This is an application for continued support for studies of cell death in the nervous system. The proposal is concerned mainly with the mechanism of action and the therapeutic potential of a cell survival peptide that was identified and synthesized during the previous period of support. This peptide, called Y-P30 because of its amino acid composition, has several interesting and potentially important properties: 1) it promotes the survival of cell lines in vitro and of cortical neurons in vivo after application to cerebral cortex lesions; 2) it inhibits the differentiation of a promonocyte cell line (HL-60) in vitro and CNS monocyte derivatives (macrophages/microglia) in vivo in the cortical lesion model; 3) it binds specifically to membrane-associated calreticulin, a rediscovered calcium binding protein that may be important for Ca signaling associated with integrins and other ligands, including those related to neurotransmission; 4) the peptide may destabilize the calreticulin at membranes via its phosphatase activity causing calreticulin release from cells; 5) intravenous-delivery of Y-P30 inhibits macrophages/microglia cells, promotes neuron survival, and causes calreticulin release from cortical cells in rats with cortical lesions, and in unoperated rats. A model has therefore evolved in which Y-P30 acts to inhibit aspects of the cascade of inflammation-neuron death that characterizes several disorders of primary neuronal degeneration. The proposed experiments will further explore mechanisms related to Y-P30 biological activities, especially as these relate to calreticulin function in the nervous system. First, Y-P30's effects on integrin-mediated functions will be tested in an in vitro chemotaxis chamber with brain and peritoneal macrophages. Second the effects of Y- P30 stimulated release of calreticulin on complement-mediated mechanisms (calreticulin as a Clq receptor homologue) will be determined in human neuroblastoma cells 5Y5Y. Third, Y-P 30 will be tested for its effects on Ca2+ handling in resting 5Y5Y cells and after stimulation with bradykinin. A major part of this research will involve further comparisons of direct application of Y-P30 to intravenous delivery in order to determine dosages and potential for long term and retrograde/transneuronal survival effects in the visual and somatosensory systems. Antibodies to the peptide and to brain calreticulin will be made for use in these studies as well as to investigate the possibility of endogenous peptide like activity in the CNS. It is expected that these experiments will provide further insights into mechanisms and potential therapies for neuron degeneration after CNS injury and in nervous system diseases.
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