Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade

<p>Abstract</p> <p>Background</p> <p>Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H₂O₂), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target.</p> <p>Methods</p> <p>NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H₂O₂-induced carboxy-H₂-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22^phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.</p> <p>Results</p> <p>Nigral dopaminergic neurons <it>in situ</it> expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22^phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H₂O₂production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H₂O₂levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.</p> <p>Conclusions</p> <p>A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.</p

Angiotensin type 1 receptor antagonist losartan, reduces MPTP-induced degeneration of dopaminergic neurons in substantia nigra

BACKGROUND: Recent attention has focused on understanding the role of the brain-renin-angiotensin-system (RAS) in stroke and neurodegenerative diseases. Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT(1)) receptor, losartan, protects dopaminergic (DA) neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary ventral mesencephalic (VM) cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice (Fig. 1). RESULTS: In the presence of exogenous Ang II, losartan reduced MPP(+ )(5 μM) induced DA neuronal loss by 72% in vitro. Mice challenged with MPTP showed a 62% reduction in the number of DA neurons in the SNpc and a 71% decrease in tyrosine hydroxylase (TH) immunostaining of the striatum, whereas daily treatment with losartan lessened MPTP-induced loss of DA neurons to 25% and reduced the decrease in striatal TH(+ )immunostaining to 34% of control. CONCLUSION: Our study demonstrates that the brain-RAS plays an important neuroprotective role in the MPTP model of PD and points to AT(1 )receptor as a potential novel target for neuroprotection

Chapter 4: The LOTUS regression model

One of the primary motivations of the LOTUS effort is to attempt to reconcile the discrepancies in ozone trend results from the wealth of literature on the subject. Doing so requires investigating the various methodologies employed to derive long-term trends in ozone as well as to examine the large array of possible variables that feed into those methodologies and analyse their impacts on potential trend results. Given the limited amount of time, the LOTUS group focused on the most common methodology of multiple linear regression and performed a number of sensitivity tests with the goal of trying to establish best practices and come to a consensus on a single regression model to use for this study. This chapter discusses the details and results of the sensitivity tests before describing the components of the final single model that was chosen and the reasons for that choice

JNK2 regulates vascular remodeling in pulmonary hypertension

Pulmonary arterial (PA) wall modifications are key pathological features of pulmonary hypertension (PH). Although such abnormalities correlate with heightened phosphorylation of c-Jun N-terminal kinases 1/2 (JNK1/2) in a rat model of PH, the contribution of specific JNK isoforms to the pathophysiology of PH is unknown. Hence, we hypothesized that activation of either one, or both JNK isoforms regulates PA remodeling in PH. We detected increased JNK1/2 phosphorylation in the thickened vessels of PH patients' lungs compared to that in lungs of healthy individuals. JNK1/2 phosphorylation paralleled a marked reduction in MAP kinase phosphatase 1 JNK dephosphorylator) expression in patients' lungs. Association of JNK1/2 activation with vascular modification was confirmed in the calf model of severe hypoxia-induced PH. To ascertain the role of each JNK isoform in pathophysiology of PH, wild-type (WT), JNK1 null (JKN1(-/-)), and JNK2 null (JNK2(-/)(-)) mice were exposed to chronic hypoxia (10% O-2 for six weeks) to develop PH. In hypoxic WT lungs, an increase in JNK1/2 phosphorylation was associated with PH-like pathology. Hallmarks of PH pathophysiology, i.e. excessive accumulation of extracellular matrix and vessel muscularization with medial wall thickening, was also detected in hypoxic JNK1(-/-) lungs, but not in hypoxia-exposed JNK2(-/-) lungs. However, hypoxia-induced increases in right ventricular systolic pressure (RVSP) and in right ventricular hypertrophy (RVH) were similar in all three genotypes. Our findings suggest that JNK2 participates in PA remodeling (but likely not in vasoconstriction) in murine hypoxic PH and that modulating JNK2 actions might quell vascular abnormalities and limit the course of PH.National Heart, Lung and Blood Institute [HL64917 MD]; A. T. Still University startup funds12 month embargo; published online: 3 July 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Mitogen-Activated Protein Kinase Phosphatase-1 Is a Key Regulator of Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Vessel Density in Lung

Although mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key deactivator of MAP kinases, known effectors of lung vessel formation, whether it plays a role in the expression of proangiogenic vascular endothelial growth factor (VEGF) in hypoxic lung is unknown. We therefore hypothesized that MKP-1 is a crucial modulator of hypoxia-stimulated vessel development by regulating lung VEGF levels. Wild-type MKP-1+/+, heterozygous MKP-1+/−, and deficient MKP-1−/− mice were exposed to sea level (SL), Denver altitude (DA) (1609 m [5280 feet]), and severe high altitude (HYP) (∼5182 m [∼17,000 feet]) for 6 weeks. Hypoxia enhanced phosphorylation of p38 MAP kinase, a substrate of MKP-1, as well as α smooth muscle actin (αSMA) expression in vessels, respiratory epithelium, and interstitium of phosphatase-deficient lung. αSMA-positive vessel (<50 μm outside diameter) densities were markedly reduced, whereas vessel wall thickness was increased in hypoxic MKP-1−/− lung. Mouse embryonic fibroblasts (MEFs) of all three genotypes were isolated to pinpoint the mechanism involved in hypoxia-induced vascular abnormalities of MKP-1−/− lung. Sustained phosphorylation of p38 MAP kinase was observed in MKP-1-null MEFs in response to hypoxia exposure. Although hypoxia up-regulated VEGF levels in MKP-1+/+ MEFs eightfold, only a 70% increase in VEGF expression was observed in MKP-1-deficient cells. Therefore, our data strongly suggest that MKP-1 might be the key regulator of vascular densities through the regulation of VEGF levels in hypoxic lung