Ndufa4l2 drives the pathological activation of Müller cells in ischemic retinopathy via the PI3K/AKT signaling pathway.

Ischemic retinopathies are major causes of irreversible blindness such as proliferative diabetic retinopathy (PDR). Müller cells, essential for retinal homeostasis, undergo profound pathological activation (reactive gliosis, aberrant proliferation, and migration) under ischemic stress, exacerbating retinal structural disruption and disease progression; however, the precise molecular mechanisms driving this transition remain incompletely understood. This study investigated the role of Ndufa4l2 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2), a hypoxia-inducible mitochondrial protein, in Müller cell pathological activation in ischemic retinopathy. Through single-cell RNA sequencing (scRNA-seq) of an oxygen-induced retinopathy (OIR) mouse model, Ndufa4l2 was identified as the most significantly upregulated gene in pathologically activated Müller cells, exhibiting a transcriptomic signature consistent with reactive gliosis. This upregulation was validated in vivo in OIR retinas and in vitro under hypoxic conditions. Gain- and loss-of-function experiments utilizing Ndufa4l2 overexpression plasmids and CRISPR/Cas9-mediated knockout demonstrated that Ndufa4l2 actively promotes Müller cell reactive gliosis, hyper-proliferation, and enhanced migration. Mechanistically, bioinformatics analyses and experimental validation revealed that Ndufa4l2 exerts its effects by activating the PI3K/AKT signaling pathway. Crucially, pharmacological inhibition of PI3K with LY294002 significantly reversed Ndufa4l2-induced reactive gliosis, proliferation, and migration. In conclusion, our findings establish Ndufa4l2 as a crucial intrinsic driver of Müller cell pathological activation and highlight the Ndufa4l2-PI3K/AKT axis as a promising therapeutic target for mitigating reactive gliosis in ischemic retinal diseases.