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Ganglioside GD3 is up-regulated in microglia and regulates phagocytosis following global cerebral ischemia.

ABSTRACT: Gangliosides, the major sialic-acid containing glycosphingolipids in the mammalian brain, play important roles in brain development and neural functions. Here, we show that the b-series ganglioside GD3 and its biosynthetic enzyme, GD3-synthase (GD3S), were up-regulated predominantly in the microglia of mouse hippocampus from 2 to 7 days following global cerebral ischemia (GCI). Interestingly, GD3S knockout (GD3S-KO) mice exhibited decreased hippocampal neuronal loss following GCI, as compared to wild-type (WT) mice. While comparable levels of astrogliosis and microglial proliferation were observed between WT and GD3S-KO mice, the phagocytic capacity of the GD3S-KO microglia was significantly compromised after GCI. At 2 and 4 days following GCI, the GD3S-KO microglia demonstrated decreased amoebic morphology, reduced neuronal material engulfment, and lower expression of the phagolysosome marker CD68, as compared to the WT microglia. Finally, by using a microglia-primary neuron co-culture model, we demonstrated that the GD3S-KO microglia isolated from mouse brains at 2 days after GCI are less neurotoxic to co-cultured hippocampal neurons than the WT-GCI microglia. Moreover, the percentage of microglia with engulfed neuronal elements in the co-cultured wells was also significantly decreased in the GD3S-KO mice after GCI. Interestingly, the impaired phagocytic capacity of GD3S-KO microglia could be partially restored by pre-treatment with exogenous ganglioside GD3. Altogether, this study provides functional evidence that ganglioside GD3 regulates phagocytosis by microglia in an ischemic stroke model. Our data also suggest that the GD3-linked microglial phagocytosis may contribute to the mechanism of delayed neuronal death following ischemic brain injury.

SUBMITTER: Wang J

PROVIDER: S-EPMC8363563 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Ganglioside GD3 is up-regulated in microglia and regulates phagocytosis following global cerebral ischemia.

Wang Jing J Zhang Quanguang Q Lu Yujiao Y Dong Yan Y Dhandapani Krishnan M KM Brann Darrell W DW Yu Robert K RK

Journal of neurochemistry 20210707 3

Gangliosides, the major sialic-acid containing glycosphingolipids in the mammalian brain, play important roles in brain development and neural functions. Here, we show that the b-series ganglioside GD3 and its biosynthetic enzyme, GD3-synthase (GD3S), were up-regulated predominantly in the microglia of mouse hippocampus from 2 to 7 days following global cerebral ischemia (GCI). Interestingly, GD3S knockout (GD3S-KO) mice exhibited decreased hippocampal neuronal loss following GCI, as compared to ...[more]

PMID: 34133773

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Project description:Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.

Project description:Microglia are activated following cerebral ischemic insult. P-glycoprotein (P-gp) is an efflux transporter on microvascular endothelial cells and upregulated after cerebral ischemia. This study evaluated the effects and possible mechanisms of P-gp on microglial polarization/activation in mice after ischemic stroke. P-gp-specific siRNA and adeno-associated virus (p-AAV) were used to silence and overexpress P-gp, respectively. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were performed in mice and cerebral microvascular endothelial cells (bEnd.3) in vitro, respectively. OGD/R-injured bEnd.3 cells were cocultured with mouse microglial cells (BV2) in Transwell. Influences on acute ischemic stroke outcome, the expression of inflammatory cytokines, and chemokines and chemokines receptors, microglial polarization, glucocorticoid receptor (GR) nuclear translocation, and GR-mediated mRNA decay (GMD) activation were evaluated via reverse transcription real-time polymerase chain reaction, western blot, or immunofluorescence. Silencing P-gp markedly alleviated experimental ischemia injury as indicated by reduced cerebral infarct size, improved neurological deficits, and reduced the expression of interleukin-6 (IL-6) and IL-12 expression. Silencing P-gp also mitigated proinflammatory microglial polarization and the expression of C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 expression, whereas promoted anti-inflammatory microglia polarization. Additionally, P-gp silencing promoted GR nuclear translocation and the expression of GMD relative proteins in endothelial cells. Conversely, overexpressing P-gp via p-AAV transfection offset all these effects. Furthermore, silencing endothelial GR counteracted all effects mediated by silencing or overexpressing P-gp. Elevated P-gp expression aggravated inflammatory response and brain damage after ischemic stroke by augmenting proinflammatory microglial polarization in association with increased endothelial CCL2 release due to GMD inhibition by P-gp.

Project description:BackgroundMonocyte-derived macrophages (MDMs) and microglia elicit neural inflammation and clear debris for subsequent tissue repair and remodeling. The role of infiltrating MDMs in the injured brain, however, has been controversial due to overlapping antigen expression with microglia. In this study, we define the origin and function of MDMs in cerebral ischemia.MethodsUsing adoptive transfer of GFP+ splenocytes into adult asplenic mice subjected to transient middle cerebral artery occlusion, we compared the role of CD11b+/CD45+/NK1.1-/Ly6G- MDMs and microglia in the ischemic brain. The phagocytic activities of MDMs and microglia were measured by the uptake of fluorescent beads both in vivo with mice infused with GFP+ splenocytes and ex vivo with cultures of isolated brain immune cells.ResultsStroke induced an infiltration of MDMs [GFP+] into the ipsilateral hemisphere at acute (3 days) and sub-acute phases (7 days) of post-stroke. At 7 days, the infiltrating MDMs contained both CD45High and CD45Low subsets. The CD45High MDMs in the injured hemisphere exhibited a significantly higher proliferation capacity (Ki-67 expression levels) as well as higher expression levels of CD11c when compared to CD45Low MDMs. The CD45High and CD45Low MDM subsets in the injured hemisphere were approximately equal populations, indicating that CD45High MDMs infiltrating the ischemic brain changes their phenotype to CD45Low microglia-like phenotype. Studies with fluorescent beads reveal high levels of MDM phagocytic activity in the post-stroke brain, but this phagocytic activity was exclusive to post-ischemic brain tissue and was not detected in circulating monocytes. By contrast, CD45Low microglia-like cells had low levels of phagocytic activity when compared to CD45High cells. Both in vivo and ex vivo studies also show that the phagocytic activity in CD45High MDMs is associated with an increase in the CD45Low/CD45High ratio, indicating that phagocytosis promotes MDM phenotype conversion.ConclusionsThis study demonstrates that MDMs are the predominant phagocytes in the post-ischemic brain, with the CD45High subset having the highest phagocytic activity levels. Upon phagocytosis, CD45High MDMs in the post-ischemic brain adopt a CD45Low phenotype that is microglia-like. Together, these studies reveal key roles for MDMs and their phagocytic function in tissue repair and remodeling following cerebral ischemia.

Project description:Ischemic stroke can induce rapid disruption of blood-brain barrier (BBB). It has been suggested that increased BBB permeability can affect the pathological progression of ischemic tissue. However, the impact of increased BBB permeability on microglial activation and synaptic structures following reperfusion after ischemia remains unclear. In this study, we investigated microglial activation, dendritic damage and plasticity of dendritic spines after increasing BBB permeability following transient global cerebral ischemia in the somatosensory cortices in mice. Bilateral common carotid artery ligation (BCAL) was used to induce transient global cerebral ischemia. Mannitol was used to increase the BBB permeability. Intravital two-photon imaging was performed to image the dendritic structures and BBB extravasation. Microglial morphology was quantitated using a skeletonization analysis method. To evaluate inflammation of cerebral cortex, the mRNA expression levels of integrin alpha M (CD11b), CD68, chemokine (C-X-C motif) ligand 10 (IP10) and tumor necrosis factor alpha (TNF-α) were measured by fluorescent quantitative PCR. Intravital two-photon imaging revealed that mannitol caused a drastic increase in BBB extravasation during reperfusion after transient global ischemia. Increased BBB permeability induced by mannitol had no significant effect on inflammation and dendritic spines in healthy mice but triggered a marked de-ramification of microglia; importantly, in ischemic animals, mannitol accelerated de-ramification of microglia and aggravated inflammation at 3 h but not at 3 days following reperfusion after ischemia. Although mannitol did not cause significant change in the percentage of blebbed dendrites and did not affect the reversible recovery of the dendritic structures, excessive extravasation was accompanied with significant decrease in spine formation and increase in spine elimination during reperfusion in ischemic mice. These findings suggest that increased BBB permeability induced by mannitol can lead to acute activation of microglia and cause excessive loss of dendritic spines after transient global cerebral ischemia.

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Ganglioside GD3 is up-regulated in microglia and regulates phagocytosis following global cerebral ischemia.

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Ganglioside GD3 is up-regulated in microglia and regulates phagocytosis following global cerebral ischemia.

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