The neuroprotective effects of milk fat globule-EGF factor 8 against oligomeric amyloid β toxicity

BACKGROUND: Phosphatidylserine receptor is a key molecule that mediates the phagocytosis of apoptotic cells. Milk fat globule-EGF factor 8 (MFG-E8) is a phosphatidylserine receptor that is expressed on various macrophage lineage cells, including microglia in the central nervous system (CNS). Targeted clearance of degenerated neurons by microglia is essential to maintain healthy neural networks. We previously showed that the CX3C chemokine fractalkine is secreted from degenerated neurons and accelerates microglial clearance of neuronal debris via inducing the release of MFG-E8. However, the mechanisms by which microglia produce MFG-E8 and the precise functions of MFG-E8 are unknown. METHODS: The release of MFG-E8 from microglia treated with conditioned medium from neurons exposed to neurotoxic substances, glutamate or oligomeric amyloid β (oAβ) was measured by ELISA. The neuroprotective effects of MFG-E8 and MFG-E8 − induced microglial phagocytosis of oAβ were assessed by immunocytochemistry. The effects of MFG-E8 on the production of the anti-oxidative enzyme hemeoxygenase-1 (HO-1) were determined by ELISA and immunocytochemisty. RESULTS: MFG-E8 was induced in microglia treated with conditioned medium from neurons that had been exposed to neurotoxicants, glutamate or oAβ. MFG-E8 significantly attenuated oAβ-induced neuronal cell death in a primary neuron − microglia coculture system. Microglial phagocytosis of oAβ was accelerated by MFG-E8 treatment due to increased CD47 expression in the absence of neurotoxic molecule production, such as tumor necrosis factor-α, nitric oxide, and glutamate. MFG-E8 − treated microglia induced nuclear factor E(2) − related factor 2 (Nrf2) − mediated HO-1 production, which also contributed to neuroprotection. CONCLUSIONS: These results suggest that microglia release MFG-E8 in response to signals from degenerated neurons and that MFG-E8 protects oAβ-induced neuronal cell death by promoting microglial phagocytic activity and activating the Nrf2-HO-1 pathway. Thus, MFG-E8 may have novel roles as a neuroprotectant in neurodegenerative conditions

Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer's disease mouse model.Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases

Molecular and functional interactions between tumor necrosis factor-alpha receptors and the glutamatergic system in the mouse hippocampus : implications for seizure susceptibility

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine acting on two distinct receptor subtypes, namely p55 and p75 receptors. TNF-alpha p55 and p75 receptor knockout mice were previously shown to display a decreased or enhanced susceptibility to seizures, respectively, suggesting intrinsic modifications in neuronal excitability. We investigated whether alterations in glutamate system function occur in these naive knockout mice with perturbed cytokine signaling that could explain their different propensity to develop seizures. Using Western blot analysis of hippocampal homogenates, we found that p55(-/-) mice have decreased levels of membrane GluR3 and NR1 glutamate receptor subunits while GluR1, GluR2, GluR6/7 and NR2A/B were unchanged as compared to wild-type mice. In p75(-/-) mice, GluR2, GluR3, GluR6/7 and NR2A/B glutamate receptor subunits were increased in the hippocampus while GluR1 and NR1 did not change. Extracellular single-cell recordings of the electrical activity of hippocampal neurons were carried out in anesthetized mice by standard electrophysiological techniques. Microiontophoretic application of glutamate increased the basal firing rate of hippocampal neurons in p75(-/-) mice versus wild-type mice, and this effect was blocked by 2-amino-5-phosphopentanoic acid and 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione denoting the involvement of N-methyl-D-aspartic acid and AMPA receptors. In p55(-/-) mice, hippocampal neurons responses to glutamate were similar to wild-type mice. Spontaneous glutamate release measured by in vivo hippocampal microdialysis was significantly decreased only in p55(-/-) mice. No changes were observed in KCl-induced glutamate release in both receptor knockout mice strains versus wild-type mice. These findings highlight specific molecular and functional interactions between p55 and p75 receptor-mediated signaling and the glutamate system. These interactions may be relevant for controlling neuronal excitability in physiological and pathological conditions.peer-reviewe

Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway

<p>Abstract</p> <p>Background</p> <p>Microglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS). In the event of neuronal stress or injury, microglial cells rapidly react and change their phenotype. This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions. We investigated the molecular mechanisms underlying triggering of microglial activation by necrotic neuronal damage.</p> <p>Methods</p> <p>Primary cultures of microglia were used to study the effect of necrotic neurons on microglial inflammatory responses and toxicity towards cerebellar granule neurons (CGN). The mouse hippocampal cell line, HT22, was used in this study as the main source of necrotic neurons to stimulate microglia. To identify the signal transduction pathways activated in microglia, primary microglial cultures were obtained from mice deficient in Toll-like receptor (TLR) -2, -4, or in the TLR adapter protein MyD88.</p> <p>Results</p> <p>Necrotic neurons, but not other necrotic cell types, induced microglial activation which was characterized by up-regulation of: i) MHC class II; ii) co-stimulatory molecules, i.e. CD40 and CD24; iii) β2 integrin CD11b; iii) pro-inflammatory cytokines, i.e. interleukin 6 (IL-6), IL-12p40 and tumor-necrosis factor (TNF); iv) pro-inflammatory enzymes such as nitric oxide synthase (iNOS, type II NOS), indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX-2) and increased microglial motility. Moreover, microglia-conditioned medium (MCM) obtained from cultures of activated microglia showed increased neurotoxicity mediated through the <it>N</it>-methyl-D-aspartate receptor (NMDAR). The activation of microglia by necrotic neurons was shown to be dependent on the TLR-associated adapter molecule myeloid differentiation primary response gene (<it>MyD88</it>). Furthermore, MyD88 mediated enhanced neurotoxicity by activated microglia through up-regulation of the expression and activity of glutaminase, an enzyme that produces glutamate, which is an NMDAR agonist.</p> <p>Conclusion</p> <p>These results show that necrotic neurons activate in microglia a MyD88-dependent pathway responsible for a pro-inflammatory response that also leads to increased neurotoxic activity through induction of glutaminase. This finding contributes to better understanding the mechanisms causing increased neuroinflammation and microglial neurotoxicity in a neurodegenerative environment.</p

The Deacetylase HDAC6 Mediates Endogenous Neuritic Tau Pathology

The initiating events that promote tau mislocalization and pathology in Alzheimer's disease (AD) are not well defined, partly because of the lack of endogenous models that recapitulate tau dysfunction. We exposed wild-type neurons to a neuroinflammatory trigger and examined the effect on endogenous tau. We found that tau re-localized and accumulated within pathological neuritic foci, or beads, comprised of mostly hypo-phosphorylated, acetylated, and oligomeric tau. These structures were detected in aged wild-type mice and were enhanced in response to neuroinflammation in vivo, highlighting a previously undescribed endogenous age-related tau pathology. Strikingly, deletion or inhibition of the cytoplasmic shuttling factor HDAC6 suppressed neuritic tau bead formation in neurons and mice. Using mass spectrometry-based profiling, we identified a single neuroinflammatory factor, the metalloproteinase MMP-9, as a mediator of neuritic tau beading. Thus, our study uncovers a link between neuroinflammation and neuritic tau beading as a potential early-stage pathogenic mechanism in AD

Increased expression of cystine/glutamate antiporter in multiple sclerosis

<p>Abstract</p> <p>Background</p> <p>Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter x_c^-, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system x_c^-in glutamate homeostasis alterations in MS pathology.</p> <p>Methods</p> <p>Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients.</p> <p>Results and discussion</p> <p>We show here that human activated monocytes release glutamate through cystine/glutamate antiporter x_c^-and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes.</p> <p>Conclusions</p> <p>Together, these results reveal that increased expression of the cystine/glutamate antiporter system x_c^-in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.</p

Galectin-1 Deactivates Classically Activated Microglia and Protects from Inflammation-Induced Neurodegeneration

SummaryInflammation-mediated neurodegeneration occurs in the acute and the chronic phases of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Classically activated (M1) microglia are key players mediating this process. Here, we identified Galectin-1 (Gal1), an endogenous glycan-binding protein, as a pivotal regulator of M1 microglial activation that targets the activation of p38MAPK-, CREB-, and NF-κB-dependent signaling pathways and hierarchically suppresses downstream proinflammatory mediators, such as iNOS, TNF, and CCL2. Gal1 bound to core 2 O-glycans on CD45, favoring retention of this glycoprotein on the microglial cell surface and augmenting its phosphatase activity and inhibitory function. Gal1 was highly expressed in the acute phase of EAE, and its targeted deletion resulted in pronounced inflammation-induced neurodegeneration. Adoptive transfer of Gal1-secreting astrocytes or administration of recombinant Gal1 suppressed EAE through mechanisms involving microglial deactivation. Thus, Gal1-glycan interactions are essential in tempering microglial activation, brain inflammation, and neurodegeneration, with critical therapeutic implications for MS

Antiinflammatory effect of Triptolide on rat microglial cells

In dieser Arbeit wurde die Wirkung von Triptolid auf Mikrogliazellen in einem in vitro Modell untersucht mit der Fragestellung, ob diese Substanz in der Therapie neurodegenerativer Erkrankungen eingesetzt werden kann. Die steigende Prävalenz neurodegenerativer Erkrankungen wie der M. Alzheimer und der M. Parkinson macht die Erforschung neuer Therapieoptionen notwendig. Neurodegenerativen Erkrankungen ist gemeinsam, dass diesen eine Dysregulation des lokalen Immunsystems zugrunde liegt, die zu einer chronischen Inflammation mit Neuronenverlust und damit Progredienz der Erkrankung führt. Eine Eindämmung der Neuroinflammation, an der hauptsächlich Mikrogliazellen beteiligt sind, ist somit ein vielversprechender Therapieansatz. Triptolid, eine bioaktive Komponente aus der chinesischen Kletterpflanze Wilfords Dreiflügelfrucht (Tripterygium wilfordii) wird traditionell in der chinesischen Medizin zur Behandlung von entzündungsvermittelten Erkrankungen, wie bspw. Arthritis, eingesetzt. In dieser Arbeit wurde Triptolid in einem neuroinflammatorischen in vitro Modell untersucht, ob es zu einer Veränderung der Zytokinsekretion, der Proliferation und Migration sowie der Phagozytoseaktivität kommt, wenn primäre Mikrogliazellen aus den Hirnen neugeborener Ratten mit LPS stimuliert und mit Triptolid koinkubiert wurden. Es wurde eine signifikante eine Reduktion der Expression von iNOS in LPS-stimulierten Mikroglia nach Koinkubation mit Triptolid nachgewiesen. Auch die Untersuchungen des Genexpressionsprofils der proinflammatorischen Zytokine in aktivierten Mikrogliazellen zeigte, dass die Expression der Zytokine IL-6, IL-1β und TNF-α durch Triptolid signifikant gesenkt wurde. Auf Proteinebene wurde ebenfalls eine signifikante Reduktion der Freisetzung von IL-6 in Mikrogliazellen nachgewiesen. Neben proinflammatorischen Zytokinen ist auch das Chemokinsystem mit dem neuroinflammatorischen und neurodegenerativem Geschehen im ZNS assoziiert. Veränderungen im Chemokinmuster kann eine Grundlage der chronischen Inflammationsreaktion darstellen. In dieser Arbeit wurde gezeigt, dass Triptolid modulierend auf das Chemokinsystem wirkt, indem es die Genexpression der Chemokine CCL2, CCL3, CCL5, CCL7 und CXCL10 in aktivierten Mikrogliazellen signifikant reduziert. Einen weniger ausgeprägten Effekt hat Triptotlid auf die Expression der Chemokinrezeptoren CCR5 und CXCR4. Hier reduziert Triptolid lediglich die Genexpression des Rezeptors CXCR4 im Vergleich zu ruhenden Mikrogliazellen. Ein weiterer Faktor, der eine Rolle in der Pathogenese neurodegenerativer Erkrankungen spielt, ist eine veränderte Phagozytoseaktivität von Mikrogliazellen. In dieser Arbeit wurde gezeigt, dass Triptolid keinen Einfluss auf die Genexpression des Phagozytoserezeptors FcγR1 in LPS-aktivierten Mikrogliazellen hat und dass es tendenziell die durch LPS-induzierte verminderte Phagozytoseaktivität von Mikrogliazellen wieder aufheben kann. Zusammenfassend ist die Neuroinflammation ein komplexer Prozess, der eine zentrale Rolle bei verschiedenen ZNS-Erkrankungen spielt. Triptolid stellt aufgrund seiner antiinflammatorischen Wirkungsweise und seinen modulierenden Effekt auf Mikrogliazellen eine vielversprechende Substanz in der Therapie neurodegenerativer Erkrankungen dar