Neuroprotective pathways in the retina

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

Novel potential interacting partners of fibronectin in spontaneous animal model of interstitial cystitis

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future

Equine recurrent uveitis - A spontaneous horse model of uveitis

Equine recurrent uveitis (ERU) is an autoimmune disease that occurs with a high prevalence (10%) in horses. ERU represents the only reliable spontaneous model for human autoimmune uveitis. We already identified and characterized novel autoantigens (malate dehydrogenase, recoverin, CRALBP) by analyzing the autoantibody-binding pattern of horses affected by spontaneous recurrent uveitis (ERU) to the retinal proteome. CRALBP also seems to be relevant to human autoimmune uveitis. Proteomic screening of vitreous and retinal samples from ERU diseased cases in comparison to healthy controls has led to the identification of a series of differentially regulated proteins, which are functionally linked to the immune system and the maintenance of the blood-retinal barrier. Copyright (c) 2008 S. Karger AG, Basel

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies

Osteopontin and Fibronectin Levels Are Decreased in Vitreous of Autoimmune Uveitis and Retinal Expression of Both Proteins Indicates ECM Re-Modeling

Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed

Unraveling the Equine Lymphocyte Proteome: Differential Septin 7 Expression Associates with Immune Cells in Equine Recurrent Uveitis

Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease

Protein expression profile of Gasterophilus intestinalis larvae causing horse gastric myiasis and characterization of horse immune reaction

Background Little information is available on the immunological aspect of parasitic Gasterophilus intestinalis (Diptera, Oestridae) larvae causing horse gastric myiasis. The objectives of this research were to analyze the protein content of larval crude extracts of the migrating second and third larvae (L2 and L3) of G. intestinalis in order to characterize the immune response of horses. Results The proteomic profile of L2 and L3, investigated by using one and two dimensional approaches, revealed a migration pattern specific to each larval stage. Furthermore, Western blots were performed with horse sera and with sera of Balb/c mice immunised with the larval crude extracts of L2 or L3, revealing a different immune reaction in naturally infected horses vs. artificially induced immune reaction in mice. The comparisons of the immunoblot profiles demonstrate that the stage L2 is more immunogenic than the stage L3 most likely as an effect of the highest enzymatic production of L2 while migrating through the host tissues. Fifteen proteins were identified by mass spectrometry. Conclusion This work provides further information into the understanding of the interaction between G. intestinalis and their host and by contributing a novel scheme of the proteomic profile of the main larval stages

The agonistic TSPO ligand XBD173 attenuates the glial response thereby protecting inner retinal neurons in a murine model of retinal ischemia

Peer reviewedPublisher PD

Retinal glia promote dorsal root ganglion axon regeneration.

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.This work was supported by a van Geest Fight for Sight Early Career Investigator Award, grant number 1868 [BL].This is the final version of the article. It first appeared at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.011599