MERRF (myoclonus epilepsy with ragged-red fibers), and MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) are maternally-inherited encephalomyopathies, almost invariably associated with mitochondrial DNA (mtDNA) point mutations. The mutant mtDNAs in MERRF and MELAS are usually """"""""heteroplasmic"""""""" in brain, muscle, and other tissues, co-existing with varying proportions of normal mtDNAs. Abnormal mitochondrial proliferation, particularly in brain arterioles and in muscle (ragged-red fibers or RRF), is an important pathologic feature in MERRF and MELAS. The pathogenetic mechanisms leading to mitochondrial proliferation are not known. Studies in proliferating cell cultures from patients provided important information on the molecular genetics of mitochondrial encephalomyopathies, but pathologic mitochondrial proliferation is difficult to study in these systems, because mitochondria segregate during mitosis. Brain and muscle are the most severely affected in MERRF and MELAS, most likely due to their high energy demand. Because neurons and muscle fibers are post-mitotic, we propose to use innervated muscle cultures - a non-proliferating system - to investigate the developmental pathobiology of these syndromes. Unlike aneurally-grown muscle, the myofibers in such cultures are remarkably mature, functional (i.e. pulsating), and can be maintained for several months. Clonal muscle cells with known proportions of mutant mtDNAs, derived from muscle satellite cells or from non-muscle cells that are converted to myoblasts with a retrovirus vector containing the muscle regulatory gene myoD, will be innervated with fetal rat spinal cord neurons. At time intervals the cultures will be studied by Southern blot analysis, or a quantitative polymerase chain reaction, and in situ hybridization, in order to assess relative copy number and distribution of mutant mtDNAs. Immunocytochemistry will be performed using antibodies against subunits of cytochrome c oxidase, to assess RRF formation and mitochondrial distribution. The presence of abnormal mitochondria will be evaluated by electron microscopy. Mitochondrial function will be assessed biochemically and cytochemically and by measuring oxygen consumption. Nerve-muscle co-cultures should provide important data on the pathogenesis of MERRF and MELAS, and could become a useful in vitro model for evaluating therapeutic strategies.
Showing the most recent 10 out of 241 publications
