Aberrant Splicing in Huntington’s Disease Accompanies Disrupted TDP-43 Activity and Altered m6A RNA Modification

Academic Background

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms. The disease is caused by a CAG repeat expansion in the HTT gene, leading to abnormal polyglutamine repeat sequences in the huntingtin (HTT) protein. Although the mutation mechanism of the HTT gene has been extensively studied, the mechanisms underlying RNA processing abnormalities in HD remain unclear. In particular, the specific role of aberrant RNA splicing in HD has not been fully elucidated. In recent years, the role of RNA-binding proteins (RBPs) and RNA modifications (such as m6A methylation) in neurodegenerative diseases has garnered increasing attention. TDP-43 (TAR DNA-binding protein 43) is an RBP that plays a critical role in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD), but its function in HD remains unclear.

This study aims to explore the regulatory roles of TDP-43 and m6A RNA modification in HD, uncovering their potential mechanisms in aberrant RNA splicing in HD.

Source of the Paper

This paper was co-authored by Thai B. Nguyen, Ricardo Miramontes, and others, with the research team from multiple institutions, including the University of California, Irvine and the Ludwig Institute for Cancer Research. The study was published in February 2025 in the journal Nature Neuroscience, with the title “Aberrant splicing in Huntington’s disease accompanies disrupted TDP-43 activity and altered m6A RNA modification.”

Research Process and Results

1. RNA Processing Abnormalities in HD Mouse Models

The study first performed RNA sequencing (RNA-seq) on the R6/2 transgenic mouse model of HD, analyzing RNA splicing changes in the striatum and cortex of 3-month-old mice. The results showed a significant increase in splicing abnormalities, particularly in exon skipping events. Using the RMATS (RNA-seq multivariate analysis of transcript splicing) tool, the researchers identified 9,033 and 10,074 significant splicing events in the cortex and striatum, respectively. Further Gene Ontology (GO) analysis revealed that these splicing abnormalities mainly involved genes related to neuronal synaptic transmission.

To validate these splicing changes, the researchers employed RASL-seq (RNA-mediated oligonucleotide annealing, selection and ligation with next-generation sequencing) to analyze splicing events in the R6/2 mouse model and two other HD models (Q150 and Q175). The results showed a significant increase in exon skipping events in HD mice, with these changes exhibiting dose-dependency and age-dependency.

2. Enrichment of TDP-43 and m6A Motifs in HD Mice

To explore the potential mechanisms underlying splicing abnormalities, the researchers conducted de novo motif analysis and found that skipped exons in HD mice were enriched for UG/GU motifs, which are binding sites for TDP-43. Additionally, the classical m6A motif DRACh (D = A/G/U, R = A/G, H = A/C/U) was identified, suggesting that m6A modification may play a role in splicing regulation in HD.

3. Discovery of Unannotated Splicing Events in HD

The researchers further used MAJIQ and LeafCutter tools to analyze unannotated splicing events in the cortex and striatum of HD mice. The results showed that approximately 50% of the splicing changes were unannotated, primarily involving genes related to neuronal development and function. Through single-cell RNA-seq and long-read sequencing (PacBio Iso-seq), the researchers validated the existence of these unannotated splicing events and found that they were closely associated with TDP-43 loss.

4. Attenuation of TDP-43 Binding in HD

To investigate the role of TDP-43 in HD, the researchers performed TDP-43 eCLIP-seq (enhanced crosslinking and immunoprecipitation sequencing) to analyze TDP-43 binding sites in the cortex and striatum of HD and control mice. The results showed a significant reduction in TDP-43 binding in HD mice, particularly in downregulated genes. Additionally, TDP-43 binding sites were closely associated with m6A modification sites, suggesting that TDP-43 function may depend on m6A modification.

5. Reduced Nuclear Localization of TDP-43 in HD Mouse and Human Brains

Through immunofluorescence staining, the researchers found a significant reduction in the nuclear localization of TDP-43 in HD mice and human patient brains, with phosphorylated TDP-43 (pTDP-43) accumulating in the cytoplasm. Furthermore, the researchers discovered a novel nuclear pTDP-43 aggregation-like structure (aggregation-like bodies, AL bodies), which co-localized with HTT protein, suggesting that abnormal aggregation of TDP-43 may be part of HD pathology.

6. Aberrant m6A Modification in HD

The researchers further analyzed changes in m6A modification in HD mice. Through m6A eCLIP-seq, they found a significant reduction in m6A modification sites in HD mice, particularly in downregulated genes. Additionally, the reduction in m6A modification was closely associated with decreased TDP-43 binding, indicating that m6A modification may regulate TDP-43 function in HD.

Conclusions and Significance

This study reveals the important roles of TDP-43 dysfunction and abnormal m6A RNA modification in aberrant RNA splicing in HD. The research shows that reduced nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 are key features of HD pathology. Furthermore, the reduction in m6A modification may regulate gene expression and splicing by affecting TDP-43 binding. These findings provide new insights into the pathological mechanisms of HD and suggest that TDP-43 and m6A modification may be potential therapeutic targets for HD.

Research Highlights

1. TDP-43 Dysfunction in HD: The study systematically reveals TDP-43 dysfunction in HD for the first time, particularly its reduced nuclear localization and cytoplasmic accumulation of phosphorylated TDP-43.
2. Abnormal m6A Modification: The study finds that m6A modification is significantly reduced in HD, and this change is closely associated with TDP-43 binding, suggesting the regulatory role of m6A modification in HD.
3. Discovery of Unannotated Splicing Events: Through single-cell RNA-seq and long-read sequencing, the researchers discovered a large number of unannotated splicing events, which are closely related to HD pathology.
4. Novel Nuclear pTDP-43 Aggregation-like Structures: The researchers identified nuclear pTDP-43 aggregation-like structures in HD patient brains for the first time, which may be related to the progression of HD pathology.

Research Value

This study not only reveals the important roles of TDP-43 and m6A modification in HD but also provides new insights into the pathological mechanisms of HD. These findings offer a theoretical foundation for developing therapeutic strategies targeting TDP-43 and m6A modification, holding significant scientific and applied value.