SHMT2 overexpression improves glaucoma by enhancing mitophagy in retinal ganglion cells through promoting the phospho of PINK1

Abstract

Background: Glaucoma is a major eye disease that causes blindness. The loss of retinal ganglion cells (RGCs) due to mitophagy impairment is a key driver of glaucoma. SHMT2 depletion leads to an increase in reactive oxygen species (ROS), but its role in regulating mitophagy remains unclear. This study aims to investigate the mechanism by which SHMT2 contributes to glaucoma through the regulation of RGC mitophagy.

Methods: The role of SHMT2 in glaucoma was evaluated through hematoxylin and eosin (H&E) staining and immunofluorescence (IF) staining of acute ocular hypertension (AOH) mouse eyeballs. Mitophagy was assessed by measuring LDH release, apoptosis, mitochondrial membrane potential, lipid ROS, and the protein levels of mitophagy-related proteins in RGCs. The underlying mechanism was investigated using co-immunoprecipitation, IF staining, and Western blot analysis.

Results: Results showed that SHMT2 expression was decreased in the AOH mouse model. NMDA inhibited mitophagy in RGCs, which was restored by SHMT2 overexpression. Moreover, SHMT2 overexpression stabilized PINK1 expression by enhancing the phosphorylation of PINK1. In vivo experiments suggested that SHMT2 overexpression increased the thickness of the retinal ganglion cell-inner plexiform layer.

Conclusion: This study confirmed that SHMT2 overexpression alleviated glaucoma by enhancing mitophagy in RGCs through the upregulation of PINK1 phosphorylation, suggesting that SHMT2 may serve as a potential therapeutic target for glaucoma.

Keywords: Glaucoma; Mitophagy; PINK1; Retinal ganglion cells; SHMT2.

Fig. 1

SHMT2 expression is decreased in AOH mouse model (A) The expression of SHMT2 in retinal tissues from mice in the sham and AOH groups was quantified using qPCR. (B) Histological changes in the eyeballs of mice from the sham and AOH groups were assessed using H&E staining. (C) The quantitative results of RGCs in mice of the sham group and the AOH group. The expression of (D) Brn3a and (E) SHMT2 in retinal tissues was analyzed by IF staining. (F-J) Western blot was performed to detect the levels of mitophagy-related cytokines LC3 II/I ratio, the protein levels of PINK1, Parkin, TIM23 and the phosphorylation level of PINK1 in mouse retina. n = 6 in each group

Fig. 2

SHMT2 overexpression restored mitophagy suppressed by NMDA in RGCs (A and B) SHMT2 mRNA expression in RGCs was quantified using qPCR. (C) Cytotoxicity was evaluated by measuring LDH release in RGCs using an LDH cytotoxicity assay kit. (D and E) Apoptosis in RGCs was analyzed by flow cytometry. (F) Mitochondrial membrane potential was assessed using a JC-1 mitochondrial membrane potential assay kit. (G) Lipid ROS levels were determined by flow cytometry following staining of RGCs with C11 BODIPY 581/591. (H-L) The protein levels of mitophagy-related markers, including LC3 II/LC3 I, PINK1, p-PINK1, Parkin, and TIM23, were examined by Western blot analysis. n = 3 in each group

Fig. 3

SHMT2 overexpression increased the ratio of p-PINK1/PINK1 by augmenting their protein stability (A-C) The interactions between SHMT2 and PINK1, Parkin, or TIM23 were investigated using co-IP assays. (D) The colocalization of SHMT2 and PINK1 in RGCs was assessed through IF staining. (E and F) The stability of PINK1 proteins and phosphorylated PINK1 was evaluated by Western blot analysis following treatment of RGCs with 10 µM cycloheximide (CHX) for 0, 8, 16, and 24 h. n = 3 in each group

Fig. 4

SHMT2 overexpression increases the thickness of GCIPL in AOH mouse model (A) The thickness of the retinal GCIPL in mice was assessed using H&E staining. (B) The quantitative results of RGCs in mice of different groups. The expression of (C) Brn3a and (D) SHMT2 in retinal tissues of mice was analyzed by IF staining. (E-I) Western blot was performed to detect the levels of mitophagy-related cytokines LC3 II/I ratio, the protein levels of PINK1, Parkin, TIM23 and the phosphorylation level of PINK1 in mouse retina. n = 6 in each group