Microglia Acquire Distinct Activation Profiles Depending on the Degree of α-Synuclein Neuropathology in a rAAV Based Model of Parkinson's Disease

Post-mortem analysis of brains from Parkinson''s disease (PD) patients strongly supports microglia activation and adaptive immunity as factors contributing to disease progression. Such responses may be triggered by α-synuclein (α-syn), which is known to be the main constituent of the aggregated proteins found in Lewy bodies in the brains of PD patients. To investigate this we used a recombinant viral vector to express human α-syn in rat midbrain at levels that induced neuronal pathology either in the absence or the presence of dopaminergic cell death, thereby mimicking early or late stages of the disease. Microglia activation was assessed by stereological quantification of Mac1+ cells, as well as the expression patterns of CD68 and MCH II. In our study, when α-syn induced neuronal pathology but not cell death, a fast transient increase in microglia cell numbers resulted in the long-term induction of MHC II+ microglia, denoting antigen-presenting ability. On the other hand, when α-syn induced both neuronal pathology and cell death, there was a delayed increase in microglia cell numbers, which correlated with long-lasting CD68 expression and a morphology reminiscent of peripheral macrophages. In addition T-lymphocyte infiltration, as judged by the presence of CD4+ and CD8+ cells, showed distinct kinetics depending on the degree of neurodegeneration, and was significantly higher when cell death occurred. We have thus for the first time shown that the microglial response differs depending on whether α-syn expression results on cell death or not, suggesting that microglia may play different roles during disease progression. Furthermore, our data suggest that the microglial response is modulated by early events related to α-syn expression in substantia nigra and persists at the long term.     

Fractalkine Signaling Regulates the Inflammatory Response in an α-Synuclein Model of Parkinson Disease

Background

Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer’s disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1 (fractalkine) and its receptor (CX3CR1) have important roles in modulating neuroinflammation.

Methods

To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an in vivo mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and in vitro phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn.

Results

We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn.

Conclusion

Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD.     

α-Synuclein induces migration of BV-2 microglial cells by up-regulation of CD44 and MT1-MMP

Although there is known to be a marked concentration of reactive microglia in the substantia nigra pars compacta (SNpc) of patients with Parkinson's disease (PD), a disorder in which α-synuclein plays a key pathogenic role, the specific roles of α-synuclein and microglia remains poorly understood. In this study, we investigated the effects of α-synuclein and the mechanisms of invasive microglial migration into the SNpc. We show that α-synuclein up-regulates the expressions of the cell adhesion molecule CD44 and the cell surface protease membrane-type 1 matrix metalloproteinase through the extracellular regulated kinases 1/2 pathway. These concurrent inductions increased the generation of soluble CD44 to liberate microglia from the surrounding extracellular matrix for migration. The effects of α-synuclein were identical in BV-2 murine microglial cells subjected to cDNA transfection and extracellular treatment. These inductions in primary microglial cultures of C57Bl/6 mice were identical to those in BV-2 cells. α-Synuclein-induced microglial migration into the SNpc was confirmed in vivo using a 6-hydroxydopamine mouse model of PD. Our data demonstrate a correlation between α-synuclein-induced phenotypic changes and microglial migration. With the recruitment of the microglial population into the SNpc during dopaminergic neurodegeneration, α-synuclein may play a role in accelerating the pathogenesis of PD.     

Reactive Macrophages Increase Oxidative Stress and Alpha-Synuclein Nitration During Death of Dopaminergic Neuronal Cells in Co-Culture: Relevance to Parkinson’s Disease

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons and a substantial decrease in the neurotransmitter dopamine in the nigro-striatal region of the brain. Increased markers of oxidative stress, activated microglias and elevated levels of pro-inflammatory cytokines have been identified in the brains of patients with PD. Although the precise mechanism of loss of neurons in PD remains unclear, these findings suggest that microglial activation may contribute directly to loss of dopaminergic neurons in PD patients. In the present study, we tested the hypothesis that activated microglia induces nitric oxide-dependent oxidative stress which subsequently causes death of dopaminergic neuronal cells in culture. We employed lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) as a reactive microglial model and SH-SY5Y cells as a model for human dopaminergic neurons. LPS stimulation of macrophages led to increased production of nitric oxide in a time and dose dependent manner as well as subsequent generation of other reactive nitrogen species such as peroxynitrite anions. In co-culture conditions, reactive macrophages stimulated SH-SY5Y cell death characterized by increased peroxynitrite concentrations and nitration of alpha-synuclein within SH-SY5Y cells. Importantly 1400W, an inhibitor of the inducible nitric oxide synthase provided protection from cell death via decreasing the levels of nitrated alpha-synuclein. These results suggest that reactive microglias could induce oxidative stress in dopaminergic neurons and such oxidative stress may finally lead to nitration of alpha-synuclein and death of dopaminergic neurons in PD.     

Alpha-synuclein aggregation and cell death triggered by energy deprivation and dopamine overload are counteracted by D2/D3 receptor activation

Progressive degeneration and intraneuronal Lewy bodies made of filamentous α-synuclein (α-syn) in dopaminergic cells of the nigrostriatal system are characteristics of Parkinson's disease (PD). Glucose uptake is reduced in some of the brain regions affected by PD neurodegenerative changes. Defects in mitochondrial activity in the substantia nigra have been observed in the brain of patients affected by PD and substantia nigra lesions can induce the onset of a secondary parkinsonism. Thus, energy starvation and consequently metabolic impairment to dopaminergic neurons may be related to the onset of PD. On this line, we evaluated the effect of nutrient starvation, reproduced ' in vitro ' by glucose deprivation (GD), in primary mesecephalic neuronal cultures and dopaminergic-differentiated SH-SY5Y cells, to evaluate if decreased glucose support to dopaminergic cells can lead to mitochondrial damage, neurodegeneration and α-syn misfolding. Furthermore, we investigated the effect of dopamine (DA) treatment in the presence of a DA-uptake inhibitor or of the D₂/D₃ receptor (D₂R/D₃R) agonist quinpirole on GD-treated cells, to evaluate the efficacy of these therapeutic compounds. We found that GD induced the formation of fibrillary aggregated α-syn inclusions containing the DA transporter in dopaminergic cells. These alterations were accompanied by dopaminergic cell death and were exacerbated by DA overload. Conversely, the block of DA uptake and D₂R/D₃R agonist treatment exerted neuroprotective effects. These data indicate that glucose starvation is likely involved in the induction of PD-related pathological changes in dopaminergic neurons. These changes may be counteracted by the block of DA uptake and by dopaminergic agonist treatment.     

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.     

Ion channel blockade attenuates aggregated alpha synuclein induction of microglial reactive oxygen species: relevance for the pathogenesis of Parkinson's disease

Brain mononuclear phagocyte (perivascular macrophage and microglia, MG) inflammatory neurotoxins play a principal role in the pathogenesis of Parkinson's disease; chief among these are reactive oxygen species (ROS). We posit that aggregated, misfolded and oxidized alpha-synuclein (a major constituent of Lewy bodies), released or secreted from dying dopaminergic neurons, induces microglial ROS production that is regulated by ion channels and as such affects disease progression. To address this hypothesis, we performed patch clamp recordings of outward ionic currents in murine microglia and characterized their links to ROS production during alpha-synuclein stimulation. Aggregated nitrated alpha-synuclein induced ROS production in a dose-dependent manner that was inhibited by voltage-gated potassium current blockade, and to a more limited degree, by chloride current blockade. Interestingly, ROS produced in MG primed with tumor necrosis factor alpha and activated with phorbol myristate acetate was attenuated by voltage-gated potassium current blockade and more completely by chloride current blockade. In contrast, amyloid beta or cell membrane extract failed to induce microglial ROS production. Similar results were obtained using bone marrow-derived macrophages. The association of ROS production with specific plasma membrane ion currents provides a link between regulation of microglial ion transport and oxygen free radical production. Understanding these linkages may lead to novel therapeutics for Parkinson's disease where modulation of redox-related stress may slow disease progression.     

Partial depletion and repopulation of microglia have different effects in the acute MPTP mouse model of Parkinson’s disease

ObjectivesParkinson''s disease (PD) is a common neurodegenerative disorder characterized by the progressive and selective degeneration of dopaminergic neurons. Microglial activation and neuroinflammation are associated with the pathogenesis of PD. However, the relationship between microglial activation and PD pathology remains to be explored.Materials and MethodsAn acute regimen of MPTP was administered to adult C57BL/6J mice with normal, much reduced or repopulated microglial population. Damages of the dopaminergic system were comprehensively assessed. Inflammation‐related factors were assessed by quantitative PCR and Multiplex immunoassay. Behavioural tests were carried out to evaluate the motor deficits in MPTP‐challenged mice.ResultsThe receptor for colony‐stimulating factor 1 inhibitor PLX3397 could effectively deplete microglia in the nigrostriatal pathway of mice via feeding a PLX3397‐formulated diet for 21 days. Microglial depletion downregulated both pro‐inflammatory and anti‐inflammatory molecule expression at baseline and after MPTP administration. At 1d post‐MPTP injection, dopaminergic neurons showed a significant reduction in PLX3397‐fed mice, but not in control diet (CD)‐fed mice. However, partial microglial depletion in mice exerted little effect on MPTP‐induced dopaminergic injuries compared with CD mice at later time points. Interestingly, microglial repopulation brought about apparent resistance to MPTP intoxication.ConclusionsMicroglia can inhibit PD development at a very early stage; partial microglial depletion has little effect in terms of the whole process of the disease; and microglial replenishment elicits neuroprotection in PD mice.     

Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus ( S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus . In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-α (TNF-α) enhances TLR2 expression in microglia, whereas interleukin-1β has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-α, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-α-induced TLR2 expression. Similar results were observed with the IKK-2 and IκB-α inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-α was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-α knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-α knockout mice. Collectively, these results indicate that in response to S. aureus , TNF-α acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.     

Role of Secondary Mediators in Caffeine-Mediated Neuroprotection in Maneb- and Paraquat-Induced Parkinson’s Disease Phenotype in the Mouse

Maneb and paraquat are known to induce Parkinson’s disease (PD) phenotype, however, caffeine offers neuroprotection. Nitric oxide (NO) acts an important mediator in PD phenotype and tyrosine kinase (TK), nuclear factor kappa B (NF-kB), p38 mitogen activated protein kinase (p38 MAPK) are known to regulate its production. The present study aimed to elucidate the role of caffeine in the regulation of NO production and microglial activation and their subsequent contribution in dopaminergic neuroprotection. The animals were treated with caffeine and/or maneb and paraquat along with controls. In a few sets of experiments, the animals were also treated with aminoguanidine, an inhibitor of inducible NO synthase, pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB, genistein, an inhibitor of TK or SB202190, an inhibitor of p38 MAPK. Tyrosine hydroxylase (TH)-immunoreactivity and anti-integrin αM (OX-42) staining were performed to assess the number of dopaminergic neurons and activation of microglia, respectively. NO was measured in terms of nitrite, however, the expressions of p38 MAPK, interleukin (IL)-1β, NF-kB and TK were checked by western blot analyses. Maneb and paraquat induced the number of degenerating dopaminergic neurons, microglial cells, nitrite content, expressions of IL-1β, p38 MAPK, NF-kB and TK and caffeine co-treatment reduced the level of such alterations. Reductions were more pronounced in the animals co-treated with aminoguanidine, PDTC, genistein or SB202190. The results obtained thus demonstrate that caffeine down-regulates NO production, neuroinflammation and microglial activation, which possibly contribute to neuroprotection.     

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.     

A Spirulina-Enhanced Diet Provides Neuroprotection in an α-Synuclein Model of Parkinson's Disease

Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson''s disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action.     

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.     

Common Mechanisms Underlying α-Synuclein-Induced Mitochondrial Dysfunction in Parkinson’s Disease

Parkinson’s disease (PD) is the most common neurological movement disorder characterized by the selective and irreversible loss of dopaminergic neurons in substantia nigra pars compacta resulting in dopamine deficiency in the striatum. While most cases are sporadic or environmental, about 10% of patients have a positive family history with a genetic cause. The misfolding and aggregation of α-synuclein (α-syn) as a casual factor in the pathogenesis of PD has been supported by a great deal of literature. Extensive studies of mechanisms underpinning degeneration of the dopaminergic neurons induced by α-syn dysfunction suggest a complex process that involves multiple pathways, including mitochondrial dysfunction and increased oxidative stress, impaired calcium homeostasis through membrane permeabilization, synaptic dysfunction, impairment of quality control systems, disruption of microtubule dynamics and axonal transport, endoplasmic reticulum/Golgi dysfunction, nucleus malfunction, and microglia activation leading to neuroinflammation. Among them mitochondrial dysfunction has been considered as the most primary target of α-syn-induced toxicity, leading to neuronal cell death in both sporadic and familial forms of PD. Despite reviewing many aspects of PD pathogenesis related to mitochondrial dysfunction, a systemic study on how α-syn malfunction/aggregation damages mitochondrial functionality and leads to neurodegeneration is missing in the literature. In this review, we give a detailed molecular overview of the proposed mechanisms by which α-syn, directly or indirectly, contributes to mitochondrial dysfunction. This may provide valuable insights for development of new therapeutic approaches in relation to PD. Antioxidant-based therapy as a potential strategy to protect mitochondria against oxidative damage, its challenges, and recent developments in the field are discussed.     

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     

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.     

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.     

Seeding induced by α-synuclein oligomers provides evidence for spreading of α-synuclein pathology

Lewy bodies, α-synuclein (α-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of α-syn in the graft is that the aggregation of α-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro -generated α-syn oligomers induce transmembrane seeding of α-syn aggregation in a dose- and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that α-syn oligomers form as a dynamic mixture of oligomer types with different properties and that α-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular α-syn oligomers can induce intracellular α-syn aggregation, therefore we hypothesize that a similar mechanism might lead to α-syn pathology propagation.     

The structure of dopamine induced α-synuclein oligomers

Inclusions of aggregated α-synuclein (α-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson’s disease (PD). In vitro, α-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of α-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on α-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive β-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of α-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the β-sheet structure found in other pre-fibrillar forms of α-syn.