Post-ischemic inhibition of the mitochondrial Ca2+ uniporter with Ru265 protects in ex vivo and in vivo models of cerebral ischemia.

Brain ischemia is a leading cause of morbidity and mortality in the aging population, yet no effective neuroprotective interventions currently exist. This therapeutic gap stems from two key challenges: the inability to deliver neuroprotective drugs before reperfusion and limited understanding of post-reperfusion events leading to delayed neurodegeneration. Zn2+ is an important contributor to ischemic brain injury, with mitochondrial Zn2+ uptake playing a central role in its neurotoxic effects. Ongoing Zn2+ accumulation occurs through the mitochondrial Ca2+ uniporter (MCU) in selectively vulnerable CA1 neurons after transient ischemia, contributing to delayed mitochondrial dysfunction. We previously demonstrated that post-ischemic administration of the non-selective MCU antagonist, ruthenium red attenuated neuronal damage in both an ex vivo oxygen-glucose deprivation (OGD) mouse brain slice model and in an in vivo rat cardiac arrest (CA) model. In the present study, we test post-ischemic treatment with Ru265, a novel selective MCU blocker, in both ischemic models. In the slice model, OGD induced delayed Zn2+ accumulation in CA1 neuronal mitochondria (as previously reported), followed by late-onset persistent mitochondrial depolarization and sustained elevation of cytoplasmic Ca2+ levels, indicating loss of Ca2+ homeostasis. Application of Ru265 after OGD termination inhibited the mitochondrial Zn2+ accumulation and largely prevented subsequent mitochondrial depolarization and loss of cytosolic Ca2+ homeostasis. In the CA model, intravenous Ru265 administration during cardiopulmonary resuscitation largely mitigated mitochondrial damage, markedly reduced neuronal injury and improved behavioral outcomes 20-24 h after ischemia. These findings suggest that post-ischemic MCU blockade may constitute a promising neuroprotective therapeutic strategy.