Targeting the IRE1α/JNK-autophagy axis in microglia attenuates retinal neovascularization in oxygen-induced...

To investigate the role of IRE1α/JNK-Autophagy Axis on neovascular retinopathy, which will ultimately lead to irreversible vision loss without timely and effective intervention. Despite recent studies indicating that Endoplasmic Reticulum Stress (ERS) and autophagy are both associated with retinal neovascularization, their interaction and underlying molecular mechanisms remain unclear.

Bioinformatics analysis of the GSE102485 dataset was conducted to evaluate gene level differences between patients with neovascular retinopathy and controls. The oxygen-induced retinopathy (OIR) mouse model was established to explore the role of IRE1α/JNK pathway in retinal microglia and its effect on neovascularization. The model was induced by exposing P7 mice to 75% oxygen for 5 days, followed by normoxia. IRE1α/JNK pathway activation was assessed using Western-blot, PCR and immunofluorescence. Primary retinal microglia were extracted and cultured under hypoxia to mimic the in vivo environment. The roles of IRE1α/JNK pathway on autophagy of retinal microglia were evaluated via Western-blot, immunofluorescence and Primary retinal microglia were transduced with an mRFP-GFP-LC3 adenovirus to visualize autophagosomes and autolysosomes. Primary microglia subjected to different treatments were co-cultured with HRMVECs, and tube formation assays were performed on the latter. In vivo, intravitreal injections of the IRE1 inhibitor 4μ8C or the IRE1 agonist IXA4, with/without the autophagy inhibitor bafilomycin A1 were administered. In vitro, primary microglia were treated with 4μ8C or IXA4, with/without bafilomycin A1.

Bioinformatics analysis revealed Gene ERN1 upregulated in patients with neovascular retinopathy, and pathways downstream of ERN1 showed enrichment in autophagy. In the OIR model, a significant activation of the IRE1α/JNK pathway was observed, reaching its peak on postnatal day 17, which coincided with increased levels of LC3AB and vascular endothelial growth factor A (VEGF-A). The compound 4μ8C was effective in suppressing IRE1α/JNK activation and autophagy activity, as well as VEGF-A level, thereby inhibiting retinal neovascularization in OIR mice. In vitro, we also observed upregulated levels of p-IRE1, p-JNK and LC3AB protein and decreased level of p62 protein in cultured retinal microglia exposed to hypoxia for 12h. However, 4μ8C attenuated these effects, leading to a decrease in VEGF-A secretion. Furthermore, IXA4, a highly selective IRE1 activator, was found to enhance autophagy activity and VEGF-A expression in retinal microglia both in vivo and in vitro. Also, IXA4 treatment further promoted retinal neovascularization in OIR mice. However, these effects were inhibited by bafilomycin A1, an autophagy inhibitor.

These findings demonstrates that activation of IRE1α/JNK pathway under hypoxic conditions in retinal microglia promotes RNV via enhancing autophagy activity., highlighting the potential of IRE1α/JNK -Autophagy Axis as a promising therapeutic target for neovascularization related retinopathy.