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S.M. Hauck, M. Harley, S. Schöffmann, C.–J. Gloeckner, M. Ueffing; Secreted Proteins from Retinal Mueller Glia Enhance Photoreceptor Survival: An Approach to Detection of New Candidates for Neuroprotection . Invest. Ophthalmol. Vis. Sci. 2004;45(13):850.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Degeneration of retinal photoreceptor cells (RPR) is a major cause of blindness. Within the retina, Mueller glia cells (RMG) support RPR survival by yet unknown means. Factors secreted by RMG in vitro have been shown to support RPR survival (Hauck et al., ARVO 2002, #3623). We show here partial purification of this survival promoting activity and attempt identification of novel candidate proteins. Methods:Secreted factors from primary RMG in vitro (conditioned medium) were collected and tested on primary porcine photoreceptor preparations for neuroprotective activity in a specially herefore developed large–scale survival assay. Subfractionation of conditioned medium by means of size exclusion and FPLC mono–Q binding ability were also tested, and the proteins from active subfractions were identified by CAP/LC–MS Q–TOF mass spectrometry. Results:RMG–conditioned medium promotes porcine RPR survival in vitro, but the secreted, active factors are lost with prolonged RMG culture conditions. Survival–promoting activity from RMG is a protein that separated into a size fraction above 10kDa and did not bind anionic affinity chromatography resins (mono–Q FPLC). Although the protein composition of flowthrough–fraction was much less complex than other fractions, it contained the survival promoting activity. Mass spectrometric protein identification in flowthrough fractions resulted in 20 identified proteins, among them novel candidates which may have neuroprotective function. Conclusions:We present here a very powerful method for identifying active components for cell–to–cell comunication in vitro. We have identified several candidates for RMG–derived survival promoting activity and their neuroprotective activity can be studied in detail.
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