Inhibition of the mitochondrial calcium uniporter complex (MCUcx) as a neuroprotective strategy for ischemic stroke

Abstract

Ischemic stroke, typically caused by a clot that blocks a major cerebral artery, is a leading cause of death and disability. Despite intense effort, a viable protective strategy has yet to emerge. Mitochondrial dysfunction, resulting in metabolic collapse and the initiation of numerous cell death pathways, is considered to play a pivotal role in ischemic brain injury. Ischemia deprives mitochondria of oxygen and glucose necessary to manufacture adenosine triphosphate that fuels ion pumping. This causes a massive rise of cytosolic calcium (Ca2+) levels that triggers toxic mitochondrial Ca2+ overloading. The mitochondrial Ca2+ uniporter complex (MCUcx) mediates high-capacity mitochondrial Ca2+ uptake responsible for ischemic cell death. This thesis therefore examined the protective effects of the MCUcx inhibitor Ru265 in models of ischemic stroke. Ru265 protected cortical neuron and astrocyte cultures from death by a lethal period of oxygen-glucose deprivation (OGD). In cortical neuron cultures, this was accompanied by preserved mitochondrial respiration and the prevention of changes in MCUcx subunit expression. Ru265 also elevated anti-inflammatory cytokine mRNA levels in astrocyte cultures and suppressed lipopolysaccharide-induced pro-inflammatory cytokine mRNA expression in astrocyte and endothelial cell cultures. In mice subjected to hypoxic/ischemic (HI) brain injury, intraperitoneal injection of Ru265 (3 mg/kg) reduced sensorimotor deficits and infarct volumes. However, Ru265 (10 and 30 mg/kg) produced convulsions by a non-MCUcx mechanism, perhaps involving P/Q-type Ca2+ channel inhibition. To reduce the pro-convulsant effects of Ru265, two strategies were tested. The first employed structural analogues of Ru265 termed Os245 and Os245’. Although these compounds preserved cell viability and mitochondrial function in the OGD model, they still caused convulsions. The second strategy utilized the phosphodiesterase 2A inhibitor PF-05180999 to oppose mitochondrial Ca2+ overloading by increasing mitochondrial Ca2+ extrusion. Combining low concentrations of Ru265 and PF-05180999 that by themselves are not neuroprotective, synergistically protected and markedly preserved mitochondrial respiration in cortical neuron cultures subjected to a lethal period of OGD. Alternative strategies to further improve the safety and efficacy of Ru265 are also discussed. These findings support the strong neuroprotective potential of Ru265 for ischemic stroke and provide methods by which to improve the safety of this promising MCUcx inhibitor.