Inflammatory Neurodegeneration and Mechanisms of Microglial Killing of Neurons

Inflammatory neurodegeneration contributes to a wide variety of brain pathologies. A number of mechanisms by which inflammatory-activated microglia and astrocytes kill neurons have been identified in culture. These include: (1) acute activation of the phagocyte NADPH oxidase (PHOX) found in microglia, (2) expression of the inducible nitric oxide synthase (iNOS) in glia, and (3) microglial phagocytosis of neurons. Activation of PHOX (by cytokines, β-amyloid, prion protein, lipopolysaccharide, ATP, or arachidonate) causes microglial proliferation and inflammatory activation; thus, PHOX is a key regulator of inflammation. However, activation of PHOX alone causes little or no death, but when combined with iNOS expression results in apparent apoptosis via peroxynitrite production. Nitric oxide (NO) from iNOS expression also strongly synergizes with hypoxia to induce neuronal death because NO inhibits cytochrome oxidase in competition with oxygen, resulting in glutamate release and excitotoxicity. Finally, microglial phagocytosis of these stressed neurons may contribute to their loss.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Nitric oxide and mitochondrial respiration.

Nitric oxide (NO) and its derivative peroxynitrite (ONOO-) inhibit mitochondrial respiration by distinct mechanisms. Low (nanomolar) concentrations of NO specifically inhibit cytochrome oxidase in competition with oxygen, and this inhibition is fully reversible when NO is removed. Higher concentrations of NO can inhibit the other respiratory chain complexes, probably by nitrosylating or oxidising protein thiols and removing iron from the iron-sulphur centres. Peroxynitrite causes irreversible inhibition of mitochondrial respiration and damage to a variety of mitochondrial components via oxidising reactions. Thus peroxynitrite inhibits or damages mitochondrial complexes I, II, IV and V, aconitase, creatine kinase, the mitochondrial membrane, mitochondrial DNA, superoxide dismutase, and induces mitochondrial swelling, depolarisation, calcium release and permeability transition. The NO inhibition of cytochrome oxidase may be involved in the physiological regulation of respiration rate, as indicated by the finding that isolated cells producing NO can regulate cellular respiration by this means, and the finding that inhibition of NO synthase in vivo causes a stimulation of tissue and whole body oxygen consumption. The recent finding that mitochondria may contain a NO synthase and can produce significant amounts of NO to regulate their own respiration also suggests this regulation may be important for physiological regulation of energy metabolism. However, definitive evidence that NO regulation of mitochondrial respiration occurs in vivo is still missing, and interpretation is complicated by the fact that NO appears to affect tissue respiration by cGMP-dependent mechanisms. The NO inhibition of cytochrome oxidase may also be involved in the cytotoxicity of NO, and may cause increased oxygen radical production by mitochondria, which may in turn lead to the generation of peroxynitrite. Mitochondrial damage by peroxynitrite may mediate the cytotoxicity of NO, and may be involved in a variety of pathologies.     

Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production

Cultured rat microglia produced extracellular superoxide at a rate of 814 +/- 52 pmol/min/million cells when stimulated with phorbol 12-myristate 13-acetate (PMA) as measured by extracellular cytochrome c reduction. This superoxide production resulted in a rapid rate of superoxide dismutase-sensitive nitric oxide (NO) breakdown (155 +/- 30 pmol of NO/min/million cells) when NO was added to PMA stimulated microglia. Lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-activated microglia produce NO at the rate of 145 +/- 42 pmol/min/million cells and activated astrocytes at the rate of 51 +/- 9 pmol/min/million cells as estimated by NO electrode. Both types of cells maintained a steady-state level of 0.5-0.7 microm NO, only in the presence of L-arginine. Addition of PMA to activated microglia (but not activated astrocytes) caused the rapid and complete disappearance of all extracellular NO (but was restored in the presence of superoxide dismutase) followed by the production of peroxynitrite (as measured by urate-sensitive oxidation of dihydrorhodamine). Co-incubation of activated microglia with cerebellar granule neurones resulted in NO inhibition of neuronal respiration, but this was rapidly removed by PMA-induced breakdown of the NO. Thus, microglial NADPH oxidase can regulate the bioavailability of NO and the production of peroxynitrite.     

Oligomeric Aβ-Induced Microglial Activation is Possibly Mediated by NADPH Oxidase

Recent studies have shown that oligomeric amyloid-β (oAβ) peptide can potentially activate microglia in addition to inducing more potent neurotoxicity compared with fibrillar Aβ (fAβ); however, its mechanisms of action remain unclear. This study was designed to investigate the possible mechanisms involved in the microglial activation induced by oAβ in BV-2 microglial cells. The results showed that oAβ induced activated properties of microglia, including higher proliferative capacity as well as increased production of reactive oxygen species, nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). NADPH oxidase inhibitors [diphenylene iodonium (DPI) and apocynin (4-hydroxy-3-methoxy-acetophenone)] prevented the microglial activation induced by oAβ, suggesting that NADPH oxidase activation was involved in microglial activation. In addition, TNF-α and IL-1β, which are massively released by activated microglia, significantly induced the activation of microglia, thereby resulting in the production of NO and proliferation of microglia, respectively. These effects could be inhibited by diphenylene iodonium and apocynin, indicating a self-cycle regulated by NADPH oxidase in microglial activation in response to oAβ. In conclusion, microglial activation induced by oAβ is possibly mediated by NADPH oxidase, suggesting that oAβ, which is normally considered a neurotoxin, may also lead to indirect neuronal damage through the pro-inflammation activation of microglia in Alzheimer’s disease and that NADPH oxidase could be a potential target to prevent oAβ-induced inflammatory neurodegeneration.     

Catalpol protects dopaminergic neurons from LPS-induced neurotoxicity in mesencephalic neuron-glia cultures

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Microglia, the resident immune cells in the central nervous system, are pivotal in the inflammatory reaction. Activated microglia can induce expression of inducible nitric-oxide synthase (iNOS) and release significant amounts of nitric oxide (NO) and TNF-alpha, which can damage the dopaminergic neurons. Catalpol, an iridoid glycoside, contained richly in the roots of Rehmannia glutinosa, was found to be neuroprotective in gerbils subjected to transient global cerebral ischemia. But the effect of catalpol on inflammation-mediated neurodegeneration has not been examined. In this study, microglia in mesencephalic neuron-glia cultures were activated with lipopolysaccharide (LPS) and the aim of the study was to examine whether catalpol could protect dopaminergic neurons from LPS-induced neurotoxicity. The results showed that catalpol significantly reduced the release of reactive oxygen species (ROS), TNF-alpha and NO after LPS-induced microglial activation. Further, catalpol attenuated LPS-induced the expression of iNOS. As determined by immunocytochemical analysis, pretreatment by catalpol dose-dependently protected dopaminergic neurons against LPS-induced neurotoxicity. These results suggest that catalpol exerts its protective effect on dopaminergic neurons by inhibiting microglial activation and reducing the production of proinflammatory factors. Thus, catalpol may possess therapeutic potential against inflammation-related neurodegenerative diseases.     

Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34

An inherited deficiency in the frataxin protein causes neurodegeneration of the dorsal root ganglia and Friedreich''s ataxia (FA). Frataxin deficiency leads to oxidative stress and inflammatory changes in cell and animal models; however, the cause of the inflammatory changes, and especially what causes brain microglial activation is unclear. Here we investigated: 1) the mechanism by which frataxin deficiency activates microglia, 2) whether a brain-localized inflammatory stimulus provokes a greater microglial response in FA animal models, and 3) whether an anti-inflammatory treatment improves their condition. Intracerebroventricular administration of LPS induced higher amounts of microglial activation in the FA mouse model vs controls. We also observed an increase in oxidative damage in the form of 8-oxoguanine (8-oxo-G) and the DNA repair proteins MUTYH and PARP-1 in cerebellar microglia of FA mutant mice. We hypothesized that frataxin deficiency increases DNA damage and DNA repair genes specifically in microglia, activating them. siRNA-mediated frataxin knockdown in microglial BV2 cells clearly elevated DNA damage and the expression of DNA repair genes MUTYH and PARP-1. Frataxin knockdown also induced a higher level of PARP-1 in MEF cells, and this was suppressed in MUTYH-/- knockout cells. Administration of the PARP-1 inhibitor PJ34 attenuated the microglial activation induced by intracerebroventricular injection of LPS. The combined administration of LPS and angiotensin II provoke an even stronger activation of microglia and neurobehavioral impairment. PJ34 treatment attenuated the neurobehavioral impairments in FA mice. These results suggest that the DNA repair proteins MUTYH and PARP-1 may form a pathway regulating microglial activation initiated by DNA damage, and inhibition of microglial PARP-1 induction could be an important therapeutic target in Friedreich''s ataxia.     

A self-propelling cycle mediated by reactive oxide species and nitric oxide exists in LPS-activated microglia

It has been widely accepted that microglia, the innate immune cells in the brain, can be chronically activated in response to neuron death, fuelling a self-renewing cycle of microglial activation followed by further neuron damage (reactive microgliosis), which has been considered as the main reason responsible for the progressive nature of neurodegenerative diseases. In the present study, it was found that LPS (lipopolysaccharide) significantly induced the activation of N9 microglia, and the increase of NO level induced by pretreatment of LPS could last after the removal of LPS. The culture medium of activated microglia significantly decreased the viability of rat primary cortical neuron. These results can be blocked by the antioxidant N-acetylcysteine (NAC) and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase inhibitor diphenyleneiodonium sulfate (DPI), suggesting that intracellular reactive oxide species (iROS) released from the activated microglial cells may continue to further activate microglia. Next, it was shown that the iROS level increased rapidly after the LPS treatment in microglia cells followed by the NO production through the regulation of iNOS (inducible nitric oxide synthase) expression. The increase of iROS could be reversed by gp91phox (the critical and catalytic subunit of NADPH oxidase) siRNA. Moreover, NO released from sodium nitroprusside (SNP) was able to increase the iROS production of N9 microglia by regulating of the activity and the expression of NADPH oxidase. In conclusion, our research suggests for the first time that there may exist a self-propelling cycle in microglial cells possibly mediated by iROS and NO when they become activated by LPS. It may be responsible partially for the ongoing microglial activation and the progressive nature of neurodegenerative diseases.     

Dual Role of Superoxide Dismutase 2 Induced in Activated Microglia: OXIDATIVE STRESS TOLERANCE AND CONVERGENCE OF INFLAMMATORY RESPONSES*

Microglia are activated quickly in response to external pathogens or cell debris and clear these substances via the inflammatory response. However, excessive activation of microglia can be harmful to host cells due to the increased production of reactive oxygen species and proinflammatory cytokines. Superoxide dismutase 2 (SOD2) is reportedly induced under various inflammatory conditions in the central nervous system. We herein demonstrated that activated microglia strongly express SOD2 and examined the role of SOD2, focusing on regulation of the microglial activity and the susceptibility of microglia to oxidative stress. When rat primary microglia were treated with LPS, poly(I:C), peptidoglycan, or CpG oligodeoxynucleotide, respectively, the mRNA and protein levels of SOD2 largely increased. However, an increased expression of SOD2 was not detected in the primary neurons or astrocytes, indicating that SOD2 is specifically induced in microglia under inflammatory conditions. The activated microglia showed high tolerance to oxidative stress, whereas SOD2 knockdown conferred vulnerability to oxidative stress. Interestingly, the production of proinflammatory cytokines was increased in the activated microglia treated with SOD2 siRNA compared with that observed in the control siRNA-treated cells. Pretreatment with NADPH oxidase inhibitors, diphenylene iodonium and apocynin, decreased in not only reactive oxygen species generation but also the proinflammatory cytokine expression. Notably, SOD2 knockdown largely potentiated the nuclear factor κB activity in the activated microglia. Taken together, increased SOD2 conferred tolerance to oxidative stress in the microglia and decreased proinflammatory cytokine production by attenuating the nuclear factor κB activity. Therefore, SOD2 might regulate neuroinflammation by controlling the microglial activities.     

Phospholipids modulate superoxide and nitric oxide production by lipopolysaccharide and phorbol 12-myristate-13-acetate-activated microglia

Microglial activation and inflammatory processes have been implicated in the pathogenesis of a number of neurodegenerative disorders. Recently, peroxynitrite (ONOO(-)), the reaction product of superoxide (O(2)(-)) and nitric oxide (NO) both of which can be generated by activated microglia, has been demonstrated to act as a major mediator in the neurotoxicity induced by activated microglia. On the other hand, phospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC) have been reported to modulate the immune function of phagocytes. We therefore evaluated the effects of liposomes which comprise both PS and PC (PS/PC liposomes) or PC only (PC liposomes) regarding the production of both O(2)(-) and NO by lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)-activated microglia using electron spin resonance (ESR) spin trap technique with a DEPMPO and Griess reaction, respectively. Pretreatment with PS/PC liposomes or PC liposomes considerably inhibited the signal intensity of O(2)(-) adduct associated with LPS/PMA-activated microglia in a dose-dependent manner. In addition, pretreatment with PS/PC liposomes also significantly reduced LPS/PMA-induced microglial NO production. In contrast, pretreatment with PC liposomes had no effect on the NO production. These results indicate that PS/PC liposomes can inhibit the microglial production of both NO and O(2)(-), and thus presumably prevent a subsequent formation of ONOO(-). Therefore, PS/PC liposomes appear to have both neuroprotective and anti-oxidative properties through the inhibition of microglial activation.     

Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase

Nitric oxide (NO) and its derivatives inhibit mitochondrial respiration by a variety of means. Nanomolar concentrations of NO immediately, specifically and reversibly inhibit cytochrome oxidase in competition with oxygen, in isolated cytochrome oxidase, mitochondria, nerve terminals, cultured cells and tissues. Higher concentrations of NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Isolated mitochondria, cultured cells, isolated tissues and animals in vivo display respiratory inhibition by endogenously produced NO from constitutive isoforms of NO synthase (NOS), which may be largely mediated by NO inhibition of cytochrome oxidase. Cultured cells expressing the inducible isoform of NOS (iNOS) can acutely and reversibly inhibit their own cellular respiration and that of co-incubated cells due to NO inhibition of cytochrome oxidase, but after longer-term incubation result in irreversible inhibition of cellular respiration due to NO or its derivatives. Thus the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen.     

Early release of arachidonic acid prevents an otherwise immediate formation of toxic levels of peroxynitrite in astrocytes stimulated with lipopolysaccharide/interferon-gamma

Addition of bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-gamma also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO-) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO- levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO- formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-gamma, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival.     

Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: Implications for neurodegenerative diseases

Microglial activation is considered as a hallmark of several neurodegenerative disorders. During microglial activation, the expression of CD11b, the beta-integrin marker of microglia, is increased. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of reactive oxygen species (ROS) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) stimulated the expression of CD11b in mouse BV-2 microglial cells and primary microglia, the effect that was blocked by antioxidants such as N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Furthermore, comicroinjection of either NAC or PDTC with LPS was also able to suppress LPS-stimulated expression of CD11b in striatum in vivo. Similarly, other neurotoxic molecules, such as interleukin-1beta (IL-1beta), IL-12 p40(2), fibrillar amyloid-beta (Abeta) peptides, HIV-1 gp120, and double-stranded RNA (poly(IC)), also stimulated the expression of CD11b in microglia through the involvement of ROS. Complete inhibition of LPS-stimulated expression of CD11b by catalase, induction of CD11b expression by H2O2 alone, and inhibition of superoxide-stimulated CD11b expression by catalase suggest that H2O2, but not superoxide, is in fact involved in the expression of CD11b. Interestingly, we also demonstrate that ROS stimulated the expression of CD11b after the induction of nitric oxide (NO) production and failed to stimulate CD11b when NO production was inhibited by either 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) or L-N6-(1-iminoethyl)-L-lysine (L-NIL). Taken together, these studies suggest that the up-regulation of CD11b in microglia is redox sensitive and that ROS up-regulates CD11b via NO.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex by the myeloperoxidase products, hypochlorous acid and mono-N-chloramine

Abstract alpha-Ketoglutarate dehydrogenase (KGDHC) complex activity is diminished in a number of neurodegenerative disorders and its diminution in Alzheimer Disease (AD) is thought to contribute to the major loss of cerebral energy metabolism that accompanies this disease. The loss of KGDHC activity appears to be predominantly due to post-translation modifications. Thiamine deficiency also results in decreased KGDHC activity and a selective neuronal loss. Recently, myeloperoxidase has been identified in the activated microglia of brains from AD patients and thiamine-deficient animals. Myeloperoxidase produces a powerful oxidant, hypochlorous acid that reacts with amines to form chloramines. The aim of this study was to investigate the ability of hypochlorous acid and chloramines to inhibit the activity of KGDHC activity as a first step towards investigating the role of myeloperoxidase in AD. Hypochlorous acid and mono-N-chloramine both inhibited purified and cellular KGDHC and the order of inhibition of the purified complex was hypochlorous acid (1x) > mono-N-chloramine (approximately 50x) > hydrogen peroxide (approximately 1,500). The inhibition of cellular KGDHC occurred with no significant loss of cellular viability at all exposure times that were examined. Thus, hypochlorous acid and chloramines have the potential to inactivate a major target in neurodegeneration.     

Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation

Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer's disease. We investigated the role of NADPH oxidase in microglial activation state using p47(phox) and gp91(phox) -deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or Aβ????. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47(phox) subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer's disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.