Biallelic AAGGG expansions in Replication Factor Complex Subunit 1 ( RFC1) are associated with cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and are increasingly recognised as a common cause of adult-onset ataxia and sensory neuropathy. However, the disease-causing mechanisms remain unclear. Here we leveraged in vitro assays, post-mortem brain tissue, patient-derived cell lines and a neuronal Drosophila model to demonstrate that AAGGG expansions are associated with tissue-specific reductions in the expression of RFC1 transcript, along with impaired RFC1 function and increased sensitivity to DNA damage from platinum-based drugs. CRISPR/Cas9 excision of the AAGGG repeat and flanking AluSx3 element normalized RFC1 expression in iPSC-derived neurons and rescued the DNA damage response, providing a framework for future therapeutic strategies. We also show that these biological findings are clinically relevant in heterozygous AAGGG expansion carriers, who display an increased risk and severity of neuropathy with platinum-based chemotherapy. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 101165557 National Ataxia Foundation, https://ror.org/03s9xxw55 Medical Research Council, MR/T001712/1 Fondazione Cariplo, https://ror.org/01gb56c55, 2019-1836 Charcot-Marie-Tooth Association, SR-202504 AFM-Telethon, 28813 Fondazione Regionale per la Ricerca Biomedica, 1751723 Muscular Dystrophy UK, 24GRO-PG24-0719-1 Guarantors of Brain, https://ror.org/00xkj2889

BackgroundHeterozygous NKX2-1 loss-of-function variants cause combinations of hyperkinetic movement disorders (MDs, particularly childhood-onset chorea), pulmonary dysfunction, and hypothyroidism. Mobile element insertions (MEIs) are potential disease-causing structural variants whose detection in routine diagnostics remains challenging. ObjectiveTo establish the molecular diagnosis of two first-degree relatives with clinically suspected NKX2-1-related disorder who had negative NKX2-1 Sanger (SS), whole-exome (WES), and whole-genome (WGS) sequencing. MethodsThe proband's WES was analyzed for MEIs. A candidate MEI in NKX2-1 underwent optimized SS after plasmid cloning. Functional studies exploring NKX2-1 haploinsufficiency at RNA and protein levels were performed. ResultsA 347-bp AluYa5 insertion with a 65-bp poly-A tail followed by a 16-bp duplication of the pre-insertion wild-type sequence in exon 3 of NKX2-1 (ENST00000354822.7:c.556_557insAlu541_556dup) segregated with the disease phenotype. ConclusionsWe identified a de novo exonic AluYa5 insertion causing NKX2-1-related disorder in SS/WES/WGS-negative cases, suggesting that MEI analysis of short-read sequencing data or targeted long-read sequencing could unmask the molecular diagnosis of unsolved MD cases. (c) 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Background and Objective Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult-onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)(n) repeat expansions in the second intron of the replication factor complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expand the genotypic spectrum of CANVAS by identifying sequence variants in RFC1-coding region associated with this condition. Methods Fifteen individuals diagnosed with CANVAS and carrying only 1 heterozygous (AAGGG)(n) expansion in RFC1 underwent whole-genome sequencing or whole-exome sequencing to test for the presence of a second variant in RFC1 or other unrelated gene. To assess the effect of truncating variants on RFC1 expression, we tested the level of RFC1 transcript and protein on patients' derived cell lines. Results We identified 7 patients from 5 unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)(n) expansion together with a second truncating variant in trans in RFC1, which included the following: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6), and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein. Discussion Our report expands the genotype spectrum of RFC1 disease. Full RFC1 sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)(n) expansions. In addition, it sheds further light on the pathogenesis of RFC1 CANVAS because it supports the existence of a loss-of-function mechanism underlying this complex neurodegenerative condition.

The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.

Corticobasal degeneration typically progresses gradually over 5–7 years from onset till death. Fulminant corticobasal degeneration cases with a rapidly progressive course were rarely reported (RP-CBD). This study aimed to investigate their neuropathological characteristics. Of the 124 autopsy-confirmed corticobasal degeneration cases collected from 14 centres, we identified 6 RP-CBD cases (4.8%) who died of advanced disease within 3 years of onset. These RP-CBD cases had different clinical phenotypes including rapid global cognitive decline ( N = 2), corticobasal syndrome ( N = 2) and Richardson’s syndrome ( N = 2). We also studied four corticobasal degeneration cases with an average disease duration of 3 years or less, who died of another unrelated illness (Intermediate-CBD). Finally, we selected 12 age-matched corticobasal degeneration cases out of a cohort of 110, who had a typical gradually progressive course and reached advanced clinical stage (End-stage-CBD). Quantitative analysis showed high overall tau burden ( p = 0.2) and severe nigral cell loss ( p = 0.47) in both the RP-CBD and End-stage-CBD groups consistent with advanced pathological changes, while the Intermediate-CBD group (mean disease duration = 3 years) had milder changes than End-stage-CBD ( p < 0.05). These findings indicated that RP-CBD cases had already developed advanced pathological changes as those observed in End-stage-CBD cases (mean disease duration = 6.7 years), but within a significantly shorter duration (2.5 years; p < 0.001). Subgroup analysis was performed to investigate the cellular patterns of tau aggregates in the anterior frontal cortex and caudate by comparing neuronal-to-astrocytic plaque ratios between six RP-CBD cases, four Intermediate-CBD and 12 age-matched End-stage-CBD. Neuronal-to-astrocytic plaque ratios of Intermediate-CBD and End-stage-CBD, but not RP-CBD, positively correlated with disease duration in both the anterior frontal cortex and caudate ( p = 0.02). In contrast to the predominance of astrocytic plaques we previously reported in preclinical asymptomatic corticobasal degeneration cases, neuronal tau aggregates predominated in RP-CBD exceeding those in Intermediate-CBD (anterior frontal cortex: p < 0.001, caudate: p = 0.001) and End-stage-CBD (anterior frontal cortex: p = 0.03, caudate: p = 0.01) as demonstrated by its higher neuronal-to-astrocytic plaque ratios in both anterior frontal cortex and caudate. We did not identify any difference in age at onset, any pathogenic tau mutation or concomitant pathologies that could have contributed to the rapid progression of these RP-CBD cases. Mild TDP-43 pathology was observed in three RP-CBD cases. All RP-CBD cases were men. The MAPT H2 haplotype, known to be protective, was identified in one RP-CBD case (17%) and 8 of the matched End-stage-CBD cases (67%). We conclude that RP-CBD is a distinct aggressive variant of corticobasal degeneration with characteristic neuropathological substrates resulting in a fulminant disease process as evident both clinically and pathologically. Biological factors such as genetic modifiers likely play a pivotal role in the RP-CBD variant and should be the subject of future research.

43 Late-onset ataxia cerebellar, or vestibular impairment, when in combination also termed cerebellar neuropathy, vestibular areflexia syndrome (CANVAS). We used non-parametric linkage analysis and genome sequencing to identify a biallelic intronic AAGGG repeat 47 expansion in the replication factor C subunit-1 ( RFC1 ) as the cause of familial CANVAS 48 and a frequent cause of late-onset ataxia, particularly if sensory neuronopathy and bilateral 49 vestibular areflexia coexisted. The expansion, which occurs in the polyA tail of an AluSx3 50 element and differs in terms of both size and nucleotide sequence from the reference 51 (AAAAG) 11 allele, does not affect RFC1 expression in patient peripheral and brain tissue 52 suggesting no overt loss-of-function. These data, along with the European expansion 53 carrier frequency of 0.7%, implies that biallelic AAGGG expansion in RFC1 is a frequent 54 cause of late-onset ataxia. 55 resonance imaging, NS not significant, SAP sensory action potential, UL upper limbs.

In the version of this article initially published, the name of author Wai Yan Yau was misspelled. The error has been corrected in the HTML and PDF versions of the article.

Fibrillar aggregates of tau form the defining pathological hallmarks of Alzheimer's disease (AD), tau variant frontotemporal dementia and tauopathies. Studies of AD, tauopathy and seizure in vivo models have demonstrated therapeutic benefit of reduction of CNS tau levels. We identified an antisense long non-coding RNA gene, MAPT-AS1, overlapping with the 5’ promoter region of the tau gene (MAPT) that potently represses tau levels by influencing ribosomal recruitment of mRNA. Noting the safety of AAV vector-based gene therapy in CNS, we are testing intracranial delivery of MAPT-AS1 transcripts and variants in the htau mouse. The htau mouse model[1] carries full-length human MAPT against a Mapt-/- background and manifests early (∼3m) somatodendritic relocalisation of tau and late (>12m) pathological and behavioural changes reminiscent of AD. We produced AAV9 vectors for CMV promoter-driven expression of a full-length MAPT-AS1 transcript (tNAT1-FL) and a minimised artificial variant as well as an inactive deletion variant (ΔM). Adult htau mice (9-12m) were injected into right hippocampus with AAV9-CMV vectors for tNAT1-FL and ΔM and AAV9-CMV-eGFP as control. Brains harvested 8 weeks later were analysed by Western blot and real-time qRT-PCR. With AAV9-CMV-eGFP, we showed extensive CNS spread of GFP without any site-specific tropism. Eight weeks post-injection, both ipsi- and contralateral sides of htau mice injected with AAV9-CMV with tNAT1-FL showed robust reduction (up to 70%) of tau protein levels that correlate with spread and levels of the tNAT1-FL transcript. In contrast, there were no changes in tau protein levels with expression of ΔM transcript. MAPT-AS1 presents a novel opportunity for therapeutic tau reduction with the advantage of exploiting physiological repression of CNS tau. The excellent safety profile and robust CNS spread and persistence of AAV has made them vector of choice for CNS-targetted gene therapy. The htau mouse model gives us the platform for the pre-clinical study of the benefits of this tau reduction in slowing pathological tau progression and alleviating behavioural deficits displayed by these transgenic mice. [1] Andorfer, C., et al., J Neurochem, 2003. 86:582-90.

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

Citation for published version: Plessy, C, Pascarella, G, Bertin, N, Akalin, A, Carrieri, C, Vassalli, A, Lazarevic, D, Severin, J, Vlachouli, C, Simone, R, Faulkner, GJ, Kawai, J, Daub, CO, Zucchelli, S, Hayashizaki, Y, Mombaerts, P, Lenhard, B, Gustincich, S & Carninci, P 2012, 'Promoter architecture of mouse olfactory receptor genes' Genome Research, vol 22, no. 3, pp. 486-497. DOI: 10.1101/gr.126201.111

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA-binding proteins and through toxic dipeptide repeat proteins generated by repeat-associated non-ATG translation. GGGGCC repeat RNA folds into a G-quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G-quadruplex RNA. We investigated their effect in C9orf72 patient iPSC-derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat-expressing Drosophila. Therefore, small molecules that target GGGGCC repeat G-quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS. These data provide proof of principle that targeting GGGGCC repeat G-quadruplexes has therapeutic potential.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and > 95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

BackgroundA hexanucleotide expansion in C9orf72 is a common cause of the fatal neurodegenerative disorder amyotrophic lateral sclerosis. We have found evidence in a Drosophila model that neurotoxicity is mediated by dipeptide repeat (DPR) proteins generated by repeat-associated non-ATG translation. Here we aimed to evaluate in models of amyotrophic lateral sclerosis caused by the C9orf72 mutation (C9orf72-ALS) whether DPR proteins cause nucleolar dysfunction and whether novel small molecules that bind C9orf72-repeat RNA reduce DPR formation and neurotoxicity.MethodsWe assessed nucleolar function in in-vivo Drosophila models. Nucleolar size was measured with immunofluorescence and confocal microscopy, using automated image analysis. Human induced pluripotent stem cells (iPSCs) from patients with C9orf72-ALS and from healthy controls were taken through neural induction and patterning to derive spinal motor neuron populations. Disease phenotypes were measured with fluorescence in-situ hybridisation for the typical RNA foci seen in C9orf72-ALS patients. Small molecules binding to C9orf72-repeat RNA were fed to C9orf72 Drosophila and applied to the human iPSC-derived spinal motor neurons to evaluate rescue of disease phenotypes.FindingsDPR proteins colocalised with nucleoli in C9orf72-Drosophila brain tissue, with effects on nucleolar morphology. C9orf72-Drosophila had significantly reduced egg-to-adult viability (p<0·05), and the bioavailability of the small molecules in Drosophila was investigated.InterpretationEmerging evidence suggests that nucleolar dysfunction is a key mechanism in C9orf72-ALS. It is crucial that this finding is validated in relevant disease models to rapidly translate findings into promising therapeutic targets. The high prevalence of C9orf72-ALS makes use of targeted therapies a compelling strategy. These experiments might provide novel mechanistic insights into a common form of amyotrophic lateral sclerosis and deliver preclinical data on an exciting therapeutic approach.FundingRB is a Leonard Wolfson Clinical Research Training Fellow and is funded by a Wellcome Trust Clinical Research Training Fellowship (107196/Z/14/Z).

G-rich sequences in DNA and RNA have a propensity to fold into stable secondary structures termed G-quadruplexes. G-quadruplex forming sequences are widespread throughout the human genome, within both, protein coding and non-coding genes, and regulatory regions. G-quadruplexes have been implicated in multiple cellular functions including chromatin epigenetic regulation, DNA recombination, transcriptional regulation of gene promoters and enhancers, and translation. Here we will review the evidence for the occurrence of G-quadruplexes both in vitro and in vivo; their role in neurological diseases including G-quadruplex-forming repeat expansions in the C9orf72 gene in frontotemporal dementia and amyotrophic lateral sclerosis and loss of the G-quadruplex binding protein FMRP in the intellectual disability fragile X syndrome. We also review mounting evidence that supports a role for G-quadruplexes in regulating the processing or function of a range of non-coding RNAs. Finally we will highlight current perspectives for therapeutic interventions that target G-quadruplexes.

Progressive supranuclear palsy is a rare parkinsonian disorder with characteristic neurofibrillary pathology consisting of hyperphosphorylated tau protein. Common variation defining the microtubule associated protein tau gene (MAPT) H1 haplotype strongly contributes to disease risk. A recent genome-wide association study (GWAS) revealed 3 novel risk loci on chromosomes 1, 2, and 3 that primarily implicate STX6, EIF2AK3, and MOBP, respectively. Genetic associations, however, rarely lead to direct identification of the relevant functional allele. More often, they are in linkage disequilibrium with the causative polymorphism(s) that could be a coding change or affect gene expression regulatory motifs. To identify any such changes, we sequenced all coding exons of those genes directly implicated by the associations in progressive supranuclear palsy cases and analyzed regional gene expression data from control brains to identify expression quantitative trait loci within 1 Mb of the risk loci. Although we did not find any coding variants underlying the associations, GWAS-associated single-nucleotide polymorphisms at these loci are in complete linkage disequilibrium with haplotypes that completely overlap with the respective genes. Although implication of EIF2AK3 and MOBP could not be fully assessed, we show that the GWAS single-nucleotide polymorphism rs1411478 (STX6) is a strong expression quantitative trait locus with significantly lower expression of STX6 in white matter in carriers of the risk allele.