Abstract

DO.09.07

Neurotrophic factors secreted by Mueller Glial cells

Stefanie M. Hauck1, Martin Irmler2, Caroline Bobe1, Patricia delRio1, Johannes Beckers2, Per Ekstrom3, Marius Ueffing1
1Abteilung für Proteinanalytik, Helmholtz Zentrum München, Neuherberg; 2Institut für Experimentelle Genetik, Helmholtz Zentrum München, Neuherberg; 3Department Ophthalmology, Lund University, Lund, Sweden

Objective
Retinal Mueller Glial cells (RMG) are recognized as intrinsic source of neuroprotective factors supporting retinal neurons. We aim at identifying as yet unknown secreted factors from RMG and validate their neuroprotective potential in order to develop future therapeutic strategies against neurodegeneration.
Methods
Primary RMG are isolated from pig retinas. Total RNA is isolated from RMG and used for gene chip analysis on porcine arrays. Additionally, for direct analysis of secreted proteins in the extracellular compartments, conditioned media from RMG are collected, proteins tryptically digested and identified by LC-MSMS mass spectrometric analyses (OrbiTrap). Expression levels are quantified by label free quantification and candidates for neuroprotective activities are validated by photoreceptor survival assays, followed by validation on retinal explant cultures.
Results
We screened primary RMG for mRNA coding for secreted proteins and found that RMG express a total of 216 such transcripts directly after preparation. Quantitative mass spectrometric measurements from extracellular compartments derived from RMG resulted in the identification of more than 250 proteins, 80 of them containing a signal peptide for classical secretion. Combination of both complementary approaches enables us to predict a RMG secreted protein profile which includes a broad spectrum of chemokines, metalloproteinases, cytokines, interleukins and constituents of extracellular matrix as well as known factors secreted by RMGs such as PEDF, SPARC and CNTF. However, in addition the dataset also includes many factors yet unknown to be secreted by RMGs which provide good candidates for transmitting neurotrophic activity to retinal neurons. We meanwhile validated the potential neurotrophic activity of several candidates on primary photoreceptors in vitro. Among them is IGF-II, which proofed to increase photoreceptor survival in vitro and showed neuroprotective potential in the retinal explant cultures of rd1 mouse model of Retinitis Pigmentosa.
Conclusions
The combined approach of transcriptome and targeted proteome analysis enables the identification of a large set of secreted proteins from primary RMG. Among these are well-known extracellular components, like SPARC, PEDF, MMPs and FGFs, as well as many novel secreted factors, some of which are currently being evaluated towards neuroprotective activity.

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