Simone Barattucci

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.

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.

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.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho‐family GTPases. It is a bi‐functional protein, acting both as a guanine exchange factor and as an RNA‐binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA ( NEFL ) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co‐localize with inclusions of TDP‐43, the major well‐known RNA‐binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss‐of‐function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP‐43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss‐of‐function of factors co‐aggregating with TDP‐43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA‐binding factors that can be co‐aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.