Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity. Electronic supplementary material The online version of this article (10.1007/s00401-018-1946-4) contains supplementary material, which is available to authorized users.

We recently described aberrantly increased cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as new hallmarks of amyotrophic lateral sclerosis (ALS). However the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep-sequencing of nuclear and cytoplasmic fractions of hiPSCs undergoing motor neurogenesis, we reveal that ALS-causing VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify >100 IRTs with increased cytoplasmic abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins (RBPs). Remarkably, TDP-43, SFPQ and FUS—RBPs known for nuclear-to-cytoplasmic mislocalization in ALS—abundantly and specifically bind to this aberrant cytoplasmic pool of IRTs, as opposed to any individual IRT. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets.

Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients.

SUMMARYWe recently described aberrant cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as a new hallmark of amyotrophic lateral sclerosis (ALS). However the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep-sequencing of nuclear and cytoplasmic fractions of hiPSCs undergoing motor neurogenesis, we reveal that ALS-causing VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify >100 IRTs with increased cytoplasmic (but not nuclear) abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins (RBPs). Remarkably, TDP-43, SFPQ and FUS – RBPs known for nuclear-to-cytoplasmic mislocalization in ALS – avidly and specifically bind to this aberrant cytoplasmic pool of IRTs, as opposed to any individual IRT. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets.Abstract Figure

Reactive astrocytes are implicated in amyotrophic lateral sclerosis (ALS), although the mechanisms controlling reactive transformation are unknown. We show that decreased intron retention (IR) is common to human-induced pluripotent stem cell (hiPSC)-derived astrocytes carrying ALS-causing mutations in VCP, SOD1 and C9orf72. Notably, transcripts with decreased IR and increased expression are overrepresented in reactivity processes including cell adhesion, stress response and immune activation. This was recapitulated in public-datasets for (i) hiPSC-derived astrocytes stimulated with cytokines to undergo reactive transformation and (ii) in vivo astrocytes following selective deletion of TDP-43. We also re-examined public translatome sequencing (TRAP-seq) of astrocytes from a SOD1 mouse model, which revealed that transcripts upregulated in translation significantly overlap with transcripts exhibiting decreased IR. Using nucleocytoplasmic fractionation of VCP mutant astrocytes coupled with mRNA sequencing and proteomics, we identify that decreased IR in nuclear transcripts is associated with enhanced nonsense mediated decay and increased cytoplasmic expression of transcripts and proteins regulating reactive transformation. These findings are consistent with a molecular model for reactive transformation in astrocytes whereby poised nuclear reactivity-related IR transcripts are spliced, undergo nuclear-to-cytoplasmic translocation and translation. Our study therefore provides new insights into the molecular regulation of reactive transformation in astrocytes.

Letters diseases on MRI and cerebrospinal fluid analysis [CSF] plus genetic testing). Canine DM has an insidious onset, followed by progressive non-painful neurodegeneration of the spinal cord. It was initially described in German shepherd dogs in 1973 (Averill 1973). The disease affects both sexes, at about eight years of age and leads to pelvic limb ataxia and paresis (often asymmetric), with preserved spinal reflexes (ie, an 'upper motor neuron' presentation). Nowadays it is identified in several breeds, for example, corgis, boxers, cavalier King Charles spaniels, Chesapeake Bay retrievers, Rhodesian ridgebacks and pugs (Zeng and others 2014). The insidious onset, slow progression and lack of spinal pain point the differential diagnosis towards a degenerative disease. Diagnosis relies on exclusion of compressive and inflammatory diseases of the spinal cord (with MRI/CSF analysis). We have an increasing understanding of DM pathology and genetics, thanks to research conducted at the University of Missouri (Dr J. Coates, Canine Genetic Diseases Network, www. caninegeneticdiseases.net/DM/basicDM. htm), which linked a mutation of the super-oxide dismutase 1 gene (SOD1)

In a recent investigation Deshaies and colleagues identify HNRNP A1-7B, an isoform of the RNA binding protein (RBP) HNRNP A1, as present in post mortem amyotrophic lateral sclerosis (ALS) spinal motor neuron inclusions (Deshaies et al., 2018). HNRNP A1 is an important player in ALS, as mutations in its low complexity domain (LCD) can be causative for the disease (Kim et al., 2013). Intriguingly, the isoform described by Deshaies et al.includes exon 7B, which produces an extension of the LCD by 52 amino acids, pointing to a link between this alternative splicing event and neuronal protein aggregation. The authors further suggest that loss of RBP TDP-43 can influence this splicing event and result in an increase of the aggregation-prone 7B isoform. TDP-43 is abnormally mislocalised in >95% of ALS cases and when mutated can cause ALS (Harrison and Shorter, 2017). TDP-43 binds RNA, preferentially at UG repeats, and is involved in alternative splicing (Buratti and Baralle, 2001). Importantly, its activity is extremely dosage sensitive, making physiological expression an essential condition for studying the effect of TDP-43 mutations on splicing. Recently, we and others have published an allelic series of novel physiological TDP-43 mouse mutant models and shown that C-terminal mutations induce RNA splicing gain of function (Fratta et al., 2018; White et al., 2018). In order to investigate the link between TDP-43, its mutations and Hnrnpa1 exon 7B splicing, we utilised recently published