The long term goal of my project is to broaden our understanding of how neurons communicate with each other and with other cells via chemical signals. Over the last several years it has become clear the exchange of information between neurons involves the cooperation of fast and slow; point-to-point and diffuse messages. One of the bases for this complexity appears to be the property of neurons to secrete a cocktail of neurotransmitters, via at least two types of secretory organelles, small synaptic vesicles [SSVs] (which are thought to contain classical neurotransmitters only) and large dense-core vesicles [LDCVs] (which contain peptides and may also contain amines). LDCVs may be seen as the equivalent organelles in neurons of secretory granules (SGs) in endocrine cells. Typical SSVs are present only in neurons, but recent evidence suggests that closely related vesicles (synaptic-like microvesicles [SLMVs]) are present also in endocrine cells. The biogenesis of SSVs and SLMVs is controversial and some authors have proposed that they are generated at the level of the plasmalemma as a result of endocytosis of LDCV and SG membranes. The working hypothesis of this research proposal is that SSVs represent the neuronal adaptation of SLMVs, that the two organelle share important functional similarities and that they have a biogenesis distant from that of LDCVs and SGs respectively. Studies aimed at testing this hypothesis are proposed. We will isolate SLMVs from anterior pituitary tissue and from naive PC12 cells and compare their biochemical characteristics with those of SSVs purified from brain. In PC12 cells we will analyze the intracellular traffic of SLMVs and characterize, with biochemical and morphological procedures, the transition from a SLMVs phenotype to a SSVs phenotype in parallel with neuronal differentiation. We will test the possibility that also SLMVs might have a secretory function. To study the biogenesis of SLMVs and SSVs we will carry out metabolic labeling experiments in PC12 cells and immunocytochemical experiments of organelles in transit to the nerve terminal in peripheral nerves. Finally, we will try to establish the relationship of SSVs and SLMVs to exocytic and endocytic pathways present in all cells by studying the expression, via cDNA transfection, of SSV proteins in fibroblastic CHO cells. I expect that the improved understanding of the mechanisms of chemical signalling between nerve cells will provide new clues towards the understanding of the molecular basis of neurological and psychiatric diseases and towards the development of novel pharmacological tools to treat such diseases.
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