See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. Ras-GAP SH3-domain-binding protein 1 (G3BP1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3’UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. G3BP1 (Ras-GAP SH3-domain-binding protein 1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3’UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

RNA binding proteins (RBPs) play a key role in cellular growth, homoeostasis and survival and are tightly regulated. A deep understanding of their spatiotemporal regulation is needed to understand their contribution to physiology and pathology. Here, we have characterized the spatiotemporal expression pattern of hnRNP A1 and its splice variant hnRNP A1B in mice. We have found that hnRNP A1B expression is more restricted to the CNS compared to hnRNP A1, and that it can form an SDS-resistant dimer in the CNS. Also, hnRNP A1B expression becomes progressively restricted to motor neurons in the ventral horn of the spinal cord, compared to hnRNP A1 which is more broadly expressed. We also demonstrate that hnRNP A1B is present in neuronal processes, while hnRNP A1 is absent. This finding supports a hypothesis that hnRNP A1B may have a cytosolic function in neurons that is not shared with hnRNP A1. Our results demonstrate that both isoforms are differentially expressed across tissues and have distinct localization profiles, suggesting that the two isoforms may have specific subcellular functions that can uniquely contribute to disease progression.