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.     

Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson's disease

The etiology of sporadic Parkinson's disease (PD) remains unknown. Increasing evidence has suggested a role for inflammation in the brain in the pathogenesis of PD. However, it has not been clearly demonstrated whether microglial activation, the most integral part of the brain inflammatory process, will result in a delayed and progressive degeneration of dopaminergic neurons in substantia nigra, a hallmark of PD. We report here that chronic infusion of an inflammagen lipopolysaccharide at 5 ng/h for 2 weeks into rat brain triggered a rapid activation of microglia that reached a plateau in 2 weeks, followed by a delayed and gradual loss of nigral dopaminergic neurons that began at between 4 and 6 weeks and reached 70% by 10 weeks. Further investigation of the underlying mechanism of action of microglia-mediated neurotoxicity using rat mesencephalic neuron-glia cultures demonstrated that low concentrations of lipopolysaccharide (0.1-10 ng/mL)-induced microglial activation and production of neurotoxic factors preceded the progressive and selective degeneration of dopaminergic neurons. Among the factors produced by activated microglia, the NADPH oxidase-mediated release of superoxide appeared to be a predominant effector of neurodegeneration, consistent with the notion that dopaminergic neurons are particularly vulnerable to oxidative insults. This is the first report that microglial activation induced by chronic exposure to inflammagen was capable of inducing a delayed and selective degeneration of nigral dopaminergic neurons and that microglia-originated free radicals play a pivotal role in dopaminergic neurotoxicity in this inflammation-mediated model of PD.     

Microglia enhance beta-amyloid peptide-induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species

The purpose of this study was to assess and compare the toxicity of beta-amyloid (Abeta) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Abeta-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron-glia cultures with Abeta(1-42) over the concentration range 0.1-6.0 microm caused concentration-dependent neurotoxicity. High concentrations of Abeta (6.0 microm for cortex and 1.5-2.0 microm for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Abeta (1.0-3.0 microm for cortex and 0.25-1.0 microm for mesencephalon) caused significant neurotoxicity in mixed neuron-glia cultures, but not in neuron- enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Abeta, including the observations that low concentrations of Abeta activated microglia morphologically in neuron-glia cultures and that addition of microglia to cortical neuron-glia cultures enhanced Abeta-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron-glia cultures. Low doses of Abeta significantly increased the production of superoxide anions, but not of tumor necrosis factor-alpha, interleukin-1beta or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Abeta toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Abeta-induced neurotoxicity in neuron-glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Abeta-induced neurotoxicity was further substantiated by a study which showed that Abeta caused less of a decrease in dopamine uptake in mesencephalic neuron-glia cultures from NADPH oxidase-deficient mutant mice than in that from wild-type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Abeta is via the production of reactive oxygen species.     

Impaired CD200-CD200R-mediated microglia silencing enhances midbrain dopaminergic neurodegeneration: roles of aging, superoxide, NADPH oxidase, and p38 MAPK

CD200-CD200R signaling holds microglia in a quiescent state. Parkinson disease (PD) neurodegeneration may be associated with impairment of CD200-CD200R-mediated microglia silencing in the substantia nigra (SN). In this study, an anti-CD200R blocking antibody (ACDR) selectively and significantly enhanced the susceptibility of dopaminergic neurons to neurotoxicity induced by rotenone (Rot) and iron (Ir) in mesencephalic neuron/glia cultures. Microglia were shown to mediate dopaminergic neurotoxicity induced by ACDR/Rot (combination of ACDR and Rot) and ACDR/Ir (combination of ACDR and Ir). ACDR significantly enhanced the microglial activation induced by Rot and Ir in neuron/glia cultures. NADPH oxidase-mediated superoxide generation was a key contributor to dopaminergic neurotoxicity induced by ACDR/Rot and ACDR/Ir. p38 MAPK contributed to NADPH oxidase activation induced by ACDR/Rot and ACDR/Ir. Interestingly, there were a decrease in CD200 expression (mRNA and protein) and an enhancement of microglial response (MHCII mRNA and ICAM-1 protein) in the rat SN with aging. ICAM-1 expression was significantly inversely correlated with CD200 expression. These results strongly indicate the participation of SN CD200-CD200R dysfunction in the etiopathogenesis of PD and provide a new insight into the molecular mechanisms underlying the involvement of aging in PD and help to elucidate the mechanisms of the combined involvement of immune/inflammatory factors, environmental substances, and aging in PD.     

NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity and proinflammatory gene expression in activated microglia

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. We have previously reported that lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons is mediated by the release of proinflammatory factors from activated microglia. Here, we report the pivotal role of NADPH oxidase in inflammation-mediated neurotoxicity, where the LPS-induced loss of nigral dopaminergic neurons in vivo was significantly less pronounced in NADPH oxidase-deficient (PHOX-/-) mice when compared with control (PHOX+/+) mice. Dopaminergic neurons in primary mensencephalic neuron-glia cultures from PHOX+/+ mice were significantly more sensitive to LPS-induced neurotoxicity in vitro when compared with PHOX-/- mice. Further, PHOX+/+ neuron-glia cultures chemically depleted of microglia failed to show dopaminergic neurotoxicity with the addition of LPS. Neuron-enriched cultures from both PHOX+/+ mice and PHOX-/- mice also failed to show any direct LPS-induced dopaminergic neurotoxicity. However, the addition of PHOX+/+ microglia to neuron-enriched cultures from either strain resulted in reinstatement of LPS-induced dopaminergic neurotoxicity, supporting the role of microglia as the primary source of NADPH oxidase-generated insult and neurotoxicity. Immunostaining for F4/80 in mensencephalic neuron-glia cultures revealed that PHOX-/- microglia failed to show activated morphology at 10 h, suggesting an important role of reactive oxygen species (ROS) generated from NADPH oxidase in the early activation of microglia. LPS also failed to elicit extracellular superoxide and produced low levels of intracellular ROS in microglia-enriched cultures from PHOX-/- mice. Gene expression and release of tumor necrosis factor alpha was much lower in PHOX-/- mice than in control PHOX+/+ mice. Together, these results demonstrate the dual neurotoxic functions of microglial NADPH oxidase: 1) the production of extracellular ROS that is toxic to dopamine neurons and 2) the amplification of proinflammatory gene expression and associated neurotoxicity.     

Urocortin modulates inflammatory response and neurotoxicity induced by microglial activation

Microglia are the major inflammatory cells in the brain. Recent studies have highlighted the reciprocal roles of other brain cells in modulating the microglial inflammatory responses. Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. In the present study, we demonstrated that expression of UCN in rat substantia nigra was found to be localized principally to dopaminergic neurons. In cell culture models, the CRH receptors were expressed in microglia, and CRHR expression was up-regulated by treatment with LPS. Thus, it might be proposed that UCN regulates cellular communication between dopaminergic neurons and microglia. We show that femtomolar concentrations of UCN could inhibit LPS-induced TNF-alpha production in cultured microglia. Investigation of the underlying signaling pathway that mediated the anti-inflammatory effect of UCN the involved PI3K/Akt and glycogen synthase kinase-3beta pathway, but not cAMP pathway. Furthermore, UCN protected dopaminergic neurons against LPS-induced neurotoxicity by inhibiting microglial activation in LPS-treated mesencephalic neuron-glia cultures. These results suggest that endogenous UCN and its receptors might be involved in a complex network of paracrine interaction between dopaminergic neurons and glia.     

Disialogangliosides induce neurodegeneration in rat mesencephalic cultures

The present study evaluated the neurotoxicity of various gangliosides against dopaminergic neurons in mesencephalic cultures. Among them, GD1a and GD1b but not GD3 and GQ1b were found to be neurotoxic against dopaminergic neurons as determined by TH immunocytochemistry and [(3)H]DA uptake. When quantified and expressed as a percentage of control values, treatment with 60-200 microg/ml GD1a and GD1b attenuated the number of TH-ip neurons by 31-47% and 37-55%, respectively, compared with non-treated control cultures. Consistent with the results of the TH immunocytochemistry, treatment with 60-200 microg/ml GD1a and GD1b reduced [(3)H]DA uptake levels by 27-56% and 41-60%, respectively, compared with non-treated control cultures. This neurotoxicity was almost completely abolished in the presence of neuraminidase, which removes the sialic acid residues from ganglioside, or in the treatment of insulin or IGF-1. Additional immunostaining also showed a significant loss of GABAergic neurons in GD1a or GD1b-treated cultures, indicating non-selective neurotoxicity of GD1a and GD1b. Moreover, these gangliosides had little effect on nitric oxide (NO) production in mesencephalic or microglia cultures. Together, these data suggest that GD1a and GD1b exert a direct neurotoxicity against dopaminergic neurons independent of NO and/or microglia.     

Microglia-mediated neurotoxicity is inhibited by morphine through an opioid receptor-independent reduction of NADPH oxidase activity

Recent studies have shown that morphine modulates the function of glia cells through both opioid receptor dependent and independent mechanisms. However, the mechanism by which morphine regulates neuronal disorders through the alteration of microglia activity remains unclear. In this study, using rat primary mesencephalic neuron-glia cultures, we report that both l-morphine and its synthetic stereoenantiomer, d-morphine, an ineffective opioid receptor agonist, significantly reduced LPS- or 1-methyl-4-phenylpyridinium-induced dopaminergic neurotoxicity with similar efficacy, indicating a nonopioid receptor-mediated effect. In addition, using reconstituted neuron and glia cultures, subpicomolar concentrations of morphine were found to be neuroprotective only in the presence of microglia, and significantly inhibited the production of inflammatory mediators from LPS-stimulated microglia cells. Mechanistic studies showed that both l- and d- morphine failed to protect dopaminergic neurons in cultures from NADPH oxidase (PHOX) knockout mice and significantly reduced LPS-induced PHOX cytosolic subunit p47(phox) translocation to the cell membrane by inhibiting ERK phosphorylation. Taken together, our results demonstrate that morphine, even at subpicomolar concentrations, exerts potent anti-inflammatory and neuroprotective effects either through the inhibition of direct microglial activation by LPS or through the inhibition of reactive microgliosis elicited by 1-methyl-4-phenylpyridinium. Furthermore, our study reveals that inhibition of PHOX is a novel site of action for the mu-opioid receptor-independent effect of morphine.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

MAPKAP kinase 2-deficiency prevents neurons from cell death by reducing neuroinflammation--relevance in a mouse model of Parkinson's disease

The inflammatory response in the brain is closely associated with the pathogenesis of degenerative neurological disorders. A role for the p38 stress-activated protein kinase/MAPK-activated protein kinase 2 (MK2) axis in inflammation and apoptosis is well documented. Here, we provide evidence that neurodegeneration can be prevented by eliminating MK2. In primary mesencephalic neuron-glia co-cultures dopaminergic neurons from MK2-deficient (MK2−/−) mice were significantly more resistant to lipopolysaccharide-induced neurotoxicity compared with cells from wild-type mice. This neuroprotection in MK2-deficient cultures was associated with a reduced inflammatory response, especially with reduced production of the inflammatory mediators tumor necrosis factor alpha, keratinocyte-derived chemokine, interleukin-6, and nitric oxide (NO). Interestingly, in primary neuron-enriched cell cultures p38 MAPK, but not MK2, also participates in NO-mediated neuronal cell death. In the MPTP mouse model for Parkinson's disease, MK2-deficient mice show a reduced neuroinflammation and less degeneration of dopaminergic neurons in the substantia nigra after MPTP lesion compared with wild-type mice. In conclusion, our results reveal that MK2 does not directly participate in neuronal cell death, but indirectly contributes to neurodegeneration by the production of neurotoxic substances, such as NO or tumor necrosis factor alpha, from activated glia cells.     

Superoxide dismutase/catalase mimetics are neuroprotective against selective paraquat-mediated dopaminergic neuron death in the substantial nigra: implications for Parkinson disease

Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquat-induced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.     

CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB₁/CB₂ agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB₂ receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety of ways, acting principally through the CB₂ receptors and microglia.     

Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [(3)H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired.     

Microglia-Derived Cytokines/Chemokines Are Involved in the Enhancement of LPS-Induced Loss of Nigrostriatal Dopaminergic Neurons in DJ-1 Knockout Mice

Mutation of DJ-1 (PARK7) has been linked to the development of early-onset Parkinson’s disease (PD). However, the underlying molecular mechanism is still unclear. This study is aimed to compare the sensitivity of nigrostriatal dopaminergic neurons to lipopolysaccharide (LPS) challenge between DJ-1 knockout (KO) and wild-type (WT) mice, and explore the underlying cellular and molecular mechanisms. Our results found that the basal levels of interferon (IFN)-γ (the hub cytokine) and interferon-inducible T-cell alpha chemoattractant (I-TAC) (a downstream mediator) were elevated in the substantia nigra of DJ-1 KO mice and in microglia cells with DJ-1 deficiency, and the release of cytokine/chemokine was greatly enhanced following LPS administration in the DJ-1 deficient conditions. In addition, direct intranigral LPS challenge caused a greater loss of nigrostriatal dopaminergic neurons and striatal dopamine content in DJ-1 KO mice than in WT mice. Furthermore, the sensitization of microglia cells to LPS challenge to release IFN-γ and I-TAC was via the enhancement of NF-κB signaling, which was antagonized by NF-κB inhibitors. LPS-induced increase in neuronal death in the neuron-glia co-culture was enhanced by DJ-1 deficiency in microglia, which was antagonized by the neutralizing antibodies against IFN-γ or I-TAC. These results indicate that DJ-1 deficiency sensitizes microglia cells to release IFN-γ and I-TAC and causes inflammatory damage to dopaminergic neurons. The interaction between the genetic defect (i.e. DJ-1) and inflammatory factors (e.g. LPS) may contribute to the development of PD.     

Ethyl pyruvate rescues nigrostriatal dopaminergic neurons by regulating glial activation in a mouse model of Parkinson's disease

This study examined whether ethyl pyruvate (EP) promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or glial fibrillary acidic protein immunoreactivity. Western blotting and immunohistochemistry showed activation of microglial NADPH oxidase and astroglial myeloperoxidase (MPO) and subsequent reactive oxygen species/reactive nitrogen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with EP prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by EP was associated with the suppression of astroglial MPO expression, NADPH oxidase-, and/or inducible NO synthase-derived reactive oxygen species/reactive nitrogen species production by activated microglia. Interestingly, EP was found to protect DA neurons from 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present findings show that EP may inhibit glial-mediated oxidative stress, suggesting that EP may have therapeutic value in the treatment of aspects of Parkinson's disease related to glia-derived oxidative damage.     

Role and Mechanism of Microglial Activation in Iron-Induced Selective and Progressive Dopaminergic Neurodegeneration

Parkinson’s disease (PD) patients have excessive iron depositions in substantia nigra (SN). Neuroinflammation characterized by microglial activation is pivotal for dopaminergic neurodegeneration in PD. However, the role and mechanism of microglial activation in iron-induced dopaminergic neurodegeneration in SN remain unclear yet. This study aimed to investigate the role and mechanism of microglial β-nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) activation in iron-induced selective and progressive dopaminergic neurodegeneration. Multiple primary midbrain cultures from rat, NOX2^+/+ and NOX2^?/? mice were used. Dopaminergic neurons, total neurons, and microglia were visualized by immunostainings. Cell viability was measured by MTT assay. Superoxide (O₂ ^·?) and intracellular reactive oxygen species (iROS) were determined by measuring SOD-inhibitable reduction of tetrazolium salt WST-1 and DCFH-DA assay. mRNA and protein were detected by real-time PCR and Western blot. Iron induces selective and progressive dopaminergic neurotoxicity in rat neuron–microglia–astroglia cultures and microglial activation potentiates the neurotoxicity. Activated microglia produce a magnitude of O₂ ^·? and iROS, and display morphological alteration. NOX2 inhibitor diphenylene iodonium protects against iron-elicited dopaminergic neurotoxicity through decreasing microglial O₂ ^·? generation, and NOX2^?/? mice are resistant to the neurotoxicity by reducing microglial O₂ ^·? production, indicating that iron-elicited dopaminergic neurotoxicity is dependent of NOX2, a O₂ ^·?-generating enzyme. NOX2 activation is indicated by the increased mRNA and protein levels of subunits P47 and gp91. Molecules relevant to NOX2 activation include PKC-σ, P38, ERK1/2, JNK, and NF-КB_P65 as their mRNA and protein levels are enhanced by NOX2 activation. Iron causes selective and progressive dopaminergic neurodegeneration, and microglial NOX2 activation potentiates the neurotoxicity. PKC-σ, P38, ERK1/2, JNK, and NF-КB_P65 are the potential molecules relevant to microglial NOX2 activation.     

Lipid-like components released from degenerating dopaminergic neurons trigger the dynamic migration of microglia

In the brain, communication between neural and non-neural cells is crucial for the proper functioning of the central nervous system. Microglia play an important role in the clearance of neural cellular corpses and debris, especially under pathological conditions. It remains, however, unclear how microglia sense the degenerating neurons at a distance in order to migrate to them. In the present study, we explored the interaction between neurons and microglia using an in vitro model of Parkinson's disease (PD). In primary mesencephalic neuronal cultures, 1-methyl-4-phenylpridinium (MPP(+)) induced the selective death of dopaminergic (DAergic) neurons in a dose- and time-dependent manner. Transmigration assay showed that the conditioned medium (CM) from mesencephalic cultures treated with MPP(+) was enough to trigger the attraction of microglia at an early as well as a late phase of neuronal damage. Microglia preferably reacted with the soluble parts separated by ultracentrifugation over the neural debris-containing pellets. This chemoattractive activity was significantly reduced by the removal of the lipidic components in CM, but not by the removal of proteins, DNA or RNA. These results suggest that as yet-unidentified lipid-like components released from dying DAergic neurons are likely to recruit microglia, and thus have a role in neuronal damage.     

α-Mangostin Inhibits α-Synuclein-Induced Microglial Neuroinflammation and Neurotoxicity

Microglia-mediated neuroinflammation induced by α-synuclein in the substantianigra likely either initiates or aggravates nigral neuro degeneration in Parkinson’s disease (PD). We aimed to explore the effects of α-mangostin (α-M), a polyphenolicxanthone derivative from mangosteen on α-synuclein-stimulated DA neurodegeneration. Primary microglia, mesencephalic neuron, mesencephalic neuron-glianeuronal cultures, and transwell co-cultures were prepared separately. Liquid scintillation counting was used to determine the radioactivity in DA uptake. Enzyme-linked immunosorbent assay (ELISA) was performed in the IL-1β, IL-6, and TNF-α assay. The expression of proteins was analyzed by Western blot. α-M inhibited the increased levels of pro-inflammatory cytokines, NO, and ROS in α-synuclein-stimulated primary microglia. Mechanistic study revealed that α-M functioned by inhibition of nuclear factor kappa B (NF-κB) and NADPH oxidase. Further, α-M protected α-synuclein-induced microglial and direct neurotoxicity. Although detailed mechanisms remain to be defined, our observations suggest a potential compound, which inhibits microglial activation induced by α-synuclein by targeting NADPH oxidase, might be a therapeutic possibility in preventing PD progression.