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Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

ABSTRACT: The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ?50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients.

SUBMITTER: Martier R

PROVIDER: S-EPMC6378669 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

Martier Raygene R Liefhebber Jolanda M JM Miniarikova Jana J van der Zon Tom T Snapper Jolanda J Kolder Iris I Petry Harald H van Deventer Sander J SJ Evers Melvin M MM Konstantinova Pavlina P

Molecular therapy. Nucleic acids 20190130

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G<sub>4</sub>C<sub>2</sub>) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases. RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed ...[more]

PMID: 30776581

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Project description:Mutations in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathogenetic mechanisms linked to this gene are a direct consequence of an aberrant intronic expansion of a GGGGCC hexanucleotide located between the 1a and 1b non-coding exons, which can be transcribed to form cytotoxic RNA foci or even translated into aggregation-prone dipeptide repeat proteins. Importantly, the abnormal length of these repeats affects also the expression levels of C9orf72 itself, which suggests haploinsufficiency as additional pathomechanism. Thus, it appears that both toxic gain of function and loss of function are distinct but still coexistent features contributing to the insurgence of the disease in case of C9orf72 mutations. In this study, we aimed at identifying a strategy to address both aspects of the C9orf72-related pathobiochemistry and provide proof-of-principle information for a better understanding of the mechanisms leading to neuronal loss. By using primary neurons overexpressing toxic poly(GA), the most abundant protein product of the GGGGCC repeats, we found that the antiarrhythmic drug propranolol could efficiently reduce the accumulation of aberrant aggregates and increase the survival of C9orf72-related cultures. Interestingly, the improved catabolism appeared to not depend on major degradative pathways such as autophagy and the proteasome. By analyzing the proteome of poly(GA)-expressing neurons after exposure to propranolol, we found that the drug increased lysosomal degradation through a mechanism directly involving C9orf72 protein, whose levels were increased after treatment. Further confirmation of the beneficial effect of the beta blocker on aggregates' accumulation and survival of hiPSC-derived C9orf72-mutant motoneurons strengthened the finding that addressing both facets of C9orf72 pathology might represent a valid strategy for the treatment of these ALS/FTD cases.

Dataset Information

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

Publications

Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.

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