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.

Dravet syndrome is a devastating genetic brain disorder caused by heterozygous loss-of-function mutation in the voltage-gated sodium channel gene SCN1A. There are currently no treatments, but the upregulation of SCN1A healthy allele represents an appealing therapeutic strategy. In this study we identified a novel, evolutionary conserved mechanism controlling the expression of SCN1A that is mediated by an antisense non-coding RNA (SCN1ANAT). Using oligonucleotide-based compounds (AntagoNATs) targeting SCN1ANAT we were able to induce specific upregulation of SCN1A both in vitro and in vivo, in the brain of Dravet knock-in mouse model and a non-human primate. AntagoNAT-mediated upregulation of Scn1a in postnatal Dravet mice led to significant improvements in seizure phenotype and excitability of hippocampal interneurons. These results further elucidate the pathophysiology of Dravet syndrome and outline a possible new approach for the treatment of this and other genetic disorders with similar etiology.

Distinct translational initiation mechanisms between prokaryotes and eukaryotes limit the exploitation of prokaryotic riboswitch repertoire for regulatory RNA circuit construction in mammalian application. Here, we explored programmed ribosomal frameshifting (PRF) as the regulatory gene expression platform for engineered ligand-responsive RNA devices in higher eukaryotes. Regulation was enabled by designed ligand-dependent conformational rearrangements of the two cis-acting RNA motifs of opposite activity in -1 PRF. Particularly, RNA elements responsive to trans-acting ligands can be tailored to modify co-translational RNA refolding dynamics of a hairpin upstream of frameshifting site to achieve reversible and adjustable -1 PRF attenuating activity. Combined with a ligand-responsive stimulator, synthetic RNA devices for synergetic translational-elongation control of gene expression can be constructed. Due to the similarity between co-transcriptional RNA hairpin folding and co-translational RNA hairpin refolding, the RNA-responsive ligand repertoire provided in prokaryotic systems thus becomes accessible to gene-regulatory circuit construction for synthetic biology application in mammalian cells.

Background Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. Methods Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. Results Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1–3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Conclusions Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

Repeat-associated non-AUG (RAN) translation was discovered in 2011 in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). This non-canonical form of translation occurs in all reading frames from both coding and non-coding regions of sense and antisense transcripts carrying expansions of trinucleotide to hexanucleotide repeat sequences. RAN translation has since been reported in 7 of the 53 known microsatellite expansion disorders which mainly present with neurodegenerative features. RAN translation leads to the biosynthesis of low-complexity polymeric repeat proteins with aggregating and cytotoxic properties. However, the molecular mechanisms and protein factors involved in assembling functional ribosomes in absence of canonical AUG start codons remain poorly characterised while secondary repeat RNA structures play key roles in initiating RAN translation. Here, we briefly review the repeat expansion disorders, their complex pathogenesis and the mechanisms of physiological translation initiation together with the known factors involved in RAN translation. Finally, we discuss research challenges surrounding the understanding of pathogenesis and future directions that may provide opportunities for the development of novel therapeutic approaches for this group of incurable neurodegenerative diseases.

Disruption to protein homeostasis caused by lysosomal dysfunction and associated impairment of autophagy is a prominent pathology in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The most common genetic cause of ALS/FTD is a G4C2 hexanucleotide repeat expansion in C9orf72 (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 repeat transcripts gives rise to dipeptide repeat (DPR) proteins that have been shown to be toxic and may contribute to disease etiology. Genetic variants in TMEM106B have been associated with frontotemporal lobar degeneration with TDP-43 pathology and disease progression in C9ALS/FTD. TMEM106B encodes a lysosomal transmembrane protein of unknown function that is involved in various aspects of lysosomal biology. How TMEM106B variants affect C9ALS/FTD is not well understood but has been linked to changes in TMEM106B protein levels. Here, we investigated TMEM106B function in the context of C9ALS/FTD DPR pathology. We report that knockdown of TMEM106B expression exacerbates the accumulation of C9ALS/FTD-associated cytotoxic DPR proteins in cell models expressing RAN-translated or AUG-driven DPRs as well as in C9ALS/FTD-derived iAstrocytes with an endogenous G4C2 expansion by impairing autophagy. Loss of TMEM106B caused a block late in autophagy by disrupting autophagosome to autolysosome maturation which coincided with impaired lysosomal acidification, reduced cathepsin activity, and juxtanuclear clustering of lysosomes. Lysosomal clustering required Rab7A and coincided with reduced Arl8b-mediated anterograde transport of lysosomes to the cell periphery. Increasing Arl8b activity in TMEM106B-deficient cells not only restored the distribution of lysosomes, but also fully rescued autophagy and DPR protein accumulation. Thus, we identified a novel function of TMEM106B in autophagosome maturation via Arl8b. Our findings indicate that TMEM106B variants may modify C9ALS/FTD by regulating autophagic clearance of DPR proteins. Caution should therefore be taken when considering modifying TMEM106B expression levels as a therapeutic approach in ALS/FTD.

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72 -repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72 -linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

Short repeated sequences of 3−6 nucleotides are causing a growing number of over 50 microsatellite expansion disorders, which mainly present with neurodegenerative features. Although considered rare diseases in relation to the relatively low number of cases, these primarily adult-onset conditions, often debilitating and fatal in absence of a cure, collectively pose a large burden on healthcare systems in an ageing world population. The pathological mechanisms driving disease onset are complex implicating several non-exclusive mechanisms of neuronal injury linked to RNA and protein toxic gain- and loss- of functions. Adding to the complexity of pathogenesis, microsatellite repeat expansions are polymorphic and found in coding as well as in non-coding regions of genes. They form secondary and tertiary structures involving G-quadruplexes and atypical helices in repeated GC-rich sequences. Unwinding of these structures by RNA helicases plays multiple roles in the expression of genes including repeat-associated non-AUG (RAN) translation of polymeric-repeat proteins with aggregating and cytotoxic properties. Here, we will briefly review the pathogenic mechanisms mediated by microsatellite repeat expansions prior to focus on the RNA helicases eIF4A, DDX3X and DHX36 which act as modifiers of RAN translation in C9ORF72-linked amyotrophic lateral sclerosis/frontotemporal dementia (C9ORF72-ALS/FTD) and Fragile X-associated tremor/ataxia syndrome (FXTAS). We will further review the RNA helicases DDX5/17, DHX9, Dicer and UPF1 which play additional roles in the dysregulation of RNA metabolism in repeat expansion disorders. In addition, we will contrast these with the roles of other RNA helicases such as DDX19/20, senataxin and others which have been associated with neurodegeneration independently of microsatellite repeat expansions. Finally, we will discuss the challenges and potential opportunities that are associated with the targeting of RNA helicases for the development of future therapeutic approaches.