CYR61 PROTECTS PHOTORECEPTORS IN THE RD1 MOUSE MODEL OF RETINITIS PIGMENTOSA THROUGH ACTIVATION OF RETINAL MÜLLER GLIA AND PIGMENT EPITHELIUM CELLS

Abstract

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 quantitative proteomics showed an increase in the secretion of several proteins involved in extracellular matrix organization. The upregulation of two of these proteins - collagen, type III alpha 1 (COL3A1) and family with sequence similarity 3, member C (FAM3C), at the transcriptional level was confirmed by qPCR. Finally, we investigated whether Cyr61 could protect PR in vivo in an RP rodent model. To this end we used the S334ter-3 rat model. Preliminary data form intravitreal injections of Cyr61 showed substantial changes in vascularization of the retina and lack of neuroprotective action on PR. Furthermore, Cyr61 significantly increased the percentage of TUNEL positive cells in the inner nuclear layer (INL). It remains to be determined whether alterations in the retinal vasculature may be a factor, which diminishes the neuroprotective potential of Cyr61 Taken together, the presented results introduce Cyr61 as a novel neuroprotective agent prolonging PR survival in an indirect way through stimulation of RMG and RPE cells. Further investigations may confirm Cyr61’s neuroprotective effect in vivo and describe molecular basis of its indirect action in more detail.

Retinitis pigmentosa wird durch eine Reihe unterschiedlicher Gendefekte verursacht, die zu einer Störung der Funktionen der Photorezeptoren (PR) führen und damit einem fortschreitenden Verlust des Sehvermögens verursachen. Die Behandlung mit einem trophischen Faktor kann unabhängig vom jeweiligen Gendefekt für die symptomatische Behandlung eingesetzt werden. In früheren Arbeiten wurde gezeigt, dass Glial cell line-derived neurotrophic factor (GDNF) die Morphologie und Funktion der PR durch Stimulation der retinalen Müller Gliazellen (RMG) erhalten kann. In Anbetracht der Wirksamkeit von GDNF wurde in dieser Arbeit das neuroprotektive Potential aktivierter RMG untersucht. GDNF Stimulation von RMG induzierte die Sekretion von Cysteine-rich heparin-binding protein 61 (Cyr61). Eine neuroprotektive Aktivität von Cyr61 ließ sich in ex vivo Retinaexplantaten von rd1 Mäusen nachweisen. Daher wurde das Wirkungsspektrum von Cyr61 in der Retina auf Zelltyp- und Singaltransduktionsebene untersucht. Cyr61 aktivierte die MAPK/Erk und JAK/Stat Signalwege in der intakten Retina, hatte aber keinen Einfluss auf den PI3K/Akt Weg. Allerdings aktivierte Cyr61 keinen dieser Transduktionswege in isolierten primären PR. Im Gegensatz dazu reagierten primäre RMG auf Cyr61 Stimulation mit einer Aktivierung aller drei Transduktionwege. Diese Ergebnisse zeigen einen positiven Rückkopplungsmechanismus von Cyr61 auf RMG und implizieren gleichzeitig einen indirekten Mechanismus des neuroprotektiven Effekts von Cyr61. Die Untersuchung des Einflusses von Cyr61 auf retinale Pigmentepithelzellen (RPE) zeigte außerdem starke Aktivierung der MAPK/Erk und PI3K/Akt. Signalwege, allerdings keine Aktivierung des JAK/Stat3 Weges. Daher ist ein indirekter Mechanismus des neuroprotektiven Effekts über RPE ebenfalls plausibel. Insgesamt charakterisieren die Ergebnisse dieser Arbeit Cyr61 als einen neuen neuroprotektiven Faktor, der das Überleben von PR durch einen indirekten Mechanismus über Aktivierung von RMG und RPE verlängern kann.