Biology subjects

Nita, I. M.

Publications and source records attributed to Nita, I. M..

5 recordsLinked to original sources

bioRxiv2026

Enhancing the detection of HTT1a with neoepitope antibodies in mouse models of Huntington's disease

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene, encoding an expanded polyglutamine tract in the huntingtin (HTT) protein. The pathogenic CAG repeat of HTT is unstable and undergoes progressive somatic expansion in specific brain cells and peripheral tissues throughout life. Genes involved in DNA mismatch repair pathways, which promote repeat expansion, have been identified as genetic modifiers of the disease. Consequently, the rate of CAG repeat expansion is a key determinant driving the age of onset and disease progression. As the CAG repeat expands, alternative processing of HTT pre-mRNA increasingly favours production of the HTT1a transcript, which encodes the highly pathogenic and aggregation-prone HTT1a protein. This process provides a mechanistic link between CAG repeat expansion and disease pathogenesis, as increased HTT1a production accelerates HTT aggregation and neuronal dysfunction. HTT1a has previously been detected in Huntingtons disease mouse models by using immunoprecipitation coupled with western blotting, homogeneous time-resolved fluorescence (HTRF) and Meso Scale Discovery (MSD) bioassays, and immunohistochemistry. These approaches were developed using MW8, a neoepitope antibody that specifically recognizes the C-terminus of HTT1a. MW8 is a relatively weak antibody with limited detection sensitivity. To generate more robust HTT1a-specific reagents, two novel recombinant antibodies, 1B12 and 11G2, have been developed for evaluation. Using an allelic series of knock-in (HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175) mice, alongside transgenic YAC128 and N171-82Q models, we extensively evaluated and compared the performance of MW8, 1B12 and 11G2. We demonstrate that 1B12 and 11G2 function as HTT1a-specific neoepitope antibodies by immunoprecipitation with western blotting, and by immunohistochemistry. To enhance HTT1a detection using HTRF and MSD technology platforms, we further evaluated the performance of 1B12 and 11G2 in HTT bioassays using cortical lysates from zQ175 and YAC128 mice. In zQ175 mice, enhanced detection of aggregated HTT1a by HTRF and MSD revealed that HTT fragments longer than HTT1a can be incorporated into HTT1a-containing aggregates. The most sensitive assays were subsequently applied across the allelic series of knock-in mice to assess the effect of polyglutamine length on bioassay performance. For optimal sensitivity, we recommend the preferential use of 1B12 for HTRF assays and 11G2 for MSD assays. Collectively, these findings establish 1B12 and 11G2 as robust antibodies to reliably detect and track HTT1a pathology in vivo and promotes the replacement of previously used MW8-based experimental approaches. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/708805v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@16d10faorg.highwire.dtl.DTLVardef@1759f41org.highwire.dtl.DTLVardef@12a8c21org.highwire.dtl.DTLVardef@55fe6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Osborne et al. used Huntingtons disease mouse models to evaluate and compare the performance of HTT1a-specific neoepitope antibodies by using immunoprecipitation with western blotting, bioassays, and immunohistochemistry. In contrast to MW8, they establish that 1B12 and 11G2 are robust antibodies to reliably detect and track HTT1a pathology in vivo.

neuroscience↗

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

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↗