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

Abstract

BackgroundReactive 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.MethodsNADPH 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 p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.ResultsNigral dopaminergic neurons in situ 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 p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.ConclusionsA 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.

Critical period for dopaminergic neuroprotection by hormonal replacement in menopausal rats

“…Interestingly, we also observed a significant increase in AT1 (and decrease in AT2) receptor expression, which may be related to a feedback mechanism for regulating levels of brain angiotensinogen (O'Callaghan et al, 2011) or to induction of further proinflammatory changes in astrocytes (Clark et al, 2008;Lanz et al, 2010). An increase in AII levels has been shown to increase oxidative stress in neurons (Joglar et al, 2009;Zawada et al, 2011) and to enhance the microglial neuroinflammatory response (Rodriguez-Pallares et al, 2008;Villar-Cheda et al, 2012). Furthermore, in the present study, we observed that estrogen, via ER-a, also induces direct neuroprotection of dopaminergic neurons; thus, in cultures of the dopaminergic neuron cell line (i.e., in the absence of glia), the ER-a agonist PPT inhibits the loss of dopaminergic cells induced by MPP þ and MPP þ þ AII, possibly by inhibition of neuronal oxidative stress and apoptosis (Brendel et al, 2013;Wang et al, 2012).…”

“…In animal models of PD, hyperactivation of the local RAS exacerbates the inflammatory microglial response, oxidative stress and dopaminergic degeneration, all of which are inhibited by angiotensin receptor blockers (for reviews, see Labandeira-Garcia et al, 2013;Wright and Harding, 2012). Recent studies have shown that AII-induced activation of the NADPHoxidase complex (Joglar et al, 2009;Rodriguez-Pallares et al, 2008;Zawada et al, 2011) and activation of the microglial RhoA/Rho kinase pathway are involved in AII-induced oxidative stress and neuroinflammation. In the present study, we observed that RAS inhibition plays a major role in the beneficial effects of estrogen, which was confirmed by the neuroprotective effect of RAS inhibition by candesartan after LTED (i.e., when the ERT is unable to decrease RAS activity).…”

Dopamine Receptors and Neurodegeneration

“…In neurotoxin-induced parkinsonism with 6-OHDA and MPTP the role of RAS has been studied and evidences indicate that in these models up-regulated levels of AII, exacerbates the DAergic neuronal death mediated by AT1Rs [86]. Experimental data also support the involvement of brain RAS in dopaminergic degeneration [110,111,112]. It was demonstrated that AII increased the neurotoxic effect induced by low doses of 6-OHDA, and the treatment with inhibitors of ACE [113,114,112] or blockage of AT1Rs [98,95,96] resulted in a significant reduction of both the loss of dopaminergic neurons and the levels of protein oxidation and lipid peroxidation induced by the neurotoxins [115].…”

Activation of the ACE2/Ang-(1–7)/Mas pathway reduces oxygen–glucose deprivation-induced tissue swelling, ROS production, and cell death in mouse brain with angiotensin II overproduction

“…Various isoforms of NADPH have been identified in neurons and glial cells of the CNS 11, 14, 65, 68, 93 and previous clinical and animal studies have shown their importance for pathogenesis of injury following ischemia 48, 53 . Compared with slices from wild type mice in control conditions, slices from R+A+ animals had similar expression levels of Nox2, but elevated expression levels of Nox4.…”