Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.     

Neuroprotection mediated by glial group-II metabotropic glutamate receptors requires the activation of the MAP kinase and the phosphatidylinositol-3-kinase pathways

The mGlu2/3 receptor agonists 4-carboxy-3-hydroxyphenylglycine (4C3HPG) and LY379268 attenuated NMDA toxicity in primary cultures containing both neurons and astrocytes. Neuroprotection was abrogated by PD98059 and LY294002, which inhibit the mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI-3-K) pathways, respectively. Cultured astrocytes lost the ability to produce transforming growth factor-beta1 (TGF-beta1) in response to mGlu2/3 receptor agonists when co-incubated with PD98059 or LY294002. As a result, the glial medium was no longer protective against NMDA toxicity. Activation of the MAPK and PI-3-K pathways in cultured astrocytes treated with 4C3HPG or LY379268 was directly demonstrated by an increase in the phosphorylated forms of ERK-1/2 and Akt. Similarly to that observed in the culture, intracerebral or systemic injections of mGlu2/3 receptor agonists enhanced TGF-beta1 formation in the rat or mouse caudate nucleus, and this effect was reduced by PD98059. PD98059 also reduced the ability of LY379268 to protect striatal neurons against NMDA toxicity. These results suggest that activation of glial mGlu2/3 receptors induces neuroprotection through the activation of the MAPK and PI-3-K pathways leading to the induction of TGF-beta.     

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.     

In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation

Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl's ligand, growth arrest-specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull-down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor-bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol-3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85's second proline-rich region and the N-terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6-stimulation of Axl-transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4− flow-through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways.     

A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.     

Inhibition of NADPH oxidase promotes alternative and anti-inflammatory microglial activation during neuroinflammation

Like macrophages, microglia are functionally polarized into different phenotypic activation states, referred as classical and alternative. The balance of the two phenotypes may be critical to ensure proper brain homeostasis, and may be altered in brain pathological states, such as Alzheimer's disease. We investigated the role of NADPH oxidase in microglial activation state using p47(phox) and gp91(phox) -deficient mice as well as apocynin, a NADPH oxidase inhibitor during neuroinflammation induced by an intracerebroventricular injection of LPS or Aβ????. We showed that NADPH oxidase plays a critical role in the modulation of microglial phenotype and subsequent inflammatory response. We demonstrated that inhibition of NADPH oxidase or gene deletion of its functional p47(phox) subunit switched microglial activation from a classical to an alternative state in response to an inflammatory challenge. Moreover, we showed a shift in redox state towards an oxidized milieu and that subpopulations of microglia retain their detrimental phenotype in Alzheimer's disease brains. Microglia can change their activation phenotype depending on NADPH oxidase-dependent redox state of microenvironment. Inhibition of NADPH oxidase represents a promising neuroprotective approach to reduce oxidative stress and modulate microglial phenotype towards an alternative state.     

Inhibition of MMP-3 or -9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and iNOS in microglia

Recently, matrix metalloproteinases (MMPs) are emerging as important molecules in neuroinflammation as well as neuronal cell death. However, the role of MMPs in activated microglia remains unclear. In the present study, we found that expressions of MMP-1, -3, -8 and -9 were significantly induced by single or combined treatment of immunostimulants lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in primary cultured microglia and BV2 microglial cells. Inhibition of MMP-3 or -9 significantly suppressed the expression of iNOS and pro-inflammatory cytokines and the activities of NF-κB, AP-1, and MAPK in LPS-stimulated microglia. The results suggest that MMP-3 and -9 both mediate LPS-induced inflammatory reactions. Inhibition of reactive oxygen species (ROS) by N-acetyl-cysteine or diphenylene iodonium significantly suppressed the expression of MMP-3, MMP-9, NO and TNF-α in LPS-stimulated microglia, suggesting that ROS is an early signaling inducer in LPS-stimulated microglial cells. MMP inhibitors also suppressed ROS production, suggesting a cross-talk between ROS and MMPs. Collectively, the present study demonstrates that MMP-3 and MMP-9 play a role as inflammatory mediators in activated microglia. Pharmacological intervention of MMPs especially MMP-3 and -9 would be a therapeutic strategy for the treatment of inflammatory diseases in the CNS caused by over-activation of microglial cells.     

Neuroprotective effect of rhaponticin against Parkinson disease: Insights from in vitro BV‐2 model and in vivo MPTP‐induced mice model

Parkinson's disease (PD) is a complex neurodegenerative illness associated with the loss or damage to neurons of the dopaminergic system in the brain. Few therapeutic approaches and considerable side effects of conventional drugs necessitate a new therapeutic agent to treat patients with PD. Rhaponticin is a natural hydroxystilbene, found in herbal plants such as Rheum rhaponticum, and known to have desirable biological activity including anti‐inflammatory properties. However, the neuroinflammation on rhaponticin levels has only been investigated partially so far. So, the current study explored whether rhaponticin could ameliorate the pathophysiology observed in both the in vitro microglial BV‐2 cells and the in vivo (1‐methyl‐4‐phenyl‐1,2,3,5‐tetrahydropyridine [MPTP])‐mediated PD model. The results show rhaponticin significantly attenuated lipopolysaccharide (LPS)‐mediated microglial activation by suppressing nitric oxide synthase in conjunction with abridged reactive oxygen species production together with proinflammatory mediator reduction. In vivo rhaponticin treatment improves motor impairments as well as the loss of dopaminergic neurons in MPTP‐treated mice possibly through suppression via mediators of inflammation. Taken together, these results offer evidence that rhaponticin exerts anti‐inflammatory effects and neuroprotection in an LPS‐induced microglial model and the MPTP‐induced mouse models of PD.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

Opposing Functions of Microglial and Macrophagic TNFR2 in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

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Novel EGF pathway regulators modulate C. elegans healthspan and lifespan via EGF receptor,PLC‐γ, and IP3R activation

Improving health of the rapidly growing aging population is a critical medical, social, and economic goal. Identification of genes that modulate healthspan, the period of mid‐life vigor that precedes significant functional decline, will be an essential part of the effort to design anti‐aging therapies. Because locomotory decline in humans is a major contributor to frailty and loss of independence and because slowing of movement is a conserved feature of aging across phyla, we screened for genetic interventions that extend locomotory healthspan of Caenorhabditis elegans. From a group of 54 genes previously noted to encode secreted proteins similar in sequence to extracellular domains of insulin receptor, we identified two genes for which RNAi knockdown delayed age‐associated locomotory decline, conferring a high performance in advanced age phenotype (Hpa). Unexpectedly, we found that hpa‐1 and hpa‐2 act through the EGF pathway, rather than the insulin signaling pathway, to control systemic healthspan benefits without detectable developmental consequences. Further analysis revealed a potent role of EGF signaling, acting via downstream phospholipase C‐γplc‐3 and inositol‐3‐phosphate receptor itr‐1, to promote healthy aging associated with low lipofuscin levels, enhanced physical performance, and extended lifespan. This study identifies HPA‐1 and HPA‐2 as novel negative regulators of EGF signaling and constitutes the first report of EGF signaling as a major pathway for healthy aging. Our data raise the possibility that EGF family members should be investigated for similar activities in higher organisms.     

Modulation of the basal activity of N-acetylglucosaminyltransferase V by phosphatidylinositol-3-kinase/protein kinase B signaling pathway in human hepatocarcinoma cells

The modulation of GnT-V activity by signaling molecules in PI-3-K/PKB pathway in human hepatocarcinoma cell line 7721 was studied. GnT-V activity was determined after the transfection of sense or antisense cDNA of PKB into the cells, as well as the addition of activators, specific inhibitors, and the antibodies to the enzyme assay system or culture medium. It was found that the basal activity of GnT-V was up regulated by the sense and down regulated by the antisense cDNA of PKB transfected into 7721 cells. GnT-V was activated by PIP₂, PIP₃ or GTP[S] added to the assay system, and the activation of PIP₂ or GTP[S] was abolished by LY2940002, a specific inhibitor of PI-3-K, but the activation of PIP₃ was not attenuated by LY2940002. In addition, GnT-V activity in cultured parental or H-ras transfected cells was inhibited by the antibody against PKB or PI-3-K. These findings demonstrated the involvement of PI-3-K/PKB signaling pathway in the regulation of GnT-V. Moreover, ET18-OCH₃, an inhibitor of Raf translocation and PI-PLC enzyme, which produces the activator of PKC, as well as the antibodies against Raf-1 or MEK also inhibited GnT-V activity in the parental and H-ras transfected cells. The inhibitory rates, however, were less in the transfected cells than those in the parental cells. These results reveal that in parental and H-ras transfected 7721 cells, the basal activity of GnT-V is also regulated by the Ras/Raf-1/MEK/MAPK cascade in addition to PI-3-K/PKB signaling pathway. The significance of these two pathways in the regulation of GnT-V and their relations to the activation of PKC previously reported by our laboratory (Ju TZ et al., 1995 Glyconjugate J 12, 767–772) was discussed.     

Prolonged Alzheimer-like tau hyperphosphorylation induced by simultaneous inhibition of phosphoinositol-3 kinase and protein kinase C in N2a cells

Co-injection of wortmannin (inhibitor of phosphatidylinositol-3 kinase, PI3K) and GF109203X(inhibitor of protein kinase C, PKC) into the rat brain was found to induce spatial memory deficiency and enhance tau hyperphosphorylation in the hippocampus of rat brain. To establish a cell model with durative Alzheimer-like tau hyperphosphorylation in this study, we treated N2a neuroblastoma cells with wortmannin and GF109203X separately and simultaneously, and measured the glycogen synthase kinase 3 (GSK-3)activity by y-32p-labeling and the level of tau phosphorylation by Western blotting. It was found that the application of wortmannin alone only transitorily increased the activity of GSK-3 (about 1 h) and the level of tau hyperphosphorylation at Ser^396/Ser^404 and Ser^199/Ser^202 sites (no longer than 3 h); however, a prolonged and intense activation of GSK-3 (over 12 h) and enhanced tau hyperphosphorylation (about 24 h) were observed when these two selective kinase inhibitors were applied together. We conclude that the simultaneous inhibition of PI3K and PKC can induce GSK-3 overactivation, and further strengthen and prolong the Alzheimerlike tau hyperphosphorylation in N2a cells, suggesting the establishment of a cell model with early pathological events of Alzheimer‘s disease.     

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

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