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S. M. Hauck, C. J. Gloeckner, M. E. Harley, S. Schoeffmann, K. Boldt, P. A. Ekström, M. Ueffing; Identification of Mueller Glia-Derived Paracrine Neuroprotective Proteins. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5840.
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© ARVO (1962-2015); The Authors (2016-present)
Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Within the retina, Mueller glial cells (RMG) are proposed to release factors that support retinal neurons, especially photoreceptors (PR). To discover such neurotrophic factors, we developed an integrated proteomic workflow towards systematic identification of neuroprotective proteins, which are, like most cytokines expressed only in minute amounts.
Towards this goal we first isolated conditioned medium from primary porcine RMG cultured in vitro and tested for survival-promoting activity using primary PR. We then developed a large-scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins towards their identification consists of several fractionation steps combined with tests for biological activity. Here, we combined 1) size fractionation, 2) ion-exchange chromatography, 3) reverse phase liquid chromatography and 4) mass spectrometry (Q-TOF-MSMS or MALDI-MSMS) for protein identification. Cloning and stable expression of identified candidate factors in HEK293 cells enabled functional validation of each protein.
We dissected RMG conditioned medium into native subfractions and found PR survival-promoting protein/s separating consistently into fractions above 7kDa and anion exchange flow-through. As a result of this integrated workflow we identified 74 different proteins from the neuroprotective subfraction after FPLC separation. Among these, the insulin-like growth factor binding proteins IGFBP-5 and IGFBP-7 and connective tissue growth factor (CTGF) were considered as likely candidates for promoting PR survival. Validation with conditioned medium derived from HEK cell lines stably expressing each factor demonstrated that IGFBP5 and CTGF, but not IGFBP7 significantly increased photoreceptor survival when added to the original RMG conditioned medium.
Identification of two survival-promoting activities secreted from RMG indicates the importacnce of new signaling pathways relevant for PR survival. The survival-promoting activity in RMG-CM is multi-factorial, with IGFBP5 and CTGF as an integral part of this activity.
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