Nuclear depletion of TDP-43 is a defining neuropathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Loss of nuclear TDP-43 function disrupts normal RNA processing and has been previously linked to the inclusion of cryptic linear exons. The current study extends this concept by identifying a role for TDP-43 in regulating circular RNA biogenesis and by demonstrating that TDP-43 dysfunction can induce the formation of previously unreported cryptic circular RNAs (c-circRNAs).
Using multiple human neuronal cell models, the authors show that normal TDP-43 activity influences the production of circular RNAs (circRNAs). Experimental perturbation of TDP-43 function led to de novo generation of c-circRNAs, indicating that TDP-43 is a direct regulator of circRNA biogenesis in these systems. The study characterizes this regulatory relationship across the cellular models employed, reporting the induction of c-circRNAs specifically associated with TDP-43 dysfunction.
Transcriptomic analysis of post-mortem brain tissue from human donors identified a subset of c-circRNAs that are present specifically in ALS and FTD cases with TDP-43 pathology. These disease-associated c-circRNAs were distinguished from constitutive circRNAs and interpreted as de novo products linked to disease-related RNA misprocessing. The authors highlight that some of these c-circRNAs localize to genomic regions that also exhibit cryptic linear splicing events, suggesting spatially restricted RNA misprocessing hotspots in affected tissues.
To detect and quantify c-circRNAs with high sensitivity, the investigators developed rolling-circle amplification–based assays tailored for circRNA detection. These assays were applied to human central nervous system (CNS) tissues and demonstrated strong discriminatory performance for TDP-43 pathology, with an area under the receiver-operating-characteristic curve (AUC) reported as 0.99. The assays are presented as a sensitive method to distinguish TDP-43 pathology in tissue samples and as a potential platform for biomarker development.
The study reports that some c-circRNAs co-occur with cryptic linear splicing events, forming complex RNA misprocessing hotspots. These hotspots are associated with reduced expression of disease-relevant proteins. Two proteins specifically noted as affected by these RNA defects are RPTOR and EHMT1. The co-occurrence of circular and linear cryptic events suggests multifaceted consequences of TDP-43 dysfunction on RNA processing and downstream protein expression.
A notable finding is the identification of a c-circRNA originating from UNC13A. UNC13A has been previously implicated in ALS/FTD through a cryptic exon that overlaps a major genome-wide association study (GWAS) signal and is under investigation as a therapeutic target using splice-switching antisense oligonucleotides (ASOs). In this work, the UNC13A-derived c-circRNA (c-circUNC13A) is reported to be co-regulated with the linear cryptic transcript.
Functional experiments showed that suppression of the UNC13A cryptic exon reduces levels of c-circUNC13A in cultured neurons and in vivo. This coordinated regulation supports the concept that c-circUNC13A could serve as a target-engagement biomarker for emerging UNC13A-directed therapies, because changes in the cryptic exon inclusion appear to track with changes in the corresponding c-circRNA.
These findings identify a previously unrecognized class of RNA species—disease-associated cryptic circular RNAs—that arise in the context of TDP-43 dysfunction in ALS and FTD. The co-occurrence of c-circRNAs with cryptic linear splicing events and the association with loss of specific proteins suggest that c-circRNAs mark loci of severe RNA misprocessing with potential pathogenic consequences. The development of a high-sensitivity rolling-circle amplification assay that discriminates TDP-43 pathology with an AUC of 0.99 indicates a promising path toward tissue-based biomarkers for TDP-43–related disease.
The UNC13A example illustrates translational potential: because suppression of the cryptic exon reduces the corresponding c-circRNA in cellular and in vivo models, c-circRNAs may be useful for monitoring the molecular effects of splice-modulating therapies and for assessing target engagement in clinical development programs.
The authors declare that PF, DD and FP filed a patent concerning TDP-43–related circRNAs. PF is founder, advisor, and holds shares in Trace Neuroscience Inc.; MJK consults for and holds shares in Trace Neuroscience Inc. The article provides links to several data repositories and accession numbers for transcriptomic datasets used in the analyses; specifics were reported in the source. The study was supported by multiple funders listed by the authors, including Target ALS, the National Institutes of Health, the Packard Center for ALS Research, Trace Neuroscience Inc., and the Medical Research Council.
Overall, this work identifies a novel molecular consequence of TDP-43 dysfunction—the induction of disease-associated cryptic circular RNAs—and proposes sensitive detection methods and a candidate biomarker (c-circUNC13A) with potential to inform therapeutic development for ALS and FTD.