Microglia-enriched Cultures Research Articles

Neuron-Microglia Contacts Govern the PGE2 Tolerance through TLR4-Mediated de Novo Protein Synthesis.

Cellular and molecular mechanisms of the peripheral immune system (e.g., macrophage and monocyte) in programming endotoxin tolerance (ET) have been well studied. However, regulatory mechanism in development of brain immune tolerance remains unclear. The inducible COX-2/PGE2 axis in microglia, the primary innate immune cells of the brain, is a pivotal feature in causing inflammation and neuronal injury, both in acute excitotoxic insults and chronic neurodegenerative diseases. This present study investigated the regulatory mechanism of PGE2 tolerance in microglia. Multiple reconstituted primary brain cells cultures, including neuron–glial (NG), mixed glial (MG), neuron-enriched, and microglia-enriched cultures, were performed and consequently applied to a treatment regimen for ET induction. Our results revealed that the levels of COX-2 mRNA and supernatant PGE2 in NG cultures, but not in microglia-enriched and MG cultures, were drastically reduced in response to the ET challenge, suggesting that the presence of neurons, rather than astroglia, is required for PGE2 tolerance in microglia. Furthermore, our data showed that neural contact, instead of its soluble factors, is sufficient for developing microglial PGE2 tolerance. Simultaneously, this finding determined how neurons regulated microglial PGE2 tolerance. Moreover, by inhibiting TLR4 activation and de novo protein synthesis by LPS-binding protein (LBP) manipulation and cycloheximide, our data showed that the TLR4 signal and de novo protein synthesis are necessary for microglia to develop PGE2 tolerance in NG cells under the ET challenge. Altogether, our findings demonstrated that neuron–microglia contacts are indispensable in emerging PGE2 tolerance through the regulation of TLR4-mediated de novo protein synthesis.

Kynurenic Acid and Its Analog SZR104 Exhibit Strong Antiinflammatory Effects and Alter the Intracellular Distribution and Methylation Patterns of H3 Histones in Immunochallenged Microglia-Enriched Cultures of Newborn Rat Brains.

Kynurenic acid (KYNA) is implicated in antiinflammatory processes in the brain through several cellular and molecular targets, among which microglia-related mechanisms are of paramount importance. In this study, we describe the effects of KYNA and one of its analogs, the brain-penetrable SZR104 (N-(2-(dimethylamino)ethyl)-3-(morpholinomethyl)-4-hydroxyquinoline-2-carboxamide), on the intracellular distribution and methylation patterns of histone H3 in immunochallenged microglia cultures. Microglia-enriched secondary cultures made from newborn rat forebrains were immunochallenged with lipopolysaccharide (LPS). The protein levels of selected inflammatory markers C–X–C motif chemokine ligand 10 (CXCL10) and C–C motif chemokine receptor 1 (CCR1), histone H3, and posttranslational modifications of histone H3 lys methylation sites (H3K9me3 and H3K36me2, marks typically associated with opposite effects on gene expression) were analyzed using quantitative fluorescent immunocytochemistry and western blots in control or LPS-treated cultures with or without KYNA or SZR104. KYNA and SZR104 reduced levels of the inflammatory marker proteins CXCL10 and CCR1 after LPS-treatment. Moreover, KYNA and SZR104 favorably affected histone methylation patterns as H3K9me3 and H3K36me2 immunoreactivities, and histone H3 protein levels returned toward control values after LPS treatment. The cytoplasmic translocation of H3K9me3 from the nucleus indicated inflammatory distress, a process that could be inhibited by KYNA and SZR104. Thus, KYNA signaling and metabolism, and especially brain-penetrable KYNA analogs such as SZR104, could be key targets in the pathway that connects chromatin structure and epigenetic mechanisms with functional consequences that affect neuroinflammation and perhaps neurodegeneration.

Quantitative morphometric and cell-type-specific population analysis of microglia-enriched cultures subcloned to high purity from newborn rat brains.

Morphological and functional characterizations of cultured microglia are essential for the improved understanding of their roles in neuronal health and disease. Although some studies (phenotype analysis, phagocytosis) can be carried out in mixed or microglia-enriched cultures, in others (gene expression) pure microglia must be used. If the use of genetically modified microglial cells is not feasible, isolation of resident microglia from nervous tissue must be carried out. In this study, mixed primary cultures were established from the forebrains of newborn rats. Secondary microglia-enriched cultures were then prepared by shaking off these cells from the primary cultures, which were subsequently used to establish tertiary cultures by further shaking off the easily detachable microglia. The composition of these cultures was quantitatively analyzed by immunocytochemistry of microglia-, astrocyte-, oligodendrocyte- and neuron-specific markers to determine yield and purity. Microglia were quantitatively characterized regarding morphological and proliferation aspects. Secondary and tertiary cultures typically exhibited 73.3% ± 17.8% and 93.1% ± 6.0% purity for microglia, respectively, although the total number of microglia in the latter was much smaller. One in seven attempts of culturing the tertiary cultures had ~99% purity for microglia. The overall yield from the number of cells plated at DIV0 to the Iba1-positive microglia in tertiary cultures was ~1%. Astrocytic and neuronal contamination progressively decreased during subcloning, while oligodendrocytes were found sporadically throughout culturing. Although the tertiary microglia cultures had a low yield, they produced consistently high purity for microglia; after validation, such cultures are suitable for purity-sensitive functional screenings (gene/protein expression).

Protective Effect of Semisynthetic and Natural Flavonoid on Aged Rat Microglia-enriched Cultures.

The ROS-mediated lysosomal dysfunction and coinciding deterioration of mitochondrial function are thought to be the prominent mechanisms responsible for aging. Microglia, the resident macrophages in the central nervous system, were postulated to belong to the major targets vulnerable to these detrimental processes, acting as principal drivers in brain aging. The present study investigated the potential protective effect of the semisynthetic flavonoid 3'-O-(3-chloropivaloyl) quercetin (CPQ) and quercetin (Q) on microglia-enriched mixed brain cultures (MBCs) established from aged Wistar rats. Both flavonoids tested suppressed the development of lipofuscin-related autofluorescence in aged cells. Further ensuing protective effects included reduction of protein oxidation markers in aged cells. Moreover, unlike Q, CPQ significantly suppressed sensitivity of aged cells to stimulation of superoxide burst. Other activation markers, cellular hypertrophy and isolectin B4 binding, were also downregulated by treatment with both CPQ and Q. In conclusion, results of our study suggest that both flavonoids tested may protect microglia with a quite comparable efficacy against aging-related accumulated alterations. The protective mechanism can include interference with the ROS-mediated vicious cycles involving lysosomal dysfunction. Nevertheless, the lipophilized quercetin, CPQ, a compound with proposed enhanced biological availability compared to parent molecule, can represent an agent potentially useful for new effective pharmaceutical intervention against brain aging, overcoming the limitations of clinical applicability of quercetin.

TGF-β1 Neuroprotection via Inhibition of Microglial Activation in a Rat Model of Parkinson's Disease.

Transforming growth factor (TGF)-β1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. Recently we have shown that TGF-β1 pretreatment in vitro protects against 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neuronal loss that characterizes in Parkinson's disease (PD). Herein, we aimed to demonstrate that TGF-β1 administration in vivo after MPP+ toxicity has neuroprotection that is achieved by a mediation of microglia. A rat model of PD was prepared by injecting MPP+ unilaterally in the striatum. At 14days after MPP+ injection, TGF-β1 was administrated in the right lateral cerebral ventricle. Primary ventral mesencephalic (VM) neurons and cerebral cortical microglia were treated by MPP+, respectively, and TGF-β1 was applied to neuronal or microglial cultures at 1h after MPP+ treatment. As expected, MPP+ resulted in decrease in TGF-β1 production in the substantia nigra and in primary VM neurons and microglia. TGF-β1 intracerebroventricular administration alleviated MPP+-induced PD-like changes in pathology, motor coordination and behavior. Meanwhile, TGF-β1 ameliorated MPP+-induced microglial activation and inflammatory cytokine production in vivo. Interestingly, TGF-β1 treatment was not able to ameliorate MPP+-induced dopaminergic neuronal loss and caspase-3/9 activation in mono-neuron cultures, but TGF-β1 alleviated MPP+-induced microglial activation and inflammatory cytokine production in microglia-enriched cultures. This effect of TGF-β1 inhibiting microglial inflammatory response was blocked by Smad3 inhibitor SIS3. Importantly, neuronal exposure to supernatants of primary microglia that had been treated with TGF-β1 reduced dopaminergic neuronal loss and caspase-3/9 activation induced by MPP+-treated microglial supernatants. These findings establish that TGF-β1 exerts neuroprotective property in PD by inhibiting microglial inflammatory response via Smad3 signaling.

P71 - Can semi-synthetic flavonoids return old microglia to their youthful state?

A number of studies have indicated that brain inflammation may deteriorate during normal aging and that neuroinflammation is amplified in age-related neurodegenerative diseases. A pivotal role in age-related neuroinflammatory pathologies is attributed to amplified and prolonged activation of microglia. In addition, microglia from the aged brain were reported as senescent displaying many functional impairments. Flavonoids were shown to be promising molecules in modulation of neuroinflammation. Quercetin, a naturally occurring flavonoid, was proven to downregulate inflammatory genes in microglia. Synthetically modified quercetin, 3´-O-(3-chloropivaloyl)quercetin (CPQ), is assumed to posses better biological availability and enhanced antioxidant properties. In the present study, the antineuroinflammatory capacity of CPQ was assessed in BV-2 microglial cells and rat primary microglia. CPQ suppressed more efficiently than its precursor quercetin LPS-induced NO production and iNOS protein expression. However, neither of the compounds tested influenced significantly phagocytosis of BV-2 cells. In addition, CPQ showed a somewhat better suppression of PMA-induced generation of superoxide than did quercetin. Unlike quercetin, CPQ caused a decline in BV-2 microglia proliferation (without any impact on cell viability) along with interference with cell cycle progression. Both compounds tested at 10uM concentration notably enhanced viability of microglia-enriched cultures prepared from 22-month-old rat brains. This was followed by suppression of lipofuscin-like autofluorescence, improvement of lysosomal function and protection of mitochondria in the old microglia. These results can highlight the therapeutic potential of CPQ as a novel antiinflammatory drug in neurodegenerative diseases. In addition, our data suggest that both natural and semisynthetic flavonoids might protect functions of old microglia [VEGA2/0031/12,1/0076/13;APVV-0052-10;ITMS26240220040].

Involvement of interleukin-1β in the autophagic process of microglia: relevance to Alzheimer’s disease

BackgroundAutophagy is a major pathway of protein and organelle degradation in the lysosome. Autophagy exists at basal constitutive level and can be induced as a defense mechanism under stress conditions. Molecular relationships between autophagy and inflammation at the periphery were recently evidenced, highlighting a role of autophagy in the regulation of inflammation. Impairment of autophagy (with accumulation of autophagic vacuoles) and substantial inflammation are found in neurodegenerative diseases such as Alzheimer’s Disease (AD). However, the links between autophagy and inflammation in AD remain to be determined.MethodsHere, we examined the inflammatory reaction and autophagy in murine tri-cultures of neurons, astrocytes, and microglia. Tri-cultures were exposed to various inflammatory stresses (lipopolysaccharide (LPS), amyloid peptide (Aβ42) with or without cytokines) for 48 hours. Furthermore, the relationships between inflammation and autophagy were also analyzed in astrocyte- and microglia-enriched cultures. Data for multiple variable comparisons were analyzed by a one-way ANOVA followed by a Newman-keuls’ test.ResultsAβ42 induced a low inflammation without accumulation of acidic vesicles contrary to moderate or severe inflammation induced by LPS or the cytokine cocktail (IL-1β, TNF-α, and IL-6) or IL-1β alone which led to co-localization of p62 and LC3, two markers of autophagy, with acidic vesicles stained with Lyso-ID Red dye. Moreover, the study reveals a major role of IL-1β in the induction of autophagy in tri-cultures in the presence or absence of Aβ42. However, the vulnerability of the autophagic process in purified microglia to IL-1β was prevented by Aβ42.ConclusionThese findings show a close relationship between inflammation and autophagy, in particular a major role of IL-1β in the induction of the microglial autophagy which could be the case in AD. New therapeutic strategies could target inflammasome and autophagy in microglia to maintain its role in the amyloid immunosurveillance.

Microglial MAC1 receptor and PI3K are essential in mediating β-amyloid peptide-induced microglial activation and subsequent neurotoxicity

Backgroundβ-Amyloid peptide (Aβ) is a major protein in the brain associated with Alzheimer's and Parkinson's diseases. The purpose of this study was to investigate the role of macrophage antigen-1 (MAC1) receptor, an integrin scavenger receptor in microglia, and subsequent signaling events in mediating Aβ-induced neurotoxicity. We have previously reported that NADPH oxidase (PHOX) on microglia and superoxide produced by PHOX are critical for Aβ-induced loss of dopaminergic neurons. However, the upstream signaling pathway of superoxide production remains unclear.MethodsFor the in vitro study, mesencephalic neuron-glia cultures and microglia-enriched cultures from mice deficient in the MAC1 receptor (MAC1-/-) and wild type controls were used to investigate the role of MAC1 receptor in Aβ-induced neurotoxicity and the role of phosphoinositide-3 kinase (PI3K) in the signal pathway between MAC1 receptor and PHOX. For the in vivo study, Aβ was injected into the substantia nigra of MAC1-/- mice and wild type mice to confirm the role of MAC1 receptor.ResultsWe found that Aβ-induced activation of microglia, activation of PHOX, generation of superoxide and other reactive oxygen species, and loss of dopaminergic neurons were decreased in MAC1-/- cultures compared to MAC1+/+ cultures. In MAC1-/- mice, dopaminergic neuron loss in response to Aβ injection into the substantia nigra was reduced relative to MAC1+/+ mice. Thus, MAC1 receptor-mediated PHOX activation and increased superoxide production are associated with Aβ-induced neurotoxicity. PI3K activation was one downstream step in MAC1 signaling to PHOX and played an important role in Aβ-induced neurotoxicity. In microglia-enriched cultures from MAC1-/- mice, Aβ-induced activation of PI3K (phosphorylation of target proteins and PIP3 production) was reduced relative to MAC1+/+ cultures.ConclusionsTaken together, our data demonstrate that Aβ activates MAC1 receptor to increase the activity of PI3K, which in turn phosphorylates p47phox, triggers the translocation of cytosolic subunits of PHOX to microglia membrane, increases PHOX activation and the subsequent production of superoxide and causes neurotoxicity.

Generation of Microglia Cultures and Mixed Glial Culture

Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during immune reaction. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu., 原代啮齿动物小胶质细胞富集的培养物是研究小胶质细胞生物学在体外的最流行的模型，并且探索免疫信号传导途径。 含有小胶质细胞和星形胶质细胞的混合胶质细胞培养物非常有用于研究免疫反应期间小神经胶质 - 星形胶质相互作用的精确机制。 这个协议已经由Hong博士实验室的各种研究人员，尤其是刘博士多年来开发和改进。

Glycogen synthase kinase‐3 negatively regulates anti‐inflammatory interleukin‐10 for lipopolysaccharide‐induced iNOS/NO biosynthesis and RANTES production in microglial cells

The inflammatory effects of glycogen synthase kinase-3 (GSK-3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK-3-mediated interleukin-10 (IL-10) inhibition on lipopolysaccharide (LPS)-induced inflammation. Treatment with GSK-3 inhibitor significantly blocked LPS-induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia-enriched cultures. Using an antibody array and enzyme-linked immunosorbent assay, we found that GSK-3-inhibitor treatment blocked LPS-induced upregulation of regulated on activation normal T-cell expressed and secreted (RANTES) and increased IL-10 expression. The time kinetics and dose-response relations were confirmed. Reverse transcription-polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK-3 using short-interference RNA, and transfecting cells with dominant-negative GSK-3beta, blocked LPS-elicited NO and RANTES expression but increased IL-10 expression. In contrast, GSK-3beta overexpression upregulated NO and RANTES but downregulated IL-10 in LPS-stimulated cells. Treating cells with anti-IL-10 neutralizing antibodies to prevent GSK-3 from downregulating NO and RANTES showed that the anti-inflammatory effects are, at least in part, IL-10-dependent. The involvement of Akt, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-kappaB that positively regulated IL-10 was demonstrated. Furthermore, inhibiting GSK-3 increased the nuclear translocation of transcription factors, that all important for IL-10 expression, including CCAAT/enhancer-binding protein beat (C/EBPbeta), C/EBPdelta, cAMP response binding element protein and NF-kappaB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK-3-mediated IL-10 downregulation.

Mutant α-Synuclein Overexpression Mediates Early Proinflammatory Activity

Microglia provide immune surveillance for the brain through both the removal of cellular debris and protection against infection by microorganisms and "foreign" molecules. Upon activation, microglia display an altered morphology and increased expression of proinflammatory molecules. Increased numbers of activated microglia have been identified in a number of neurodegenerative diseases including Parkinson's disease (PD). What remains to be determined is whether activated microglia result from ongoing cell death or are involved in disease initiation and progression. To address this question we utilized a transgenic mouse model that expresses a mutated form of a key protein involved in Parkinson's disease, alpha-synuclein. Herein, we report an increase in activated microglia and proinflammatory molecules in 1-month-old transgenic mice well before cell death occurs in this model. Frank microglial activation is resolved by 6 months of age while a subset of proinflammatory molecules remain elevated for 12 months. Both tyrosine hydroxylase mRNA expression and alpha-synuclein protein are decreased in the striatum of older animals evidence of dystrophic neuritic projections. To determine whether mutated alpha-synuclein could directly activate microglia primary microglia-enriched cell cultures were treated with exogenous mutated alpha-synuclein. The data reveal an increase in activated microglia and proinflammatory molecules due to direct interaction with mutated alpha-synuclein. Together, these data demonstrate that mutated alpha-synuclein mediates a proinflammatory response in microglia and this activity may participate in PD pathogenesis.

Luteolin protects dopaminergic neurons from inflammation-induced injury through inhibition of microglial activation

Parkinson's disease is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra. Accumulating evidence has suggested that inflammation in the brain participates in the pathogenesis of Parkinson's disease. Luteolin, a polyphenolic compound found in foods of plant origin, belongs to the flavone subclass of flavonoids, and has been shown to possess antimutagenic, antitumorigenic, antioxidant and antiinflammatory properties. In this study, we found that luteolin concentration-dependently attenuated the lipopolysaccharide (LPS)-induced decrease in [ 3H]dopamine uptake and loss of tyrosine hydroxylase-immunoreactive neurons in primary mesencephalic neuron–glia cultures. Moreover, luteolin also significantly inhibited LPS-induced activation of microglia and excessive production of tumor necrosis factor-α, nitric oxide and superoxide in mesencephalic neuron–glia cultures and microglia-enriched cultures. Our results demonstrate that luteolin may protect dopaminergic neurons from LPS-induced injury and its efficiency in inhibiting microglia activation may underlie the mechanism.

Upregulation of p21Cip1 in activated glial cells

The cdk inhibitor p21(Cip1), also named p21(Cip1/Waf1), is intimately involved in coupling growth arrest to cellular differentiation in several cell types. p21(Cip1) is a multifunctional protein that might regulate cell-cycle progression at different levels. In a recent study, we found no differences in the rate of proliferation between glial cells from wild-type and p21(Cip1-/-) mice. In the present study, we examined differences in glial activation between glial cells from wild-type and p21(Cip1-/-) mice, using mixed glial cultures, microglia-enriched cultures, and astrocyte-enriched cultures. We compared the effect of lipopolysaccharide and two forms (oligomeric and fibrillar) of the 1-42 beta-amyloid peptide on glial activation. We observed an attenuation of nuclear translocation of the nuclear factor kappa-B in p21(Cip1-/-) glial cells, when compared with glial cells from wild-type mice. In contrast, tumor necrosis factor-alpha release was enhanced in p21(Cip1-/-)microglial cells. In addition glial activation induced by lipopolysaccharide and the fibrillar form of the 1-42 beta-amyloid peptide upregulated p21(Cip1). Our results support a role for p21(Cip1) in the activation of glial cells, particularly in microglia.

Protective effect of isoflavones from Trifolium pratense on dopaminergic neurons

In the present study, protective effect of five isoflavones (formononetin, daidzein, pratensein, calycosin and irilone) from Trifolium pratense on lipopolysaccharide-induced dopaminergic neurodegeneration was studied for the first time. The results showed that all five isoflavones attenuated LPS-induced decrease in dopamine uptake and the number of dopaminergic neurons in a dose-dependent manner in rat mesencephalic neuron-glia cultures. Moreover, they also significantly inhibited LPS-induced activation of microglia and production of tumor necrosis factor-α, nitric oxide and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures. In addition, the rank order of protective potency of five isoflavones was: pratensein > daidzein > calycosin > formononetin > irilone. This study suggested that all five isoflavones protected dopaminergic neurons against LPS-induced injury through inhibition of microglia activation and proinflammatory factors generation.

Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

BackgroundPrevious studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.MethodsMesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.ResultsOur results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.ConclusionWe demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-γ activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

MAC1 mediates LPS‐induced production of superoxide by microglia: The role of pattern recognition receptors in dopaminergic neurotoxicity

Microglia-derived superoxide is critical for the inflammation-induced selective loss of dopaminergic (DA) neurons, but the underlying mechanisms of microglial activation remain poorly defined. Using neuron-glia and microglia-enriched cultures from mice deficient in the MAC1 receptor (MAC1-/-), we demonstrate that lipopolysaccharide (LPS) treatment results in lower TNFalpha response, attenuated loss of DA neurons, and absence of extracellular superoxide production in MAC1-/- cultures. Microglia accumulated fluorescently labeled LPS in punctate compartments associated with the plasma membrane, intracellular vesicles, and the Golgi apparatus. Cytochalasin D (CD), an inhibitor of phagocytosis, blocked LPS internalization. However, microglia derived from Toll-like receptor 4 deficient mice and MAC1-/- mice failed to show a significant decrease in intracellular accumulation of labeled LPS, when compared with controls. Pretreatment with the scavenger receptor inhibitor, fucoidan, inhibited 79% of LPS accumulation in microglia without affecting superoxide, indicating that LPS internalization and superoxide production are mediated by separate phagocytosis receptors. Together, these data demonstrate that MAC1 is essential for LPS-induced superoxide from microglia, implicating MAC1 as a critical trigger of microglial-derived oxidative stress during inflammation-mediated neurodegeneration.

Cadmium-Induced Toxicity in Rat Primary Mid-brain Neuroglia Cultures: Role of Oxidative Stress from Microglia

This study examined the role of oxidative stress in neurotoxic effects of cadmium chloride (Cd) in rat primary mid-brain neuron-glia cultures. Cd accumulated in neuron-glia cultures and produced cytotoxicity in a dose-dependent manner, with IC(50) of 2.5microM 24 h after exposure. (3)H-dopamine uptake into neuron-glia cultures was decreased 7 days after Cd exposure, with IC(50) of 0.9microM, indicative of the sensitivity of dopaminergic neurons to Cd toxicity. To investigate the role of microglia in Cd-induced toxicity to neurons, microglia-enriched cultures were prepared. Cd significantly increased intracellular reactive oxygen species production in microglia-enriched cultures, as evidenced by threefold increases in 2',7'-dichlorofluorescein signals. Using 5,5-dimethyl-1-pyrroline N-oxide as a spin-trapping agent, Cd increased electron spin resonance signals by 3.5-fold in microglia-enriched cultures. Cd-induced oxidative stress to microglia-enriched cultures was further evidenced by activation of redox-sensitive transcription factor nuclear factor kappa B and activator protein-1 (AP-1), and the increased expression of oxidative stress-related genes, such as metallothionein, heme oxygenase-1, glutathione S-transferase pi, and metal transport protein-1, as determined by gel-shift assays and real-time reverse transcription-PCR, respectively, in microglia-enriched cultures. In conclusion, Cd is toxic to neuron-glia cultures, and the oxidative stress from microglia may play important roles in Cd-induced damage to dopaminergic neurons.

Biochanin A protects dopaminergic neurons against lipopolysaccharide-induced damage through inhibition of microglia activation and proinflammatory factors generation

Activation of microglia and consequent release of proinflammatory factors, are believed to contribute to neurodegeneration in Parkinson's disease (PD). Hence, identification of compounds that prevent microglial activation is highly desirable in the search for therapeutic agents for inflammation-mediated neurodegenerative diseases. In this study, we reported that biochanin A, one of the predominant isoflavones in Trifolium pratense, attenuated lipopolysaccharide (LPS)-induced decrease in dopamine uptake and the number of dopaminergic neurons in a dose-dependent manner in rat mesencephalic neuron-glia cultures. Moreover, biochanin A also significantly inhibited LPS-induced activation of microglia and production of tumor necrosis factor-α, nitric oxide and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures. This study suggested for the first time that biochanin A protected dopaminergic neurons against LPS-induced damage through inhibition of microglia activation and proinflammatory factors generation.

Reactive microgliosis participates in MPP+‐induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor

It has been reported that extracellular matrix (ECM) molecules regulate monocyte activation by binding with a 67 kDa nonintegrin laminin receptor (LR). As microgliosis is a pivotal factor in propelling the progress of chronic neurodegeneration in the brain, we hypothesized that LR may regulate the microgliosis and subsequent neurotoxicity. Using 1-methyl-4-phenylpyridinium (MPP+) -treated C57 mice primary mesencephalic neuron-glia cultures as an in vitro Parkinson's disease (PD) model, we observed that MPP+ treatment increased LR expression only in the mixed neuron-glia but not in microglia-enriched or microglia-depleted cultures, indicating that MPP+-induced increase of LR expression is associated with neuron-microglia interaction. Using confocal microscopic examination, we found that LR was localized in the microglia, which were F4/80 positive. Treatment with the antibody (Ab) against LR (LR-Ab) or YIGSR, a synthetic pentapeptide inhibitor for LR, significantly attenuated the MPP+-increased F4/80 immunoreactivity (24 h) and dopaminergic (DA) neurotoxicity. LR-Ab also attenuated MPP+-increased microglial phagocytotic activity (48 h) and the superoxide production (4 days). Further study demonstrated that exogenous laminin (1-10 microg/ml) treatment induced microglial activation and DA neurotoxicity, in a dose-dependent manner, which was partially attenuated by the LR-Ab. We concluded that by regulating cell-ECM interaction, LR plays important roles in mediating microgliosis and subsequent DA neurotoxicity. Laminin is a potential ligand for activating this LR receptor. This study also suggests that laminin/LR is a potential target for developing new therapeutic drugs against neurodegenerative disorders such as PD.

Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca 2+ disturbance in microglia activation-mediated dopaminergic cell degeneration

Increasing evidences suggest that activated microglia may contribute to neurodegeneration in Parkinson's disease (PD). In the present study, primary ventral mesencephalic (VM) cultures from E14 rats and PC12 cells were utilized as in vitro models to examine the mechanism underlying microglia activation mediated dopaminergic neurodegeneration. Using lipopolysaccharide (LPS) (1–100 ng/ml) as a tool, we observed that microglia activation-mediated a selective dopaminergic neurodegeneration in VM neuron-glia cultures, which was supported by the further study showing that conditioned medium (CM) from microglia-enriched cultures treated with LPS (10–100 ng/ml) decreased PC12 cell viability. The results from antibody neutralization, NO inhibition and superoxide neutralization suggested that the dopaminergic cell death was due to the production of microglia-derived proinflammatory factors (TNF-α, NO and superoxide), among which reactive oxygen species (ROS) might outweigh proinflammatory cytokines. Apoptosis assay on PC12 cells and primary dopaminergic neurons showed that apoptosis was a mechanism for both microglia activation-mediated dopaminergic cell death. Through Western blot and immunocytochemistry, we found that caspase-3 activation was involved in both dopaminergic cell injuries. Finally, the results from laser scanning confocal microscope demonstrated that PC12 cell intracellular free Ca 2+ ([Ca 2+] i ) increased early after CM treatment. [Ca 2+] i increase involved influx of calcium from the extracellular milieu and release from intracellular stores and participated in the CM-induced PC12 cell apoptosis. Further investigations indicated that TNF-α, IL-1β, NO and superoxide contributed at different degrees to CM-induced [Ca 2+] i increase and apoptosis in PC12 cells. Using primary VM cultures and PC12 cells, our study shows the roles of proinflammatory factors, apoptosis, caspase-3 activation and Ca 2+ disturbance in microglia activation-mediated dopaminergic cell degeneration. Understanding the mechanism for microglia activation-mediated dopaminergic neurodegeneration may contribute to the development of new neuroprotective strategies against PD.