AbstractsBiology & Animal Science

The term retinitis pigmentosa (RP) describes a group of retinal degenerations, in which inherited mutations lead to disturbed physiological functions of photoreceptors (PR) or retinal pigment epithelium (RPE) cells and progressive visual loss. Although gene therapy of RP can prevent loss of vision, the high heterogeneity of gene mutations restricts one gene therapy only to a subset of patients. In contrast, trophic factor delivery cannot be curative for RP, but protects PR irrespective of the underlying gene defect, leading to a significant delay in PR death and temporally maintaining residual vision. In experiments on rodent models of RP, subretinally injected glial cell line-derived neurotrophic factor (GDNF) significantly preserved morphology and function of rod cells. This neuroprotective effect is postulated to be indirect through retinal Müller glial (RMG) cells, which release a variety of factors upon GDNF stimulation. These factors in turn bind to receptors on the PR membrane to stimulate signal transduction pathways, which are crucial for their survival. Encouraged by very promising results of GDNF’s indirect prosurvival action in many studies of rodent retina, we focused on the investigation of the neuroprotective potential of RMG cells. To achieve this aim, primary mouse RMG cells were stimulated with GDNF and seven secreted factors were found to be highly upregulated. Out of this group, cysteine-rich heparin-binding protein 61 (Cyr61), a molecule with proved prosurvival activity in the context of cancer cells, was chosen for further investigation. As anticipated, Cy61 did indeed increase PR survival in ex vivo experiments on short-term and long-term rd1 mouse retinal explants, , confirming its neuroprotective activity. Subsequently, we focused on the mechanism of Cyr61’s prosurvival action on PR. Stimulation of the whole retinal tissue revealed activation of mitogen-activated protein kinase (MAPK)/Erk and janus kinase (JAK)/Stat pathways, but not phosphoinositide 3-kinase (PI3K)/Akt. Furthermore, analysis of very pure PR cultures treated with Cyr61 showed no activation of any of mentioned pathways, suggesting lack of a direct prosurvival effect of Cyr61. In contrast, stimulations of primary porcine RMG cells showed an increase in phosphorylation of all investigated proteins: Erk1/2, Stat3 and Akt. These results uncovered a positive feedback loop of Cyr61 action on RMG, implying at the same time an indirect mechanism for Cyr61 mediated neuroprotection. Investigation of Cyr61 treatment of primary porcine RPE cells demonstrated a very strong activation of MAPK/Erk and PI3K/Akt but not JAK/Stat3, suggesting indirect protection of PR also through RPE cells stimulation. Treatments of the human RMG and RPE cell lines MIO-M1 and ARPE19 with Cyr61 additionally revealed nuclear translocation of activated MAPK/Erk. To determine the consequences of MAPK/Erk, JAK/Stat and PI3K/Akt pathways stimulation in RMG cells, we focused on Cyr61 induced secretome changes. Secretome analysis using LC-MS/MS based…