May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Presence Of Prohormone Convertase 2 and its Substrate Neuropeptides in the Secretory Pathway of Retinal Ganglion Cells
Author Affiliations & Notes
  • A. Zhou
    Robert S. Dow Neurobiology, Legacy Research, Portland, Oregon
  • U. K. Abeywarna
    Chemistry, Oregon State University, Corvallis, Oregon
  • A. White
    Robert S. Dow Neurobiology, Legacy Research, Portland, Oregon
  • B. Arbogast
    Chemistry, Oregon State University, Corvallis, Oregon
  • D. F. Barofsky
    Chemistry, Oregon State University, Corvallis, Oregon
  • G. A. Cioffi
    Devers Eye Institute, Portland, Oregon
  • Footnotes
    Commercial Relationships A. Zhou, None; U.K. Abeywarna, None; A. White, None; B. Arbogast, None; D.F. Barofsky, None; G.A. Cioffi, None.
  • Footnotes
    Support NIH Grant EY017345-01
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 241. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: 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.

Methods:: 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.

Results:: 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.

Conclusions:: 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.

Keywords: protein modifications-post translational • neuropeptides • proteomics 
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