Project description:HeLa cells lacking MORC2 generated through CRISPR/Cas9-mediated gene disruption were reconstituted with either wild-type or R252W mutant MORC2, and re-repression of HUSH target genes assessed by RNA-seq
Project description:We have generated CRISPR edited versions of hESC line MShef11 to produce MFN2 R94Q/+ and MFN2 R94Q/R94Q lines as a model for Charcot Marie Tooth Disease (CMT) 2A. This were differentiated to limb innervating motor neurons, the predominantly affected cell time in CMT2A and RNA was examined to investigate differences in cell lines.
Project description:Charcot-Marie-Tooth (CMT) disease can be caused by mutations in Aminoacyl-tRNA-Synthetases, including G240R mutation in Glycyl-tRNA-Synthetase (GARS). Ribo-seq generates snapshots of translating ribosomes on mRNA and therefore allows analysis of ribosome pausing mRNA. Here we performed Ribo-seq on lysates of HEK293T cells overexpressing GARS, WT or G240R, to dissect mechanism of CMT linked with translation. We found that GARS G240R causes pausing of ribosomes with glycine codons in A-site. The effect is specific for 21 nt ribosome-protected fragments, produced by ribosomes with empty A-sites, suggestive of the deficit of charged Glycyl-tRNA in GARS G240R-CMT.
Project description:Mutations in the Microrchidia CW-Type Zinc Finger 2 (MORC2) GHKL ATPase module cause Charcot Marie Tooth type 2Z or a broad range of neuropathy, but etiology and therapeutic strategy are not fully defined. Previously, we reported that the Morc2a p.S87L mouse model led to neuropathy and muscular dysfunction through DNA damage accumulation. This study revealed that Morc2a p.S87L caused a protein synthesis defect, resulting in the loss of function of Morc2a and weakening its function of maintaining DNA integrity and hydroxyl radical scavenging in the GHKL ATPase domain. Morc2a GHKL ATPase domain was considered a therapeutic target based on its function of simultaneously complementing hydroxyl radical scavenging and ATPase activity. Adeno-associated virus PHP.eB serotype that has high central nervous system transduction efficiency was applied to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. AAV gene therapy improved neuropathy and muscular dysfunction with single-time treatment. The loss of function characteristics due to protein synthesis defect in Morc2a p.S87L was also observed in human MORC2 p.S87L or p.R252W variant, suggesting a relevance between mouse and human pathogenesis. Here, we demonstrate Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy and could be rescued through AAV-based gene therapy.
Project description:The goal of this study was to identify deregulated genes in Schwann cells of Pmp22 transgenic rats in comparison to wildtype rats. Three timepoints in the course of peripheral nerve myelination were chosen (embryonic day [E] 21, perinatal day [P]6 and P18) in order to reveal mechanistic insight into early pathological processes of Charcot-Marie-Tooth disease 1A (CMT1A).
Project description:GDAP1 is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. Gdap1 knockout mice, mimicking genetic alterations of patients suffering from severe CMT forms, develop an age-related, hypomyelinating peripheral neuropathy. We used microarrays to determine changes in the expression profiles in the peripheral nervous system before a phenotype was detectable in the animal model (2 month of age).
