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.
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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 of seizure-like behaviours and the duration of clonic convulsions. 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 potential therapeutic utility of MCU inhibition in the acute management of ischemic stroke but also indicate that such clinical translation will require drug delivery strategies which mitigate the pro-convulsant effects of Ru265.
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- Award ID(s):
- 1750295
- NSF-PAR ID:
- 10147792
- Date Published:
- Journal Name:
- Journal of Cerebral Blood Flow & Metabolism
- ISSN:
- 0271-678X
- Page Range / eLocation ID:
- 0271678X2090852
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Objective Organophosphates (OPs) such as diisopropylfluorophosphate (DFP) and soman are lethal chemical agents that can produce seizures, refractory status epilepticus (SE), and brain damage. There are few optimal treatments for late or refractory SE. Phenobarbital is a second‐line drug for SE, usually after lorazepam, diazepam, or midazolam have failed to stop SE. Practically, 40 minutes or less is often necessary for first responders to arrive and assist in a chemical incident. However, it remains unclear whether administration of phenobarbital 40 minutes after OP intoxication is still effective. Here, we investigated the efficacy of phenobarbital treatment at 40 minutes postexposure to OP intoxication.
Methods Acute refractory SE was induced in rats by DFP injection as per a standard paradigm. After 40 minutes, subjects were given phenobarbital intramuscularly (30‐100 mg/kg) and progression of seizure activity was monitored by video‐EEG recording. The extent of brain damage was assessed 3 days after DFP injections by neuropathology analysis of neurodegeneration and neuronal injury by unbiased stereology.
Results Phenobarbital produced a dose‐dependent seizure protection. A substantial decrease in SE was evident at 30 and 60 mg/kg, and a complete seizure termination was noted at 100 mg/kg within 40 minutes after treatment. Neuropathology findings showed significant neuroprotection in 100 mg/kg cohorts in brain regions associated with SE. Although higher doses resulted in greater protection against refractory SE and neuronal damage, they did not positively correlate with improved survival rate. Moreover, phenobarbital caused serious adverse effects including anesthetic or comatose state and even death.
Significance Phenobarbital appears as an alternate anticonvulsant for OP‐induced refractive SE in hospital settings. A careful risk‐benefit analysis is required because of negative outcomes on survival and cardio‐respiratory function. However, the need for sophisticated support and critical monitoring in hospital may preclude its use as medical countermeasure in mass casualty situations.
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Hypoxic-Ischemic Encephalopathy (HIE) in the brain is the leading cause of morbidity and mortality in neonates and can lead to irreparable tissue damage and cognition. Thus, investigating key mediators of the HI response to identify points of therapeutic intervention has significant clinical potential. Brain repair after HI requires highly coordinated injury responses mediated by cell-derived extracellular vesicles (EVs). Studies show that stem cell-derived EVs attenuate the injury response in ischemic models by releasing neuroprotective, neurogenic, and anti-inflammatory factors. In contrast to 2D cell cultures, we successfully isolated and characterized EVs from whole brain rat tissue (BEV) to study the therapeutic potential of endogenous EVs. We showed that BEVs decrease cytotoxicity in an ex vivo oxygen glucose deprivation (OGD) brain slice model of HI in a dose- and time-dependent manner. The minimum therapeutic dosage was determined to be 25 μg BEVs with a therapeutic application time window of 4–24 h post-injury. At this therapeutic dosage, BEV treatment increased anti-inflammatory cytokine expression. The morphology of microglia was also observed to shift from an amoeboid, inflammatory phenotype to a restorative, anti-inflammatory phenotype between 24–48 h of BEV exposure after OGD injury, indicating a shift in phenotype following BEV treatment. These results demonstrate the use of OWH brain slices to facilitate understanding of BEV activity and therapeutic potential in complex brain pathologies for treating neurological injury in neonates.more » « less
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Abstract The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca2+uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca2+. The best‐known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X‐ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1177/0271678X20908523
