Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone.

Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoKATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoKATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients. © 2016 American Physiological Society. Compr Physiol 6:1529-1548, 2016.

Depolarization of mitochondria in neurons promotes activation of nitric oxide synthase and generation of nitric oxide.

The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.

Dynamics of enhanced mitochondrial respiration in female compared with male rat cerebral arteries.

Mitochondrial respiration has never been directly examined in intact cerebral arteries. We tested the hypothesis that mitochondrial energetics of large cerebral arteries ex vivo are sex dependent. The Seahorse XFe24 analyzer was used to examine mitochondrial respiration in isolated cerebral arteries from adult male and female Sprague-Dawley rats. We examined the role of nitric oxide (NO) on mitochondrial respiration under basal conditions, using N(ω)-nitro-l-arginine methyl ester, and following pharmacological challenge using diazoxide (DZ), and also determined levels of mitochondrial and nonmitochondrial proteins using Western blot, and vascular diameter responses to DZ. The components of mitochondrial respiration including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity were elevated in females compared with males, but increased in both male and female arteries in the presence of the NOS inhibitor. Although acute DZ treatment had little effect on mitochondrial respiration of male arteries, it decreased the respiration in female arteries. Levels of mitochondrial proteins in Complexes I-V and the voltage-dependent anion channel protein were elevated in female compared with male cerebral arteries. The DZ-induced vasodilation was greater in females than in males. Our findings show that substantial sex differences in mitochondrial respiratory dynamics exist in large cerebral arteries and may provide the mechanistic basis for observations that the female cerebral vasculature is more adaptable after injury.

The mechanistic target of rapamycin (mTOR) pathway and S6 Kinase mediate diazoxide preconditioning in primary rat cortical neurons.

We examined the role of the mechanistic target of rapamycin (mTOR) pathway in delayed diazoxide (DZ)-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for 3 days with 500 µM DZ or feeding medium and then exposed to 3 h of continuous normoxia in Dulbecco's modified eagle medium with glucose or with 3 h of oxygen-glucose deprivation (OGD) followed by normoxia and feeding medium. The OGD decreased viability by 50%, depolarized mitochondria, and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Neuroprotection by DZ was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). The mTOR inhibitors rapamycin and Torin-1, as well as S6K-targeted siRNA abolished the protective effects of DZ. The effects of DZ on mitochondrial membrane potential and reactive oxygen species production were not affected by rapamycin. Preconditioning with DZ also changed mitochondrial and non-mitochondrial oxygen consumption rates. We conclude that in addition to reducing reactive oxygen species (ROS) production and mitochondrial membrane depolarization, DZ protects against OGD by activation of the Akt-mTOR-S6K pathway and by changes in mitochondrial respiration. Ischemic strokes have limited therapeutic options. Diazoxide (DZ) preconditioning can reduce neuronal damage. Using oxygen-glucose deprivation (OGD), we studied Akt/mTOR/S6K signaling and mitochondrial respiration in neuronal preconditioning. We found DZ protects neurons against OGD via the Akt/mTOR/S6K pathway and alters the mitochondrial and non-mitochondrial oxygen consumption rate. This suggests that the Akt/mTOR/S6k pathway and mitochondria are novel stroke targets.

Sustained mitochondrial functioning in cerebral arteries after transient ischemic stress in the rat: a potential target for therapies.

The objective of the present study was to determine whether mitochondrial function in the cerebral vasculature is maintained after transient middle cerebral artery (MCA) occlusion (tMCAO) in rats. Sprague-Dawley rats were exposed to 90 min of tMCAO followed by 4 or 48 h of reperfusion. MCAs from ischemic (ipsilateral) and nonischemic (contralateral) sides were compared with control MCAs from sham-operated rats. We determined 1) vasoreactivity to diazoxide (DZ; a mitochondrial ATP-activated K(+) channel opener), ACh, bradykinin (BK), serotonin, and sodium nitroprusside; 2) levels of mitochondrial and nonmitochondrial proteins and mitochondrial DNA; and 3) vascular levels of tetramethylrhodamine ethyl ester (an indicator of mitochondrial membrane potential). All dilator responses, including those with DZ, were intact 4 h post-tMCAO. Dilator responses to ACh, BK, and sodium nitroprusside were reduced in ipsilateral MCAs at 48 h compared with contralateral MCAs, but DZ responses were comparable with control MCAs. Surprisingly, contralateral responses to ACh, BK, and serotonin were reduced compared with control MCAs at 48 h. Ipsilateral vasodilation to DZ at 48 h was eliminated by endothelial denudation and endothelial nitric oxide synthase (eNOS) inhibition but was only reduced in control MCAs. Mitochondrial proteins, phosphorylated eNOS, mitochondrial DNA, and mitochondrial membrane potential were higher in ipsilateral compared with contralateral MCAs. In conclusion, contrary to conventional wisdom, mitochondria remain functional for at least 48 h after severe ischemic stress in MCAs, and DZ-induced dilation is preserved due to maintained mitochondrial mass, probably in the endothelium, and eNOS signaling. Our findings support the concept that functioning vascular mitochondria are an unexpected target for novel stroke therapies.

Increased susceptibility to amyloid-ß toxicity in rat brain microvascular endothelial cells under hyperglycemic conditions.

We hypothesized that hyperglycemia-induced mitochondrial dysfunction and oxidative stress are closely associated with amyloid-ß peptide (Aß) toxicity in endothelial cells. Brain microvascular endothelial cells from rat (RBMEC) and mice (MBMEC) were isolated from adult Sprague-Dawley rats and homozygous db/db (Leprdb/Leprdb) and heterozygous (Dock7m/Leprdb) mice, and cultured under normo- and hyperglycemic conditions for 7 d followed by 24 h exposure to Aß1-40. Some experiments were also performed with two mitochondrial superoxide (O2•-) scavengers, MitoTempo and Peg-SOD. Cell viability was measured by the Alamar blue assay and mitochondrial membrane potential (ΔΨm) by confocal microscopy. Mitochondrial O2•- and hydrogen peroxide (H2O2) production was assessed by fluorescence microscopy and H2O2 production was confirmed by microplate reader. Hyperglycemia or Aß1-40 alone did not affect cell viability in RBMEC. However, the simultaneous presence of high glucose and Aß1-40 reduced cell viability and ΔΨm, and enhanced mitochondrial O2•- and H2O2 production. MitoTempo and PEG-SOD prevented Aß1-40 toxicity. Interestingly, MBMEC presented a similar pattern of alterations with db/db cultures presenting higher susceptibility to Aß1-40. Overall, our results show that high glucose levels increase the susceptibility of brain microvascular endothelial cells to Aß toxicity supporting the idea that hyperglycemia is a major risk factor for vascular injury associated with AD.

Mitochondrial dynamics associated with oxygen-glucose deprivation in rat primary neuronal cultures.

Our objective was to investigate the mitochondrial dynamics following oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. We documented changes in morphology, protein expression, and DNA levels in mitochondria following OGD and examined the roles of mitochondrial fission [dynamin-related protein 1 (Drp1), fission protein-1 (Fis1)] and fusion [mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy-1 protein (OPA1)] proteins on mitochondrial biogenesis and morphogenesis. We tested the effects of two Drp1 blockers [15-deoxy-Δ12,14-Prostaglandin J2 (PGJ2) and Mitochondrial Division Inhibitor (Mdivi-1)] on mitochondrial dynamics and cell survival. One hour of OGD had minimal effects on neuronal viability but mitochondria appeared condensed. Three hours of OGD caused a 60% decrease in neuronal viability accompanied by a transition from primarily normal/tubular and lesser number of rounded mitochondria during normoxia to either poorly labeled or small and large rounded mitochondria. The percentage of rounded mitochondria remained the same. The mitochondrial voltage-dependent anion channel, Complex V, and mitoDNA levels increased after OGD associated with a dramatic reduction in Drp1 expression, less reduction in Mfn2 expression, an increase in Mfn1 expression, with no changes in either OPA1 or Fis1. Although PGJ2 increased polymerization of Drp1, it did not reduce cell death or alter mitochondrial morphology following OGD and Mdivi-1 did not protect neurons against OGD. In summary, mitochondrial biogenesis and maintained fusion occurred in neurons along with mitochondrial fission following OGD; thus Mfn1 but not Drp1 may be a major regulator of these processes.