Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD.

Perm1 promotes cardiomyocyte mitochondrial biogenesis and protects against hypoxia/reoxygenation-induced damage in mice.

Normal contractile function of the heart depends on a constant and reliable production of ATP by cardiomyocytes. Dysregulation of cardiac energy metabolism can result in immature heart development and disrupt the ability of the adult myocardium to adapt to stress, potentially leading to heart failure. Further, restoration of abnormal mitochondrial function can have beneficial effects on cardiac dysfunction. Previously, we identified a novel protein termed Perm1 (PGC-1 and estrogen-related receptor (ERR)-induced regulator, muscle 1) that is enriched in skeletal and cardiac-muscle mitochondria and transcriptionally regulated by PGC-1 (peroxisome proliferator-activated receptor gamma coactivator 1) and ERR. The role of Perm1 in the heart is poorly understood and is studied here. We utilized cell culture, mouse models, and human tissue, to study its expression and transcriptional control, as well as its role in transcription of other factors. Critically, we tested Perm1's role in cardiomyocyte mitochondrial function and its ability to protect myocytes from stress-induced damage. Our studies show that Perm1 expression increases throughout mouse cardiogenesis, demonstrate that Perm1 interacts with PGC-1α and enhances activation of PGC-1 and ERR, increases mitochondrial DNA copy number, and augments oxidative capacity in cultured neonatal mouse cardiomyocytes. Moreover, we found that Perm1 reduced cellular damage produced as a result of hypoxia and reoxygenation-induced stress and mitigated cell death of cardiomyocytes. Taken together, our results show that Perm1 promotes mitochondrial biogenesis in mouse cardiomyocytes. Future studies can assess the potential of Perm1 to be used as a novel therapeutic to restore cardiac dysfunction induced by ischemic injury.

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The transcription factor NR4A3 controls CD103+ dendritic cell migration.

The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.

Intravenous (-)-epicatechin reduces myocardial ischemic injury by protecting mitochondrial function.

BACKGROUND: Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (-)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated. METHODS AND RESULTS: Ischemia was induced in rats via a 45 min occlusion of the left anterior descending coronary artery followed by 1 h, 48 h, or 3 week reperfusion. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48 h and 28% at 3 week. Double dose treatment further decreased infarct size by 80% at 48 h, and 52% by 3 weeks. The protective effect of EPI on mitochondrial function was evident after 1h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca2+ load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation. CONCLUSIONS: IV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility.

AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function.

Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2(+/-)) mice had no evidence of increased renal disease, and Ampka2(-/-) mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity.