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P. del Rio, S. M. Hauck, M. Irmler, P. Tripathi, J. Beckers, M. Goetz, M. Ueffing; Gene Expression Analysis of GDNF-induced Mueller Glial Cell-derived Neuroprotective Factors: Possible Candidates for Photoreceptor Survival. Invest. Ophthalmol. Vis. Sci. 2009;50(13):691.
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Retinal Mueller Glial cells (RMG) are the major type of glia in the mammalian retina. Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is a powerful neuroprotective factor for photoreceptors in the retina which acts via RMG cells (Hauck et al., 2006). Upon stimulation with GDNF, RMG cells secretion neuroprotective factors, which enhance survival of rods, cones and other retinal neurons. Unfortunately, those GDNF-induced neuroprotective factors have not been identified to date. The purpose of this work is to identify GDNF-induced neuroprotective factors from RMG.
Explanted retinae from hGFAP: eGFP mice were directly stimulated with GDNF for 24h and the RNA was isolated. Additionally, isolated RMG were prepared by FACS sorting and then stimulated with GDNF for 24h. From all experiments (including controls) total RNA was isolated and expression profiling was performed with Illumina microarrays. Results were confirmed by real-time PCR and selected candidate genes were further validated in a photoreceptor survival bioassay.
A total of 37 genes were found upregulated in response to GDNF stimulation from total mouse retina and from FACS sorted RMG cells. Among these genes, several 12 transcripts are expected to code for secreted proteins and were thus included in the confirmation experiments. GDNF-induced upregulation was confirmed by RT-PCR for all selected genes. One of these candidates (osteopontin) was obtained as purified protein and tested for survival promoting activity on primary photoreceptors in vitro. We found that osteopontin enhances survival of photoreceptors in a concentration-dependent manner.
Gene expression analysis showed that the stimulation of explanted retinae and primary RMG cells in culture with GDNF specifically changes gene expression profiles. Among GDNF-induced transcripts are novel candidate factors for neuroprotective activity. We are currently testing a set of prioritized candidates for their therapeutic properties towards future clinical application.
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