Grx2 activates the AMPK/PGC1α/NRF1 pathway to preserve PACS2-dependent MAM integrity and mitochondrial function in diabetic cataract

High glucose (HG)-induced mitochondrial dysfunction in lens epithelial cells (LECs) is a key mechanism in the pathogenesis of diabetic cataract (DC). The integrity of mitochondria-associated endoplasmic reticulum membranes (MAMs) is closely linked to mitochondrial function. Our previous studies confirmed that glutaredoxin (Grx) deficiency increased protein S-glutathionylation (PSSG) in lenses, leading to oxidative damage in LECs and accelerating DC progression. However, the underlying mechanisms remain unclear. Streptozotocin (STZ)-induced diabetic mouse models with Grx2 knock-in (KI) or knockout (KO), lenses from Grx2 KI/KO mice, as well as HLE-B3 cells were utilized to explore the protective action of Grx2 against HG‑triggered injury in LECs. MAM integrity and mitochondrial function were assessed by measuring ATP, mitochondrial membrane potential, and reactive oxygen species (ROS) levels in Grx2-downregulated or overexpressing LECs. NRF1 coupled with peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) on phosphofurin acidic cluster sorting protein 2 (PACS2) promoter was validated using co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter gene assays. Rescue experiments further verified the functional roles and underlying molecular mechanisms of Grx2 on HG-induced HLE-B3 cells damage. Grx2 KO aggravated PSSG accumulation, MAM disruption, and mitochondrial dysfunction in DC mice, whereas Grx2 KI rescued these defects. In vitro, Grx2 overexpression suppressed HG-induced mitochondrial fission and restored MAM integrity, whereas Grx2 knockdown produced opposite effects. Mechanistically, Grx2 activated AMP-activated protein kinase (AMPK) by S-deglutathionylation of AMPK, subsequently activating the PGC1α/NRF1 axis to drive PACS2 transcription. Rescue experiments confirmed that Grx2 protects LECs from HG-induced oxidative damage by activating the AMPK/PGC1α/NRF1 pathway, which restores PACS2-dependent MAM integrity and mitochondrial function. Ex vivo lenses organ culture further verified Grx2-mediated AMPK activation ameliorated HG-induced pathological damage in the lens. These findings reveal that the Grx2/AMPK/PACS2 axis maintains MAM integrity and mitochondrial homeostasis, offering novel therapeutic strategies for DC intervention.