Aberrant splicing exonizes C9orf72 repeat expansion in ALS/FTD

Neurology Basic Medical Sciences Cell Biology Biochemistry Genetics Life Sciences Medicine
aberrant splicing C9orf72 repeat expansion ALS frontotemporal dementia DPR proteins therapeutic targets

New Pathway for ALS/FTD-Related C9orf72 Pathogenesis Revealed by Latest Nature Neuroscience Study

Academic Background and Research Motivation

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are among the most challenging neurodegenerative diseases in clinical medicine, with complex pathogenesis that remains insufficiently explained. In recent years, the expansion of a G4C2 (ggggcc) hexanucleotide repeat within the first intron of the C9orf72 gene has been confirmed as one of the most common hereditary causes of ALS/FTD. In patients, the repeat number can expand from less than 12 in healthy individuals to hundreds or more, greatly increasing the prevalence of these diseases.

However, the C9orf72 repeat expansion is located in a non-coding (intronic) region. Traditional theory suggests that intronic RNAs are degraded or retained in the nucleus after splicing, and rarely exported to the cytoplasm for translation. Although previous studies have demonstrated that C9orf72 repeat expansions can, through a repeat-associated non-AUG (RAN) translation mechanism, encode multiple pathogenic dipeptide repeat proteins (DPRs), it has remained an open question as to how an intronic RNA is permitted for export and cytoplasmic translation. This fundamental challenge has long lacked a breakthrough mechanistic explanation.

This research was conducted to address this gap, aiming to elucidate the molecular mechanisms by which C9orf72 intronic hexanucleotide repeat expansion RNAs (NRE, Nucleotide Repeat Expansion) acquire the ability for cytoplasmic translation, to investigate whether this capability may be mediated through RNA processing or splicing, and to provide new targets for therapeutic interventions in ALS/FTD.

Paper Information and Authors

This article, entitled “Aberrant splicing exonizes c9orf72 repeat expansion in als/ftd”, was published in Nature Neuroscience, October 2025, Volume 28 (pages 2034-2043), with an online publication date of August 11, 2025. The authors are from the Yale University School of Medicine, Interdepartmental Neuroscience Program at Yale, Mayo Clinic Graduate School of Biomedical Sciences & Department of Neuroscience, University of Alabama at Birmingham School of Medicine, and other top-notch institutions. The lead corresponding author is Junjie U. Guo (junje.guo@yale.edu).

Detailed Research Process and Innovative Methods

1. Research Methodology and Innovative Experimental Workflow

1.1 Research Subjects and Grouping

The study employed multiple types of samples, including patient-derived fibroblasts, iPSC-derived motor neurons (MNs), and brain tissue samples. The subjects were classified into two groups: C9orf72 hexanucleotide expansion patients (c9 nre+) and non-expanded controls (c9 nre–). Sample sizes included numerous patient cases, as well as human brain samples from the New York Genome Center/Target ALS Consortium database (105 c9 nre+ and 503 c9 nre–).

1.2 Sample Processing and Molecular Detection Workflow

The workflow was highly innovative, developing a dedicated NRE capture sequencing technique (nre-capture-seq) to identify the scarcely enriched C9orf72 NRE RNA. Key steps included:

  • ASO-based nre-capture-seq
    The team used 5’-biotinylated antisense oligonucleotides (ASOs) targeting the C9orf72 hexanucleotide repeat region ((ccccgg)3). After extracting cytoplasmic RNAs from patient fibroblasts, motor neurons, and brain tissues, hybridization followed. Streptavidin magnetic bead capture and RNase H digestion released target NRE RNA, followed by low-input RNA-seq library prep and high-throughput sequencing.

  • Subcellular Fractionation and RT-qPCR
    To accurately localize NRE RNA in nucleus and cytoplasm, the team applied fractionation technologies, separating nuclear and cytoplasmic RNAs for NRE enrichment and qPCR quantification.

  • Detection and Analysis of Novel Splice Sites
    Utilizing highly sensitive sequencing and bioinformatic strategies (e.g., STAR, samtools markdup, etc.), they systematically analyzed splice isoforms and locus reads, quantitatively profiling novel aberrant splice site usage.

  • Functional Intervention Experiments
    Through siRNAs targeting splicing factors and NRE-binding proteins (such as SRSF1), and gapmer antisense oligonucleotides (targeting aberrant splice isoforms), the team evaluated regulatory effects on splicing and DPR protein levels. Protein expression was quantified with highly sensitive Meso Scale Discovery sandwich immunoassays.

1.3 Constructing Model Systems and Splicing Mechanism Assays

  • Luciferase Dual Reporter System and Artificial Splicing Models
    Various lengths of C9orf72 (ggggcc) repeats were inserted into custom luciferase reporter plasmids (with/without introns). By monitoring transcription and translation, the team explored repeat region effects on splicing and translation, and, via in vitro transcription (IVT) and RNA transfection experiments, distinguished direct splicing effects from effects on export/translation.

  • siRNA Screening and Functional Factor Identification
    A large-scale screen of known NRE RNA-binding proteins identified key mediators of aberrant splicing.

2. Key Experimental Findings and Data Interpretation

2.1 NRE RNA Capture and Isoform Characterization

After nre-capture-seq, C9orf72 NRE RNA was dramatically enriched in samples compared to controls, with DPR products rising by nearly 1000-fold. Control cells showed almost no C9 locus reads, indicating high specificity and sensitivity of this method.

2.2 Aberrant Splicing Leads to “Exonization” of the NRE Region

Key finding: The expansion of the C9orf72 hexanucleotide repeat alters intron 1 splicing behavior, activating several downstream cryptic 5’ splice sites, leading to the NRE region being incorporated as part of an extended exon 1. Three main isoforms result, each using different 5’ splice sites (ex1b, ex1c, ex1d) but sharing the same 3’ splice site, thereby generating cytoplasmic, translatable new mRNAs. These isoforms prominently accumulate in ALS/FTD brain tissues, and their formation is promoted by the splicing factor SRSF1.

2.3 Aberrant Isoforms Efficiently Export, and Serve as DPR Translation Templates

Subcellular fractionation and qPCR confirmed that the vast majority of NRE “exonized” splicing isoforms were efficiently exported to the cytoplasm, comparable in cytoplasmic distribution to normal C9 mRNA (40–60%). Knockdown of exon 2 via siRNA demonstrated that these splice isoforms are effective templates for DPR translation, causing significant reductions in downstream PolyGA and PolyGP dipeptides.

2.4 Cell-Type Specific Regulation of Aberrant Splicing

In patient iPSC-derived motor neurons, the ex1c–ex2 aberrant splice isoform became the dominant NRE RNA form, significantly exceeding levels in fibroblasts, indicating cell-type-specific splicing regulation. Targeted gapmer ASO intervention against the ex1c–ex2 splice site markedly reduced DPR products, reflecting promising therapeutic potential.

2.5 Validation of Aberrant Splicing Mechanism in Large Human Brain Cohorts

Analysis of brain tissue data from the New York Genome Center revealed widespread elevation of aberrant splice isoforms (ex1c–ex2, ex1d–ex2, ex1e–ex2) in c9 nre+ regions (cerebellum, frontal cortex, motor cortex), with low-level occurrences in non-NRE patients as well. This indicates that NRE expansion strongly activates the mechanism, with minimal spontaneous occurrence in non-pathogenic states.

Further analyses confirmed using TDP-43 nuclear depletion experiments that this aberrant splicing is a direct NRE-mediated event, not a downstream effect.

2.6 Repeat-Length Dependent Mechanism for Splice Activation

Luciferase reporter engineering experiments confirmed that as the ggggcc repeat number increased to pathological thresholds (>33×), novel cryptic 5’ splice site activation became pronounced. Splicing did not affect classic intron splice site initiation efficiency but directly promoted use of downstream cryptic splice sites, with effect strictly “cis” and minimal impact on endogenous transcriptomes, demonstrating high specificity.

2.7 Critical Role of Splicing Factor SRSF1

siRNA knockdown screening revealed that SRSF1 is pivotal in regulating NRE aberrant splicing. Knockdown markedly reduced aberrant splice isoforms, cytoplasmic NRE RNA export, and DPR products, without affecting C9 transcription and nuclear pre-mRNA levels. This points to SRSF1 as a potential therapeutic target, possibly acting through the “exonization” mechanism to facilitate NRE RNA translation and cytoplasmic export.

3. Conclusions, Scientific Significance, and Application Value

3.1 Mechanism Elucidation

This study was the first to reveal that C9orf72 hexanucleotide expansions are not translated via full intron retention or lariat ring structures, but by activating multiple downstream cryptic 5’ splice sites, incorporating the NRE region into an extended exon 1, producing complete 5’-capped, spliced, and 3’-polyadenylated mRNA, thereby promoting cytoplasmic export and efficient RAN translation. The splicing mechanism depends on repeat length and is regulated by splicing factors such as SRSF1.

3.2 Application and Scientific Innovation Value

  • New Targets for ALS/FTD Intervention: Targeting aberrant splicing factors (e.g., SRSF1) or using antisense oligonucleotides against aberrant splice isoforms can effectively lower pathogenic DPR protein levels, potentially opening new avenues for gene therapy.
  • Breakthrough in Splicing Mechanism Theory: This is the first clear demonstration of “exonization” as a central process in hereditary repeat expansion neurodegenerative diseases, providing referential value for mechanistic studies of other non-coding repeat disorders (e.g., HD, DM1/2).
  • Methodological Innovation and Broad Applicability: The nre-capture-seq protocol shows highly sensitive capture of rare disease-associated RNAs, applicable to other disease research. ASO and siRNA interventions are highly flexible.

3.3 Research Highlights

  • Revealed a new molecular mechanism underlying the most common genetic cause of ALS/FTD, solving a longstanding scientific puzzle about how non-coding RNAs are exported for cytoplasmic translation.
  • Identified aberrant exonization splicing mechanism and cell-type specificity, suggesting directions for precision clinical therapy.
  • Developed high-efficiency NRE RNA capture and analysis technology, opening new avenues for investigation of elusive RNA molecules.
  • Proposed SRSF1 and associated splicing factors as novel drug development targets, providing molecular disease pathway maps.

Other Valuable Information

  • The study suggests the possibility of similar “exonization” mechanisms in antisense NRE RNAs and other repeat expansion diseases, warranting development of analogous analytical technologies.
  • The major databases and analytical workflows employed (e.g., STAR, samtools) serve as useful references for transcriptome analysis in other neurogenetic diseases.
  • For NRE reporter construct experiments, the authors caution that future RAN translation mechanism studies must verify for aberrant splicing or other expression artifacts to avoid misinterpretation.

This groundbreaking study published in Nature Neuroscience (2025) revealed that RNA from ALS/FTD-associated C9orf72 repeat expansions can be incorporated into exons by aberrant splicing, gaining capacity for cytoplasmic export and translation, clarified its molecular mechanism, and presented important therapeutic intervention targets—a major advance for the field of neurogenetic disease research.