AGEs-RAGE mediated up-regulation of connexin43 in activated human microglial CHME-5 cells

Microglial activation is a significant contributor to the pathogenesis of many neurodegenerative diseases. Microglia respond to a range of stimuli including pathogenic protein deposits such as advanced glycation endproducts (AGEs). AGEs are prominent inflammatory stimuli that accumulate in the ageing brain. AGEs can activate microglia, leading to the production of excessive amounts of inflammatory cytokines and coupling via gap junction proteins especially connexin43 (Cx43). The literature on the expression of microglial Cx43 during inflammation is controversial. Many cellular effects of AGEs are thought to be mediated by the receptor RAGE. There is however, no evidence suggesting Cx43 is a downstream effector of AGEs-RAGE interaction in microglia. In addition, most of the AGEs-related studies have been undertaken using rodent microglia; the information on human microglia is sparse. Microglia of human and rodent origin respond differently to certain stimuli. The aims of this study were to investigate the AGEs-RAGE-mediated activation of human microglia and establish if Cx43 is one of the downstream effectors of AGEs-RAGE interaction in these cells. Human microglial CHME-5 cells were treated with different doses of AGEs for a selected time-period and microglial activation studied using specific markers. The protein expression of RAGE, Cx43 and TNF-α-receptors (RI and RII) was analysed in response to AGEs in the absence/presence of various doses of anti-RAGE Fabs. TNF-α levels in media were measured using ELISA. TNF-α-induced opening of gap junctional channels was assessed by dye uptake assays and the effect of neutralising TNFRII on Cx43 levels was also studied. CHME-5 cells showed an up-regulation of RAGE, TNF-α, TNFRs (especially TNFRII) and Cx43 upon AGEs treatment and a significant dose-dependent drop in the levels of TNF-α, TNFRII and Cx43 in the presence of anti-RAGE Fabs. TNF-α induced gap junctional/hemichannel opening whereas blocking TNFRII inhibited TNF-α-induced increase in Cx43 levels. Results suggested that TNF-α, TNFRII and Cx43 are downstream effectors of the AGEs-RAGE interaction in human microglial CHME-5 cells.     

Microglial cell death induced by glycated bovine serum albumin: nitric oxide involvement

Nonenzymatic glycation results in the formation of advanced glycation end products (AGEs) through a nonenzymatic multistep reaction of reducing sugars with proteins. AGEs have been suspected to be involved in the pathogenesis of several chronic clinical neurodegenerative complications including Alzheimer's disease, which is characterized with the activation of microglial cells in neuritic plaques. To find out the consequence of this activation on microglial cells, we treated the cultured microglial cells with different glycation levels of Bovine Serum Albumin (BSA) which were prepared in vitro. Extent of glycation of protein has been characterized during 16 weeks of incubation with glucose. Treatment of microglial cells with various levels of glycated albumin induced nitric oxide (NO) production and consequently cell death. We also tried to find out the mode of death in AGE-activated microglial cells. Altogether, our results suggest that AGE treatment causes microglia to undergo NO-mediated apoptotic and necrotic cell death in short term and long term, respectively. NO production is a consequence of iNOS expression in a JNK dependent RAGE signalling after activation of RAGE by AGE-BSA.     

Effects of macrophage colony-stimulating factor on microglial responses to lipopolysaccharide and beta amyloid

A challenge for studies involving microglia cultures is obtaining sufficient cells for downstream experiments. Macrophage colony-stimulating factor (M-CSF) has been used to improve yield of microglia in culture. However, the effects of M-CSF on activation profiles of microglia cultures are still unclear. Microglia activation is characterised by upregulation of co-stimulatory molecules and an inflammatory phenotype. The aim of this study is to demonstrate whether M-CSF supplementation alters microglial responses in resting and activated conditions. Microglia derived from mixed glia cultures and the BV-2 microglia cell line were cultivated with/without M-CSF and activated with lipopolysaccharide (LPS) and beta amyloid (Aβ). We show M-CSF expands primary microglia without affecting microglial responses to LPS and Aβ, as shown by the comparable expression of MHC class II and CD40 to microglia grown without this growth factor. M-CSF supplementation in BV-2 cells had no effect on nitric oxide (NO) production. Therefore, M-CSF can be considered for improving microglia yield in culture without introducing activation artefacts.     

Oligomeric Aβ-Induced Microglial Activation is Possibly Mediated by NADPH Oxidase

Recent studies have shown that oligomeric amyloid-β (oAβ) peptide can potentially activate microglia in addition to inducing more potent neurotoxicity compared with fibrillar Aβ (fAβ); however, its mechanisms of action remain unclear. This study was designed to investigate the possible mechanisms involved in the microglial activation induced by oAβ in BV-2 microglial cells. The results showed that oAβ induced activated properties of microglia, including higher proliferative capacity as well as increased production of reactive oxygen species, nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). NADPH oxidase inhibitors [diphenylene iodonium (DPI) and apocynin (4-hydroxy-3-methoxy-acetophenone)] prevented the microglial activation induced by oAβ, suggesting that NADPH oxidase activation was involved in microglial activation. In addition, TNF-α and IL-1β, which are massively released by activated microglia, significantly induced the activation of microglia, thereby resulting in the production of NO and proliferation of microglia, respectively. These effects could be inhibited by diphenylene iodonium and apocynin, indicating a self-cycle regulated by NADPH oxidase in microglial activation in response to oAβ. In conclusion, microglial activation induced by oAβ is possibly mediated by NADPH oxidase, suggesting that oAβ, which is normally considered a neurotoxin, may also lead to indirect neuronal damage through the pro-inflammation activation of microglia in Alzheimer’s disease and that NADPH oxidase could be a potential target to prevent oAβ-induced inflammatory neurodegeneration.     

Scavenger receptor control of chromogranin A-induced microglial stress and neurotoxic cascades

Chromogranin A (CgA), a neuroactive glycoprotein, is associated with microglial activation cascades implicated in neurodegeneration. Here we show that CgA-dependent inducible nitric oxide synthase (iNOS) expression and stress responses in microglia involved signalling via scavenger receptors (SR), since SR class-A (SR-A) ligands blocked iNOS expression, mitochondrial depolarisation, apoptosis and glutamate release. Furthermore, block of SR-A ameliorated CgA-induced microglial neurotoxicity. In contrast, block of CD36, or the receptor for advanced glycation end products (RAGE) did not prevent CgA-induced microglial activation and neurotoxicity. Thus, manipulation of specific scavenger receptor-coupled signalling pathways may provide avenues for therapeutic intervention in neurodegenerative diseases implicating microglial activation with chromogranin peptides.     

Upregulation of IFN-β induced by Sema4D-dependent partial Erk1/2 inhibition promotes NO production in microglia

Interactions between Sema4D and its receptors, PlexinB1 and CD72, induce various functions, including axon guidance, angiogenesis, and immune activation. Our previous study revealed that Sema4D is involved in the upregulation of nitric oxide production in microglia after cerebral ischemia. In this study, we investigated the underlying mechanisms of the enhancement of microglial nitric oxide production by Sema4D. Primary microglia expressed PlexinB1 and CD72, and cortical microglia expressed CD72. Sema4D promoted nitric oxide production and slightly inhibited Erk1/2 phosphorylation in microglia. Partial Erk1/2 inhibition enhanced microglial nitric oxide production. Inhibition of Erk1/2 phosphorylation induced the expression of Ifn-β mRNA, and IFN-β promoted nitric oxide production in microglia. In the ischemic cortex, the expression of Ifn-β mRNA was downregulated by Sema4D deficiency. These findings indicated that the enhancement of nitric oxide production by Sema4D is involved in partial Erk1/2 inhibition and upregulation of IFN-β.     

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

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

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1β promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.     

Receptor for advanced glycation end-products promotes premature senescence of proximal tubular epithelial cells via activation of endoplasmic reticulum stress-dependent p21 signaling

Premature senescence is a key process in the progression of diabetic nephropathy (DN). In our study, we hypothesized that receptors for advanced glycation end-products (RAGE) mediate endoplasmic reticulum (ER) stress to induce premature senescence via p21 signaling activation in diabetic nephropathy. Here, we demonstrated that elevated expression of RAGE, ER stress marker glucose-regulated protein 78 (GRP78), and cell-cycle regulator p21 was all positively correlated with enhanced senescence-associated-β-galactosidase (SA-β-gal) activity in DN patients. In addition, the fraction of SA-β-gal or cells in the G₀G₁ phase were enhanced in cultured mouse proximal tubular epithelial cells (PTECs) and the expression of RAGE, GRP78 and p21 was up-regulated by advanced glycation end-products (AGEs) in a dose- and time-dependent manner. Interestingly, ER stress inducers or RAGE overexpression mimicked AGEs induced-premature senescence, and this was significantly suppressed by p21 gene silencing. However, RAGE blocking successfully attenuated AGEs-induced ER stress and p21 expression, as well as premature senescence. Moreover, ER stress inducers directly caused p21 activation, premature senescence, and also enhanced RAGE expression by positive feedback. These observations suggest that RAGE promotes premature senescence of PTECs by activation of ER stress-dependent p21 signaling.     

Metabolic impairment induces oxidative stress, compromises inflammatory responses, and inactivates a key mitochondrial enzyme in microglia

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.     

Induction of neuronal death by microglial AGE-albumin: implications for Alzheimer's disease

Advanced glycation end products (AGEs) have long been considered as potent molecules promoting neuronal cell death and contributing to neurodegenerative disorders such as Alzheimer's disease (AD). In this study, we demonstrate that AGE-albumin, the most abundant AGE product in human AD brains, is synthesized in activated microglial cells and secreted into the extracellular space. The rate of AGE-albumin synthesis in human microglial cells is markedly increased by amyloid-β exposure and oxidative stress. Exogenous AGE-albumin upregulates the receptor protein for AGE (RAGE) and augments calcium influx, leading to apoptosis of human primary neurons. In animal experiments, soluble RAGE (sRAGE), pyridoxamine or ALT-711 prevented Aβ-induced neuronal death in rat brains. Collectively, these results provide evidence for a new mechanism by which microglial cells promote death of neuronal cells through synthesis and secretion of AGE-albumin, thereby likely contributing to neurodegenerative diseases such as AD.     

Anti-Inflammatory Effects of Ginsenoside-Rh2 Inhibits LPS-Induced Activation of Microglia and Overproduction of Inflammatory Mediators Via Modulation of TGF-β1/Smad Pathway

Microglia activation plays an important role in neuroinflammation and contributes to several neurological disorders. Hence, inhibition of both microglia activation and pro-inflammatory cytokines may lead to the effective treatment of neurodegenerative diseases. In this study, we found that GRh2 inhibited the inflammatory response to lipopolysaccharide (LPS) and prevented the LPS-induced neurotoxicity in microglia cells. GRh2 significantly decreased the generation of nitric oxide production, and tumor necrosis factor-α, interleukin (IL)-6, IL-1β, cyclooxygenase-2 and inducible nitric oxide synthase in LPS-induced activated microglia cells. Furthermore, GRh2 (20 and 50 μM) significantly increased TGF-β1 expression and reduced the expression of Smad. These results suggest that GRh2 effectively inhibits microglia activation and production of pro-inflammatory cytokines via modulating the TGF-β1/Smad pathway.     

Aucubin suppresses lipopolysaccharide-induced pro-inflammatory responses by blocking the NF-κB translocation signaling pathways in activated microglial cells

Aucubin is an iridoid glycoside with demonstrable hepatoprotective and anti-osteoporotic effects. Herein, using microglial cells and lipopolysaccharide (LPS) to induce inflammatory responses, we studied the signaling pathways involved in the anti-inflammatory action of aucubin and their influence on the expression of several genes known to be involved in inflammation. Aucubin inhibited LPS-stimulated pro-inflammatory responses by suppressing the production of nitric oxide and prostaglandin E₂. Furthermore, aucubin inhibited inducible nitric oxide synthase and cyclooxygenase-2 at both the protein and mRNA levels. In addition, aucubin inhibited pro-inflammatory cytokine production in LPS-stimulated BV-2 microglial cells. Subsequent mechanistic studies revealed that aucubin inhibited the LPS-induced activation of nuclear factor-kappa B (NF-κB) translocation and phosphorylation of phosphatidylinositol 3-kinases (PI3K)/Akt as well as of mitogen-activated protein kinases (MAPKs), which are upstream molecules responsible for controlling inflammatory reactions. These results suggest that aucubin may exert anti-neuroinflammatory responses by suppressing the LPS-induced expression of pro-inflammatory mediators by blocking the activation of NF-κB, PI3K/Akt, and MAPK signaling pathways in microglial cells.     

AGEs Induce Cell Death via Oxidative and Endoplasmic Reticulum Stresses in Both Human SH-SY5Y Neuroblastoma Cells and Rat Cortical Neurons

Advanced glycation endproducts (AGEs) are elevated in aging and neurodegenerative diseases such as Alzheimer??s disease (AD), and they can stimulate the generation of reactive oxygen species (ROSs) via NADPH oxidase, induce oxidative stress that lead to cell death. In the current study, we investigated the molecular events underlying the process that AGEs induce cell death in SH-SY5Y cells and rat cortical neurons. We found: (1) AGEs increase intracellular ROSs; (2) AGEs cause cell death after ROSs increase; (3) oxidative stress-induced cell death is inhibited via the blockage of AGEs receptor (RAGE), the down-regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the increase of scavenging by anti-oxidant alpha-lipoic acid (ALA); (4) endoplasmic reticulum (ER) stress was triggered by AGE-induced oxidative stress, resulting in the activation of C/EBP homologous protein (CHOP) and caspase-12 that consequently initiates cell death, taurine-conjugated ursodeoxycholic acid (TUDCA) inhibited AGE-induced ER stress and cell death. Blocking RAGE?CNADPH oxidase, and RAGE?CNADPH oxidase?CROSs and ER stress scavenging pathways could efficiently prevent the oxidative and ER stresses, and consequently inhibited cell death. Our results suggest a new prevention and or therapeutic approach in AGE-induced cell death.     

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

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

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3‐inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.