The long-term goal of this research program is to use a forward genetic approach in mice to identify genes required for the maturation and maintenance of neuromuscular junctions and the associated motor neurons, Schwann cells, and muscle fibers. Toward this end, we have identified two novel mutations in mice that cause postnatal neuromuscular dysfunction. The first has been identified as a mutation in glycyl tRNA synthetase (Gars), the same gene that is mutated in human Charcot Marie Tooth 2D (CMT2D) neuropathy. The mice have myelination defects, distal axonopathy, and synaptic defects that are similar to, but more severe than, the human disease. We will address the genetic and cellular mechanism of the phenotype using mouse genetics. The mutations are dominant in mice and humans, and we will first determine if the phenotype is caused by the simple loss of one allele (haplo-insufficiency), or if a more complicated genetic mechanism, such as a dominant negative or neomorph function, may be involved. To do this, we will create new loss-of-function and transgenic alleles. Next we will determine the cellular basis for phenotype and whether dysfunction derives from a developmental defect, from a defect intrinsic to Schwann cells, motor axons, or both, and if the phenotype is caused by a defect in the mitochondrial or cytosolic functions of the protein. Finally, we will introduce the human mutations into the mouse to study allelic variability in the severity of the phenotype. In addition to studies on Gars, we will also identify the gene underlying the second neuromuscular mutation by positional cloning. This mutation has a synaptic phenotype at the neuromuscular junction, is recessive, and results in a shortened 3-6 month lifespan. We will extend our phenotypic analysis to the level of individual motor units to determine the physiological basis for the neuromuscular dysfunction, specifically whether the defects arise at the level of the whole motor neuron, the individual synapse, or the individual muscle fiber. These studies will improve our understanding of the basic biology of how neuromuscular connections are maintained and provide mechanistic insights into diseases such as CMT Neuropathies and myasthenias. This work has relevance to public health because it examines a mouse model of a human disease, Charcot Marie Tooth 2D. These studies will determine the genetic and cellular mechanisms of the disease and potentially aid in identifying and testing therapeutic approaches. In addition, this work will identify a second model of neuromuscular dysfunction that will improve our understanding of the genes that may give rise to hereditary motor neuropathies and myasthenias.
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