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SRSF1-Dependent Nuclear Export Inhibition of C9ORF72 Repeat Transcripts Prevents Neurodegeneration and Associated Motor Deficits

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ARTICLE Received 4 Dec 2016 | Accepted 24 May 2017 | Published 5 Jul 2017 DOI: 10.1038/ncomms16063 OPEN SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits Guillaume M. Hautbergue1,*,**, Lydia M. Castelli1,*, Laura Ferraiuolo1,*, Alvaro Sanchez-Martinez2, Johnathan Cooper-Knock1, Adrian Higginbottom1, Ya-Hui Lin1, Claudia S. Bauer1, Jennifer E. Dodd1, Monika A. Myszczynska1, Sarah M. Alam2, Pierre Garneret1, Jayanth S. Chandran1, Evangelia Karyka1, Matthew J. Stopford1, Emma F. Smith1, Janine Kirby1, Kathrin Meyer3, Brian K. Kaspar3, Adrian M. Isaacs4, Sherif F. El-Khamisy5, Kurt J. De Vos1, Ke Ning1, Mimoun Azzouz1, Alexander J. Whitworth2,** & Pamela J. Shaw1,** Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection. 1 Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, 385a Glossop Road, Sheffield S10 2HQ, UK. 2 MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK. 3 Nationwide Children’s Research Institute, Department of Pediatrics, The Ohio State University, 700 Children’s Drive, Rm. WA3022, Columbus, Ohio 43205, USA. 4 Department of Neurodegenerative Disease, UCL Institute of Neurology, London WC1N 3BG, UK. 5 Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Sheffield S10 2TN, UK. * These authors contributed equally to this work. ** These authors jointly supervised this work. Correspondence and requests for materials should be addressed to G.M.H. (email: g.hautbergue@sheffield.ac.uk) or to A.J.W. (email: [email protected]). NATURE COMMUNICATIONS | 8:16063 | DOI: 10.1038/ncomms16063 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms16063 myotrophic lateral sclerosis (ALS) and frontotemporal nuclear export receptor NXF1, remodel NXF1 in an open dementia (FTD) are fatal adult-onset neurodegenerative conformation in concert with subunits of the TREX Adiseases, which respectively cause progressive death of (Transcription-Export) complex to increase its affinity for motor neurons with escalating failure of the neuromuscular mature mRNAs and trigger the process of mRNA nuclear system and characteristic alterations of cognitive function and export35–39. The remodelling of NXF1 offers a control personality features. Neuroprotective treatment options are mechanism to retain unprocessed transcripts in the currently extremely limited and the anti-glutamatergic agent nucleus37,40. Knockdowns of ALYREF in Caenorhabditis riluzole prolongs survival in ALS patients by only approximately elegans41 and Drosophila melanogaster42 are dispensable to the 3 months. The most commonly identified genetic cause of ALS global nuclear export of mRNA and development. Notably, only a and FTD involves polymorphic repeat expansions, composed of partial block of nuclear export is induced upon ALYREF hundreds to thousands of the GGGGCC hexanucleotide-repeat depletion in human cells43, suggesting the existence of other sequence (hereafter abbreviated G4C2) in the first intron of the proteins with redundant export function. Consistent with this, C9ORF72 gene, with autosomal dominant inheritance and multiple conserved human nuclear export adaptors were incomplete penetrance1–4. The repeat DNA sequences are bi- found to interact with the RNA-binding domain of NXF1 directionally transcribed leading to the characteristic formation of (refs 34,37,44,45) and can simultaneously interact with the same G4C2-sense and C4G2-antisense RNA foci both in ALS and FTD mRNA molecules44. A recent study further showed that whilst cases5,6. The expression levels and splicing of transcripts involved each of the seven SR-rich splicing factors SRSF1-7 bind thousands in multiple cellular pathways are affected in ALS models of transcripts, the nuclear export of only a small proportion of and human post-mortem tissues leading to dysregulation of transcripts (o0.5–2% mRNAs) is affected upon individual RNA metabolism, mitochondrial dysfunction, oxidative stress, knockdown of the SRSF1-7 proteins, clearly highlighting excitotoxicity, apoptosis, altered mechanisms of autophagy, redundancy and/or cooperation in the NXF1-dependent nuclear protein clearance, axonal transport and motor neuron-astrocyte export adaptor function46. We refer to references47,48 for recent cross-talk (for reviews, see references1,3). Consistent with this, reviews on TREX and the nuclear export of mRNA. widespread alterations of alternative splicing (48,000) and Here, we hypothesized that: (i) Excessive binding of nuclear alternative polyadenylation site usage (41,000) were recently export adaptor(s) onto C9ORF72 repeat transcripts might force identified in biosamples of cerebellum from C9ORF72-ALS interactions with NXF1 and override the nuclear retention patients5. We have also reported that alteration of splicing mechanisms; (ii) depletion of sequestered export factors that consistency correlates with faster disease progression in may inappropriately license the nuclear export of intron-retaining C9ORF72-ALS cases independently of the DNA repeat repeat transcripts might in turn confer neuroprotection. We used expansion length7. an established Drosophila model of C9ORF72-related disease The pathophysiology mediated by C9ORF72 repeat expansions which exhibits both neurodegeneration and locomotor deficits16 potentially involves three extensively-studied mechanisms which to identify potential nuclear export adaptor(s) involved in the may all contribute to neuronal injury and disease progression: nuclear export of C9ORF72 repeat transcripts. We also used a (i) RNA toxic gain-of-function by sequestration of RNA-binding combination of neuronal N2A cells and ALS patient-derived factors8–12; (ii) protein toxic gain-of-function due to repeat- neurons and astrocytes to validate our in vivo findings and dissect associated non-ATG (RAN) translation that occurs in all sense the molecular mechanisms driving the nuclear export of repeat and antisense reading frames to produce five dipeptide-repeat transcripts and their associated neurotoxicity. In this study, proteins (DPRs)6,13–16; (iii) haploinsufficiency due to decreased we demonstrate that sequestration of SRSF1 onto C9ORF72 expression of the C9ORF72 protein2,17,18 which has recently been repeat transcripts triggers their NXF1-related nuclear export shown to play a key role in the Rab GTPase-dependent regulation independently of splicing which leads to the subsequent RAN of autophagy19–21. We refer to references22–26 for recent reviews translation of neurotoxic levels of DPRs. Moreover, we show that on the mechanisms of C9ORF72-mediated neurotoxicity. the partial depletion of SRSF1 does not alter expression level, The splicing of the first intron of C9ORF72 does not appear intron-1 splicing or nuclear export of the wild-type C9ORF72 to be affected by the presence of the hexanucleotide repeat transcripts while it specifically prevents C9ORF72-mediated expansions as the proportion of unspliced transcripts measured neurodegeneration and in vivo associated motor deficits. by the exon1–intron1 junction remains similar in control and patient-derived neurons or post-mortem brain tissues27. A small proportion of C9ORF72 repeat transcripts retaining pathological Results repeat expansions in intron-1 escape nuclear retention ALYREF and SRSF1 directly bind G4C2 and C4G2 repeat RNA. mechanisms and were detected in the cytoplasm of patient- We performed in vitro UV-crosslinking assays using purified derived lymphoblasts28 where they can subsequently be translated recombinant proteins and synthetic G4C2x5 and C4G2x5 into DPRs. Interestingly, nucleocytoplasmic transport defects of RNA probes to investigate direct protein:RNA interactions. proteins and RNA were recently highlighted in Drosophila, yeast Recombinant hexa-histidine-tagged human ALYREF, SRSF1 and human neuronal models of C9ORF72-related ALS29–32. amino-acids 11-196 which retains wild-type ability to bind RNA In particular, a loss-of-function screen in Drosophila identified and NXF1 (ref. 36), and MAGOH, a control protein which ALYREF (Aly/REF export factor) and NXF1 (nuclear export does not bind RNA49, were purified by ion metal affinity factor 1), two components of the mRNA nuclear export chromatography in high salt to disrupt potential interactions
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