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).
Project description:Heterozygous mutations in six tRNA synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT-mutant glycyl- or tyrosyl-tRNA synthetases inhibit global protein synthesis by an unknown mechanism, independent of aminoacylation activity. We report that tRNAGly overexpression rescues protein synthesis and peripheral neuropathy phenotypes in Drosophila and mouse models of CMT caused by glycyl-tRNA synthetase (GlyRS) mutations (CMT2D). Kinetic experiments revealed that CMT-mutant GlyRS bind tRNAGly, but display markedly slow release rates. This tRNAGly sequestration may deplete the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome and translation deficit.
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: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). To seek changes in gene expression patterns of Gdap1-/- mice, we isolated motoneurons by laser dissection and took sciatic nerve lysates of two-month-old mice and purified RNA of five mice. Based on the amount and quality of the isolated RNA we selected three samples per tissue (sciatic nerve lysate and motoneruons) and genotype (GDAP1-/- or wild type). Note: the probe sets for Gdap1 lie within exon 4 and exon 6, thus no apparent loss of Gdap1 mRNA expression is present in Gdap1-/- animals.
Project description:Charcot-Marie-Tooth 1A is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), which produces muscle weakness and loss of sensation in the hands and feet. A recent case-only genome wide association study by the Inherited Neuropathy Consortium identified a strong association between variants in signal induced proliferation associated 1 like 2 (SIPA1L2) and strength of foot dorsiflexion. To validate SIPA1L2 as a candidate modifier, and to assess its potential as a therapeutic target, we engineered mice with a deletion in SIPA1L2 and crossed them to the C3-PMP22 mouse model of CMT1A. We performed neuromuscular phenotyping and identified an interaction between Sipa1l2 deletion and muscular endurance decrements assayed by wire-hang duration in C3-PMP22 mice, as well as several interactions in femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggested an involvement of Sipa1l2 in cholesterol biosynthesis, which was also implicated in C3-PMP22 mice. Though several interactions between Sipa1l2 deletion and CMT1A-associated phenotypes were identified, validating a genetic interaction, the overall effect on neuropathy was small.
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:To determine transcriptome changes in motor neurons induced by the GarsC201R mutation. How mutations in broadly expressed housekeeping genes lead to neurodegeneration in specific cell types remains unclear. Mutations in ubiquitously expressed tRNA synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth disease. Genetic evidence in mouse and Drosophila models suggests a neomorphic gain-of-function mechanism. Here, we use in vivo, cell-type-specific transcriptional and translational profiling of affected peripheral neurons to show that mutant tRNA synthetases impair translation and activate the integrated stress response (ISR) through the sensor kinase, GCN2. The chronic activation of the ISR contributes to the pathophysiology, and genetic deletion of Gcn2 alleviates the peripheral neuropathy. The activation of GCN2 by tRNA synthetase mutations indicates their neomorphic activity is still related to translation and suggests inhibiting GCN2 or the ISR as a therapeutic strategy.
Project description:To determine transcriptome changes in pre-disease onset motor neurons induced by the GarsC201R mutation. How mutations in broadly expressed housekeeping genes lead to neurodegeneration in specific cell types remains unclear. Mutations in ubiquitously expressed tRNA synthetase genes cause axonal peripheral neuropathy, accounting for at least six forms of Charcot-Marie-Tooth disease. Genetic evidence in mouse and Drosophila models suggests a neomorphic gain-of-function mechanism. Here, we use in vivo, cell-type-specific transcriptional and translational profiling of affected peripheral neurons to show that mutant tRNA synthetases impair translation and activate the integrated stress response (ISR) through the sensor kinase, GCN2. The chronic activation of the ISR contributes to the pathophysiology, and genetic deletion of Gcn2 alleviates the peripheral neuropathy. The activation of GCN2 by tRNA synthetase mutations indicates their neomorphic activity is still related to translation and suggests inhibiting GCN2 or the ISR as a therapeutic strategy.