All proteins destined for fast axoplasmic transport are processed through the Golgi apparatus (GA) and an intact organelle is essential for neuronal function. Our laboratory was the first to identify the disruption of the GA in a high percentage of motor neurons in ALS and in both asymptomatic and symptomatic transgenic mice expressing the G93A mutation of the human Cu/Zn superoxide dismutase (SOD1) gene found in familial Amyotrophic Lateral Sclerosis. The fragmentation of the GA in ALS and G93A transgenes is identical to its dispersion in cells undergoing mitosis, and in cells treated with microtubule depolymerizing drugs as we discovered in 1964. We propose to evaluate the contribution of the GA in the pathogenesis of ALS using human tissues, animal models of motor neuronopathies, and in vitro (cell culture) systems. Specifically, the GA, the microtubules, and the proteins linking them with the GA will be examined by immunocytochemistry and morphometry of motor neurons of the spinal cord, cranial nerve nuclei and motor cortex in ALS (reviewer 2), in G93A transgenics, in transgenic mice overexpressing the human heavy neurofilament subunit, and in the wobbler mouse, a model of infantile spinal muscular atrophy (reviewer 1). All ALS spinal cord motor neurons with ubiquitin positive inclusions have a fragmented GA and a possible correlation with ubiquitin-positive Mallory bodies in hepatocytes will be examined (reviewer 2). Recent studies have proposed that the point mutations of the SOD1 protein cause neuronal degeneration by a gain of function mechanism. Pursuing this hypothesis the yeast-two hybrid system will be used to identify proteins that interact with the G93A mutant. The specificity of the interaction of the putative proteins with G93A will be examined by in vitro binding assays using CHO cells and post-mitotic human neurons (NT2N) inducibly expressing the G93A mutant and the putative binding proteins by immunocytochemistry and immunoprecipitations with appropriate antibodies. In parallel experiments the GA will be investigated in cells expressing G93A after oxidative stress induced by H202, paraquat and peroxynitrite. The structure of the GA will be examined by light and ultrastructural immunocytochemistry and its function will be evaluated using as a marker the glycosylation, sialylation and immunolocalization of MG160, a Golgi membrane sialoglycoprotein identified, cloned and expressed in our laboratory.
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