The goals of this proposal are to develop organelle specific antibodies and to identify, isolate and characterize novel intrinsic membrane polypeptides of the GA of neurons from rat and human brain so that: a) we can better understand the structure and function of this organelle in a highly-specialized cell; and b) we can study the involvement of the neuronal GA in experimental and human neuropathologic conditions. We will continue our studies on MG-160, a membrane sialoglycoprotein of the medial cisternae of the GA of rat brain neurons, PC12 and certain other cells, which was isolated in our laboratory. The codistribution in the rat neuronal GA of MG-160, Sialyl and galactosyltransferases will be studied by immunoelectron microscopy. Similar methods will be employed to investigate a possible relationship between the neuronal GA and smooth cisternae in neuronal processes. In order to obtain partial or complete sequences of MG-160, cDNA or expression libraries will be screened with synthetic oligonucleotides or antibodies respectively. Antibody and DNA probes will be used in metabolic and developmental studies to define how MG-160 is regulated during neuron migration, neuritic sprouting, synaptogenesis, and Nerve Growth Factor-induced """"""""differentiation"""""""" in PC12 cells. Monoclonal antibody 10 A8 binds to MG-160. Microinjection of 10 A8, or of mRNA from 10 A8, into PC12 cells will be used to examine a possible effect of 10 A8 on Golgi functions. New intrinsic membrane polypeptides of the rat and human neuronal GA will be identified and isolated with monoclonal antibodies raised to Golgi fractions from PC12 and human melanoma. Antibodies against the human GA will be used to study the organelle in Amyotrophic Lateral Sclerosis (ALS). Preliminary studies of the GA of motor neurons in ALS have disclosed fragmentation of the organelle similar to that observed after microtubule disrupting agents. We will investigate in the rat visual system whether microtubule disruption or neuronal deafferentation produces fragmentation of the neuronal GA. Monoclonal antibody 2Hl, reacting with a 68 kDA polypeptide of membranes of the rough endoplasmic reticulum (RER) of rat neurons and PC12 cells, will be used for the characterization and isolation of this novel protein and in experimental studies to establish whether changes of the neuronal GA is an organelle specific event or part of a general reaction.
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