SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits

Abstract

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.

SRSF1-dependent nuclear export of C9ORF72 repeat transcripts: targeting toxic gain-of-functions induced by protein sequestration as a selective therapeutic strategy for neuroprotection

“…Since SRSF1 is involved in both splicing and nuclear export functions, the reporter constructs were also designed without splicing consensus sites or intronic sequences to allow for specific investigation of the SRSF1-dependent nuclear export potential of G4C2 and C4G2 repeat sequences in absence of functional coupling to splicing. Interestingly, the expression of uninterrupted G4C2x38 or C4G2x39 repeat transcripts which lead to the production of DPRs, but not of G4C2/C4G2x15 repeat transcripts which do not, is much more potent at inhibiting neuronal cell proliferation (by approximately 50%, 48 hours post transfection) [45] than transfection (not shown) or stable integration [47] of interrupted transcripts harboring 102 repeats of the G4C2 sequence -in direct correlation with the expression levels of DPRs. Depletion of ~70% of the SRSF1 protein in neuronal N2A cells transfected with the DPR-reporter constructs further led to an efficient inhibition of the nuclear export and subsequent RAN translation of sense/ antisense repeat transcripts which correlated with a suppression of the C9ORF72 repeat-mediated cytotoxicity in cell proliferation assays.…”

“…In our recent study, we also showed that SRSF1-m4 directly interact with G4C2x5 and C4G2x5 synthetic RNA probes and is sequestered in a length dependent manner onto G4C2-sense and C4G2-antisense repeat transcripts in neuronal N2A cells. Expression of the SRSF1-m4 mutant protein also resulted in nuclear retention of the repeat transcripts and in reduced DPR production [45] , has not yet been investigated. It might involve recruitment of the TREX complex by uncharacterised nuclear adaptor(s) (NEA) and/or coupling to other SR-rich NEAs such as SRSF3 or SRSF7 (SR) during splicing.…”

“…In support of this, ALYREF and NXF1 have been identified by another group as modifiers of the C9ORF72 hexanucleotide-repeat mediated neurotoxicity in a Drosophila loss-of-function screen [44] . In the Nature Communications study [45] which forms the basis for this research highlight review, we showed that recombinantly-purified ALYREF and SRSF1 proteins directly interact with 5 repetitions of G4C2-sense and C4G2-antisense synthetic RNA oligonucleotides. These findings are consistent with the co-localisation of ALYREF [8] and SRSF1 [45] with nuclear RNA foci in human post mortem spinal motor neurons from C9ORF72-ALS cases.…”

“…In the Nature Communications study [45] which forms the basis for this research highlight review, we showed that recombinantly-purified ALYREF and SRSF1 proteins directly interact with 5 repetitions of G4C2-sense and C4G2-antisense synthetic RNA oligonucleotides. These findings are consistent with the co-localisation of ALYREF [8] and SRSF1 [45] with nuclear RNA foci in human post mortem spinal motor neurons from C9ORF72-ALS cases. To investigate the potential involvement of ALYREF and SRSF1 in the nuclear export of hexanucleotide repeat transcripts which are substrates of cytoplasmic RAN translation, we crossed transgenic ALYREF-RNAi and SRSF1-RNAi Drosophila lines with flies expressing 36 uninterrupted G4C2 repeats and exhibiting DPR-mediated neurotoxicity [46] .…”

“…Only the de-phosphorylated forms of SRSF1,3,7 proteins interact with NXF1 upon completion of splicing [35] and the SRSF1 region encompassing the two RRMs confers the same NXF1-binding efficiency as wild type SRSF1 -the linker peptide constituting the NXF1-binding site [37] . Substitution of 4 arginines by alanines in the linker region impairs the interaction of SRSF1-m4 with NXF1 in both HEK and N2A cells [37,45] ( Figure 2). In our recent study, we also showed that SRSF1-m4 directly interact with G4C2x5 and C4G2x5 synthetic RNA probes and is sequestered in a length dependent manner onto G4C2-sense and C4G2-antisense repeat transcripts in neuronal N2A cells.…”