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.     

Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons

Cell death induced by 6-hydroxydopamine (6-OHDA) is thought to be caused by reactive oxygen species (ROS) derived from 6-OHDA autooxidation and by a possible direct effect of 6-OHDA on the mitochondrial respiratory chain. However, the process has not been totally clarified. In rat primary mesencephalic cultures, we observed a significant increase in dopaminergic (DA) cell loss 24 h after administration of 6-OHDA (40 micromol/L) and a significant increase in NADPH subunit expression, microglial activation and superoxide anion/superoxide-derived ROS in DA cells that were decreased by the NADPH inhibitor apocynin. Low doses of 6-OHDA (10 micromol/L) did not induce a significant loss of DA cells or a significant increase in NADPH subunit expression, microglial activation or superoxide-derived ROS. However, treatment with the NADPH complex activator angiotensin II caused a significant increase in all the latter. Forty-eight hours after intrastriatal 6-OHDA injection in rats, there was still no loss of DA neurons although there was an increase in NADPH subunit expression and NADPH oxidase activity. The results suggest that in addition to the autooxidation-derived ROS and the inhibition of the mitochondrial respiratory chain, early microglial activation and NADPH oxidase-derived ROS act synergistically with 6-OHDA and constitute a relevant and early component of the 6-OHDA-induced cell death.     

The role of NADPH oxidase (NOX) enzymes in neurodegenerative disease

Recently, mounting evidence implicating reactive oxygen species (ROS) generated by NADPH oxidase (NOX) enzymes in the pathogenesis of several neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Alzheimer’s (AD), Parkinson’s (PD) and polyglutamine disease, have arisen. NOX enzymes are transmembrane proteins and generate reactive oxygen species by transporting electrons across lipid membranes. Under normal healthy conditions, low levels of ROS produced by NOX enzymes have been shown to play a role in neuronal differentiation and synaptic plasticity. However, in chronic neurodegenerative diseases over-activation of NOX in neurons, as well as in astrocytes and microglia, has been linked to pathogenic processes such as oxidative stress, exitotoxicity and neuroinflammation. In this review, we summarize the current knowledge about NOX functions in the healthy central nervous system and especially the role of NOX enzymes in neurodegenerative disease processes.     

大鼠黑质多巴胺能神经元的二型性

成年哺乳动物的某些脑区存在性别差异,即二型性,但中脑黑质是否存在性分化目前不清楚。本文旨在探讨成年大鼠中脑黑质是否存在二型性。将60只成年大鼠分成5组：（1）正常雌鼠对照组：（2）正常雄鼠对照组：（3）去卵巢组;（4）去睾丸组;（5）去卵巢后回补雌激素组,该组大鼠在去卵巢后的第7天开始连续3d给予生理剂量的雌激素回补。所有大鼠在右侧黑质埋置记录电极,在清醒和安静的生理状态下连续14d记录黑质的P50听觉诱发电位（P50）,之后作黑质酪氨酸羟化酶（tyrosine hydroxylase,TH）免疫组织化学染色,检查TH阳性（TH^＋）细胞数量和形态变化。结果表明,正常成年雄鼠黑质的TH^＋细胞数量较雌鼠少22．47％（P〈0．05）,P50的T／C值也低34．72％（P〈0．01）,提示正常成年大鼠黑质在结构和功能上存在二型性。与正常雄鼠相比,去睾丸大鼠黑质的TH^＋细胞数量、形态和P50的T／C值无显著性变化（P〉0．05）。与正常雌鼠相比,去卵巢大鼠黑质TH^＋细胞数量减少28．09％（P〈0．01）,P50的T／C值降低30．85％（P〈0．01）。在大鼠去卵巢后的短时间内给予3d生理剂量的雌激素,15-20d后可观察到其黑质TH^＋细胞数量、形态和P50的T／C值基本恢复到去卵巢前水平。结果提示,大鼠中脑黑质的多巴胺能神经元在数量、结构和功能活动上存在性别差异：内源性雌激素在维持黑质多巴胺系统完整性及调节其功能活动中起重要作用。     

In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats

We investigated the effect of an injection of 6-hydroxydopamine (6-OHDA) into the rat medial forebrain bundle (MFB) on the degeneration and the function of the dopaminergic cell bodies in the substantia nigra (SN) 3 and 5 weeks after lesioning. After injection of 6-OHDA into the MFB a complete loss of dopamine content was apparent in the striatum 3 weeks after lesioning. In the SN the amount of tyrosine hydroxylase-immunoreactive dopamine cells decreased gradually, with a near-complete lesion (> 90%) obtained only after 5 weeks, indicating that neurodegeneration of the nigral cells was still ongoing when total dopamine denervation of the striatum had already been achieved. Baseline dialysate and extracellular dopamine levels in the SN, as determined by in vivo microdialysis, were not altered by the lesion. A combination of compensatory changes of the remaining neurones and dopamine originating from the ventral tegmental area may maintain extracellular dopamine at near-normal levels. In both intact and lesioned rats, the somatodendritic release was about 60% tetrodotoxin (TTX) dependent. Possibly two pools contribute to the basal dopamine levels in the SN: a fast sodium channel-dependent portion and a TTX-insensitive one originating from diffusion of dopamine. Amphetamine-evoked dopamine release and release after injection of the selective dopamine reuptake blocker GBR 12909 were attenuated after a near-complete denervation of the SN (5 weeks after lesioning). So, despite a 90% dopamine cell loss in the SN 5 weeks after an MFB lesion, extracellular dopamine levels in the SN are kept at near-normal levels. However, the response to a pharmacological challenge is severely disrupted.     

Distribution of zinc in the substantia nigra of rats treated with 6-hydroxydopamine

To study the relationship between tissue accumulation of Zinc (Zn) and neurodegeneration in the nigrostriatal dopaminergic pathway,⁶⁵Zn distribution in this pathway was examined after unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rats. When⁶⁵ZnCl₂ was intravenously injected 4 days after treatment with 6-OHDA,⁶⁵Zn was concentrated in the ipsilateral substantia nigra 6 days after⁶⁵Zn injection. On the other hand, 19 d after treatment with 6-OHDA,⁶⁵Zn distribution in the ipsilateral substantia nigra was decreased to the level of the contralateral one. When NH₄ ⁹⁹TcO₄, which cannot go through the blood-brain barrier, was injected into rats 4 d after treatment with 6-OHDA,⁹⁹Tc was concentrated in the ipsilateral substantia nigra 30 min after⁹⁹Tc injection, but no longer detectable 6 d after injection. These results suggest that Zn is necessary for a repair process called replacement gliosis after the death of neurons and that excess Zn does not accumulate in the lesion after completion of the gliosis.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

The lesion of the rat substantia nigra pars compacta dopaminergic neurons as a model for Parkinson's disease memory disabilities

1. In this article we review the studies of memory disabilities in a rat model o Parkinson's disease (PD).2. Intranigral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to rats causes a partial lesion in the substantia nigra, compact part (SNc) and a specific loss of dopamine and its metabolites in the striatum of rats.3. These animals present learning and memory deficits but no sensorimotor impairments, thus modeling the early phase of PD when cognitive impairments are observed but the motor symptoms of the disease are barely present.4. The cognitive deficits observed in these animals affect memory tasks proposed to model habit learning (the cued version of the water maze task and the two-way active avoidance task) and working memory (a working memory version of the water maze), but spare long-term spatial memory (the spatial reference version of the Morris water maze).5. The treatment of these animals with levodopa in a dose that restores the striatal level of dopamine does not reverse these memory impairments, probably because this treatment promotes a high level of dopamine in extrastriatal brain regions, such as the prefrontal cortex and the hippocampus.6. On the other hand, the adenosine receptor antagonist, caffeine, partly reverse the memory impairment effect of SNc lesion in these rats. This effect may be due to caffeine action on nigrostriatal neurons, since it induces dopamine release and modulates the interaction between adenosine and dopamine receptor activity.7. These results suggest that the MPTP SNc-lesioned rats are a good model to study memory disabilities related to PD and that caffeine and other selective A(2A) adenosine receptor antagonists are promising drugs to treat this symptoms in PD patients.     

Participation of protease-activated receptor-1 in thrombin-induced microglial activation

Activation of microglia, the resident macrophages in the CNS, plays a significant role in neuronal death or degeneration in a broad spectrum of CNS disorders. Recent studies indicate that nanomolar concentrations of the serine protease, thrombin, can activate microglia in culture. However, in contrast to other neural cells responsive to thrombin, the participation of novel protease-activated receptors (PARs), such as the prototypic thrombin receptor PAR1, in thrombin-induced microglial activation was cast in doubt. In this report, by utilizing primary microglial cultures from PAR1 knockout (PAR1-/-) mice, application of the PAR1 active peptide TRAP-6 (SFLLRN) in comparison to a scrambled peptide (LFLNR), we have unambiguously demonstrated that murine microglia constitutively express PAR1 mRNA that is translated into fully functional protein. Activation of the microglial PAR1 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 mitogen-activated protein kinases. Moreover, although in part, this PAR1 activation directly contributes to thrombin-induced microglial proliferation. Furthermore, although not directly inducing tumor necrosis factor-alpha (TNF-alpha) release, PAR1 activation up-regulates microglial CD40 expression and potentiates CD40 ligand-induced TNF-alpha production, thus indirectly contributing to microglial activation. Taken together, these results demonstrate an essential role of PAR1 in thrombin-induced microglial activation. In addition, strategies aimed at blocking thrombin signaling through PAR1 may be therapeutically valuable for diseases associated with cerebral vascular damage and significant inflammation with microglial activation.     

Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-alpha

Activated microglia are implicated in the pathogenesis of disease-, trauma- and toxicant-induced damage to the CNS, and strategies to modulate microglial activation are gaining impetus. A novel action of the tetracycline derivative minocycline is the ability to inhibit inflammation and free radical formation, factors that influence microglial activation. Minocycline is therefore being tested as a neuroprotective agent to alleviate CNS damage, although findings so far have yielded mixed results. Here, we showed that administration of a single low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH), a paradigm that causes selective degeneration of striatal dopaminergic nerve terminals without affecting the cell body in substantia nigra, increased the expression of mRNAs encoding microglia-associated factors F4/80, interleukin (IL)-1alpha, IL-6, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor (TNF)-alpha. Minocycline treatment attenuated MPTP- or METH-mediated microglial activation, but failed to afford neuroprotection. Lack of neuroprotection was shown to be due to the inability of minocycline to abolish the induction of TNF-alpha and its receptors, thereby failing to modulate TNF signaling. Thus, TNF-alpha appeared to be an obligatory component of dopaminergic neurotoxicity. To address this possibility, we examined the effects of MPTP or METH in mice lacking genes encoding IL-6, CCL2 or TNF receptor (TNFR)1/2. Deficiency of either IL-6 or CCL2 did not alter MPTP neurotoxicity. However, deficiency of both TNFRs protected against the dopaminergic neurotoxicity of MPTP. Taken together, our findings suggest that attenuation of microglial activation is insufficient to modulate neurotoxicity as transient activation of microglia may suffice to initiate neurodegeneration. These findings support the hypothesis that TNF-alpha may play a role in the selective vulnerability of the nigrostriatal pathway associated with dopaminergic neurotoxicity and perhaps Parkinson's disease.     

Angiotensin receptor II is present in dopaminergic cell line of rat substantia nigra and it is down regulated by aminochrome

Angiotensin receptor II mRNA was found to be expressed in dopaminergic neuronal cell line RCSN3 of rat substantia nigra using RT-PCR reaction. Aminochrome (150 M), a metabolite of the dopamine oxidative pathway, was found to down regulate the expression of angiotensin receptor mRNA in RCSN3 cells by 83% (p < 0.05).     

雌激素对6-羟基多巴胺损伤黑质多巴胺能神经元的保护作用

本研究以P50听觉诱发电位(P50 auditory evoked potential, P50)和酪氨酸羟化酶(tyrosine hydroxylase, TH)阳性细胞计数作为黑质功能和形态学指标,动态追踪研究雌激素对6-羟基多巴胺(6-hydroxydopamine, 6-OHDA)损伤黑质多巴胺(dopamine, DA)能神经元的作用.将大鼠分为4组:(1)正常雌性大鼠对照组;(2)单纯帕金森氏病(Parkinson's disease, PD)模型组;(3)双侧去卵巢PD模型组;(4)去卵巢回补3d雌激素的PD模型组.在大鼠清醒和安静的生理状态下连续14d记录黑质的P50,并检测黑质TH 细胞数目的变化.结果显示:单纯PD模型大鼠黑质P50的T/C值较正常雌鼠降低40.60%(P<0.01),其损伤侧黑质TH 细胞数目减少64.74%(P<0.01);去卵巢PD模型大鼠黑质P50的T/C值较单纯PD模型大鼠进一步降低45.88%(P<0.01),同时其黑质TH 细胞数目值也进一步减少57.26%(P<0.01),表明急性缺乏生理水平性腺雌激素将增大6-OHDA损伤黑质DA能神经元的程度,同时使黑质的感觉门控(sensory gating, SG)功能明显受损;去卵巢后回补3d生理剂量雌激素,可明显改善大鼠黑质的SG功能,提高TH 细胞数量(与去卵巢PD模型大鼠比较,P<0.01),其黑质损伤程度与单纯PD模型大鼠相当.以上结果提示,生理水平的雌激素具有提高黑质DA能神经元对伤害性刺激耐受性的神经保护作用.缺乏性腺源性的雌激素时,及时给予生理剂量的雌激素可以减轻神经毒素6-OHDA对黑质DA能神经元结构和功能的损伤.     

Alpha-synuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP

Mutations in alpha-synuclein cause a form of familial Parkinson's disease (PD), and wild-type alpha-synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for alpha-synuclein in PD. Thus far, however, little is known about the importance of alpha-synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein-1, the rodent homologue of human alpha-synuclein, in both normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein-positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein-positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP-induced dopaminergic neurodegeneration. In these MPTP-injected mice, there was also a dramatic increase in the number of synuclein-immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein-positive neurons were tyrosine hydroxylase-positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up-regulated following MPTP-induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.     

Expression of cyclin-dependent kinase 5 and its activator p35 in models of induced apoptotic death in neurons of the substantia nigra in vivo

Cyclin-dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6-hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co-labeling with a neuron-specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.     

NADPH oxidase 4 regulate the glycolytic metabolic reprogramming of microglial cells to promote M1 polarization

This work aimed to investigate the role and mechanism of NADPH oxidase 4 (NOX4) in the polarization of microglial cells. Microglial cells were transfected with the NOX4 overexpression plasmid (pGL3-NOX4), and later treated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce its M1 polarization. Later, the F4/80 + CD86 + cell proportion was detected by flow cytometry (FCM), the inflammatory factor expression levels were analyzed through enzyme-linked immunosorbent assay (ELISA), while ionized calcium binding adapter molecule 1 (IBA-1) and PKM2 expression were measured by immunofluorescence (IF) staining. In addition, dichlorodihydrofluorescein diacetate probe was utilized to detect the reactive oxygen species (ROS) levels, glucose uptake, and glycolysis, as well as lactic acid level. The expression of glycolytic enzymes PKM2, HK2, and citrate (Si)-synthas (CS) was detected by Western-blot (WB) assay. Moreover, the polarization level of microglial cells was detected after ROS expression was suppressed by the ROS inhibitor N-acetylcysteine (NAC). In mouse experiments, LPS was applied in inducing central neuroinflammation in NOX4 knockdown mouse model (KO) and wild-type mice (WT). Thereafter, the inflammatory factor levels and lactic acid level in mouse tissues were detected; IBA-1 and CD86 expression in mice was measured by IF staining; and the expression of glycolytic enzymes PKM2, HK2, and CS in the central nervous system (CNS) was also detected. After NOX4 overexpression in microglial cells, the M1 polarization level was upregulated, the F4/80 + CD86 + cell proportion increased, and inflammatory factors were upregulated. At the same time, the expression of glycolytic enzymes PKM2, HK2, and CS was upregulated. NAC pretreatment suppressed the effects of NOX4, reduced the F4/80 + CD86 + cell proportion, and suppressed the expression of PKM2, HK2, and CS. In the mouse model, the expression levels of CD86 in KO group decreased, and the inflammatory factors were also downregulated. NOX4 promotes glycolysis of microglial cells via ROS, thus accelerating M1 polarization and inflammatory factor expression. In this regard, NOX4 is promising as a new target for the treatment of neuroinflammation.     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

NADPH oxidase activation is required for migration by LIGHT in human monocytes

Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for herpes virus entry mediator (HVEM; TR2) (LIGHT), a ligand of herpes virus entry mediator (HVEM), increased reactive oxygen species (ROS) and enhanced the destruction of bacteria in human monocytes. In this study, rhLIGHT was found to increase the expression of the chemokine receptors, chemokine receptor 1 (CCR1) and CCR2, as well as to accelerate the migration activity of human monocytes. Additionally, rhLIGHT was found to increase ROS via NADPH oxidase p47^phox phosphorylation, which was found to be required for LIGHT-induced NF-κB activation, CCR1 and CCR2 expression, migration and IL-8 and TNF-α production. Taken together, these results indicate that NADPH oxidase activation is required for rhLIGHT-induced migration in human monocytes.