CASK mediates methylglyoxal-induced mitochondria-associated cell death in retinal Müller cells through modulating the ROS-p38-SOCE signalling pathway and antioxidant enzymes.

Diabetic retinopathy (DR), a major cause of blindness, is partly driven by methylglyoxal (MGO), a glycolytic byproduct with cytotoxic properties. Retinal Müller cells (MCs), which preserve retinal integrity and function, are highly susceptible to MGO-induced damage. Calcium/calmodulin-dependent serine protein kinase (CASK), a scaffold protein widely expressed in the retina, has an unidentified role in MCs and DR progression. In murine rMC1 cells, CASK was detected in both the nucleus and cytosol, with strong mitochondrial localization. Knockdown of CASK markedly reduced MGO-induced apoptosis, mitochondrial reactive oxygen species (mtROS) accumulation, mitochondrial membrane potential collapse, and impairment of oxidative phosphorylation. The cytotoxic effects were abolished by the ROS scavengers NAC and MitoTEMPO. Notably, silencing CASK also elevated basal antioxidant proteins, including SOD2, GPX4, and catalase. Furthermore, CASK depletion prevented MGO-induced increases in cytosolic and mitochondrial Ca²⁺, as well as Ca²⁺ influx through ER Ca²⁺ store depletion. Pharmacological inhibition of store-operated Ca²⁺ entry (SOCE), the mitochondrial calcium uniporter (MCU), or CASK kinase activity suppressed MGO-induced Ca2 + overload and cell death without altering mtROS production. Mechanistically, CASK is associated with STIM1 to facilitate Orai1 clustering, thereby enhancing SOCE activity. Inhibition of p38 signaling similarly reduced Ca2+ accumulation and apoptosis. Transcriptomic analysis revealed that CASK silencing upregulated genes involved in mitochondrial respiration and oxidative phosphorylation, particularly complexes I and V. Collectively, these findings demonstrate that CASK promotes MGO-induced apoptosis through kinase-independent disruption of mitochondrial and antioxidant defenses, and kinase-dependent activation of SOCE, identifying CASK as a potential therapeutic target in DR.