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A. Zhou, U. K. Abeywarna, A. White, B. Arbogast, D. F. Barofsky, G. A. Cioffi; Presence Of Prohormone Convertase 2 and its Substrate Neuropeptides in the Secretory Pathway of Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):241.
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The generation of active secretory neuropeptides requires the action of a set of processing enzymes that have been well-characterized in the brain but uncharacterized in the retina. The goals of this project were to assess RGC for the presence of prohormone convertase 2 (PC2, a key processing enzymes in the brain) and the presence of a regulated secretory pathway, hence providing a mechanism for the regulation of RGC function under normal and stressed conditions.
RGC-5 cells, a cell line derived from the rat retina, were differentiated with 0.1µM staurosporine (SS). Expressions of PC2 protein and representative PC2 substrate neuropeptides were analyzed by Western blotting and/or immunocytochemistry (ICC). The enzymatic activity of PC2 was determined using a synthetic substrate. A proteomic comparison of cellular proteins from SS-treated and non-treated cells was performed by mass spectrometry (MS) and bioinformatic analyses.
Western blot analyses showed the presence of a 65kDa PC2 protein, presumably the active form of PC2, and several dynorphin-related peptides in RGC-5 cells. PC2 activity was detected in cell extracts. ICC analyses demonstrated that SS-induced differentiation resulted in a re-distribution of NPY, α-MSH, and α-adaptin (a mediator of secretory vesicle trafficking) in RGC-5 cells, i.e. from the cell body to the tips of cellular processes - a location where mature vesicles of the regulated secretory pathway typically reside in neurons. MS analyses identified a group of proteins in differentiated RGC-5 cells that are known to be involved in the biogenesis of secretory vesicles and neurotransmission.
Active PC2 protein and its substrate neuropeptides appear to be present in RGC-5 cells as does the induction of expression of secretory pathway proteins. A quantitative proteomic comparison of neuropeptides in the retina of PC2-null mice with that of wildtype mice is in progress. It remains to be established that silencing PC2 expression in RGC-5 cells may attenuate the processing of neuropeptides.
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