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Hauck, Stefanie (2005): Neuroprotective pathways in the retina: Analysis of GDNF-mediated signalling in retinal Mueller glial cells (RMG) and screening for RMG-derived neurotrophic factors. Dissertation, LMU München: Faculty of Biology
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Abstract

A major cause of blindness in the Western world is degeneration of photoreceptors as a result of point mutations in genes coding for either phototransduction-related proteins or other proteins important for retinal function. Despite the diversity of mutated genes and proteins involved in this heterogeneous group of progressive retinal dystrophies with homologous phenotypes, the final event leading to blindness is apoptosis of photoreceptors. This has led to intensive studies of the effects of neuroprotective agents on the survival of photoreceptors in animal models of retinitis pigmentosa. One such effective molecule discovered to date to exert substantial rescue of retinal photoreceptors is glial cell line-derived neurotrophic factor (GDNF). However, the molecular mechanism of action underlying GDNF-mediated neuroprotection remains unresolved. This dissertation and the herein described studies were carried out with the goal of elucidating neuroprotective mechanisms using the porcine retina as a model. This species was selected due to its morphological and anatomical similarities to human retina. In order to clarify possible cellular mechanisms involved in neuroprotection, the initial studies involved analysis of GDNF action in porcine retina. It soon became evident that the GDNF-receptive cell in retina was not the photoreceptor itself but rather retinal Mueller glial cells (RMG), which are the major retinal glial cells. Thus, primary RMG cell cultures prepared from porcine retina were established and characterised to analyse this cell type without extraneous effects from the retinal environment. Proteomic profiling revealed profound changes in expression of RMG-specific marker proteins as an effect of in vitro conditions. Thus, the in vitro experiments for studying GDNF-induced signalling were performed with primary RMG cultures in an early state (two weeks in vitro) in order to study cells resembling the in vivo phenotype. GDNF was found to induce the ERK, SAPK and PKB/AKT pathways, as well as upregulating basic fibroblast growth factor (bFGF). Application of bFGF to primary porcine photoreceptors in vitro promoted a concentration-dependent rescue. Therefore a model of RMG-mediated indirect survival promoting mechanism induced by GDNF could be proposed. The finding that RMG are mediators of photoreceptor survival prompted further screenings for RMG-specific, secreted molecules promoting photoreceptor survival. A large-scale primary photoreceptor survival assay (96well format) was developed, in which RMG-conditioned medium (RMG-CM) was tested for survival activity. Conditioned medium was observed as having specific photoreceptor survival-promoting activity stemming from previously unidentified protein/s. Reducing the complexity of RMG-CM by anionic chromatography revealed that the activity does not bind to anionic resins. Mass spectrometric identifications of the mono-Q flow-through identified 23 different proteins from the active fraction, among them three potential new candidates for neuroprotective activity in the context of photoreceptor survival: connective tissue growth factor (CTGF), insulin-like growth factor binding protein 5 (IGFBP5) and insulin-like growth factor binding protein 7 (IGFBP7). Expression cloning and re-testing of these candidates for their ability to promote photoreceptor survival revealed that CTGF and IGFBP5 were effective in protecting photoreceptors when applied in combination with the RMG-conditioned media. Taken together, these results indicate that such survival-promoting activity is multi-factorial. RMG are likely to support photoreceptors by either cell to cell-mediated paracrine signalling or by secreting factors into the intercellular space between retina and retinal pigment epithelium, which consists of a complex matrix of proteins and polysaccharides. This matrix, designated as interphotoreceptor matrix (IPM), directly borders three cell types: photoreceptors, RMG and the retinal pigment epithelium and predisposes the IPM to function as repository of neuroprotective molecules possibly secreted from adjacent cells to protect and support photoreceptors. In order to identify such novel neuroprotective substances, the composition of IPM was investigated in this thesis by comparative proteomics. Over 140 different proteins were identified, the majority of which had never been previously detected in the IPM. Among these, 13 candidates were found, which in other tissue systems have been already reported to have a functional role in neuroprotection.