The goal of this project is to define the molecular mechanisms responsible for packaging and secretion of peptides by neuronal cells, using the PC12 cell line as a model system. We have shown that packaging of peptides involves specific sorting, such that some proteins are secreted by a regulated pathway, and others by a constitutive pathway. The current project focuses on defining the specificity of this sorting, and on determining whether there are additional sorting steps along the path from the Golgi apparatus to the cell surface.
The specific aims of this project are to determine whether a foreign protein (human growth hormone) is packaged into the endogenous, neurotransmitter containing vesicles; to purify two distinct populations of cytoplasmic vesicles in order to illuminate their role in the neurosecretory process; to generate monoclonal antibody reagents to characterize the molecular components of these vesicles; and to examine the packaging and secretion of two proteins, acetyl cholinesterase and globin, in relationship to these distinct pathways. To accomplish these goals, this project combines several experimental approaches. Light level studies using immunofluorescent localization of vesicle antigens will be extended to the resolution of individual vesicles through the use of critical point dried whole mount preparation of cells, grown individually on EM grids and examined in the high voltage electron microscope. Sub-cellular fractionation will be carried out to purify the secretory vesicles, using goth biochemical and immunological tools, including fluorescence-activated cell sorting applied to sub-cellular organelles. Monoclonal antibodies will be generated and used both for characterization and purification of vesicle sub-types and vesicle components. Molecular genetic manipulations will be applied to this project by introducing a useful marker protein, a form of globin which has been modified to generate a secretory protein, into the PC12 cells and selecting clones that stably incorporate and express the foreign gene. By bringing these different experimental approaches to bear on the question of how neural cells package and secrete proteins, the cellular machinery responsible for neurosecretion will be defined at a level that is not possible to accomplish from a single experimental approach. These studies will result in a better understanding at a fundamental level of how nerve cells secrete peptides. Such information provides a basis for the rational approach to neuro- degenerative diseases, affective disorders, deficits in synaptic transmission, and maintenance and regeneration of synaptic contacts.
