TIAM1-RAC1 Signalling Axis-mediated Activation of NADPH Oxidase-2 Initiates Mitochondrial Damage in the Development of Diabetic Retinopathy

Aims/hypothesis: In diabetes, increased retinal oxidative stress is seen before the mitochondria are damaged. Phagocyte-like NADPH oxidase-2 (NOX2) is the predominant cytosolic source of reactive oxygen species (ROS). Activation of Ras-related C3 botulinum toxin substrate 1 (RAC1), a NOX2 holoenzyme member, is necessary for NOX2 activation and ROS generation. In this study we assessed the role of T cell lymphoma invasion and metastasis (TIAM1), a guanine nucleotide exchange factor for RAC1, in RAC1 and NOX2 activation and the onset of mitochondrial dysfunction in in vitro and in vivo models of glucotoxicity and diabetes.

Methods: RAC1 and NOX2 activation, ROS generation, mitochondrial damage and cell apoptosis were quantified in bovine retinal endothelial cells exposed to high glucose concentrations, in the retina from normal and streptozotocin-induced diabetic rats and mice, and the retina from human donors with diabetic retinopathy.

Results: High glucose activated RAC1 and NOX2 (expression and activity) and increased ROS in endothelial cells before increasing mitochondrial ROS and mitochondrial DNA (mtDNA) damage. N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine, trihydrochloride (NSC23766), a known inhibitor of TIAM1-RAC1, markedly attenuated RAC1 activation, total and mitochondrial ROS, mtDNA damage and cell apoptosis. An increase in NOX2 expression and membrane association of RAC1 and p47(phox) were also seen in diabetic rat retina. Administration of NSC23766 to diabetic mice attenuated retinal RAC1 activation and ROS generation. RAC1 activation and p47(phox) expression were also increased in the retinal microvasculature from human donors with diabetic retinopathy.

Conclusions/interpretation: The TIAM1-RAC1-NOX2 signalling axis is activated in the initial stages of diabetes to increase intracellular ROS leading to mitochondrial damage and accelerated capillary cell apoptosis. Strategies targeting TIAM1-RAC1 signalling could have the potential to halt the progression of diabetic retinopathy in the early stages of the disease.