Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons

A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells.     

Comparison of Ionized Calcium-binding Adapter Molecule 1-Immunoreactive Microglia in the Spinal Cord Between Young Adult and Aged Dogs

Microglia are main form of active immune defense, and they are constantly moving and analyzing the CNS for damaged neurons and infectious agents. In this study, we compared microglia in the spinal cord of the young adult (1–2 years old) and aged (10–12 years old) German Shepherd dogs via immunohistochemistry and western blot analysis for ionized calcium-binding adapter molecule 1 (Iba-1), a microglial marker. In addition, we also observed the interferon-γ (IFN-γ), a pro-inflammatory cytokine, and interleukin-1β (IL-1β), produced by activated microglia/macrophage, protein levels in these groups. At first, we found that neuronal nuclei (NeuN, a neuronal marker)-immunoreactive neurons were distributed throughout the grey mate of the spinal cord, and there were no significant differences between the adult and aged groups. Most of Iba-1-immunoreactive microglia were morphologically ramified microglia (resting form) in the adult group, while some Iba-1-immunoreactive microglia were morphologically activated microglia in the aged group. In western blot analysis, Iba-1, IFN-γ and IL-1β expression were increased in the aged group. This result may be associated with age-dependent changes in the spinal cord.     

Benfotiamine Attenuates Inflammatory Response in LPS Stimulated BV-2 Microglia

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.     

NPP1 is responsible for potent extracellular ATP hydrolysis as NTPDase1 in primary cultured murine microglia

The movement of microglia is regulated mainly by P1 and P2 purinergic receptors, which are activated by various nucleotides and their metabolites. Recently, such purinergic signalling has been spotlighted because of potential roles in the pathophysiologies of neurodegenerative and neuropsychiatric disorders. To understand the characteristics of microglia in relation of P1 and P2 signalling, we investigated the ectoenzymes expressed in microglia. At first, we profiled the expression of all known ectoenzymes in cultured microglia. We found that, like NTPDase1 (ectonucleoside triphosphate diphosphohydrolase 1, CD39), NPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, PC-1) is also highly expressed in primary cultured murine microglia. Knockdown of NPP1 significantly reduced ATP hydrolysis and P_i production in cultured microglia. In addition, the knockdown of NPP1 enhanced basal nucleotide-stimulating responses of cultured microglia, such as phagocytosis and cell migration, and these results were very similar to NTPDase1 knockdown results. Moreover, inhibition of the adenosine receptors by caffeine treatment reduced phagocytosis of NPP1 knock downed-cultured microglia. In conclusion, we suggest that these potent ectoenzymes of primary cultured murine microglia, NPP1 together with CD73 (ecto-5′-nucleotidase) maintain the adenosine levels for triggering nucleotide-stimulating responses.     

Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia

Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-alpha secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-alpha release, resulting from TNF-alpha mRNA expression in rat cultured brain microglia. The TNF-alpha release was maximally elicited by 1 mM ATP and also induced by a P2X(7) receptor-selective agonist, 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, suggesting the involvement of P2X(7) receptor. ATP-induced TNF-alpha release was Ca(2+)-dependent, and a sustained Ca(2+) influx correlated with the TNF-alpha release in ATP-stimulated microglia. ATP-induced TNF-alpha release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca(2+). These results indicate that extracellular ATP triggers TNF-alpha release in rat microglia via a P2 receptor, likely to be the P2X(7) subtype, by a mechanism that is dependent on both the sustained Ca(2+) influx and ERK/p38 cascade, regulated independently of Ca(2+) influx.     

Hydroxysafflor Yellow A Attenuates Neuron Damage by Suppressing the Lipopolysaccharide-Induced TLR4 Pathway in Activated Microglial Cells

Microglia activation initiates a neurological deficit cascade that contributes to substantial neuronal damage and impairment following ischemia stroke. Toll-like receptor 4 (TLR4) has been demonstrated to play a critical role in this cascade. In the current study, we tested the hypothesis that hydroxysafflor yellow A (HSYA), an active ingredient extracted from Flos Carthami tinctorii, alleviated inflammatory damage, and mediated neurotrophic effects in neurons by inducing the TLR4 pathway in microglia. A non-contact Transwell co-culture system comprised microglia and neurons was treated with HSYA followed by a 1 mg/mL lipopolysaccharide (LPS) stimulation. The microglia were activated prior to neuronal apoptosis, which were induced by increasing TLR4 expression in the activated microglia. However, HSYA suppressed TLR4 expression in the activated microglia, resulting in less neuronal damage at the early stage of LPS stimulation. Western blot analysis and immunofluorescence indicated that dose-dependently HSYA down-regulated TLR4-induced downstream effectors myeloid differentiation factor 88 (MyD88), nuclear factor kappa b (NF-κB), and the mitogen-activated protein kinases (MAPK)-regulated proteins c-Jun NH2-terminal protein kinase (JNK), protein kinase (ERK) 1/2 (ERK1/2), p38 MAPK (p38), as well as the LPS-induced inflammatory cytokine release. However, HSYA up-regulated brain-derived neurotrophic factor (BDNF) expression. Our data suggest that HSYA could exert neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathway-mediated signaling.     

Chemical constituents isolated from Polygala japonica leaves and their inhibitory effect on nitric oxide production in vitro

A methanolic extract of dried leaves of Polygala japonica Houtt (Polygalaceae) significantly attenuated nitric oxide production in lipopolysaccharide-simulated BV2 microglia. Five anthraquinones chrysophanol (1), emodin (2), aloe-emodin (3), emodin 8-O-beta-D-glucopyranoside (4) and trihydroxy anthraquinone (5), and four flavonoids kaempferol (6), chrysoeriol (7), kaempferol 3-gentiobioside (8) and isorhamnetin (9) were isolated from the methanolic extract using bioactivity-guided fractionation. Among them, compounds 1-4, 6 and 7 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia at the concentrations ranging from 1.0 to 100.0 microM.     

Fibrillar amyloid-beta peptides activate microglia via TLR2: implications for Alzheimer's disease

Microglial activation is an important pathological component in brains of patients with Alzheimer's disease (AD), and fibrillar amyloid-beta (Abeta) peptides play an important role in microglial activation in AD. However, mechanisms by which Abeta peptides induce the activation of microglia are poorly understood. The present study underlines the importance of TLR2 in mediating Abeta peptide-induced activation of microglia. Fibrillar Abeta1-42 peptides induced the expression of inducible NO synthase, proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6), and integrin markers (CD11b, CD11c, and CD68) in mouse primary microglia and BV-2 microglial cells. However, either antisense knockdown of TLR2 or functional blocking Abs against TLR2 suppressed Abeta1-42-induced expression of proinflammatory molecules and integrin markers in microglia. Abeta1-42 peptides were also unable to induce the expression of proinflammatory molecules and increase the expression of CD11b in microglia isolated from TLR2(-/-) mice. Finally, the inability of Abeta1-42 peptides to induce the expression of inducible NO synthase and to stimulate the expression of CD11b in vivo in the cortex of TLR2(-/-) mice highlights the importance of TLR2 in Abeta-induced microglial activation. In addition, ligation of TLR2 alone was also sufficient to induce microglial activation. Consistent to the importance of MyD88 in mediating the function of various TLRs, antisense knockdown of MyD88 also inhibited Abeta1-42 peptide-induced expression of proinflammatory molecules. Taken together, these studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar Abeta peptide-induced activation of microglia.     

Dalesconols B inhibits lipopolysaccharide induced inflammation and suppresses NF-κB and p38/JNK activation in microglial cells

Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. Dalesconols B, also termed as TL2, is a newly found polyketide from a mantis-associated fungus and has been reported to exert potent immunosuppressive effects. In the present study, the anti-inflammatory effects of TL2 was investigated in lipopolysaccharide (LPS)-treated BV2 microglia and primary microglia cells. Our observations indicated that pretreatment with TL2 significantly inhibited the production of NO and PGE2 and suppressed the expression of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), COX-2, TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in LPS-stimulated BV2 microglia. The nuclear translocation of NF-κB and the phosphorylation level of Akt, p38 and JNK MAP kinase pathways were also inhibited by TL2 in LPS-treated BV2 microglia. Moreover, TL2 also decreased Aβ-induced production of TNF-α, IL-1β and IL-6 in BV2 microglia. Additionally, TL2 protected primary cortical neurons against microglia-mediated neurotoxicity. Overall, our findings suggested that TL2 might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.     

Transient receptor potential vanilloid subtype 1 mediates microglial cell death in vivo and in vitro via Ca2+-mediated mitochondrial damage and cytochrome c release

The present study examined the expression of transient receptor potential vanilloid subtype 1 (TRPV1) in microglia, and its association with microglial cell death. In vitro cell cultures, RT-PCR, Western blot analysis, and immunocytochemical staining experiments revealed that rat microglia and a human microglia cell line (HMO6) showed TRPV1 expression. Furthermore, exposure of these cells to TRPV1 agonists, capsaicin (CAP) and resiniferatoxin (RTX), triggered cell death. This effect was ameliorated by the TRPV1 antagonists, capsazepine and iodo-resiniferatoxin (I-RTX), suggesting that TRPV1 is directly involved. Further examinations revealed that TRPV1-induced toxicity was accompanied by increases in intracellular Ca(2+), and mitochondrial damage; these effects were inhibited by capsazepine, I-RTX, and the intracellular Ca(2+) chelator BAPTA-AM. Treatment of cells with CAP or RTX led to increased mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase-3. In contrast, the caspase-3 inhibitor z-DEVD-fmk protected microglia from CAP- or RTX-induced toxicity. In vivo, we also found that intranigral injection of CAP or 12-hydroperoxyeicosatetraenoic acid, an endogenous agonist of TRPV1, into the rat brain produced microglial damage via TRPV1 in the substantia nigra, as visualized by immunocytochemistry. To our knowledge, this study is the first to demonstrate that microglia express TRPV1, and that activation of this receptor may contribute to microglial damage via Ca(2+) signaling and mitochondrial disruption.     

An in vitro cell model to study microglia activation in diabetic retinopathy

Microglial activation has been studied extensively in diabetic retinopathy. We have previously detected activation and migration of microglia in 8-week-old diabetic rat retinas. It is widely acknowledged that microglia-mediated inflammation contributes to the progression of diabetic retinopathy. However, existing cell models do not explore the role of activated microglia in vitro. In this study, microglia were subject to various conditions mimicking diabetic retinopathy, including high glucose, glyoxal, and hypoxia. Under high glucose or glyoxal treatment, microglia demonstrated only partially functional changes, while under hypoxia, microglia became fully activated showing enlarged cell bodies, enhanced migration and phagocytosis as well as increased production of pro-inflammatory factors such as cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS). The data indicate that hypoxia-treated microglia is an optimal in vitro model for exploration of microglia activation in diabetic retinopathy.     

Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood–brain barrier damage

Microglial activation by blood-borne factors following blood–brain barrier damage may play a significant role in subsequent neuropathogenesis of several neurodegenerative diseases. Exposure of primary cultured rat brain microglia to pure, fatty acid- and lipid-deficient rat serum albumin or fraction V, (fatty acid and lipid-containing rat serum albumin), caused inducible nitric oxide synthase (iNOS) expression, glutamate release, tumour necrosis factor alpha (TNFα) and transforming growth factor-beta1 release. iNOS expression was attenuated by the MAPK/extracellular signal-regulated kinase pathway inhibitor U0126 and the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 were detectable in microglia treated with albumin or fraction V. Glutamate release was prevented by l -α-aminoadipate and glutathione levels in microglia rose on exposure to albumin. Conditioned medium from microglia exposed to albumin or fraction V was neurotoxic. Peripheral macrophages were resistant to the effects of albumin but both microglia and macrophages responded to lipopolysaccharide, which induced interleukin-1 beta and tumour necrosis factor alpha release, cyclooxygenase-2 and iNOS expression in both cell types, indicating a discrete desensitised pathway in macrophages for albumin which was not desensitised in microglia. Thus, exposure of microglia in the brain to albumin may contribute to neuronal damage following blood–brain barrier breakdown and point to resident microglia rather than infiltrating macrophages as therapeutic targets.     

Selective activation of microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse

Neuronal plasticity along the pathway for sensory transmission including the spinal cord and cortex plays an important role in chronic pain, including inflammatory and neuropathic pain. While recent studies indicate that microglia in the spinal cord are involved in neuropathic pain, a systematic study has not been performed in other regions of the central nervous system (CNS). In the present study, we used heterozygous Cx3cr1 ^GFP/+mice to characterize the morphological phenotypes of microglia following common peroneal nerve (CPN) ligation. We found that microglia showed a uniform distribution throughout the CNS, and peripheral nerve injury selectively activated microglia in the spinal cord dorsal horn and related ventral horn. In contrast, microglia was not activated in supraspinal regions of the CNS, including the anterior cingulate cortex (ACC), prefrontal cortex (PFC), primary and secondary somatosensory cortex (S1 and S2), insular cortex (IC), amygdala, hippocampus, periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Our results provide strong evidence that nerve injury primarily activates microglia in the spinal cord of adult mice, and pain-related cortical plasticity is likely mediated by neurons.     

Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response

Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1β (IL1β) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1β. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.