highly pathogenic HTT1a protein. This may be a mechanism through which somatic CAG repeat expansion exerts its pathogenic effects, as the longer the CAG repeat, the more HTT1a and HTT1a is produced. The allelic series of knock-in mouse models: HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175 with polyQ expansions of 20, 50, 80, 111 140 and [~]190 can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. By western blot of cortical lysates, we found that mutant HTT levels decreased with increasing CAG repeat length; mutant HTT was only 23% and 10% of wild-type levels in CAG140 and zQ175 cortices, respectively. To identify the optimal bioassays for detecting the full-length HTT and HTT1a isoforms, we interrogated the pairwise combinations of seven well-characterized antibodies on both the HTRF and MSD platforms. In total we tested 32 HTRF and 32 MSD assays to detect full-length mutant HTT, HTT1a, total mutant HTT (full-length HTT and HTT1a) and total full-length HTT (mutant and wild type). None of these assays recapitulated the full-length mutant HTT levels as measured by western blot. We recommend using isoform- and species-specific assays that detect either full-length mutant HTT, HTT1a or wild-type HTT as opposed to those that detect more than one isoform simultaneously. Our finding that as the CAG repeat expands, full-length mutant HTT levels decrease, whilst HTT1a and HTT1a levels increase has implications for therapeutic strategies. If mutant HTT levels in cells containing (CAG)200 are only 10% of wild-type, HTT-lowering strategies targeting full-length HTT at sequences 3 to intron 1 HTT will predominantly lower wild-type HTT, as mutant HTT levels in these cells are already depleted. These data support a therapeutic strategy that lowers HTT1a and depletes levels of the HTT1a protein.

neuroscience↗

bioRxiv2023

A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease

The Huntingtons disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable, and expansions of hundreds of CAGs have been detected in Huntingtons disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntingtons disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntingtons disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntingtons disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterised. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knock-out backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntingtons disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

neuroscience↗

bioRxiv2023

m6A RNA modification of mHtt intron 1 regulates the generation of Htt1a in Huntington's Disease

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expanded, somatically unstable CAG repeat in the first exon of the huntingtin gene (HTT). In the presence of an expanded CAG repeat, huntingtin mRNA undergoes an aberrant processing that generates HTT1a transcripts with exon 1 and intron 1 sequences, which encodes the aggregation-prone and pathogenic HTTexon 1 protein. The regulatory mechanisms that contribute to the production of HTT1a are not fully understood. In a previous transcriptome-wide m6A landscape study performed in Hdh+/Q111 knock-in mice, we have found that the proximal region of intron 1 to exon1-intron 1 splice site in Htt RNA is highly modified by m6A. Several pieces of evidence have demonstrated that m6A is involved in RNA processing and splicing. Therefore, in this study we set out to explore the impact of m6A RNA modifications in the generation of Htt1a. We show in the striatum of Hdh+/Q111 mice that m6A is enriched in intronic sequences 5 to the cryptic poly (A) sites (IpA1 and IpA2) at 680 and 1145 bp into intron 1 as well as in Htt1a polyadenylated mRNA. We also verified the presence of specific m6A-modified sites near the 5 exon1-intron1 splice donor site. Intronic HTT m6A methylation was recapitulated in human samples showing a significantly increased methylation ratio in HD putamen post-mortem samples and in HD fibroblast cell lines from pre-symptomatic and symptomatic patients. In order to test the hypothesis that the m6A modification is involved in mutant Htt RNA processing, we performed a pharmacological inhibition of METTL3 and a targeted demethylation of Htt intron 1 in HD cells using a dCas13-ALKBH5 system. We found that Htt1a transcript levels in HD cells are regulated by METTL3 and by methylation status in Htt intron 1. Site-specific manipulation with an RNA editing system resulted in decreased expression levels of Htt1a, which was accompanied by a reduction in DNA damage, a major hallmark in HD. Finally, we propose that m6A methylation in intron 1 is likely dependent on the expanded CAG repeats. These findings provide insight into the role of m6A in the generation of the aberrantly spliced mutant Htt transcripts with important implications for therapeutic strategies.

neuroscience↗

bioRxiv2021

Correlative light and electron microscopy reveals that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease

Huntingtons disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (i) multivesicular bodies (MVBs), or amphisomes and (ii) autolysosomes or residual bodies. The organelles were often found in close proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

neuroscience↗

bioRxiv2019

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington's disease

Phosphorylation of the N-terminal domain of the Huntingtin (HTT) protein (at T3, S13, and S16) has emerged as a key regulator of HTT stability, clearance, localization, aggregation and toxicity. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that specifically and efficiently phosphorylates both wild-type and mutant full-length or N-terminal fragments of HTT in vitro (S13/S16) and in cell/ neuronal cultures (S13). We show that overexpression of TBK1 in mammalian cells, primary neurons and a Caenorhabditis elegans model of Huntingtons Disease (HD) increases mutant HTTex1 phosphorylation, lowers its levels, increases its nuclear localization and significantly reduces its aggregation and cytotoxicity. Our mechanistic studies demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTTex1 aggregation and an increase in autophagic flux. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/869586v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1368abeorg.highwire.dtl.DTLVardef@1cecff4org.highwire.dtl.DTLVardef@b6b7c5org.highwire.dtl.DTLVardef@1daf53d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

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