Microglial Reactive Oxygen Species Research Articles

Microglial Hv1 proton channel promotes cuprizone-induced demyelination through oxidative damage.

NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) production in inflammatory cells including microglia plays an important role in demyelination and free radical-mediated tissue injury in multiple sclerosis (MS). However, the mechanism underlying microglial ROS production and demyelination remains largely unknown. The voltage-gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain. In the present study, we sought to determine the role of microglial Hv1 proton channels in a mouse model of cuprizone-induced demyelination, a model for MS. Following cuprizone exposure, wild-type mice presented obvious demyelination, decreased myelin basic protein expression, loss of mature oligodendrocytes, and impaired motor coordination in comparison to mice on a normal chow diet. However, mice lacking Hv1 (Hv1(-/-) ) are partially protected from demyelination and motor deficits compared with those in wild-type mice. These rescued phenotypes in Hv1(-/-) mice in cuprizone-induced demyelination is accompanied by reduced ROS production, ameliorated microglial activation, increased oligodendrocyte progenitor cell (NG2) proliferation, and increased number of mature oligodendrocytes. These results demonstrate that the Hv1 proton channel is required for cuprizone-induced microglial oxidative damage and subsequent demyelination. Our study suggests that the microglial Hv1 proton channel is a unique target for controlling NOX-dependent ROS production in the pathogenesis of MS.

CCL11 enhances excitotoxic neuronal death by producing reactive oxygen species in microglia.

The chemokine CCL11 (also known as eotaxin-1) is a potent eosinophil chemoattractant that mediates allergic diseases such as asthma, atopic dermatitis, and inflammatory bowel diseases. Previous studies demonstrated that concentrations of CCL11 are elevated in the sera and cerebrospinal fluids (CSF) of patients with neuroinflammatory disorders, including multiple sclerosis. Moreover, the levels of CCL11 in plasma and CSF increase with age, and CCL11 suppresses adult neurogenesis in the central nervous system (CNS), resulting in memory impairment. However, the precise source and function of CCL11 in the CNS are not fully understood. In this study, we found that activated astrocytes release CCL11, whereas microglia predominantly express the CCL11 receptor. CCL11 significantly promoted the migration of microglia, and induced microglial production of reactive oxygen species by upregulating nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), thereby promoting excitotoxic neuronal death. These effects were reversed by inhibition of NOX1. Our findings suggest that CCL11 released from activated astrocytes triggers oxidative stress via microglial NOX1 activation and potentiates glutamate-mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders.

Microglial ROS production in an electrical rat post-status epilepticus model of epileptogenesis

Reactive oxygen species and inflammatory signaling have been identified as pivotal pathophysiological factors contributing to epileptogenesis. Considering the development of combined anti-inflammatory and antioxidant treatment strategies with antiepileptogenic potential, a characterization of the time course of microglial reactive oxygen species generation during epileptogenesis is of major interest. Thus, we isolated microglia cells and analyzed the generation of reactive oxygen species by flow cytometric analysis in an electrical rat post-status epilepticus model.Two days post status epilepticus, a large-sized cell cluster exhibited a pronounced response with excessive production of reactive oxygen species upon stimulation with phorbol-myristate-acetate. Neither in the latency phase nor in the chronic phase with spontaneous seizures a comparable cell population with induction of reactive oxygen species was identified.We were able to demonstrate in the electrical rat post-status-epilepticus model, that microglial ROS generation reaches a peak after the initial insult, is only marginally increased in the latency phase, and returns to control levels during the chronic epileptic phase. The data suggest that a combination of anti-inflammatory and radical scavenging approaches might only be beneficial during a short time window after an epileptogenic brain insult.

Somatostatin prevents lipopolysaccharide-induced neurodegeneration in the rat substantia nigra by inhibiting the activation of microglia.

Somatostatin (SST) is a neuromodulator which is abundant throughout the central nervous system (CNS) and has a crucial role in neurodegenerative disorders. However, little is known about the effects and mechanisms of SST in dopaminergic (DA) neurons in the context of Parkinson’s disease (PD). In the present study, a model of PD was generated by injecting lipopolysaccharide (LPS) into the substantia nigra (SN) of rats in order to investigate the effects of SST on LPS-induced degeneration of DA in vivo. Intramural injection of LPS resulted in a significant loss of DA neurons, while reduction of neuronal death by SST pretreatment was confirmed using immunohistochemical staining for tyrosine hydroxylase and Nissl. In parallel, immunohistochemical detection of OX-42 and hydroethidine staining were employed to determine the activation of microglia and production of reactive oxygen species (ROS), respectively. It was found that SST inhibited the LPS-induced microglial activity and ROS production. ELISA revealed a decreased production of pro-inflammatory mediators, including tumor necrosis factor-α, interleukin-1β and prostaglandin E2 when SST was administered prior to LPS treatment. Western blot analysis showed that LPS-induced expression of inducible nitric oxide synthase, cyclooxygenase-2 and nuclear factor κB (NF-κB) p-p65 was attenuated by administration of SST prior to LPS application. The results indicated that LPS-induced loss of nigral DA neurons was inhibited by SST and the observed effects of SST on neuroprotection were associated with suppression of microglial activation and the NF-κB pathway, ensuing decreases of neuroinflammation and oxidative stress. The present study therefore suggested that SST is beneficial for treating neurodegenerative diseases, such as PD, through inhibiting the activation of microglia.

Abstract T P229: Deletion of Voltage Gated Proton Channel in Microglial Cells is Neurovascular Protective After Ischemic Brain Injury

Despite the failure of antioxidant treatments in clinical trials, the undoubted role of reactive oxygen species (ROS) in neurovascular damage after ischemic stroke calls for a more targeted approach. ROS production by microglia, the primary resident immune cells in the brain, is a key event of this process in ischemic stroke. Voltage gated proton channel, Hv1, is localized primarily to microglia and sustains NADPH oxidase activity. Deletion of Hv1 is neuroprotective after permanent middle cerebral artery occlusion (MCAO). We hypothesized that Hv1-mediated microglial ROS generation is also critical for vascular integrity and contributes to reperfusion injury after transient ischemic stroke. The wildtype (WT) and Hv1 knockout (KO) rats (n=4) were subjected to permanent or 3/24 h transient MCAO. The neurological deficiency, infarct, hemorrhagic transformation, and edema ratio were assessed. We found that in both permanent and transient MCAO model, KO rats develop smaller infarct, less vascular injury, edema, and hemorrhagic transformation, resulting in better short-term functional outcome. These results suggest that deletion of microglial Hv1 channel is vasculoprotective after ischemia/reperfusion and the underlying mechanisms need to be further studied.

Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways.

Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 µg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91phox, which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways.

Regulatory effects of fisetin on microglial activation.

Increasing evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play a key role in neurodegeneration. Fisetin, a plant flavonol commonly found in fruits and vegetables, is frequently added to nutritional supplements due to its antioxidant properties. In the present study, treatment with fisetin inhibited microglial cell migration and ROS (reactive oxygen species) production. Treatment with fisetin also effectively inhibited LPS plus IFN-γ-induced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in microglial cells. Furthermore, fisetin also reduced expressions of iNOS and NO by stimulation of peptidoglycan, the major component of the Gram-positive bacterium cell wall. Fisetin also inhibited the enhancement of LPS/IFN-γ- or peptidoglycan-induced inflammatory mediator IL (interlukin)-1 β expression. Besides the antioxidative and anti-inflammatory effects of fisetin, our study also elucidates the manner in fisetin-induced an endogenous anti-oxidative enzyme HO (heme oxygenase)-1 expression. Moreover, the regulatory molecular mechanism of fisetin-induced HO-1 expression operates through the PI-3 kinase/AKT and p38 signaling pathways in microglia. Notably, fisetin also significantly attenuated inflammation-related microglial activation and coordination deficit in mice in vivo. These findings suggest that fisetin may be a candidate agent for the development of therapies for inflammation-related neurodegenerative diseases.

Insulin‐like growth factor‐1 abrogates microglial oxidative stress and TNF‐α responses to spreading depression

Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine, occurs with increased oxidative stress (OS). SD increases reactive oxygen species (ROS), and ROS, in turn, can signal to increase neuronal excitability,which includes increased SD susceptibility. SD also elevates tumor necrosis factor-α (TNF-α), which increases neuronal excitability. Accordingly, we probed for the cellular origin of OS from SD and its relationship to TNF-α, which might promote SD, using rat hippocampal slice cultures. We observed significantly increased OS from SD in astrocytes and microglia but not in neurons or oligodendrocytes. Since insulin-like growth factor-1 (IGF-1) mitigates OS from SD, we determined the cell types responsible for this effect. We found that IGF-1 significantly decreased microglial but not astrocytic OS from SD. We also show that IGF-1 abrogated the SD-induced TNF-α increase. Furthermore, TNF-α application increased microglial but not astrocytic OS, an effect abrogated by IGF-1. Next,we showed that SD increased SD susceptibility, and does so via TNF-α. This work suggests that microglia promote SD via increased and interrelated ROS and TNF-α signaling. Thus, IGF-1 mitigation of microglial ROS and TNF-α responses maybe targets for novel therapeutics development to prevent SD, and perhaps migraine.

NADPH oxidase and aging drive microglial activation, oxidative stress, and dopaminergic neurodegeneration following systemic LPS administration

Parkinson's disease is characterized by a progressive degeneration of substantia nigra (SN) dopaminergic neurons with age. We previously found that a single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) injection caused a slow progressive loss of tyrosine hydroxylase immunoreactive (TH+IR) neurons in SN associated with increasing motor dysfunction. In this study, we investigated the role of NADPH oxidase (NOX) in inflammation-mediated SN neurotoxicity. A comparison of control (NOX2(+/+) ) mice with NOX subunit gp91(phox) -deficient (NOX2(-/-) ) mice 10 months after LPS administration (5 mg/kg, i.p.) resulted in a 39% (P < 0.01) loss of TH+IR neurons in NOX2(+/+) mice, whereas NOX2(-/-) mice did not show a significant decrease. Microglia (Iba1+IR) showed morphological activation in NOX2(+/+) mice, but not in NOX2(-/-) mice at 1 hr. Treatment of NOX2(+/+) mice with LPS resulted in a 12-fold increase in NOX2 mRNA in midbrain and 5.5-6.5-fold increases in NOX2 protein (+IR) in SN compared with the saline controls. Brain reactive oxygen species (ROS), determined using diphenyliodonium histochemistry, was increased by LPS in SN between 1 hr and 20 months. Diphenyliodonium (DPI), an NOX inhibitor, blocked LPS-induced activation of microglia and production of ROS, TNFα, IL-1β, and MCP-1. Although LPS increased microglial activation and ROS at all ages studied, saline control NOX2(+/+) mice showed age-related increases in microglial activation, NOX, and ROS levels at 12 and 22 months of age. Together, these results suggest that NOX contributes to persistent microglial activation, ROS production, and dopaminergic neurodegeneration that persist and continue to increase with age.

Transient receptor potential vanilloid subtype 1 contributes to mesencephalic dopaminergic neuronal survival by inhibiting microglia-originated oxidative stress

The present study examined whether capsaicin (CAP), an agonist of transient receptor potential vanilloid subtype 1 (TRPV1) can prevent 1-methyl-4-phenylpyridinium (MPP(+))-induced dopaminergic (DA) neuronal death in the substantia nigra (SN). Unilateral injection of MPP(+) into the median forebrain bundle of rat brain resulted in a significant loss of nigral DA neurons, assessed by tyrosine hydroxylase (TH) immunostaining. In parallel, activation of microglia, visualized by OX-42 and OX-6 immunostaining were also observed in the SN, where degeneration of nigral neurons was found. By contrast, MPP(+) neurotoxicity was partially inhibited by co-treatment with MPP(+) and CAP. Interestingly, CAP significantly decreased not only immunoreactivity of OX-42 and OX-6 but also production of microglia-derived reactive oxygen species (ROS) in the SN of MPP(+)-treated rats. In experiments designed to further verify effectiveness of CAP against microglia-derived neurotoxicity, CAP inhibited ROS production and blocked MPP(+)-induced death of DA neurons in co-cultures of mesencephalic neurons and microglia, but not in microglia-free, neuron-enriched mesencephalic cultures. This beneficial effect was reversed by capsazepine, an antagonist of TRPV1, expressed in microglia, indicating TRPV1 involvement. Our data demonstrate for the first time that CAP may inhibit microglial activation-mediated oxidative stress via TRPV1, suggesting that CAP and its analogs may have therapeutic value by inhibiting microglial activation and/or ROS generation that occurs in Parkinson's disease.

Modulation of Experimental Herpes Encephalitis-Associated Neurotoxicity through Sulforaphane Treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection.

Alsin and SOD1G93A Proteins Regulate Endosomal Reactive Oxygen Species Production by Glial Cells and Proinflammatory Pathways Responsible for Neurotoxicity

Recent studies have implicated enhanced Nox2-mediated reactive oxygen species (ROS) by microglia in the pathogenesis of motor neuron death observed in familial amyotrophic lateral sclerosis (ALS). In this context, ALS mutant forms of SOD1 enhance Rac1 activation, leading to increased Nox2-dependent microglial ROS production and neuron cell death in mice. It remains unclear if other genetic mutations that cause ALS also function through similar Nox-dependent pathways to enhance ROS-mediate motor neuron death. In the present study, we sought to understand whether alsin, which is mutated in an inherited juvenile form of ALS, functionally converges on Rac1-dependent pathways acted upon by SOD1(G93A) to regulate Nox-dependent ROS production. Our studies demonstrate that glial cell expression of SOD1(G93A) or wild type alsin induces ROS production, Rac1 activation, secretion of TNFα, and activation of NFκB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1(G93A) in glial cells attenuated these proinflammatory indicators and protected motor neurons in co-culture, although shRNAs against Nox1 and Nox4 had little effect. SOD1(G93A) expression dramatically enhanced TNFα-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1(G93A)-induced endosomal ROS and Rac1 activation. SOD1(G93A) expression enhanced recruitment of alsin to the endomembrane compartment in glial cells, suggesting that these two proteins act to modulate Nox2-dependent endosomal ROS and proinflammatory signals that modulate NFκB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS.

Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia

Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimer's disease. Although it is recognized that amyloid-β (Aβ) plays a major role in inducing and regulating microglial ROS production in Alzheimer's disease, to date little is known about cellular mechanisms underlying Aβ-stimulated ROS production. Here, we identified ion channels involved in Aβ-induced microglial ROS production and in Aβ-induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aβ(1-42) (fAβ(1-42) ) or soluble Aβ(1-42) (sAβ(1-42) ) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5-iodo-resiniferatoxin (I-RTX), but were unaffected by inhibition of K(+) channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAβ(1-42) or sAβ(1-42) induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAβ(1-42) or sAβ(1-42) remained unaffected by TRPV1 channel inhibition with I-RTX. However, sAβ(1-42) -induced priming was inhibited by CTX and margatoxin, but not by TRAM-34 or paxilline, indicating a role of Kv1.3 voltage-gated K(+) channels, but not of Ca(2+) -activated K(+) channels, in the priming process. In summary, our data suggest that in microglia Aβ-induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K(+) channels may provide potential therapeutic targets to reduce microglia-induced oxidative stress in Alzheimer's disease.

Microglial Cells Contribute to Endogenous Brain Defenses after Acute Neonatal Focal Stroke

Macrophages are viewed as amplifiers of ischemic brain injury, but the origin of injury-producing macrophages is poorly defined. The role of resident brain macrophages-microglial cells-in stroke remains controversial. To determine whether microglial cells exert injurious effects after neonatal focal stroke, we selectively depleted these cells with intracerebral injection of liposome-encapsulated clodronate before transient middle cerebral artery occlusion in postnatal day 7 rats. Phagocytosis of apoptotic neurons by activated microglia was poor in animals with unmanipulated microglia, and depletion of these cells did not increase the number of apoptotic neurons. Lack of microglia increased the brain levels of several cytokines and chemokines already elevated by ischemia-reperfusion, and also increased the severity and volume of injury, suggesting that microglial cells contribute to endogenous protection during the subacute injury phase. Then, to determine whether accumulation of reactive oxygen species in microglia adversely affects phagocytosis of dying neurons and contributes to injury, we delivered reduced glutathione (GSH) into microglia, again using liposomes. Remarkably, pharmacologically increased intracellular GSH concentrations in microglia induced superoxide accumulation in lipid rafts in these cells, further increased the brain levels of macrophage chemoattractants, and exacerbated injury. Together, these data show that microglia are part of the endogenous defense mechanisms and that, while antioxidants can protect the injured neonatal brain, high levels of reducing equivalents in activated microglia, GSH, trigger superoxide production, favor the reorganization of lipids, amplify local inflammation and exacerbate injury.

Microglial Activation and Chronic Neurodegeneration

Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurodegenerative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including tumor necrosis factor-α, nitric oxide, interleukin-1β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (e.g., lipopolysaccharide or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss with time. Although the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson's disease. We review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype.

Stimulus-dependent requirement of ion channels for microglial NADPH oxidase-mediated production of reactive oxygen species

Reactive oxygen species (ROS) produced by activated microglial cells play a pivotal role in the pathogenesis of neuro-degenerative and neuro-inflammatory diseases. Here we demonstrate that the pro-inflammatory lipid lysophosphatidylcholine (LPC) is capable of inducing microglial ROS production, which is mediated by the activity of NADPH oxidase. Inhibition of TRPV1 non-selective cation channels abolished ROS production in LPC-stimulated microglia, whereas inhibitors of K + channels, H + channels and Cl − channels had no significant effects. In contrast, activity of all four ion channel types was required for PMA-induced NADPH oxidase-mediated ROS generation, suggesting a differential, stimulus-dependent regulation of microglial ROS production by ion channel activity.

Importance of the non-selective cation channel TRPV1 for microglial reactive oxygen species generation

Activated microglial cells generate reactive oxygen species (ROS), which have detrimental effects in neuroinflammatory and neurodegenerative diseases. In the present study, we have identified a novel mechanism involved in microglial NADPH oxidase-mediated ROS production. In PMA-stimulated microglia, ROS production was substantially reduced upon inhibition of the non-selective cation channel TRPV1 with La 3+, ruthenium red, capsazepine and 5-iodo-resinferatoxin. Furthermore, sustained membrane depolarization, a hallmark of NADPH oxidase activity in phagocytes, was found to induce non-selective cation/TRPV1 channel activity in microglia. Together, our data suggest that TRPV1 channels are involved in regulating NADPH oxidase-mediated ROS generation in microglia.

CLIC1 Function Is Required for β-Amyloid-Induced Generation of Reactive Oxygen Species by Microglia

The Alzheimer's disease (AD) brain is characterized by plaques containing beta-amyloid (Abeta) protein surrounded by astrocytes and reactive microglia. Activation of microglia by Abeta initiates production of reactive oxygen species (ROS) by the plasmalemmal NADPH oxidase; the resultant oxidative stress is thought to contribute to neurodegeneration in AD. We have previously shown that Abeta upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia. We now demonstrate that Abeta promotes the acute translocation of CLIC1 from the cytosol to the plasma membrane of microglia, where it mediates a chloride conductance. Both the Abeta induced Cl(-) conductance and ROS generation were prevented by pharmacological inhibition of CLIC1, by replacement of chloride with impermeant anions, by an anti-CLIC1 antibody and by suppression of CLIC1 expression using siRNA. Thus, the CLIC1-mediated Cl(-) conductance is required for Abeta-induced generation of neurotoxic ROS by microglia. Remarkably, CLIC1 activation is itself dependent on oxidation by ROS derived from the activated NADPH oxidase. We therefore propose that CLIC1 translocation from the cytosol to the plasma membrane, in response to redox modulation by NADPH oxidase-derived ROS, provides a feedforward mechanism that facilitates sustained microglial ROS generation by the NAPDH oxidase.

Synergistic microglial reactive oxygen species generation induced by pesticides lindane and dieldrin

Elevated environmental exposure to pesticides is a known risk factor to the development of sporadic Parkinson's disease resulting from the degeneration of nigral dopamine neurons. Among the suspected agents are the highly persistent and bioaccumulative organochlorinated pesticides (OCPs). We report here that lindane and dieldrin, two widely present OCPs that are found enriched in the nigra of postmortem Parkinson's disease brains synergistically induced the production of reactive oxygen species (ROS) in microglia. Inhibitor studies indicated that the lindane and dieldrin-induced ROS generation was mediated by NADPH oxidase. As microglial ROS is a key contributor to the degeneration of the oxidative damage-vulnerable dopamine neurons, our findings shed significant light on the role of OCPs in the development of Parkinson's disease.

Pharmacological and genetic inhibition of NADPH oxidase does not reduce brain damage in different models of perinatal brain injury in newborn mice

Background Inflammation and reactive oxygen species (ROS) are important in the development of perinatal brain injury. The ROS-generating enzyme NADPH oxidase (Nox2) is present in inflammatory cells and contributes to brain injury in adult animal models. Hypothesis NADPH oxidase contributes to ROS formation and development of injury in the immature brain and inhibition of NADPH oxidase attenuates perinatal brain injury. Methods We used animal models of term hypoxia–ischemia (HI) (P9 mice) as well as ibotenate-induced excitotoxic injury (P5 mice) mimicking features of periventricular leukomalacia in preterm infants. In vitro microglia cell cultures were used to investigate NADPH oxidase-dependent ROS formation. In vivo we determined the impact 1) of HI on NADPH oxidase gene expression 2) of genetic (gp91-phox/Nox2 knock-out) and 3) of pharmacological NADPH oxidase inhibition on HI-induced injury and NMDA receptor-mediated excitotoxic injury, respectively. Endpoints were ROS formation, oxidative stress, apoptosis, inflammation and extent of injury. Results Hypoxia–ischemia increased NADPH oxidase subunits mRNA expression in total brain tissue in vivo. In vitro ibotenate increased NADPH oxidase-dependent formation of reactive oxygen species in microglia. In vivo the inhibition of NADPH oxidase did not reduce the extent of brain injury in any of the animal models. In contrast, the injury was increased by inhibition of NADPH oxidase and genetic inhibition was associated with an increased level of galectin-3 and IL-1β. Conclusion NADPH oxidase is upregulated after hypoxia–ischemia and activated microglia cells are a possible source of Nox2-derived ROS. In contrast to findings in adult brain, NADPH oxidase does not significantly contribute to the pathogenesis of perinatal brain injury. Results obtained in adult animals cannot be transferred to newborns and inhibition of NADPH oxidase should not be used in attempts to attenuate injury.