KATP channels are necessary for glucose-dependent increases in amyloid-ß and Alzheimer's disease-related pathology.

Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-ß (Aß) release, offering a mechanistic link between type 2 diabetes and Alzheimer's disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aß pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aß, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aß pathology in patients with diabetes or prediabetes.

An Acidic Environment Induces APOL1-Associated Mitochondrial Fragmentation.

BACKGROUND: Apolipoprotein L1 gene (APOL1) G1 and G2 kidney-risk variants (KRVs) cause CKD in African Americans, inducing mitochondrial dysfunction. Modifying factors are required, because a minority of individuals with APOL1 high-risk genotypes develop nephropathy. Given that APOL1 function is pH-sensitive and the pH of the kidney interstitium is <7, we hypothesized the acidic kidney interstitium may facilitate APOL1 KRV-induced mitochondrial dysfunction. METHODS: Human embryonic kidney (HEK293) cells conditionally expressing empty vector (EV), APOL1-reference G0, and G1 or G2 KRVs were incubated in media pH 6.8 or 7.4 for 4, 6, or 8 h. Genotype-specific pH effects on mitochondrial length (µm) were assessed using confocal microscopy in live cells and Fiji derivative of ImageJ software with MiNA plug-in. Lower mitochondrial length indicated fragmentation and early dysfunction. RESULTS: After 6 h doxycycline (Dox) induction in pH 6.8 media, G2-expressing cells had shorter mitochondria (6.54 ± 0.40) than cells expressing EV (7.65 ± 0.72, p = 0.02) or G0 (7.46 ± 0.31, p = 0.003). After 8 h Dox induction in pH 6.8 media, both G1- (6.21 ± 0.26) and G2-expressing cells had shorter mitochondria (6.46 ± 0.34) than cells expressing EV (7.13 ± 0.32, p = 0.002 and p = 0.008, respectively) or G0 (7.22 ± 0.45, p = 0.003 and p = 0.01, respectively). Mitochondrial length in cells incubated in pH 7.4 media were comparable after 8 h Dox induction regardless of genotype. APOL1 mRNA expression and cell viability were comparable regardless of pH or genotype after 8 h Dox induction. CONCLUSION: Acidic pH facilitates early mitochondrial dysfunction induced by APOL1 G1 and G2 KRVs in HEK293 cells. We propose that the acidic kidney interstitium may play a role in APOL1-mediated mitochondrial pathophysiology and nephropathy.

APOL1 Kidney-Risk Variants Induce Mitochondrial Fission.

INTRODUCTION: APOL1 G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of APOL1 and elucidate potential mechanisms in APOL1-nephropathy. METHODS: A global gene expression analysis was performed in human primary renal tubule cell lines derived from 50 African American individuals. Follow-up gene knock out, cell-based rescue, and microscopy experiments were performed. RESULTS: APOL1 genotypes did not alter APOL1 expression levels in the global gene expression analysis. Expression quantitative trait locus (eQTL) analysis in polyinosinic-polycytidylic acid (poly IC)-stimulated renal tubule cells revealed that single nucleotide polymorphism (SNP) rs513349 adjacent to BAK1 was a trans eQTL for APOL1 and a cis eQTL for BAK1; APOL1 and BAK1 were co-expressed in cells. BAK1 knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on APOL1 expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various APOL1 genotypes. Mitochondria in APOL1 wild-type (G0G0) tubule cells maintained elongated morphology when stimulated by low-dose poly IC, whereas those with G1G1, G2G2, and G1G2 genotypes appeared to fragment. HEK293 Tet-on cells overexpressing APOL1 G0, G1, and G2 were created; G0 cells appeared to promote mitochondrial fusion, whereas G1 and G2 induced mitochondrial fission. The mitochondrial dynamic regulator Mdivi-1 significantly preserved cell viability and mitochondrial cristae structure and reversed mitochondrial fission induced by overexpression of G1 and G2. CONCLUSION: Results suggest the mitochondrial fusion/fission pathway may be a therapeutic target in APOL1-nephropathy.

APOL1 Risk Variants Impair Multiple Mitochondrial Pathways in a Metabolomics Analysis.

Background: Kidney risk variants (KRVs) in the APOL1 gene are associated with mitochondrial dysfunction. However, the molecular spectrum of metabolites affected by the G1 and G2 KRVs, and the downstream mitochondrial pathways they affect, remain unknown. Methods: We performed a metabolomics analysis using HEK293 Tet-on cells conditionally expressing APOL1 G0, G1, and G2 KRVs to determine the patterns of metabolites and pathways potentially involved in nephropathy. The Welch two-sample t test, matched-pairs t test, and two-way repeated measures ANOVA were used to identify differential metabolites. Random forest, a supervised classification algorithm that uses an ensemble of decision trees, and the mean-decrease-accuracy metric were applied to prioritize top metabolites. Results: Alterations in the tricarboxylic acid cycle, increased fatty acid oxidation, and compromised redox homeostasis were the major pathways affected by overexpression of APOL1 KRVs. Conclusions: Impairment of mitochondrial membrane respiratory chain complex I appeared to account for critical metabolic consequences of APOL1 KRVs. This finding supports depletion of the mitochondrial membrane potential, as has been reported.

JC polyoma viruria associates with protection from chronic kidney disease independently from apolipoprotein L1 genotype in African Americans.

Background: Viral infections can trigger chronic kidney disease (CKD) and the urine virome may inform risk. The Natural History of APOL1-Associated Nephropathy Study (NHAANS) reported that urine JC polyomavirus (JCPyV) associated with a lower risk of APOL1-associated nephropathy in African Americans. Herein, association was assessed between urine JCPyV with CKD in African Americans independent from the APOL1 genotype. Methods: Quantitative polymerase chain reaction was performed for urinary detection of JCPyV and BK polyoma virus (BKPyV) in 200 newly recruited nondiabetic African Americans. A combined analysis was performed in these individuals plus 300 NHAANS participants. Results: In the 200 new participants, urine JCPyV was present in 8.8% of CKD cases and 45.8% of nonnephropathy controls (P = 3.0 × 10-8). In those with APOL1 renal-risk genotypes, JCPyV was detected in 5.1% of cases and 40.0% of controls (P = 0.0002). In those lacking APOL1 renal-risk genotypes, JCPyV was detected in 12.2% of cases and 48.8% of controls (P = 8.5 × 10-5). BKPyV was detected in 1.3% of cases and 0.8% of controls (P = 0.77). In a combined analysis with 300 NHAANS participants (n = 500), individuals with urine JCPyV had a 63% lower risk of CKD compared with those without urine JCPyV (odds ratio 0.37; P = 4.6 × 10-6). RNA fluorescence in situ hybridization confirmed the presence of JCPyV genomic DNA and JCPyV messenger RNA (mRNA) in nondiseased kidney. Conclusions: Inverse relationships exist between JCPyV viruria and non-diabetic CKD. Future studies should determine whether renal inflammation associated with CKD is less permissive for JCPyV reactivation/replication or whether JCPyV is a marker of reduced host immune responsiveness that diminishes immune pathologic contributions to CKD.

APOL1 Renal-Risk Variants Induce Mitochondrial Dysfunction.

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.

Characterization of circulating APOL1 protein complexes in African Americans.

APOL1 gene renal-risk variants are associated with nephropathy and CVD in African Americans; however, little is known about the circulating APOL1 variant proteins which reportedly bind to HDL. We examined whether APOL1 G1 and G2 renal-risk variant serum concentrations or lipoprotein distributions differed from nonrisk G0 APOL1 in African Americans without nephropathy. Serum APOL1 protein concentrations were similar regardless of APOL1 genotype. In addition, serum APOL1 protein was bound to protein complexes in two nonoverlapping peaks, herein referred to as APOL1 complex A (12.2 nm diameter) and complex B (20.0 nm diameter). Neither of these protein complexes associated with HDL or LDL. Proteomic analysis revealed that complex A was composed of APOA1, haptoglobin-related protein (HPR), and complement C3, whereas complex B contained APOA1, HPR, IgM, and fibronectin. Serum HPR was less abundant on complex B in individuals with G1 and G2 renal-risk variant genotypes, relative to G0 (P = 0.0002-0.037). These circulating complexes may play roles in HDL metabolism and susceptibility to CVD.

Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines.

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

An ACACB variant implicated in diabetic nephropathy associates with body mass index and gene expression in obese subjects.

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m(2) (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m(2) (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m(2); p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m(2) for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.

Depolarization of mitochondria in endothelial cells promotes cerebral artery vasodilation by activation of nitric oxide synthase.

OBJECTIVE: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation. APPROACH AND RESULTS: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS. CONCLUSIONS: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Insulin-induced generation of reactive oxygen species and uncoupling of nitric oxide synthase underlie the cerebrovascular insulin resistance in obese rats.

Hyperinsulinemia accompanying insulin resistance (IR) is an independent risk factor for stroke. The objective is to examine the cerebrovascular actions of insulin in Zucker obese (ZO) rats with IR and Zucker lean (ZL) control rats. Diameter measurements of cerebral arteries showed diminished insulin-induced vasodilation in ZO compared with ZL. Endothelial denudation revealed vasoconstriction to insulin that was greater in ZO compared with ZL. Nonspecific inhibition of nitric oxide synthase (NOS) paradoxically improved vasodilation in ZO. Scavenging of reactive oxygen species (ROS), supplementation of tetrahydrobiopterin (BH(4)) precursor, and inhibition of neuronal NOS or NADPH oxidase or cyclooxygenase (COX) improved insulin-induced vasodilation in ZO. Immunoblot experiments revealed that insulin-induced phosphorylation of Akt, endothelial NOS, and expression of GTP cyclohydrolase-I (GTP-CH) were diminished, but phosphorylation of PKC and ERK was enhanced in ZO arteries. Fluorescence studies showed increased ROS in ZO arteries in response to insulin that was sensitive to NOS inhibition and BH(4) supplementation. Thus, a vicious cycle of abnormal insulin-induced ROS generation instigating NOS uncoupling leading to further ROS production underlies the cerebrovascular IR in ZO rats. In addition, decreased bioavailability and impaired synthesis of BH(4) by GTP-CH induced by insulin promoted NOS uncoupling.

Impaired vascular responses of insulin-resistant rats after mild subarachnoid hemorrhage.

Insulin resistance (IR) impairs cerebrovascular responses to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after subarachnoid hemorrhage (SAH) have not been described in IR. We hypothesized that IR worsens vascular reactions after a mild SAH. Hemolyzed blood (300 µl) or saline was infused (10 µl/min) into the cisterna magna of 11-13-wk-old ZO (n = 25) and Zucker lean (ZL) rats (n = 25). One day later, dilator responses of the basilar artery (BA) and its side branch (BA-Br) to acetylcholine (ACh, 10(-6) M), cromakalim (10(-7) M, 10(-6) M), and sodium nitroprusside (10(-7) M) were recorded with intravital videomicroscopy. The baseline diameter of the BA was increased both in the ZO and ZL rats 24 h after the hemolysate injection. Saline-injected ZO animals showed reduced dilation to ACh (BA = 9 ± 3 vs. 22 ± 4%; and BA-Br = 23 ± 5 vs. 37 ± 7%) compared with ZL rats. Hemolysate injection blunted the response to ACh in both the ZO (BA = 4 ± 2%; and BA-Br = 12 ± 3%) and ZL (BA = 7 ± 2%; and BA-Br = 11 ± 3%) rats. Cromakalim (10(-6) M)-induced dilation was significantly reduced in the hemolysate-injected ZO animals compared with the saline control (BA = 13 ± 3 vs. 26 ± 5%; and BA-Br = 28 ± 8 vs. 44 ± 9%) and in the hemolysate-injected ZL rats compared with their saline control (BA = 24 ± 4 vs. 32 ± 4%; but not BA-Br = 39 ± 6 vs. 59 ± 9%). No significant difference in sodium nitroprusside reactivity was observed. Western blot analysis of the BA showed a lower baseline level of neuronal nitric oxide synthase expression and an enhanced cyclooxygenase-2 level in the hemolysate-injected ZO animals. In summary, cerebrovascular reactivity to both endothelium-dependent and -independent stimuli is severely compromised by SAH in IR animals.

Rosuvastatin induces delayed preconditioning against L-glutamate excitotoxicity in cultured cortical neurons.

We tested whether rosuvastatin (RST) protected against excitotoxic neuronal cell death in rat primary cortical neuronal cultures. L-glutamate (200 microM, 1h) reduced neuronal viability (% of naive controls, mean+/-SEM, n=8-32, *p<0.05) from 100+/-2% to 60+/-1%*, but pretreatment with RST (0.5 microM, 3 days) increased survival to 88+/-2%*. RST-induced neuroprotection was not affected by co-application with mevalonate (10 microM), although the same dose of mevalonate fully prevented the neurotoxic effects of a high dose (20 microM) of RST. RST (0.5 microM) pretreatment did not affect mitochondrial membrane potential or superoxide anion levels in quiescent neurons. However, RST pretreatment blunted elevations in free intracellular Ca(2+) and reduced increases in superoxide anion levels following glutamate exposure. Manganese superoxide dismutase (SOD), copper-zinc SOD, catalase, and reduced glutathione levels were unaffected by RST pretreatment. In contrast, acute, one time RST application did not affect either baseline or L-glutamate-induced increases in superoxide levels. In summary, three-day RST pretreatment induces resistance to the excitotoxic effect of L-glutamate in cultured neurons apparently by a mechanism that is independent of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibition. The delayed neuroprotection by RST against excitotoxicity does not involve sustained mitochondrial depolarization or superoxide anion production as initiating events, although it is associated with reduced Ca(2+) influx and superoxide anion production upon L-glutamate challenge.

N-methyl-D-aspartate induces cortical hyperemia through cortical spreading depression-dependent and -independent mechanisms in rats.

OBJECTIVE: N-methyl-d-aspartate (NMDA) is a powerful cerebrovascular dilator in vivo. Cortical spreading depression (CSD) has recently been shown to contribute to the pial arteriolar dilation in mice. Our main aim was to examine the participation of CSD in the overall cerebrovascular response to NMDA in the rat. METHODS: Anesthetized Wistar rats (eight weeks old) were equipped with a closed cranial window to allow topical application of NMDA (10(-5)-10(-3) M) to the parietal cortex. Cortical blood flow (CoBF) under and outside the cranial window was simultaneously monitored by using a two-channel laser-Doppler flowmeter. CSDs were detected by recording the changes in the cortical DC potential. RESULTS: Concentrations of 10(-4) and 10(-3) M of NMDA evoked single CSDs associated with rapid, transient hyperemia, followed by a sustained, but reduced, increase in CoBF. The latency and magnitude of the CoBF responses were dose dependent. The higher dose resulted in shorter latency (100+/-5* vs. 146+/-11 seconds, *P<0.05; mean+/-standard error of the mean) and larger overall flow response (77+/-12* vs. 28+/-3% from baseline) under, but not outside, the cranial window. CONCLUSIONS: NMDA elicits dose-dependent increases in CoBF that are composed of CSD-dependent and -independent components in rats.

Immediate neuronal preconditioning by NS1619.

The objectives of our present experiments were to determine whether the BK(Ca) channel agonist NS1619 is able to induce immediate preconditioning in cultured rat cortical neurons and to elucidate the role of BK(Ca) channels in the initiation of immediate preconditioning. NS1619 depolarized mitochondria and increased reactive oxygen species (ROS) generation, but neither of these effects was inhibited by BK(Ca) channel antagonists. NS1619 also activated the extracellular signal-regulated kinase signaling pathways. One-hour treatment with NS1619 induced immediate protection against glutamate excitotoxicity (viability 24 h after glutamate exposure: control, 58.45+/-0.95%; NS1619 50 microM, 78.99+/-0.90%; NS1619 100 microM, 86.89+/-1.20%; NS1619 150 microM, 93.23+/-1.23%; mean+/-SEM; p<0.05 vs. control; n=16-32). Eliminating ROS during the preconditioning phase effectively blocked the development of cytoprotection. In contrast, the BK(Ca) channel blockers iberiotoxin and paxilline, the phosphoinositide 3-kinase inhibitor wortmannin, the protein kinase C blocker chelerythrine, and the mitogen activated protein kinase antagonist PD98059 were unable to antagonize the immediate neuroprotective effect. Finally, preconditioning with NS1619 reduced the calcium load and ROS surge upon glutamate exposure and increased superoxide dismutase activity. Our results indicate that NS1619 is an effective inducer of immediate neuronal preconditioning, but the neuroprotective effect is independent of the activation of BK(Ca) channels.

Neuroprotective effect of adenoviral catalase gene transfer in cortical neuronal cultures.

Reduced availability of reactive oxygen species is a key component of neuroprotection against various toxic stimuli. Recently we showed that the hydrogen peroxide scavenger catalase plays a central role in delayed preconditioning induced by the mitochondrial ATP-sensitive potassium channel opener BMS-191095. The purpose of the experiments discussed here was to investigate the neuroprotective effect of catalase in vitro using a recombinant adenoviral catalase gene transfer protocol. To induce catalase overexpression, cultured rat cortical neurons were infected with the adenoviral vector Ad5CMVcatalase and control cells were incubated with Ad5CMVntLacZ for 24 h. Gene transfer effectively increased catalase protein levels and activity, but did not influence other antioxidants tested. Ad5CMVcatalase, with up to 10 plaque forming units (pfu) per neuron, did not affect cell viability under control conditions and did not protect against glutamate excitotoxicity or oxygen-glucose deprivation. In contrast, catalase overexpression conferred a dose-dependent protection against exposure to hydrogen peroxide (viability: control, 33.02+/-1.09%; LacZ 10 pfu/cell, 32.85+/-1.51%; catalase 1 pfu/cell, 62.09+/-4.17%*; catalase 2 pfu/cell, 98.71+/-3.35%*; catalase 10 pfu/cell, 99.68+/-1.99%*; *p<0.05 vs. control; mean+/-SEM). Finally, the protection could be antagonized using the catalase inhibitor 3-aminotriazole. Our results support the view that enhancing cellular antioxidant capacity may play a crucial role in neuroprotective strategies.

Impaired mitochondria-dependent vasodilation in cerebral arteries of Zucker obese rats with insulin resistance.

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive K+ (K ATP) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial K ATP channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was 38 +/- 3% in ZL vs. 21 +/- 4% in ZO rats (P < 0.05). Iberiotoxin, a Ca2+-activated K+ channel inhibitor, or manganese(III) tetrakis(4-benzoic acid)porphyrin chloride, an SOD mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating Ca2+-activated K+ channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endothelium-denuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and SODs in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial K(ATP) channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity.

Rosuvastatin induces delayed preconditioning against oxygen-glucose deprivation in cultured cortical neurons.

We tested whether rosuvastatin (RST) protected against oxygen-glucose deprivation (OGD)-induced cell death in primary rat cortical neuronal cultures. OGD reduced neuronal viability (%naive controls, mean +/- SE, n = 24-96, P < 0.05) to 44 +/- 1%, but 3-day pretreatment with RST (5 microM) increased survival to 82 +/- 2% (P < 0.05). One-day RST treatment was not protective. RST-induced neuroprotection was abolished by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by cholesterol coapplication. Furthermore, RST-induced decreases in neuronal cholesterol levels were abolished by mevalonate but not by GGPP. Reactive oxygen species (ROS) levels were reduced in RST-preconditioned neurons after OGD, and this effect was also reversed by both mevalonate and GGPP. These data suggested that GGPP, but not cholesterol depletion, were responsible for the induction of neuroprotection. Therefore, we tested whether 3-day treatments with perillic acid, a nonspecific inhibitor of both geranylgeranyl transferase (GGT) GGT 1 and Rab GGT, and the GGT 1-specific inhibitor GGTI-286 would reproduce the effects of RST. Perillic acid, but not GGTI-286, elicited robust neuronal preconditioning against OGD. RST, GGTI-286, and perillic acid all decreased mitochondrial membrane potential and lactate dehydrogenase activity in the cultured neurons, but only RST and perillic acid reduced neuronal ATP and membrane Rab3a protein levels. In conclusion, RST preconditions cultured neurons against OGD via depletion of GGPP, leading to decreased geranylgeranylation of proteins that are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection.

ROS-independent preconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095.

Previously, we have shown that the selective mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel opener BMS-191095 (BMS) induces neuronal preconditioning (PC); however, the exact mechanism of BMS-induced neuroprotection remains unclear. In this study, we have identified key components of the cascade resulting in delayed neuronal PC with BMS using isolated rat brain mitochondria and primary cultures of rat cortical neurons. BMS depolarized isolated mitochondria without an increase in reactive oxygen species (ROS) generation and induced rapid phosphorylation of Akt and glycogen synthase kinase-3beta. Long-term (3 days) treatment of neurons with BMS resulted in sustained mitochondrial depolarization, decreased basal ROS generation, and elevated ATP levels. This treatment also elicited almost complete protection against glutamate excitotoxicity, which could be abolished using the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, but not with the superoxide dismutase (SOD) mimetic M40401. Long-term BMS treatment induced a PI3K-dependent increase in the expression and activity of catalase without affecting manganese SOD and copper/zinc-dependent SOD. Finally, the catalase inhibitor 3-aminotriazole dose-dependently antagonized the neuroprotective effect of BMS-induced PC. In summary, BMS depolarizes mitochondria without ROS generation, activates the PI3K-Akt pathway, improves ATP content, and increases catalase expression. These mechanisms appear to play important roles in the neuroprotective effect of BMS.

Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels.

1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), a potent activator of the large conductance Ca2+ activated potassium (BK(Ca)) channel, has been demonstrated to induce preconditioning (PC) in the heart. The aim of our study was to test the delayed PC effect of NS1619 in rat cortical neuronal cultures against oxygen-glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. Furthermore, we tested the activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase-3/7. NS1619 dose-dependently protected the cells against the toxic insults, and the protection was completely blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BK(Ca) channel inhibitors. Application of NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. However, only the effect on the cell membrane potential was antagonized by BK(Ca) channel blockers. NS1619 inhibited the activation of capase-3/7. In summary, NS1619 is a potent inducer of delayed neuronal PC. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BK(Ca) channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation.