Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.The authors thank Michael Howell and the High-Throughput Screening Platform at the Francis Crick Institute for valuable assistance. R Balendra is NIHR Academic Clinical Lecturer in Neurology at UCL and has received funding from a Wellcome Trust Research Training Fellowship [107196/Z/14/ Z] and the UCL Leonard Wolfson Experimental Neurology Centre for this work. She was funded by an Academy of Medical Sciences Starter Grant for Clinical Lecturers (SGL027\1022). This work was funded by the Motor Neurone Disease Association (to AM Isaacs), Alzheimer’s Research UK (ARUK-PG2016A6; ARUK-EXT2019A-002) (to AM Isaacs), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (648716—C9ND) (to AM Isaacs), and the UK Dementia Research Institute (to AM Isaacs), which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society, and Alzheimer’s Research UK. HM Odeh was supported by an AstraZeneca post-doctoral fellowship and an Alzheimer’s Association Research Fellowship. J Shorter was supported by ALSA, Target ALS, AFTD, and the Packard Foundation for ALS Research at JHU

C9orf72-mediated ALS and FTD: multiple pathways to disease

The discovery that repeat expansions in the C9orf72 gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has revolutionized our understanding of these diseases. Substantial headway has been made in characterizing C9orf72-mediated disease and unravelling its underlying aetiopathogenesis. Three main disease mechanisms have been proposed: loss of function of the C9orf72 protein and toxic gain of function from C9orf72 repeat RNA or from dipeptide repeat proteins produced by repeat-associated non-ATG translation. Several downstream processes across a range of cellular functions have also been implicated. In this article, we review the pathological and mechanistic features of C9orf72-associated FTD and ALS (collectively termed C9FTD/ALS), the model systems used to study these conditions, and the probable initiators of downstream disease mechanisms. We suggest that a combination of upstream mechanisms involving both loss and gain of function and downstream cellular pathways involving both cell-autonomous and non-cell-autonomous effects contributes to disease progression

Molecular mechanisms and therapeutic strategies in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia

Background A hexanucleotide expansion in C9orf72 is a common cause of the fatal neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. 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 and frontotemporal dementia caused by the C9orf72 mutation (C9FTD/ALS) whether DPR proteins cause nucleolar dysfunction and whether small molecules that bind C9orf72 G-quadruplex repeat RNA reduce disease phenotypes. Methods We assessed nucleolar function in Drosophila models. Nucleolar volume 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 and an immunoassay for DPRs. Small molecules binding to C9orf72-repeat RNA were applied to the human iPSC-derived spinal motor neurons and fed to C9orf72 Drosophila to evaluate rescue of disease phenotypes. Findings The DPR poly-GR led to increased nucleolar volumes in Drosophila brain. The DPR poly-GA also led to increased nucleolar volume, but to a much lesser extent. Small molecules binding to G-quadruplex GGGGCC RNA showed efficacy in C9orf72 patient-derived neurons and in C9orf72 Drosophila. Interpretation Emerging evidence suggests that nucleolar dysfunction is a key mechanism in C9FTD/ALS. We have shown that DPR proteins lead to nucleolar dysfunction in C9orf72 Drosophila and in C9FTLD patient frontal cortex. The high prevalence of C9orf72-ALS makes use of targeted therapies a compelling strategy. Evidence for amelioration of C9orf72 phenotypes in patient-derived neurons and in Drosophila suggests that targeting G-quadruplex GGGGCC repeat RNA is a potential strategy for treating disease, which requires further optimisation and validation

Specific biomarkers for C9orf72

A hexanucleotide repeat expansion in the C9orf72 gene is a common genetic cause of ALS and FTD. The repeats are translated into five different dipeptide repeat proteins (DPRs). In this issue, Lehmer et al (2017) demonstrate that one of these DPRs, poly(GP), can be measured in the CSF of individuals with C9orf72 mutations. In conjunction with the findings from another recent study (Gendron et al, 2017), these DPR biomarkers may prove to be extremely valuable in the quest for effective therapies for C9FTD/ALS

Specific biomarkers for C9orf72 FTD/ALS could expedite the journey towards effective therapies

A hexanucleotide repeat expansion in the C9orf72 gene is a common genetic cause of ALS and FTD. The repeats are translated into five different dipeptide repeat proteins (DPRs). In this issue, Lehmer et al (2017) demonstrate that one of these DPRs, poly(GP), can be measured in the CSF of individuals with C9orf72 mutations. In conjunction with the findings from another recent study (Gendron et al, 2017), these DPR biomarkers may prove to be extremely valuable in the quest for effective therapies for C9FTD/ALS

Disease-modifying pharmacological treatments for amyotrophic lateral sclerosis/motor neuron disease: an overview of intervention reviews

Objectives: This is a protocol for a Cochrane Review (overview). The objectives are as follows:. To summarise the evidence from Cochrane and non-Cochrane systematic reviews of pharmacological disease-modifying treatments for amyotrophic lateral sclerosis/motor neuron disease

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity

Estimating clinical stage of amyotrophic lateral sclerosis from the ALS functional rating scale

ALS is a progressive neurodegenerative disease. The stage of disease reached can be described using a simple system based on the number of central nervous system regions involved. Historically, datasets have not attempted to record clinical stage, but being able to re-analyse the data by stage would have several advantages. We therefore explored the possibility of using an algorithm based on the revised ALS Functional Rating Scale (ALSFRS-R), which is commonly used in clinical practice, to estimate clinical stage. We devised an algorithm to convert ALSFRS-R score into clinical stage. ALSFRS-R domains were mapped to equivalent CNS regions. Stage 4 is reached when gastrostomy or non- invasive ventilation is needed, but as a proxy we used provision. We collected ALSFRS-R from clinic visits, and compared the estimation of clinical stage from the ALSFRS-R with the actual stage. Results showed that the agreement between staging by the two methods was excellent with an intraclass correlation coefficient of 0.92 (95% confidence interval 0.88-0.94). There was no systematic bias towards over-staging or under-staging using the algorithm. In conclusion, we have shown that clinical stage in ALS can be reliably estimated using the ALSFRS-R in historical data and in current data where stage has not been recorded