TDP-43 Loss Induces Cryptic Polyadenylation in ALS/FTD

Background Introduction

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two severe neurodegenerative diseases affecting hundreds of thousands of people worldwide. Extensive research has shown that the RNA-binding protein TDP-43 (TAR DNA-binding protein 43) displays abnormal nuclear depletion and cytoplasmic aggregation in these diseases, serving not only as a cellular hallmark of ALS but also being highly correlated in FTD. Additionally, TDP-43 pathology has been detected in over 50% of Alzheimer’s disease patient brain tissues. Normally, TDP-43 is located in the nucleus and participates in multiple processes, including pre-mRNA splicing, transport, and polyadenylation (APA), which are essential for regulating gene expression and protein synthesis.

In the past decade, it has become clear that loss of nuclear TDP-43 lifts the repression of “cryptic splicing,” leading to abnormal inclusion of pre-mRNA fragments (known as “cryptic exons”) into mature transcripts. This can result in decreased protein expression due to nonsense-mediated decay (NMD) or the production of aberrant protein fragments that serve as pathological markers and potential therapeutic targets. Such splicing events have become a hot topic in TDP-43 inactivation research.

On the other hand, polyadenylation (APA) at the 3’ end of gene transcripts profoundly affects RNA maturation, stability, localization, and translation. APA can be categorized into three major types: alternative last exon selection (ALE), 3’ untranslated region (3’UTR) extension (3’ext) or shortening, and intronic polyadenylation (IPA). Many genes undergo APA at different positions, resulting in transcript diversity. However, APA abnormalities induced by TDP-43 inactivation have received much less attention compared to splicing events, potentially obscuring a significant class of pathological mechanisms and therapeutic targets.

Therefore, this study aims to systematically reveal the landscape of cryptic APA events induced by TDP-43 inactivation in neurons, explore their molecular features, mechanisms, and contributions to ALS/FTD pathology, and fill this gap in our understanding of the field.

Paper Source and Author Information

The paper, entitled “TDP-43 loss induces cryptic polyadenylation in ALS/FTD,” was published in the renowned scientific journal Nature Neuroscience (volume 28, November 2025, pp. 2190–2200), DOI: 10.1038/s41593-025-02050-w. The work was conducted by Sam Bryce-Smith, Anna-Leigh Brown, Max Z. Y. J. Chien, Dario Dattilo, and others. The authors are affiliated with top research institutes, including University College London (UCL), The Francis Crick Institute, the National Institutes of Health (NIH), Icahn School of Medicine at Mount Sinai, and the New York Genome Center, bringing together leading experts in ALS and FTD research.

Detailed Analysis of the Research Workflow

Data Collation and Model Construction

The authors first built a large database of RNA sequencing (RNA-seq) datasets, covering both publicly available and newly generated data under TDP-43 depletion conditions, from human neuron-like cells and some brain tissues. They developed an innovative bioinformatics analysis workflow to systematically identify and classify APA events:

1. Raw Data Collection: The authors gathered and integrated multiple bulk RNA-seq datasets, including those from induced pluripotent stem cell (iPSC)-derived neurons, cell lines, and human brain tissues.
2. APA Event Identification Workflow: They used StringTie to assemble transcripts (identifying novel last exons), combined with the polyAsite database and inherent polyadenylation signal hexamer filtering to exclude false positives. Salmon was used to quantify transcript expression, and DexSeq for differential exon usage analysis. Ultimately, APA events were categorized into ALE, IPA, and 3’ext types, with “cryptic” events defined as those with control group average usage <10% and >10% change after TDP-43 depletion.
3. Molecular Mechanism Exploration and Experimental Validation:
   • iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation) experiments were used to detect TDP-43 binding distribution, especially focusing on ALE and 3’ext events.
   • Hexamer enrichment analysis (using the innovative PEKA algorithm) was applied to explore the sequence features and regulatory capabilities of TDP-43 binding regions.
   • Reporter gene systems (e.g., ELK1 3’UTR extension) were constructed, manipulating UG dinucleotide content to experimentally control TDP-43 binding/regulation, and quantitatively analyzing APA site activity changes.
   • A variety of molecular biology and omics experiments, including 3’RACE (rapid amplification of RNA 3’ ends), Nanopore sequencing (direct RNA sequencing), SLAM-seq (metabolic labeling to detect RNA stability), Ribo-seq (ribosome profiling), FISH (fluorescence in situ hybridization), Western blot, and subcellular fractionation (nuclear-cytoplasmic separation and qPCR), were rigorously used to validate the molecular and functional effects of various events.

Main Experimental Processes and Innovative Methodologies

• APA Event Discovery and Classification: After stringent screening and differential analysis, the study identified a total of 227 cryptic APA events activated under TDP-43 depletion, including 92 ALEs, 108 3’exts (86 newly discovered 3’UTR extensions, 20 cases of 3’UTR shortening), 20 IPAs, and 9 complex events. Some overlapped with known cryptic exons (e.g., STMN2, ARHGAP32, RSF1); many were novel APA sites, independently verified by 3’RACE and Nanopore sequencing, demonstrating an accurate workflow.
• TDP-43 Binding and Regulatory Mechanism: iCLIP experiments revealed that TDP-43 is significantly enriched downstream of the splice acceptor sites and APA sites of ALE events, proving TDP-43’s dual regulatory ability (can repress or enhance APA site activity). Through the ELK1 reporter system and editing of the UG dinucleotide region, the study demonstrated that changes in the TDP-43 binding region directly affected APA usage, validating mechanistic causality.
• Detection in Clinical Samples: In FTD and ALS patient brain tissues, the authors performed FACS sorting (TDP-43 positive/negative neurons) and NYGC ALS Consortium cohort analysis, finding about 54 cryptic APA events with significantly increased expression in TDP-43-depleted neurons, especially ALE and 3’ext events. This provides a series of molecular evidence for clinical relevance of abnormal splicing and APA events.
• Functional Effects Detection: Through co-analysis of RNA-seq and Ribo-seq, most cryptic APA events were found to accompany significant changes in transcript expression (both upregulation and downregulation), but APA type was correlated with effect direction: ALE and IPA events mostly led to decreased expression, while a subset of 3’ext events (especially in transcription factors such as ELK1, SIX3, TLX1) showed significant upregulation of protein expression and functional enhancement. SLAM-seq demonstrated that these 3’ext events confer greater stability on transcripts (longer half-lives), and FISH and fractionation experiments showed that their RNAs migrate to the cytoplasm, enhancing protein synthesis.
• Transcription Factor Functional Changes: The study focused on ELK1, SIX3, and TLX1 transcription factors, finding that their cryptic 3’UTR extensions led to increased protein levels after TDP-43 depletion, with ELK1 target gene expression also showing marked changes. This suggests that cryptic APA can promote protein overexpression by stabilizing transcription factor RNAs, potentially participating in disease pathogenesis.

Key Results and Logical Reasoning

1. Discovery of widespread cryptic APA induced by TDP-43 depletion. Previously, only splicing was a major research focus; the study systematically identified multiple APA types, completing the landscape of RNA abnormal processing under TDP-43 loss.
2. TDP-43 has dual regulatory capacity in APA. Combined analysis from iCLIP showed TDP-43 can both repress and enhance usage of certain APA sites, with specific mechanisms linked to binding sequence position and the UG region.
3. Cryptic APA expression is closely related to ALS/FTD pathology. Numerous cryptic APA events were detectable in patient brain tissues and cell models. Well-known events like STMN2 further support their potential as molecular biomarkers.
4. Cryptic 3’UTR extension confers greater RNA stability and promotes protein upregulation, particularly in neural transcription factors ELK1/SIX3/TLX1, altering downstream gene expression and function.
5. Potential pathological mechanisms and new application targets. The logical link between cryptic APA event detection, abnormal RNA processing, and protein dysregulation clarifies new pathogenic mechanisms for ALS/FTD and provides rich cues for biomarker and therapeutic target development.

Conclusions, Significance and Application Value

Through cross-disciplinary development of computational biology workflows and rigorous multi-omics validation, this study systematically revealed the widespread phenomenon of cryptic polyadenylation induced by nuclear TDP-43 depletion in neurons, filling a critical gap in the molecular pathology of neurodegenerative diseases such as ALS and FTD.

Scientific significance: - Broadens our view from the previously emphasized splicing errors under TDP-43 loss, extending into the new field of 3’ end modification; - Innovatively developed and validated APA event detection and quantification pipelines, enhancing RNA-seq data mining resolution; - Revealed the molecular basis for TDP-43’s bidirectional regulation of APA (both repression and enhancement), guiding future mechanistic studies; - Thoroughly detailed how cryptic 3’UTR extension boosts transcription factor RNA stability and protein expression, complementing the classic view by showing RNA processing errors can also result in protein overexpression (not just loss or aberrant expression); - Established the strong clinical relevance of cryptic APA in brain tissue and patient molecular markers, creating new opportunities for diagnostics, monitoring, and therapeutic target development.

Application value: - Cryptic APA and related protein products can serve as molecular markers of TDP-43 pathology, improving diagnostic sensitivity for ALS/FTD; - For cryptic 3’ext events like ELK1, the study not only supports mechanisms for research and targeted therapy, but also suggests new approaches in molecular detection and RNA-targeted drug development; - The rich database resources and reusable computing/experimental tools serve the wider industry as both data and methodological assets.

Highlights and Unique Features

• Algorithm and Workflow Innovation: The self-developed APA detection and analysis system enables comprehensive classification and quantitative analysis of new RNA processing event types (ALE, IPA, 3’ext), surpassing the limitations of traditional tools restricted to only certain event types.
• Robust Multiplatform Multi-omics Integration: The study integrates RNA-seq, Ribo-seq, SLAM-seq, iCLIP, and more across clinical cohorts and cell models, ensuring broad data scope and reliable conclusions.
• Comprehensive Mechanistic and Functional Experiments: Using gene reporter systems, FISH, protein interaction assays and more, the research progressively and clearly reveals molecular regulatory pathways and functional consequences.
• Outstanding Clinical Significance: Extensive detection of cryptic APA events in patient brain tissue, not just in lab models, verifies their role in disease development and boosts clinical application potential.

Other Valuable Content

• The paper cross-references contemporary studies on APA published in the same issue of Nature Neuroscience (Zeng et al. 2025), as well as several authoritative works on TDP-43 inactivation and abnormal splicing, providing mutual support for its conclusions and highlighting cryptic APA as a new research hotspot in ALS/FTD.
• The authors have made their data and analysis pipeline available for subsequent research reuse and expansion, opening collaborative innovation space for the field.

Summary

This study represents a major breakthrough in understanding the molecular mechanisms of TDP-43 loss in ALS/FTD. Through innovative workflows and in-depth functional validation, it opens new perspectives on the involvement of RNA processing errors in neurodegenerative diseases. By revealing widespread cryptic polyadenylation, its regulation and functional consequences, the research paves the way for advances in diagnostics, biomarker screening, and targeted intervention strategies, holding promise to benefit patients and exerting an important impact on neurobiology and molecular medicine.