Matthew J. Keuss

TDP-43 nuclear depletion is a defining pathological feature of amyotrophic lateral sclerosis and frontotemporal dementia that induces widespread cryptic splicing defects in hundreds of transcripts ^1–8 . Despite this, whether TDP-43 loss affects synaptic function has remained unclear. Here we show that TDP-43 loss induces a predegenerative state in which neurons remain structurally intact but suffer a substantial breakdown in synaptic communication. Genetic correction of a single cryptic splicing event in UNC13A was sufficient to rescue these deficits. We developed splice-switching antisense oligonucleotides that correct UNC13A cryptic splicing in human cortical and spinal motor neurons and in a humanized mouse model. In human neurons this intervention restored spontaneous and evoked synaptic vesicle release, release probability at individual synapses and wider network synchrony. Together, this establishes UNC13A splice correction as a mechanistically defined and genetically validated therapeutic strategy relevant to roughly 97% of patients with amyotrophic lateral sclerosis and roughly 50% of patients with frontotemporal dementia with TDP-43 pathology.

The cell-intrinsic capacity of neurons to regenerate axons requires widespread coordination of the transcriptome, activation of multiple kinases, and reorganization of the cytoskeleton. Axonal repair is also influenced by extrinsic activating factors, such as neurotrophins. Here, we found that the neurotrophin BDNF amplifies multiple neuron-intrinsic programs to foster axonal regeneration in human iPSC-derived lower motor neurons (i ³ LMNs). Metabolic RNA sequencing (SLAM-seq) and phosphoproteomic profiling of i ³ LMNs revealed that BDNF temporally regulated the expression and RNA stability of functionally distinct transcriptional programs that included regeneration-associated gene sets, further enhancing their expression. BDNF also regulated the phosphorylation of multiple proteins involved in cytoskeletal dynamics. In compartmentalized cultures of neurons, in which microfluidic chambers isolate somata from their axons, BDNF-induced regeneration depended on axon-specific activation of the ERK-RSK-S6K kinase pathway. The findings show that extrinsic BDNF signaling coordinates intrinsic axon-regeneration programs and highlight the role of spatially regulated kinase activation in this process.

TDP-43 nuclear depletion in amyotrophic lateral sclerosis (ALS) causes de-repression of cryptic exons (CEs) in multiple transcripts, including UNC13A and STMN2 , disrupting synaptic transmission and neurite outgrowth. We developed a therapeutic U7 snRNA (tU7) approach that suppresses TDP-43-dependent mis-splicing, restores target gene expression, rescues neuronal functions in human iPSC-derived neurons, and shows target engagement in vivo , positioning tU7-mediated splicing correction as a promising therapeutic strategy for ALS.

Cryptic splicing caused by TDP-43 proteinopathy is a hallmark of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, which cryptic splicing events (CEs) are the most sensitive to TDP-43 depletion, where CEs localise within cells, and how specific CEs are in human tissues is poorly defined. Analyses of in vitro TDP-43 knockdowns and postmortem RNA-seq datasets revealed that a small subset out of thousands of CEs are specific markers for TDP-43 proteinopathy in vivo . Nonsense-mediated decay (NMD) masked a portion of CEs, influencing their subcellular localization and detectability in tissue. Dose-dependent TDP-43 depletion identified “early-responsive” CEs, which possess stronger splice sites and denser, more canonical TDP-43 binding motifs. Finally, we developed a composite cryptic burden score that effectively captured TDP-43 pathology across heterogeneous tissues and correlated with regional vulnerability and genetic background. Our work identifies robust biomarkers and offers new insights into TDP-43-mediated splicing dysregulation in neurodegeneration.

Motor neuron hyperexcitability is a broadly observed yet poorly understood feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear depletion and cytoplasmic aggregation of the RNA splicing protein TAR DNA-binding protein 43 (TDP-43) are observed in most ALS and FTD patients. Here we show that TDP-43 dysfunction causes mis-splicing of KCNQ2, which encodes a voltage-gated potassium channel (K_v7.2) that regulates neuronal excitability. Using iPSC-derived neurons and postmortem ALS/FTD brain and spinal cord tissue we find widespread, disease-specific and TDP-43-specific skipping of an exon encoding the KCNQ2 pore domain. The mis-spliced mRNA escapes degradation and is translated into a nonfunctional protein with severely reduced ion conductance that aggregates in the endoplasmic reticulum and causes intrinsic hyperexcitability in ALS neuronal models. This event, which correlates with higher phosphorylated TDP-43 levels and earlier age of disease onset in patients, can be rescued by splice-modulating antisense oligonucleotides that dampen hyperexcitability in induced pluripotent stem cell cortical neurons and spinal motor neurons with TDP-43 depletion. Our work reveals that nuclear TDP-43 maintains the fidelity of KCNQ2 expression and function and provides a mechanistic link between established excitability disruption in ALS/FTD patients and TDP-43 dysfunction.

Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 are cellular hallmarks of amyotrophic lateral sclerosis (ALS). TDP-43 nuclear loss causes de-repression of cryptic exons, yet cryptic alternative polyadenylation (APA) events have been largely overlooked. In this study, we developed a bioinformatic pipeline to reliably identify alternative last exons, 3’ untranslated region (3’UTR) extensions and intronic polyadenylation APA event types, and we identified cryptic APA sites induced by TDP-43 loss in induced pluripotent stem cell (iPSC)-derived neurons. TDP-43 binding sites are enriched at sites of these cryptic events, and TDP-43 can both repress and enhance APA. All categories of cryptic APA were also identified in ALS and frontotemporal dementia (FTD) postmortem brain tissue. RNA sequencing (RNA-seq), thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) and ribosome profiling (Ribo-seq) revealed that distinct cryptic APA categories have different downstream effects on transcript levels and that cryptic 3’UTR extensions can increase RNA stability, leading to increased translation. In summary, we demonstrate that TDP-43 nuclear depletion induces cryptic APA, expanding the palette of known consequences of TDP-43.

Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders.

TDP-43 loss of function induces multiple splicing changes, including a cryptic exon in the amyotrophic lateral sclerosis and fronto-temporal lobar degeneration risk gene UNC13A, leading to nonsense-mediated decay of UNC13A transcripts and loss of protein. UNC13A is an active zone protein with an integral role in coordinating pre-synaptic function. Here, we show TDP-43 depletion induces a severe reduction in synaptic transmission, leading to an asynchronous pattern of network activity. We demonstrate that these deficits are largely driven by a single cryptic exon in UNC13A. Antisense oligonucleotides targeting the UNC13A cryptic exon robustly rescue UNC13A protein levels and restore normal synaptic function, providing a potential new therapeutic approach for ALS and other TDP-43-related disorders.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43–depleted human iPSC–derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43–depleted human iPSC–derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43–depleted human iPSC–derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of ALS, occurring in over 97% of cases. A key consequence of TDP-43 nuclear loss is the de-repression of cryptic exons. Whilst TDP-43 regulated cryptic splicing is increasingly well catalogued, cryptic alternative polyadenylation (APA) events, which define the 3’ end of last exons, have been largely overlooked, especially when not associated with novel upstream splice junctions. We developed a novel bioinformatic approach to reliably identify distinct APA event types: alternative last exons (ALE), 3’UTR extensions (3’Ext) and intronic polyadenylation (IPA) events. We identified novel neuronal cryptic APA sites induced by TDP-43 loss of function by systematically applying our pipeline to a compendium of publicly available and in house datasets. We find that TDP-43 binding sites and target motifs are enriched at these cryptic events and that TDP-43 can have both repressive and enhancing action on APA. Importantly, all categories of cryptic APA can also be identified in ALS and FTD post mortem brain regions with TDP-43 proteinopathy underlining their potential disease relevance. RNA-seq and Ribo-seq analyses indicate that distinct cryptic APA categories have different downstream effects on transcript and translation. Intriguingly, cryptic 3’Exts occur in multiple transcription factors, such as ELK1 , SIX3, and TLX1, and lead to an increase in wild-type protein levels and function. Finally, we show that an increase in RNA stability leading to a higher cytoplasmic localisation underlies these observations. In summary, we demonstrate that TDP-43 nuclear depletion induces a novel category of cryptic RNA processing events and we expand the palette of TDP-43 loss consequences by showing this can also lead to an increase in normal protein translation.