Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains

Ly, Socheata; Didiot, Marie-Cécile; Ferguson, Chantal; Coles, Andrew H.; Miller, Rachael; Chase, Kathryn; Echeverria, Dimas; Wang, Feng; Sadri‐Vakili, Ghazaleh; Aronin, Neil; Khvorova, Anastasia

doi:10.1093/braincomms/fcac248

Cited by 9 publications

(13 citation statements)

References 87 publications

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“…The exact role of mutant transcripts in pathogenic mechanisms in polyQ diseases is being unraveled [10][11][12][13]. RNA-related studies have demonstrated so far: incomplete splicing of HTT mRNA [14], abnormal interactions of RNAs containing expanded CAG repeats with proteins [15,16], and RNA foci formation [17,18], and potential importance of alternative polyadenylation of various polyQ transcripts [19]. Furthermore, mutant polyQ disease transcripts have been shown to be promising therapeutic targets in strategies that aim to downregulate mutant gene expression [2,9,20].…”

Section: Introductionmentioning

confidence: 99%

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

et al. 2023

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Background The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington’s disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts. Results To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice. Conclusions Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.

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Section: Introductionmentioning

confidence: 99%

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

et al. 2023

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“…Furthermore, we detected the m6A methylation in polyadenylated Htt1a mRNAs, indicating its persistence during RNA maturation and potential roles in Htt1a mRNA fate. For instance, it has been shown that Htt1a can be found in the nucleus in the form of RNA foci in YAC128 mice 7 and in HD post-mortem brains which are likely caused by somatic expansion of the CAG repeats 8 . Although the direct function of m6A modifications in partitioning mRNAs into stress granules in vivo is unclear, it has been suggested that m6A, particularly in longer mRNAs containing multiple numbers of heavily modified m6A sites, can be involved in stress granule recruitment 53 .…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the observed effect in our experiment could be driven by a reduction in the production of the toxic 90 aa N-terminal HTT-exon1 protein as a consequence of the downregulation of Htt1a transcripts. However, in line with the potential role of Htt1 a in disease progression by formation of RNA foci at transcriptional sites 8 , it is also possible that reduced methylation in Htt1a would reduce the interaction with other Htt transcripts in RNA clusters avoiding for instance, sequestering of RNA binding proteins involved in DNA damage 80 .…”

Section: Discussionmentioning

confidence: 99%

“…In the context of expanded CAGs, it has been described that besides full length (FL) HTT mRNA isoforms, two small transcripts containing exon 1 and a 5’ region of intron 1 sequences ( HTT1a ) can be generated by incomplete splicing due to aberrant polyadenylation at cryptic polyA sites within intron 1 followed by premature termination of transcription 4,5 . HTT1a not only encodes the aggregation-prone HTTexon1 protein that is known to be highly pathogenic 6 but it can also form mRNA nuclear clusters which are resistant to treatment with HTT antisense oligonucleotides (ASOs) 7,8 . As the alternative processing of HTT mRNA, and consequently the levels of HTT1a and HTTexon1, increase with increasing CAG repeat length 9 it has been suggested that HTT1a and HTTexon1 may be the pathogenic contributor of somatic CAG repeat expansion in HD 10 .…”

Section: Introductionmentioning

confidence: 99%

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m6A RNA modification of mHttintron 1 regulates the generation ofHtt1ain Huntington’s Disease

Pupak,

Navarro,

Sathasivam

et al. 2023

Preprint

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Huntington’s 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 generatesHTT1atranscripts 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 ofHTT1aare not fully understood. In a previous transcriptome-wide m6A landscape study performed inHdh+/Q111knock-in mice, we have found that the proximal region of intron 1 to exon1-intron 1 splice site inHttRNA 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 ofHtt1a. We show in the striatum ofHdh+/Q111mice 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 inHtt1apolyadenylated mRNA. We also verified the presence of specific m6A-modified sites near the 5’ exon1-intron1 splice donor site. IntronicHTTm6A methylation was recapitulated in human samples showing a significantly increased methylation ratio in HD putamenpost-mortemsamples 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 mutantHttRNA processing, we performed a pharmacological inhibition of METTL3 and a targeted demethylation ofHttintron 1 in HD cells using a dCas13-ALKBH5 system. We found thatHtt1atranscript levels in HD cells are regulated by METTL3 and by methylation status inHttintron 1. Site-specific manipulation with an RNA editing system resulted in decreased expression levels ofHtt1a, 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 mutantHtttranscripts with important implications for therapeutic strategies.

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“…Recent studies indicate that uninterrupted CAG repeat length, not polyQ length (encoded by CAA-CAG), affects the rate of pathogenesis in HD patients, suggesting the potential importance of mutant HTT DNA / mRNA in HD ( 4 ). Indeed, in mouse models of HD, introducing a copy of huntingtin containing the mutant expanded CAG tract leads to HD-like phenotypes in vivo ( 5 – 7 ). The ability of different compounds to modulate not only the protein but also mutant HTT mRNA needs to be considered.…”

Section: Introductionmentioning

confidence: 99%

mRNA nuclear clustering leads to a difference in mutant huntingtin mRNA and protein silencing by siRNAsin vivo

Allen,

O’Reilly,

Miller

et al. 2024

Preprint

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Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence HTT mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a di-valent scaffold and delivered to two mouse models of HD. In both models, di-valent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild- type Htt protein was observed in both models. Subsequent fluorescent in situ hybridization (FISH) analysis shows that di-valent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

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Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains

Cited by 9 publications

References 87 publications

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

m6A RNA modification of mHttintron 1 regulates the generation ofHtt1ain Huntington’s Disease

mRNA nuclear clustering leads to a difference in mutant huntingtin mRNA and protein silencing by siRNAsin vivo

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