Microglial MT1 activation inhibits LPS‐induced neuroinflammation via regulation of metabolic reprogramming

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Although its pathogenesis remains unclear, a number of studies indicate that microglia‐mediated neuroinflammation makes a great contribution to the pathogenesis of PD. Melatonin receptor 1 (MT1) is widely expressed in glia cells and neurons in substantia nigra (SN). Neuronal MT1 is a neuroprotective factor, but it remains largely unknown whether dysfunction of microglial MT1 is involved in the PD pathogenesis. Here, we found that MT1 was reduced in microglia of SN in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced PD mouse model. Microglial MT1 activation dramatically inhibited lipopolysaccharide (LPS)‐induced neuroinflammation, whereas loss of microglial MT1 aggravated it. Metabolic reprogramming of microglia was found to contribute to the anti‐inflammatory effects of MT1 activation. LPS‐induced excessive aerobic glycolysis and impaired oxidative phosphorylation (OXPHOS) could be reversed by microglial MT1 activation. MT1 positively regulated pyruvate dehydrogenase alpha 1 (PDHA1) expression to enhance OXPHOS and suppress aerobic glycolysis. Furthermore, in LPS‐treated microglia, MT1 activation decreased the toxicity of conditioned media to the dopaminergic (DA) cell line MES23.5. Most importantly, the anti‐inflammatory effects of MT1 activation were observed in LPS‐stimulated mouse model. In general, our study demonstrates that MT1 activation inhibits LPS‐induced microglial activation through regulating its metabolic reprogramming, which provides a mechanistic insight for microglial MT1 in anti‐inflammation.     

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.     

Identifying lysophosphatidic acid receptor subtype 1 (LPA1) as a novel factor to modulate microglial activation and their TNF-α production by activating ERK1/2

Microglia regulate immune responses in the brain, and their activation is key to the pathogenesis of diverse neurological diseases. Receptor-mediated lysophosphatidic acid (LPA) signaling has been known to regulate microglial biology, but it is still unclear which receptor subtypes guide the biology, particularly, microglial activation. Here, we investigated the pathogenic aspects of LPA receptor subtype 1 (LPA₁) in microglial activation using a systemic lipopolysaccharide (LPS) administration-induced septic mouse model in vivo and LPS-stimulated rat primary microglia in vitro. LPA₁ knockdown in the brain with its specific shRNA lentivirus attenuated the sepsis-induced microglia activation, morphological transformation, and proliferation. LPA₁ knockdown also resulted in the downregulation of TNF-α, at both mRNA and protein levels in septic brains, but not IL-1β or IL-6. In rat primary microglia, genetic or pharmacological blockade of LPA₁ attenuated gene upregulation and secretion of TNF-α in LPS-stimulated cells. In particular, the latter was associated with the suppressed TNF-α converting enzyme (TACE) activity. We reaffirmed these biological aspects using a BV2 microglial cell line in which LPA₁ expression was negligible. LPA₁ overexpression in BV2 cells led to significant increments in TNF-α production upon stimulation with LPS, whereas inhibiting LPA₁ reversed the production. We further identified ERK1/2, but not p38 MAPK or Akt, as the underlying effector pathway after LPA₁ activation in both septic brains and stimulated microglia. The current findings of the novel role of LPA₁ in microglial activation along with its mechanistic aspects could be applied to understanding the pathogenesis of diverse neurological diseases that involve microglial activation.     

Neuroprotection by pramipexole against dopamine- and levodopa-induced cytotoxicity

Pramipexole, a novel non-ergoline dopamine (DA) agonist, has been applied successfully for treatment of Parkinson's disease (PD). We report here that pramipexole can protect dopaminergic cell line Mes23.5 against dopamine- and levodopa-induced cytotoxicity possibly through a mechanism related to antioxidant activity. In the MES 23.5 cultures, DA and L-DOPA induce a dose- and time-dependent cytotoxicity, as determined by tetrazolium salt and trypan blue assays. Furthermore, an in situ terminal deoxynucleotidyl transferase assay demonstrates that DA-induced cell death is apoptotic. Pretreatment with pramipexole in a concentration range (4-100 microM) significantly attenuates DA- or L-DOPA-induced cytotoxicity and apoptosis, an action which is not blocked by D3 antagonist U-99194 A or D2 antagonist raclopride. Pramipexole also protects MES 23.5 cells from hydrogen peroxide-induced cytotoxicity in a dose-dependent manner. In cell-free system, pramipexole can effectively inhibit the formation of melanin, an end product resulting from DA or L-DOPA oxidation. These results indicate that pramipexole exerts its neuroprotective effect possibly through a mechanism, which is independent of DA receptors but related to antioxidation or scavenging of free radicals (e.g. hydrogen peroxide). As a direct DA agonist and potentially neuroprotective agent, pramipexole remains attractive in the treatment of PD.     

Synergistic dopaminergic neurotoxicity of manganese and lipopolysaccharide: differential involvement of microglia and astroglia

Overexposure to manganese is known to cause damage to basal ganglial neurons and the development of movement abnormalities. Activation of microglia and astrocytes has increasingly been associated with the pathogenesis of a variety of neurological disorders. We have recently shown that microglial activation facilitates manganese chloride (MnCl₂, 10–300 μM)‐induced preferential degeneration of dopamine (DA) neurons. In this study, we report that combinations of MnCl₂ (1–30 μM) and endotoxin lipopolysaccharide (LPS, 0.5–2 ng/mL), at minimally effective concentrations when used alone, induced synergistic and preferential damage to DA neurons in rat primary neuron‐glia cultures. Mechanistically, MnCl₂ significantly potentiated LPS‐induced release of tumor necrosis factor‐alpha and interleukin‐1 beta in microglia, but not in astroglia. MnCl₂ and LPS were more effective in inducing the formation of reactive oxygen species and nitric oxide in microglia than in astroglia. Furthermore, MnCl₂ and LPS‐induced free radical generation, cytokine release, and DA neurotoxicity was significantly attenuated by pre‐treatment with potential anti‐inflammatory agents minocycline and naloxone. These results demonstrate that the combination of manganese overexposure and neuroinflammation is preferentially deleterious to DA neurons. Moreover, these findings not only shed light on the understanding of manganese neurotoxicity but may also bear relevance to the potentially multifactorial etiology of Parkinson’s disease.     

杨梅黄酮减弱MPP+ 诱导的MES23.5 细胞的细胞毒性

目的:探讨杨梅黄酮对MPP+处理的MES23.5细胞的保护作用。方法:实验分为空白对照组,MPP+处理组,MPP+和杨梅黄酮共处理组,分别处理MES23.5细胞24h后,应用Hoechst 33258染色观察各组细胞细胞核形态的改变,应用RT-PCR技术观察Bcl-2和Bax mRNA表达的改变。结果:杨梅黄酮能明显抑制MPP+引起的MES23.5细胞核固缩,核碎裂等形态学的改变,杨梅黄酮还可以对抗MPP+处理造成的MES23.5细胞的Bcl-2/Bax比率的降低。结论:杨梅黄酮对MPP+处理的MES23.5细胞具有保护作用。     

MicroRNA-27a Negatively Modulates the Inflammatory Response in Lipopolysaccharide-Stimulated Microglia by Targeting TLR4 and IRAK4

microRNA, a family of small non-coding RNA, plays significant roles in regulating gene expression, mainly via binding to the 3′-untranslated region of target genes. Although the role of miRNA in regulating neuroinflammation via the innate immune pathway has been studied, its role in the production of inflammatory mediators during microglial activation is poorly understood. In this study, we investigated the effect of miR-27a on lipopolysaccharide (LPS)-induced microglial inflammation. miR-27a expression was found to be rapidly decreased in microglia by real-time polymerase chain reaction (real-time PCR) after LPS stimulation. Over-expression of miR-27a significantly decreased the production of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), whereas knockdown of miR-27a increased the expression of these inflammatory factors. We also demonstrated by loss- and gain-of-function studies that miR-27a directly suppressed the expression of toll-like receptor 4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4)—a pivotal adaptor kinase in the TLR4/MyD88 signaling pathway—by directly binding their 3′-UTRs: knocking down TLR4 or IRAK4 in microglia significantly decreased TLR4 or IRAK4 expression and inhibited the downstream production of inflammatory mediators. Moreover, the inflammatory cytokines IL-6 and IL-1β were regulated by IRAK4, whereas TNF-α and NO were more dependent on TLR4 activation. Thus, miR-27a might regulate the LPS-induced production of inflammatory cytokines in microglia independently of TLR4 and IRAK4. Taken together, our results suggest that miR-27a is associated with microglial activation and the inflammatory response.     

The role of MAC1 in diesel exhaust particle‐induced microglial activation and loss of dopaminergic neuron function

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease‐like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron‐glia cultures were treated with either nanometer‐sized DEP (< 0.22 μM; 50 μg/mL), ultrafine carbon black (ufCB, 50 μg/mL), or DEP extracts (eDEP; from 50 μg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H₂O₂ production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP‐induced H₂O₂ production in microglia. However, pre‐treatment with the MAC1/CD11b inhibitor antibody blocked microglial H₂O₂ production in response to DEP. MAC1^?/? mesencephalic neuron‐glia cultures were protected from DEP‐induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP‐induced microglial H₂O₂ production and loss of DA neuron function.     

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.     

Jmjd3 is essential for the epigenetic modulation of microglia phenotypes in the immune pathogenesis of Parkinson's disease

Classical activation (M1 phenotype) and alternative activation (M2 phenotype) are the two polars of microglial activation states that can produce either detrimental or beneficial effects in the central nervous system (CNS). Harnessing the beneficial properties of microglia cells by modulating their polarization states provides great potential for the treatment of Parkinson''s disease (PD). However, the epigenetic mechanism that regulates microglia polarization remains elusive. Here, we reported that histone H3K27me3 demethylase Jumonji domain containing 3 (Jmjd3) was essential for M2 microglia polarization. Suppression of Jmjd3 in N9 microglia inhibited M2 polarization and simultaneously exaggerated M1 microglial inflammatory responses, which led to extensive neuron death in vitro. We also observed that the suppression of Jmjd3 in the substantia nigra (SN) in vivo dramatically caused microglial overactivation and exacerbated dopamine (DA) neuron death in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD. Moreover, we showed that the Jmjd3 level was lower in the midbrain of aged mice, which was accompanied by an elevated level of H3K27me3 and an increased ratio of M1 to M2 markers, suggesting that aging is an important factor in switching the microglia phenotypes. Overall, our studies indicate that Jmjd3 is able to enhance the polarization of M2 microglia by modifying histone H3K27me3, and therefore it has a pivotal role in the switch of microglia phenotypes that may contribute to the immune pathogenesis of PD.     

DJ-1 inhibits microglial activation and protects dopaminergic neurons in vitro and in vivo through interacting with microglial p65

Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by progressive neurodegeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). DJ-1 acts essential roles in neuronal protection and anti-neuroinflammatory response, and its loss of function is tightly associated with a familial recessive form of PD. However, the molecular mechanism of DJ-1 involved in neuroinflammation is largely unclear. Here, we found that wild-type DJ-1, rather than the pathogenic L166P mutant DJ-1, directly binds to the subunit p65 of nuclear factor-κB (NF-κB) in the cytoplasm, and loss of DJ-1 promotes p65 nuclear translocation by facilitating the dissociation between p65 and NF-κB inhibitor α (IκBα). DJ-1 knockout (DJ-1^−/−) mice exhibit more microglial activation compared with wild-type littermate controls, especially in response to lipopolysaccharide (LPS) treatment. In cellular models, knockdown of DJ-1 significantly upregulates the gene expression and increases the release of LPS-treated inflammatory cytokines in primary microglia and BV2 cells. Furthermore, DJ-1 deficiency in microglia significantly enhances the neuronal toxicity in response to LPS stimulus. In addition, pharmacological blockage of NF-κB nuclear translocation by SN-50 prevents microglial activation and alleviates the damage of DA neurons induced by microglial DJ-1 deficiency in vivo and in vitro. Thus, our data illustrate a novel mechanism by which DJ-1 facilitates the interaction between IκBα and p65 by binding to p65 in microglia, and thus repressing microglial activation and exhibiting the protection of DA neurons from neuroinflammation-mediated injury in PD.Subject terms: Cell death in the nervous system, Parkinson''s disease     

A novel synthetic isothiocyanate ITC-57 displays antioxidant,anti-inflammatory,and neuroprotective properties in a mouse Parkinson’s disease model

The degenerative process of the nigral dopamine(DA)rgic neurons in Parkinson’s disease (PD) involves both oxidative stress and neuroinflammation. In the present study, we aimed at developing a novel antioxidant and anti-inflammatory agent for PD therapy. Toward this end, we screened a novel focused library of isothiocyanate derivatives that we have generated for an anti-inflammatory property. We obtained a novel compound ITC-57 and found that ITC-57 effectively induced gene expression of the antioxidant enzymes NAD(P)H quinone oxidoreductase-1, the catalytic and modulatory subunits of glutamylcysteine ligase, and HO-1 in DAergic neuronal CATH.a cells and protected CATH.a cells from oxidative damages. The compound also induced the same antioxidant enzymes in microglial BV-2 cells and suppressed the production of the proinflammatory molecules nitric oxide, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in lipopolysaccharide-activated BV-2 cells. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-elicited mouse model of PD, ITC-57 protected the DAergic neurons from degeneration, induced HO-1, lowered TNF-α, and suppressed microglial activation in the nigra. Furthermore, ITC-57 prevented the PD-associated motor deficits from occurring. Taken together, ITC-57 would be useful toward development of a disease-modifying therapy for PD.     

VIP enhances phagocytosis of fibrillar beta-amyloid by microglia and attenuates amyloid deposition in the brain of APP/PS1 mice

Vasoactive intestinal peptide (VIP) is a multifunctional neuropeptide with demonstrated immunosuppressive and neuroprotective activities. It has been shown to inhibit Amyloid beta (Aβ)-induced neurodegeneration by indirectly suppressing the production and release of a variety of inflammatory and neurotoxic factors by activated microglia. We demonstrated that VIP markedly increased microglial phagocytosis of fibrillar Aβ42 and that this enhanced phagocytotic activity depended on activation of the Protein kinase C (PKC) signaling pathway. In addition, VIP suppressed the release of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) from microglia activated by combined treatment with fibrillar Aβ42 and low dose interferon-γ (IFN-γ). We utilized an adenovirus-mediated gene delivery method to overexpress VIP constitutively in the hippocampus of APPswPS1 transgenic mice. The Aβ load was significantly reduced in the hippocampus of this animal model of Alzheimer's disease, possibly due to the accumulation and activation of cd11b-immunoactive microglial cells. The modulation of microglial activation, phagocytosis, and secretion by VIP is a promising therapeutic option for the treatment of Alzheimer's disease (AD).     

β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway

The role of the β2 adrenergic receptor (β2AR) in the regulation of chronic neurodegenerative inflammation within the CNS is poorly understood. The purpose of this study was to determine neuroprotective effects of long-acting β2AR agonists such as salmeterol in rodent models of Parkinson's disease. Results showed salmeterol exerted potent neuroprotection against both LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium-induced dopaminergic neurotoxicity both in primary neuron-glia cultures (at subnanomolar concentrations) and in mice (1-10 μg/kg/day doses). Further studies demonstrated that salmeterol-mediated neuroprotection is not a direct effect on neurons; instead, it is mediated through the inhibition of LPS-induced microglial activation. Salmeterol significantly inhibited LPS-induced production of microglial proinflammatory neurotoxic mediators, such as TNF-α, superoxide, and NO, as well as the inhibition of TAK1-mediated phosphorylation of MAPK and p65 NF-κB. The anti-inflammatory effects of salmeterol required β2AR expression in microglia but were not mediated through the conventional G protein-coupled receptor/cAMP pathway. Rather, salmeterol failed to induce microglial cAMP production, could not be reversed by either protein kinase A inhibitors or an exchange protein directly activated by cAMP agonist, and was dependent on β-arrestin2 expression. Taken together, our results demonstrate that administration of extremely low doses of salmeterol exhibit potent neuroprotective effects by inhibiting microglial cell activation through a β2AR/β-arrestin2-dependent but cAMP/protein kinase A-independent pathway.     

Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants

The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.     

Parkin regulates microglial NLRP3 and represses neurodegeneration in Parkinson's disease

Microglial hyperactivation of the NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome contributes to the pathogenesis of Parkinson's disease (PD). Recently, neuronally expressed NLRP3 was demonstrated to be a Parkin polyubiquitination substrate and a driver of neurodegeneration in PD. However, the role of Parkin in NLRP3 inflammasome activation in microglia remains unclear. Thus, we aimed to investigate whether Parkin regulates NLRP3 in microglia. We investigated the role of Parkin in NLRP3 inflammasome activation through the overexpression of Parkin in BV2 microglial cells and knockout of Parkin in primary microglia after lipopolysaccharide (LPS) treatment. Immunoprecipitation experiments were conducted to quantify the ubiquitination levels of NLRP3 under various conditions and to assess the interaction between Parkin and NLRP3. In vivo experiments were conducted by administering intraperitoneal injections of LPS in wild-type and Parkin knockout mice. The Rotarod test, pole test, and open field test were performed to evaluate motor functions. Immunofluorescence was performed for pathological detection of key proteins. Overexpression of Parkin mediated NLRP3 degradation via K48-linked polyubiquitination in microglia. The loss of Parkin activity in LPS-induced mice resulted in excessive microglial NLRP3 inflammasome assembly, facilitating motor impairment, and dopaminergic neuron loss in the substantia nigra. Accelerating Parkin-induced NLRP3 degradation by administration of a heat shock protein (HSP90) inhibitor reduced the inflammatory response. Parkin regulates microglial NLRP3 inflammasome activation through polyubiquitination and alleviates neurodegeneration in PD. These results suggest that targeting Parkin-mediated microglial NLRP3 inflammasome activity could be a potential therapeutic strategy for PD.     

Antagonizing peroxisome proliferator‐activated receptor γ facilitates M1‐to‐M2 shift of microglia by enhancing autophagy via the LKB1–AMPK signaling pathway

Microglia‐mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator‐activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)‐induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS‐stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL‐4, IGF‐1, TGF‐β1, TGF‐β2, TGF‐β3, G‐CSF, and GM‐CSF, and reduced the expression of M1 markers, such as CD86, Cox‐2, iNOS, IL‐1β, IL‐6, TNF‐α, IFN‐γ, and CCL2, thereby inhibiting NFκB–IKKβ activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3‐II/LC3‐I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1–STRAD–MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1‐to‐M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1–AMPK signaling and inhibited NFκB–IKKβ activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1‐to‐M2 phenotypic shift in LPS‐induced microglia, which might be due to improved autophagy via the activation of the LKB1–AMPK signaling pathway.     

Microglial activation induced by neurodegeneration: a proteomic analysis

Neuroinflammation mediated by microglial activation appears to play an essential role in the pathogenesis of Parkinson disease; however, the mechanisms by which microglia are activated are not fully understood. Thus, we first evaluated the effects of two parkinsonian toxicants, manganese ethylene bisdithiocarbamate (Mn-EBDC) and 1-methyl-4-phenylpyridine (MPP+), on microglial activation as well as associated dopaminergic (DAergic) neurotoxicity in primary cell culture systems. The results demonstrated that, when rat primary mesencephalic neuron-enriched or neuron-microglia mixed cultures were treated with Mn-EBDC at 2-8 microm or MPP+ at 0.25-5 microm, respectively, for 7 days, both toxicants were capable of inducing DAergic neurodegeneration as well as activating microglia via a mechanism secondary to DAergic neurodegeneration. Furthermore activated microglia subsequently enhanced DAergic neurotoxicity induced by Mn-EBDC or MPP+. Detailed scrutiny of neuron-microglia interactions identified a fraction of the conditioned media derived from a DAergic cell line treated with Mn-EBDC or MPP+ that potently activated microglia. To further define potential mediators leading to microglial activation secondary to neurodegeneration, we utilized a quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of MPP+-treated cellular fraction that mediated microglial activation as compared with controls. The search revealed numerous novel proteins that are potentially important in neurodegeneration-mediated microglial activation, a process believed to be critical in Parkinson disease progression.