Translocator protein 18 kDa regulates retinal neuron apoptosis and pyroptosis in glaucoma

Abstract

Glaucoma is the leading cause of blindness worldwide. However, our insufficient understanding of the pathogenesis of glaucoma has limited the development of effective treatments. Because recent research has highlighted the importance of non-coding RNAs (ncRNAs) in various diseases, we investigated their roles in glaucoma. Specifically, we detected expression changes of ncRNAs in cell and animal models of acute glaucoma. Further analysis revealed that the Ier2/miR-1839/TSPO axis was critical to cell loss and retinal damage. The knockdown of Ier2, the overexpression of miR-1839, and the silencing of TSPO effectively prevented retinal damage and cell loss. Furthermore, we found that the Ier2/miR-1839/TSPO axis regulated the pyroptosis and apoptosis of retinal neurons through the NLRP3/caspase1/GSDMD, cleaved-caspase3 pathways. In addition to high expression in the retina, TSPO expression was found to be significantly higher in the dorsal lateral geniculate nucleus (DLG) of the brain in the pathologically high intraocular pressure (ph-IOP) rat model, as well as in the peripheral blood mononuclear cells (PBMCs) of glaucoma patients with high IOP. These results indicate that TSPO, which is regulated by Ier2/miR-1839, plays an important role in the pathogenesis of glaucoma, and this study provides a theoretical basis and a new target for the diagnosis and treatment of glaucoma.

Keywords: Apoptosis; Glaucoma; LncRNA; Pyroptosis; Retina ganglion cell; TSPO.

Fig. 1

Identification of lncRNA-Ier2 as a candidate regulatory lncRNA for the ph-IOP damage. a-b: RNA-seq revealed alterations in lncRNA expression in cell and animal acute glaucoma models; c: q-PCR revealed that the expression level of Ier2 was elevated after ph-IOP injury (n = 3 independent experiments); d: q-PCR revealed that the expression level of Ier2 was elevated after OGD/R (n = 3 independent experiments); e: FISH results showed that Ier2 was mainly distributed in the cytoplasm (n = 3 independent experiments), scale bar = 20 μm; f-g: Flow cytometry results showed about 50% cell damage at 3 h after OGD/R (n = 3 independent experiments); h–i: HE staining results showed about 50% cell loss in the GCL of the retina at 24 h after ph-IOP injury (n = 3 independent experiments), scale bar = 50 μm. Ctr: Control; OGD/R: oxygen-glucose deprivation/reperfusion; ph-IOP: pathologically high IOP. The comparison was based on ANOVA analysis. P-values of <0.05, <0.01, <0.001, and <0.0001 are indicated by *, **, ***, and ****, respectively.

Fig. 2

LncRNA-Ier2 regulates apoptosis and pyroptosis in R28 in vitro. a–b: Flow cytometry results showed that silencing Ier2 effectively rescued OGD/R-induced cell death (n = 3 independent experiments); c: Silencing Ier2 reduced ROS levels in the OGD/R model, scale bar = 100 μm; d: TEM showed a break in the cell membrane and pore formation after OGD/R. Silencing Ier2 improved cell morphology (n = 3 independent experiments); e–g: q-PCR results showed that NLRP3/caspase1/GSDMD mRNA expression was significantly increased by OGD/R induction while silencing Ier2 could reverse this trend (n = 3 independent experiments); h–l: Western blotting results showed that silencing Ier2 reverses the changes in pyroptosis protein expression (NLRP3/cleaved-caspase1/N-GSDMD) and increases apoptosis-related protein (cleaved-caspase3) expression (n = 3 independent experiments). Ctr: control; OGD/R: oxygen-glucose deprivation/reperfusion; NC: negative control; si-Ier2: Silencing Ier2. The comparison was based on ANOVA analysis. P-values of <0.05, <0.01, <0.001, and <0.0001 are indicated by *, **, ***, and ****, respectively.

Fig. 3

LncRNA-Ier2 regulates cell apoptosis and pyroptosis in retina a: HE staining showed that ph-IOP-induced cell loss in the GCL of the retina could be rescued by knocking down Ier2 (n = 3 independent experiments), scale bar = 50 μm; b: RBPMS staining showed that RGCs were significantly reduced after ph-IOP injury and that the number of RGCs was restored after knocking down Ier2 (n = 3 independent experiments), scale bar = 50 μm; c: Iba1 staining showed that ph-IOP could induce a significant increase in the number of microglia and that the number of microglia was significantly reduced by knocking down Ier2 (n = 3 independent experiments), scale bar = 50 μm; d: Quantitative HE staining results showed that ph-IOP induced cell loss in the GCL and that silencing Ier2 rescued cell loss in the GCL of the retina; e: The number of RBPMS-positive cells; f: Number of Iba1 positive cells; g: ph-IOP-induced increase in intracellular ROS levels could be eliminated by the knockdown of Ier2 (n = 3 independent experiments); h: TEM results showed the cell morphology in the GCL of the retina, revealing that ph-IOP damage could lead to cell membrane breakage and vacuole formation in some cells, with the significant recovery of cell morphology after the knockdown of Ier2 (n = 3 independent experiments); i: Western blotting results showed that the knockdown of Ier2 rescued ph-IOP-induced changes in NLRP3/cleaved-caspase1/N-GSDMD and cleaved-caspase3 protein expression (n = 3 independent experiments); j: q-PCR results showed that knockdown of Ier2 could reduce ph-IOP-induced NLRP3 elevation (n = 3 independent experiments); k-n: Quantitative analysis of WB results; o-p: dIer2 rescued ph-IOP-induced elevations of IL-18 and IL-1β (n = 3 independent experiments). ph-IOP: pathologically high IOP; dIer2: knockdown Ier2; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. The comparison was based on ANOVA analysis. P-values of <0.05, <0.01, and <0.001 are indicated by *, **, and ***, respectively.

Fig. 4

Ier2 functions via a ceRNA mechanism. a: ceRNA regulatory network. Red diamonds represent protein-coding genes, blue boxes represent miRNAs, and red hexagons represent lncRNAs. Black lines indicate lncRNA-miRNA-mRNA interactions; b: OGD/R can induce an increase in TSPO, while silencing Ier2 can downregulate TSPO (n = 3 independent experiments); c: ph-IOP can induce an increase in TSPO, and knocking down Ier2 can downregulate TSPO (n = 3 independent experiments); d-e: Dual luciferase assay shows that Ier2 can bind to miR-1839 (n = 3 independent experiments); f-g: Dual luciferase assay shows that TSPO can bind to miR-1839 (n = 3 independent experiments). Ctr: control; OGD/R: oxygen-glucose deprivation/reperfusion; ph-IOP: pathologically high IOP; NC: negative control. The comparison was based on ANOVA analysis. P-values of <0.05, <0.01, <0.001 and < 0.0001 are indicated by *, **, ***, and ****, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Ier2 functioned in the ceRNET during OGD/R-induced cell death a. Flow cytometry revealed that the overexpression of miR-1839 and the silencing of TSPO increased the protective effect of silencing Ier2, as well as that antagonizing miR-1839 eliminates the cytoprotective effect induced by silencing Ier2 (n = 3 independent experiments); b: Ier2/miR-1839/TSPO can regulate intracellular ROS levels (n = 3 independent experiments), scale bar = 100 μm; c: TEM showed that the overexpression of miR-1839 and silencing of TSPO further abrogate OGD/R-induced cell scorching, as well as that antagonizing miR-1839 exacerbates these OGD/R-induced changes (n = 3 independent experiments); d. Quantitative analysis of flow cytometry results; e–g: Ier2/miR-1839/TSPO can regulate NLRP3/caspase1/GSDMD mRNA expression levels (n = 3 independent experiments);h–l: Ier2/miR-1839/TSPO regulates NLRP3/cleaved-caspase1/N-GSDMD (pyropto sis-related) and cleaved-caspase3 (apoptosis-related) protein expression levels (n = 3 independent experiments). OGD/R: oxygen-glucose deprivation/reperfusion; dIer2: silenced Ier2; mimic: miR-1839 mimic; inhibitor: miR-1839 inhibitor; dTSPO: silenced TSPO; the comparison was based on ANOVA analysis. P-values of <0.05, <0.01, and <0.001 are indicated by *, **, and ***, respectively.

Fig. 6

Ier2 functioned in the ceRNET during ph-IOP induced retinal damage a. Overexpression of miR-1839 and silencing of TSPO can increase the protective effect of Ier2 knockdown on RGCs by further inhibiting ph-IOP-induced cell loss in the GCL, and antagonizing miR-1839 eliminated the retinal protective effect caused by Ier2 knockdown (n = 3 independent experiments), scale bar = 50 μm; b. The number of RBPMS-positive cells can be further increased by the overexpression of miR-1839, and silencing TSPO further increased cell counts while antagonizing miR-1839 promoted the loss of RBPMS-positive cells (n = 3 independent experiments), scale bar = 100 μm; c. Overexpression of miR-1839 and silencing TSPO could further reduce the number of microglia-positive cells, while antagonizing miR-1839 increased the number of microglia-positive cells (n = 3 independent experiments), scale bar = 50 μm; d. As compared with the ph-IOP group, the overexpression of miR-1839 and silencing of TSPO both protected RGCs from pyroptosis, and antagonizing miR-1839 exacerbated ph-IOP-induced pyroptosis (n = 3 independent experiments); e. NLRP3 mRNA expression levels are regulated by Ier2/miR-1839/TSPO.; f. ROS contents can be reduced by the overexpression of miR-1839 and the silencing of TSPO, and antagonizing miR-1839 will further increase ph-IOP-induced retinal ROS accumulation (n = 3 independent experiments); f-k. Ier2/miR-1839/TSPO regulates NLRP3/cleaved -caspase1/N-GSDMD (pyroptosis-related) and cleaved-caspase3 (apoptosis-related) protein expression levels (n = 3 independent experiments); l-m: Ier2/miR-1839/TSPO can regulate the expression levels of IL-18 and IL-1β in the retina (n = 3 independent experiments). ph-IOP: pathologically high IOP; dIer2: Ier2 knockdown; Ago: miR-1839 agomir; Antago: miR-1839 antagomir; dTSPO: silence TSPO; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. The comparison was based on ANOVA analysis. P-values of <0.05, <0.01, and<0.001 are indicated by *, **, and ***, respectively.

Fig. 7

TSPO can be used as a target for glaucoma diagnosis and treatment a: TSPO expression in the DLG of the brain was significantly elevated in ph-IOP animal models;b：ph-IOP injury can induce increased expression levels of TSPO in the retina (n = 3 independent experiments), scale bar = 50 μm; c: TSPO mRNA expression levels in PBMCs were significantly higher in glaucoma patients with high IOP than in age-related cataract patients with normal IOP (n = 15). Each dot represents a data point for individual patients; (n = 3 independent experiments), scale bar = 100 μm. ph-IOP: pathologically high IOP. The comparison was based on a Student's two-sided t-test. P-values of <0.05, <0.01, and <0.001 are indicated by *, **, and ***, respectively.

Fig. 8

Ier2/miR-1839/TSPO is involved in the regulation of neuronal damage in APACG.