Eng-Ang Ling

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.

F3/Contactin Acts as a Functional Ligand for Notch during Oligodendrocyte Maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.

Blood–retinal barrier in hypoxic ischaemic conditions: Basic concepts, clinical features and management

The blood-retinal barrier (BRB) plays an important role in the homeostatic regulation of the microenvironment in the retina. It consists of inner and outer components, the inner BRB (iBRB) being formed by the tight junctions between neighbouring retinal capillary endothelial cells and the outer barrier (oBRB) by tight junctions between retinal pigment epithelial cells. Astrocytes, Müller cells and pericytes contribute to the proper functioning of the iBRB. In many clinically important conditions including diabetic retinopathy, ischaemic central retinal vein occlusion, and some respiratory diseases, retinal hypoxia results in a breakdown of the iBRB. Disruption of the iBRB associated with increased vascular permeability, results in vasogenic oedema and tissue damage, with consequent adverse effects upon vision. Factors such as enhanced production of vascular endothelial growth factor (VEGF), NO, oxidative stress and inflammation underlie the increased permeability of the iBRB and inhibition of these factors is beneficial. Experimental studies in our laboratory have shown melatonin to be a protective agent for the iBRB in hypoxic conditions. Although oBRB breakdown can occur in conditions such as accelerated hypertension and the toxaemia of pregnancy, both of which are associated with choroidal ischaemia and in age-related macular degeneration (ARMD), and is a feature of exudative (serous) retinal detachment, our studies have shown that the oBRB remains intact in hypoxic/ischaemic conditions. Clinically, anti-VEGF therapy has been shown to improve vision in diabetic maculopathy and in neovascular ARMD. The visual benefit in both conditions appears to arise from the restoration of BRB integrity with a reduction of retinal oedema.

Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

Silent information regulator-2 (SIR2) proteins regulate lifespan of diverse organisms, but their distribution and roles in the CNS remain unclear. Here, we show that sirtuin 2 (SIRT2), a mammalian SIR2 homolog, is an oligodendroglial cytoplasmic protein and localized to the outer and juxtanodal loops in the myelin sheath. Among cytoplasmic proteins of OLN-93 oligodendrocytes, α-tubulin was the main substrate of SIRT2 deacetylase. In cultured primary oligodendrocyte precursors (OLPs), SIRT2 emergence accompanied elevated α-tubulin acetylation and OLP differentiation into the prematurity stage. Small interfering RNA knockdown of SIRT2 increased the α-tubulin acetylation, myelin basic protein expression, and cell arbor complexity of OLPs. SIRT2 overexpression had the opposite effects, and counteracted the cell arborization-promoting effect of overexpressed juxtanodin. SIRT2 mutation concomitantly reduced its deacetylase activity and its impeding effect on OLP arborization. These results demonstrated a counterbalancing role of SIRT2 against a facilitatory effect of tubulin acetylation on oligodendroglial differentiation. Selective SIRT2 availability to oligodendroglia may have important implications for myelinogenesis, myelin–axon interaction, and brain aging.

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.

Hypoxia-ischemia and retinal ganglion cell damage

Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs) occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1α and its target genes such as vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS). Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca2+ which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.

Hypoxia‐induced astrocytic reaction and increased vascular permeability in the rat cerebellum

Hypoxia is an important factor linked to induction of vascular leakage and formation of brain edema. In this connection, astrocytes associated closely with the blood vessels are deemed to be involved. This study investigated the response of astrocytes to hypoxia in the adult rat cerebellum, and along with this, the integrity of the blood–brain barrier (BBB) was assessed using fluorescent and electron dense tracers. In rats subjected to hypoxia, mRNA and protein expression of hypoxia inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), and aquaporin‐4 (AQ4) was significantly increased. VEGF and AQ4 immunoreactive cells were identified as astrocytes by double immunofluorescence labeling. Increased VEGF tissue concentration and astrocytic swelling as observed in hypoxic rats were reduced after melatonin administration. Following intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in hypoxic rats but not in the controls indicating that functional integrity of BBB is compromised in hypoxia/reoxygenation. Enhanced gene and protein expression of VEGF may contribute to increased permeability of blood vessels. AQ4, a water transporting protein, is upregulated in astrocytes in hypoxia suggesting the cells are involved in edema formation. To this end, melatonin may be beneficial in reducing edema as it reduced VEGF concentration and, hence, vascular permeability.

Expression profile of embryonic stem cell-associated genes Oct4, Sox2 and Nanog in human gliomas.

Guo Y, Liu S, Wang P, Zhao S, Wang F, Bing L, Zhang Y, Ling E‐A, Gao J & Hao A
(2011) Histopathology59, 763–775

OCT4 IS EXPRESSED IN HUMAN GLIOMAS AND PROMOTES COLONY FORMATION IN GLIOMA CELLS

There is increasing evidence that self‐renewal capacity of cancer cells is critical for carcinogenesis; hence, it is vital to examine the expression and involvement of self‐renewal regulatory genes in these cells. Here, we reported that Oct4, a well‐known regulator of self‐renewal in embryonic stem cells, was highly expressed in human gliomas and glioma cell lines, and the expression levels were increased in parallel with increasing glioma grades. In in vitro cell cultures, Oct4 was only expressed in rat C6 glioma cells and rat neural stem cells but not in rat brain differentiated cells. Downregulation of Oct4 expression by RNA interference in C6 cells was associated with reduced cell proliferation and colony formation. Further analysis revealed that Oct4 could upregulate phosphorylation of Stat3 to promote tumor cell proliferation. Overexpression of Oct4 in C6 cells increased the expression of nestin but decreased the expression of GFAP suggesting that Oct4 might inhibit the differentiation of glioma cells. Our findings may provide further evidence for the stem cell theory of carcinogenesis. In contrast, the results might also imply that Oct4 contributes to the existence of undifferentiated cells in gliomas.

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.