Abstract
Purpose :
Retinal pigment epithelial cells (RPE) play an important role in maintaining the homeostasis of retina and the development of macular degeneration. In our preliminary study, we found that the expression of suppressor of cytokine signaling 2 (SOCS2) was increased significantly in primary RPE of macular degeneration primate model. The aim of this study is to investigate the biological function of SOCS2 in RPE, and elucidate the role in the development of macular degeneration using SOCS2 knockout (KO) mouse.
Methods :
All animal experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Use C57BL/6J strain to generate the SOCS2 KO mouse using CRISPR/CAS9 technology. Primary RPE cells were cultured from SOCS2 KO mice. SOCS2 plasmid DNA was transfected into ARPE-19 cells using Lipofectamine 3000 reagent. The expression levels were detected by real-time PCR or western blot. The co-localization was detected by immunoprecipitation and immunofluorescence staining. The phagocytotic function of RPE was examined by pHrodo phagocytosis kit. Autophagic flux was measured by subretinal injection of AAV2-mRFP-GFP-LC3. Immunohistochemical and electron microscopic analyses were performed for phenotypic study. Student’s t-test or One-way ANOVA was used for statistical analysis. p<0.05 was considered statistically significant.
Results :
Drusen-like formation and Bruch’s membrane abnormality were observed in SOCS2 KO mice (n=6) compared to wild-type control. SOCS2 increased expression of Rac1 (~2-fold, p<0.05) resulting promotes phagocytosis (~3-fold, p<0.05) in RPE cells. SOCS2 were also enhanced autophagy by increasing LC3 (~2-fold, p<0.01) expression. Moreover, acts as the ubiquitin ligase, SOCS2 increased protein ubiquitination as expected in RPE. In addition, our data demonstrated that SOCS2 were co-localized with autophagic proteins LC3 and p62, as well as ubiquitinated proteins. On the other hand, that SOCS2 interacts and degradation of phosphrylated mTOR proteins resulting in promotes autophagy.
Conclusions :
SOCS2 participate in formation of autophagosome and recognize the ubiquitinated proteins that could be degraded by selective autophagy. Our data suggests that SOCS2 protect RPE function by enhancing ubiquitin-dependent selective autophagy through mTOR signaling pathway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.