S. Thameem Dheen

Microglial Activation and its Implications in the Brain Diseases

An inflammatory process in the central nervous system (CNS) is believed to play an important role in the pathway leading to neuronal cell death in a number of neurodegenerative diseases including Parkinsons disease, Alzheimers disease, prion diseases, multiple sclerosis and HIV-dementia. The inflammatory response is mediated by the activated microglia, the resident immune cells of the CNS, which normally respond to neuronal damage and remove the damaged cells by phagocytosis. Activation of microglia is a hallmark of brain pathology. However, it remains controversial whether microglial cells have beneficial or detrimental functions in various neuropathological conditions. The chronic activation of microglia may in turn cause neuronal damage through the release of potentially cytotoxic molecules such as proinflammatory cytokines, reactive oxygen intermediates, proteinases and complement proteins. Therefore, suppression of microglia-mediated inflammation has been considered as an important strategy in neurodegenerative disease therapy. Several anti-inflammatory drugs of various chemical ingredients have been shown to repress the microglial activation and to exert neuroprotective effects in the CNS following different types of injuries. However, the molecular mechanisms by which these effects occur remain unclear. In recent years, several research groups including ours have attempted to explain the potential mechanisms and signaling pathways for the repressive effect of various drugs, on activation of microglial cells in CNS injury. We provide here a comprehensive review of recent findings of mechanisms and signaling pathways by which microglial cells are activated in CNS inflammatory diseases. This review article further summarizes the role of microglial cells in neurodegenerative diseases and various forms of potential therapeutic options to inhibit the microglial activation which amplifies the inflammation-related neuronal injury in neurodegenerative diseases.

Interactions of Chemokines and Chemokine Receptors Mediate the Migration of Mesenchymal Stem Cells to the Impaired Site in the Brain After Hypoglossal Nerve Injury

Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal‐cell‐derived factor 1 (SDF‐1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF‐1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF‐1 alpha (rhSDF‐1α) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G‐protein‐dependent manner. Intracerebral injection of rhSDF‐1α has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine‐CX3CR1 and SDF‐1–CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.

Retinoic acid inhibits expression of TNF-α and iNOS in activated rat microglia

The release of proinflammatory mediators such as tumor necrosis factor‐α (TNF‐α) and nitric oxide by microglia has been implicated in neurotoxicity in chronic neurodegenerative diseases such as Alzheimers disease. As all‐trans‐retinoic acid (RA) has been reported to exert anti‐inflammatory actions in various cell types, we have examined its effects on the expression of TNF‐α and inducible nitric oxide synthase (iNOS) in microglia activated by β‐amyloid peptide (Aβ) and lipopolysaccharide (LPS). Exposure of primary cultures of rat microglial cells to Aβ or LPS stimulated the mRNA expression level of TNF‐α (6–116‐fold) and iNOS (8–500‐fold) significantly. RA acted in a dose‐dependent manner (0.1–10 μM) by attenuating both TNF‐α (29–97%) and iNOS (61–96%) mRNA expression in microglia exposed to Aβ or LPS. RA‐induced inhibition of TNF‐α and iNOS mRNA expression in activated microglia was accompanied by the concomitant reduction in release of iNOS and TNF‐α proteins as revealed by nitrite assay and ELISA, respectively. The anti‐inflammatory effects of RA were correlated with the enhanced expression of retinoic acid receptor‐β, and transforming growth factor‐β1 as well as the inhibition of NF‐κB translocation. These results suggest that RA may inhibit the neurotoxic effect of activated microglia by suppressing the production of inflammatory cytokines and cytotoxic molecules.

Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia

BackgroundHypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury.MethodsOne day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining.ResultsTLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1β and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1β, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1β and iNOS on microglia post-hypoxia.ConclusionActivated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.

Expression of chemokine receptors CXCR4, CCR2, ccr5 and CX3CR1 in neural progenitor cells isolated from the subventricular zone of the adult rat brain

We have studied the expression of chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 at the mRNA and protein levels in adult neural progenitor cells (NPCs) in neurosphere cultures using RT-PCR and immunocytochemistry methods. NPCs were isolated from the subventricular zone of adult rat brain and propagated in vitro as neurospheres. The neurospheres showed immunoactivity of nestin, an intermediate filament marker for NPCs. NPCs in the neurosphere cultures differentiated into NeuN-, GFAP-, or GalC-positive cells in vitro. Using cultured cortical microglial cells as positive control, we demonstrated the mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in neurospheres by RT-PCR. Double immunofluorescent staining further confirmed the co-localization of nestin with either CXCR4, CCR2, CCR5, or CX3CR1 on neurospheres. These results suggest that adult NPCs in the neurosphere cultures express chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1.

Dexamethasone suppresses monocyte chemoattractant protein‐1 production via mitogen activated protein kinase phosphatase‐1 dependent inhibition of Jun N‐terminal kinase and p38 mitogen‐activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein‐1 (MCP‐1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti‐inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP‐1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP‐1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)‐treated microglial culture. This study also revealed that the down‐regulation of the MCP‐1 mRNA expression by Dex in activated microglial cells was mediated via mitogen‐activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N‐terminal kinase (JNK) and p38 MAP kinases as well as c‐jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP‐1 production in activated microglial cells was confirmed as there was an attenuation of MCP‐1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase‐1 (MKP‐1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP‐1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP‐1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP‐1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP‐1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP‐1 and MCP‐1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.

BackgroundCongenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice.Methods and resultsWe analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos.ConclusionIt is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

Dexamethasone inhibits the Nox-dependent ROS production via suppression of MKP-1-dependent MAPK pathways in activated microglia

BackgroundNox-2 (also known as gp91phox), a subunit component of NADPH oxidases, generates reactive oxygen species (ROS). Nox-dependent ROS generation and nitric oxide (NO) release by microglia have been implicated in a variety of diseases in the central nervous system. Dexamethasone (Dex) has been shown to suppress the ROS production, NO release and inflammatory reaction of activated microglial cells. However, the underlying mechanisms remain unclear.ResultsThe present study showed that the increased ROS production and NO release in activated BV-2 microglial cells by LPS were associated with increased expression of Nox-2 and iNOS. Dex suppressed the upregulation of Nox-2 and iNOS, as well as the subsequent ROS production and NO synthesis in activated BV-2 cells. This inhibition caused by Dex appeared to be mediated by upregulation of MAPK phosphatase-1 (MKP-1), which antagonizes the activity of mitogen-activated protein kinases (MAPKs). Dex induced-suppression of Nox-2 and -upregulation of MKP-1 was also evident in the activated microglia from corpus callosum of postnatal rat brains. The overexpression of MKP-1 or inhibition of MAPKs (by specific inhibitors of JNK and p38 MAPKs), were found to downregulate the expression of Nox-2 and iNOS and thereby inhibit the synthesis of ROS and NO in activated BV-2 cells. Moreover, Dex was unable to suppress the LPS-induced synthesis of ROS and NO in BV-2 cells transfected with MKP-1 siRNA. On the other hand, knockdown of Nox-2 in BV-2 cells suppressed the LPS-induced ROS production and NO release.ConclusionIn conclusion, it is suggested that downregulation of Nox-2 and overexpression of MKP-1 that regulate ROS and NO may form the potential therapeutic strategy for the treatment of neuroinflammation in neurodegenerative diseases.

Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells

Notch‐1 receptor signaling pathway is involved in neuronal and glial differentiation. Its involvement in microglial functions, however, has remained elusive. This study reports the localization of Notch‐1 receptor immunoreactivity in the amoeboid microglial cells (AMC) in the postnatal rat brain. By immunofluorescence, Notch‐1 receptor was colocalized with its ligands, Jagged‐1 and Delta‐1, in the AMC in the corpus callosum and subventricular zone. Notch‐1 immunopositive cells were confirmed to be microglia labeled by OX42 and lectin. Immunoexpression of Notch‐1 receptor was progressively reduced with age. Western blot analysis showed that Notch‐1 protein level in the corpus callosum in which the AMC were heavily populated was concomitantly decreased. In postnatal rats challenged with lipopolysaccharide (LPS), Notch‐1 receptor immunofluorescence in AMC was noticeably enhanced. Furthermore, Notch‐1 protein level in the corpus callosum was increased as revealed by Western blotting analysis. In primary microglial culture treated with LPS, mRNA expression of Notch‐1 and its ligand Jagged‐1 was upregulated but that of Delta‐1 was reduced. The expression pattern of Notch‐1 and its ligands was confirmed in murine BV‐2 cells. Furthermore, Notch‐1 neutralization with its antibody reduced its protein expression. More importantly, neutralization of Notch‐1 concomitantly suppressed the mRNA expression of IL‐6, IL‐1, M‐CSF, and iNOS; TNF‐α, mRNA expression, however, was enhanced. Western blot confirmed the changes of protein level of the above except for IL‐6, which remained relatively unaltered. It is concluded that Notch‐1 signaling in the AMC and LPS‐activated microglia/BV‐2 cells modulates the expression of proinflammatory cytokines and nitric oxide.

The induction of epigenetic regulation of PROS1 gene in lung fibroblasts by gold nanoparticles and implications for potential lung injury

Advances in nanotechnology have given rise to the rapid development of novel applications in biomedicine. However, our understanding in the risks and health safety of nanomaterials is still not complete and various investigations are ongoing. Here, we show that gold nanoparticles (AuNPs) significantly altered the expression of 19 genes in human fetal lung fibroblasts (using the Affymetrix Human Gene 1.0 ST Array). Among the differentially expressed genes, up-regulation of microRNA-155 (miR-155) was observed concomitant with down-regulation of the PROS1 gene. Silencing of miR-155 established PROS1 as its possible target gene. DNA methylation profiling analysis of the PROS1 gene revealed no changes in the methylation status of this gene in AuNP-treated fibroblasts. At the ultrastructural level, chromatin condensation and reorganization was observed in the nucleus of fibroblasts exposed to AuNPs. The findings provide further insights into the molecular mechanisms underlying toxicity of AuNPs and their impact on epigenetic processes.