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Project description:DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. This study presents a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways and applies it to the neurodegenerative disease gene C9orf72. A double strand break induced by CRISPR/cas9 in the promoter of C9orf72 is sufficient to induce DNA methylation, and methylation can be precisely targeted through the process of homology directed repair (HDR) via delivery of an in vitro methylated exogenous repair template. Long methylated double stranded DNA templates induce more methylation than shorter templates and with higher efficiency than a dCas9-DNMT3a fusion protein construct. Genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HDR, whereas the dCas9-DNMT3a fusion construct causes significant off-target methylation at over 67,000 sites. This method is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that exhibits stable DNA methylation patterns similar to those seen in patients. Using this model, it’s shown for the first time that DNA methylation of the 5’ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.
2019-10-11 | GSE134995 | GEO
Project description:DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. This study presents a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways and applies it to the neurodegenerative disease gene C9orf72. A double strand break induced by CRISPR/cas9 in the promoter of C9orf72 is sufficient to induce DNA methylation, and methylation can be precisely targeted through the process of homology directed repair (HDR) via delivery of an in vitro methylated exogenous repair template. Long methylated double stranded DNA templates induce more methylation than shorter templates and with higher efficiency than a dCas9-DNMT3a fusion protein construct. Genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HDR, whereas the dCas9-DNMT3a fusion construct causes significant off-target methylation at over 67,000 sites. This method is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that exhibits stable DNA methylation patterns similar to those seen in patients. Using this model, it’s shown for the first time that DNA methylation of the 5’ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.
2019-10-11 | GSE134994 | GEO
Project description:Targeted DNA methylation of the ALS/FTD gene C9orf72 via endogenous DNA damage repair pathways
| PRJNA557106 | ENA
Project description:Targeted DNA methylation of the ALS/FTD gene C9orf72 via endogenous DNA damage repair pathways (II)
| PRJNA557107 | ENA
Project description:Targeted DNA methylation of the ALS/FTD gene C9orf72 via endogenous DNA damage repair pathways (I)
| PRJNA557108 | ENA
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport "biosensors", composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype-persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects-resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.
| S-EPMC8616043 | biostudies-literature
Project description:BACKGROUND:The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated. METHODS AND RESULTS:Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls. CONCLUSIONS:Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.
| S-EPMC7041170 | biostudies-literature
Project description:GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, we identified a number of genetic modifiers of poly(GR) toxicity. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.
| S-EPMC6511021 | biostudies-literature
Project description:Mutations in C9orf72 leading to hexanucleotide expansions are the most common genetic causes for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A phenotype resembling ALS and FTD is seen in transgenic mice overexpressing the hexanucleotide expansions, but is absent in C9orf72-deficient mice. Thus, the exact function of C9orf72 in neurons and how loss of C9orf72 may contribute to neuronal dysfunction remains to be clearly defined. Here, we showed that primary hippocampal neurons cultured from c9orf72 knockout mice have reduced dendritic arborization and spine density. Quantitative proteomic analysis identified C9orf72 as a component of the macroautophagy/autophagy initiation complex composed of ULK1-RB1CC1-ATG13-ATG101. The association was mediated through the direct interaction with ATG13 via the isoform-specific carboxyl-terminal DENN and dDENN domain of C9orf72. Furthermore, c9orf72 knockout neurons showed reduced LC3-II puncta accompanied by reduced ULK1 levels, suggesting that loss of C9orf72 impairs basal autophagy. Conversely, wild-type neurons treated with a ULK1 kinase inhibitor showed a dose-dependent reduction of dendritic arborization and spine density. Furthermore, expression of the long isoform of human C9orf72 that interacts with the ULK1 complex, but not the short isoform, rescues autophagy and the dendritic arborization phenotypes of c9orf72 knockout neurons. Taken together, our data suggests that C9orf72 has a cell-autonomous role in neuronal and dendritic morphogenesis through promotion of ULK1-mediated autophagy.
| S-EPMC6526867 | biostudies-literature
Project description:A GGGGCC hexanucleotide repeat expansion in the first intron of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Compelling evidence suggests that gain of toxicity from the bidirectionally transcribed repeat expanded RNAs plays a central role in disease pathogenesis. Two potential mechanisms have been proposed including RNA-mediated toxicity and/or the production of toxic dipeptide repeat proteins. In this review, we focus on the role of RNA mediated toxicity in ALS/FTD caused by the C9orf72 mutation and discuss arguments for and against this mechanism. In addition, we summarize how G4C2 repeat RNAs can elicit toxicity and potential therapeutic strategies to mitigate RNA-mediated toxicity.
| S-EPMC7572710 | biostudies-literature