Mitochondria are dynamic organelles that continually fuse and divide. By remodeling mitochondrial membranes and contents, these processes are important for mitochondrial function. They control the morphology of mitochondria, are essential for normal levels of respiration and cell growth, and regulate programmed cell death. Mitochondrial fusion requires the nuclearly encoded mitofusins Mfn1 and Mfn2, outer membrane GTPases. Mutations in Mfn2 cause the dominantly inherited peripheral neuropathy Charcot-Marie-Tooth subtype 2A (CMT2A), but the cellular mechanisms of nerve degeneration remain unclear. Clinical data from mitochondrial encephalomyopathies indicate that mitochondrial fusion is especially critical in skeletal muscle and neurons, but again little is known about the underlying mechanisms due to lack of experimental systems. To address these issues, this application proposes the following specific aims. (1) To use mice carrying conditional alleles of Mfn1 and Mfn2 to elucidate their roles in post-embryonic tissues. Results indicate that these mice are excellent models to study mitochondrial fusion in skeletal muscle and the cerebellum. In addition to phenotypic analysis of mutant mice, cell culture systems will be developed to study mitochondrial dynamics in developing cerebellar neurons and in skeletal muscle. (2) To develop and analyze mouse models of CMT2A. CMT2A models have been generated through both gene-targeting of knock-in constructs and transgenesis. These models will allow the study of CMT2A pathogenesis. In parallel, mice without Mfn1 or Mfn2 function in motor neurons will be analyzed to address the requirement of mitochondrial fusion in such cells. (3) To develop cell culture systems to understand how Mfn2 mutants in CMT2A cause functional defects in motor neurons. The effects of CMT2A mutations will be studied in mouse and human fibroblasts to understand their effects on mitochondrial morphology and fusion. Moreover, their effects in motor neurons will be studied by differentiating mouse embryonic stem cells containing CMT2A knock-in mutations into motor neurons. These studies will lead to an understanding of the pathogenesis CMT2A at the cellular and whole-animal level. In addition, they will likely impact the understanding of the pathogenesis of mitochondrial encephalomyopathies. ? ? ?
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