Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Dinuclear nitrido-bridged osmium complexes inhibit the mitochondrial calcium uniporter and protect cortical neurons against lethal oxygen-glucose deprivation.

Dysregulation of mitochondrial calcium uptake mediated by the mitochondrial calcium uniporter (MCU) is implicated in several pathophysiological conditions. Dinuclear ruthenium complexes are effective inhibitors of the MCU and have been leveraged as both tools to study mitochondrial calcium dynamics and potential therapeutic agents. In this study, we report the synthesis and characterization of Os245 ([Os2(µ-N)(NH3)8Cl2]3+) which is the osmium-containing analogue of our previously reported ruthenium-based inhibitor Ru265. This complex and its aqua-capped analogue Os245' ([Os2(µ-N)(NH3)8(OH2)2]5+) are both effective inhibitors of the MCU in permeabilized and intact cells. In comparison to the ruthenium-based inhibitor Ru265 (k obs = 4.92 × 10-3 s-1), the axial ligand exchange kinetics of Os245 are two orders of magnitude slower (k obs = 1.63 × 10-5 s-1) at 37 °C. The MCU-inhibitory properties of Os245 and Os245' are different (Os245 IC50 for MCU inhibition = 103 nM; Os245' IC50 for MCU inhibition = 2.3 nM), indicating that the axial ligands play an important role in their interactions with this channel. We further show that inhibition of the MCU by these complexes protects primary cortical neurons against lethal oxygen-glucose deprivation. When administered in vivo to mice (10 mg kg-1), Os245 and Os245' induce seizure-like behaviors in a manner similar to the ruthenium-based inhibitors. However, the onset of these seizures is delayed, a possible consequence of the slower ligand substitution kinetics for these osmium complexes. These findings support previous studies that demonstrate inhibition of the MCU is a promising therapeutic strategy for the treatment of ischemic stroke, but also highlight the need for improved drug delivery strategies to mitigate the pro-convulsant effects of this class of complexes before they can be implemented as therapeutic agents. Furthermore, the slower ligand substitution kinetics of the osmium analogues may afford new strategies for the development and modification of this class of MCU inhibitors.

Neuronal mitochondrial calcium uniporter deficiency exacerbates axonal injury and suppresses remyelination in mice subjected to experimental autoimmune encephalomyelitis.

High-capacity mitochondrial calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter (MCU) is strategically positioned to support the survival and remyelination of axons in multiple sclerosis (MS) by undocking mitochondria, buffering Ca2+ and elevating adenosine triphosphate (ATP) synthesis at metabolically stressed sites. Respiratory chain deficits in MS are proposed to metabolically compromise axon survival and remyelination by suppressing MCU activity. In support of this hypothesis, clinical scores, mitochondrial dysfunction, myelin loss, axon damage and inflammation were elevated while remyelination was blocked in neuronal MCU deficient (Thy1-MCU Def) mice relative to Thy1 controls subjected to experimental autoimmune encephalomyelitis (EAE). At the first sign of walking deficits, mitochondria in EAE/Thy1 axons showed signs of activation. By contrast, cytoskeletal damage, fragmented mitochondria and large autophagosomes were seen in EAE/Thy1-MCU Def axons. As EAE severity increased, EAE/Thy1 axons were filled with massively swollen mitochondria with damaged cristae while EAE/Thy1-MCU Def axons were riddled with late autophagosomes. ATP concentrations and mitochondrial gene expression were suppressed while calpain activity, autophagy-related gene mRNA levels and autophagosome marker (LC3) co-localization in Thy1-expressing neurons were elevated in the spinal cords of EAE/Thy1-MCU Def compared to EAE/Thy1 mice. These findings suggest that MCU inhibition contributes to axonal damage that drives MS progression.

The cell-permeable mitochondrial calcium uniporter inhibitor Ru265 preserves cortical neuron respiration after lethal oxygen glucose deprivation and reduces hypoxic/ischemic brain injury.

The mitochondrial calcium (Ca2+) uniporter (MCU) mediates high-capacity mitochondrial Ca2+ uptake implicated in ischemic/reperfusion cell death. We have recently shown that inducible MCU ablation in Thy1-expressing neurons renders mice resistant to sensorimotor deficits and forebrain neuron loss in a model of hypoxic/ischemic (HI) brain injury. These findings encouraged us to compare the neuroprotective effects of Ru360 and the recently identified cell permeable MCU inhibitor Ru265. Unlike Ru360, Ru265 (2-10 µM) reached intracellular concentrations in cultured cortical neurons that preserved cell viability, blocked the protease activity of Ca2+-dependent calpains and maintained mitochondrial respiration and glycolysis after a lethal period of oxygen-glucose deprivation (OGD). Intraperitoneal (i.p.) injection of adult male C57Bl/6 mice with Ru265 (3 mg/kg) also suppressed HI-induced sensorimotor deficits and brain injury. However, higher doses of Ru265 (10 and 30 mg/kg, i.p.) produced dose-dependent increases in the frequency and duration of seizure-like behaviours. Ru265 is proposed to promote convulsions by reducing Ca2+ buffering and energy production in highly energetic interneurons that suppress brain seizure activity. These findings support the therapeutic potential of MCU inhibition in the treatment of ischemic stroke but also indicate that such clinical translation will require drug delivery strategies which mitigate the pro-convulsant effects of Ru265.