Kai Sun

Abstract 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.

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.

Abstract 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 reveal that brain-derived neurotrophic factor (BDNF) amplifies multiple neuron-intrinsic programs to foster axonal regeneration in human motor neurons. Through metabolic RNA sequencing and phosphoproteomic profiling, we elucidate BDNF signalling and its role in axonal regeneration. We discover that BDNF controls RNA stability and transcriptional programs that converge with regeneration-associated gene (RAG) sets. We further unveil that BDNF governs the phosphorylation of multiple proteins essential for cytoskeletal dynamics, a major determinant of effective nerve regeneration. Using compartmentalized neuronal cultures, we demonstrate that the regeneration driven by BDNF depends on the axon-specific activation of ERK/RSK/S6K kinase pathway. We propose a model in which BDNF augments neuron-intrinsic pathways to drive axonal regeneration in human motor neurons. Teaser BDNF aids nerve repair by fine-tuning the metabolism of RNA and by changing the building blocks of the nerve cell cytoskeleton.

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.

Abstract The polarised and complex morphology of neurons pose massive challenges for efficient cargo delivery between the axon and soma, a process termed axonal transport. We have previously shown that the retrograde axonal transport of pro-survival, neurotrophic signalling endosomes relies on Rab7 in motor neurons, and that their trafficking is impaired in the early stages of amyotrophic lateral sclerosis (ALS) pathogenesis. Here, we report the effect of Rab7 phosphorylation on the transport of these signalling endosomes. We show that the ALS-linked kinase TBK1 phosphorylates Rab7 at S72 in neurons, altering its binding to cytoplasmic dynein adaptors. Accordingly, both TBK1 knockdown and the expression of a loss-of-function Rab7 mutant (S72E) induce aberrant bidirectional movement of signalling endosomes without modifying neuronal polarity or endosomal sorting. This alteration is specific for signalling endosomes, as axonal transport of lysosomes and mitochondria remain unaffected. We have therefore discovered a new TBK1 function that ensures the unidirectional transport of signalling endosomes, suggesting that reduced TBK1 activity determines retrograde transport dysfunctions and long-range signalling impairments. Summary Signalling endosomes travel from the distal axon to the cell body, exerting pro-survival functions. TBK1, an ALS-linked kinase, governs the directionality of signalling endosome transport in motor neurons by phosphorylating Rab7. Thus, loss of TBK1 function results in long-range signalling impairment.