The neurotoxicity of methylmercury (MeHg) is well documented in both humans and animals. MeHg causes acute and chronic damage to multiple organs, most profoundly the central nervous system (CNS). Microglial cells are derived from macrophage cell lineage, making up approximately 12% of cells in the CNS, yet their role in MeHg-induced neurotoxicity is not well defined. The purpose of the present study was to characterize microglial vulnerability to MeHg and their potential adaptive response to acute MeHg exposure. We examined the effects of MeHg on microglial viability, reactive oxygen species (ROS) generation, glutathione (GSH) level, redox homeostasis, and Nrf2 protein expression. Our data showed that MeHg (1-5 microM) treatment caused a rapid (within 1 min) concentration- and time-dependent increase in ROS generation, accompanied by a statistically significant decrease in the ratio of GSH and its oxidized form glutathione disulfide (GSSG) (GSH:GSSG ratio). MeHg increased the cytosolic Nrf2 protein level within 1 min of exposure, followed by its nuclear translocation after 10 min of treatment. Consistent with the nuclear translocation of Nrf2, quantitative real-time PCR revealed a concentration-dependent increase in the messenger RNA level of Ho-1, Nqo1, and xCT 30 min post MeHg exposure, whereas Nrf2 knockdown greatly reduced the upregulation of these genes. Furthermore, we observed increased microglial death upon Nrf2 knockdown by the small hairpin RNA approach. Taken together, our study has demonstrated that microglial cells are exquisitely sensitive to MeHg and respond rapidly to MeHg by upregulating the Nrf2-mediated antioxidant response.

SUMMARYDuring vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling.

Microglial cells elaborate trophic factors and cytokines and remove toxins and debris from the extracellular space, acting analogously to peripheral macrophages. Over the past two decades increased attention has been directed at the role of microglia, not only in normal physiology, but also in mediating neurotoxicity. Activation of microglia is inherent to multiple neurodegenerative disorders and exposure to toxic compounds. In large measure, these revelations have come about as a result of technologies that enable researchers to obtain high-yield and -purity primary cultures of rodent microglia. The mechanical isolation protocol discussed in this protocol offers an economical method to isolate large amount of microglia in a short and not too labor intensive manner. Most importantly, it assures a high yield of cells with great reproducibility. Given the ever increasing importance of microglia to the field of neurotoxicology research, the ability to isolate high yield of primary microglia makes it possible to investigate the role and mechanisms associated with microglial modulation of neurotoxicity. We provide a detailed description on the methods that is routinely used in our laboratory for the isolation and culturing of microglia, with emphasis on the steps which are deemed most critical for obtaining pure and healthy cultures.

As the two major glial cell types in the brain, astrocytes and microglia play pivotal but different roles in maintaining optimal brain function. Although both cell types have been implicated as major targets of methylmercury (MeHg), their sensitivities and adaptive responses to this metal can vary given their distinctive properties and physiological functions. This study was carried out to compare the responses of astrocytes and microglia following MeHg treatment, specifically addressing the effects of MeHg on cell viability, reactive oxygen species (ROS) generation and glutathione (GSH) levels, as well as mercury (Hg) uptake and the expression of NF-E2-related factor 2 (Nrf2). Results showed that microglia are more sensitive to MeHg than astrocytes, a finding that is consistent with their higher Hg uptake and lower basal GSH levels. Microglia also demonstrated higher ROS generation compared to astrocytes. Nrf2 and its downstream genes were upregulated in both cell types, but with different kinetics (much faster in microglia). In summary, microglia and astrocytes each exhibit a distinct sensitivity to MeHg, resulting in their differential temporal adaptive responses. These unique sensitivities appear to be dependent on the cellular thiol status of the particular cell type.

Methylmercury (MeHg) preferentially accumulates in glia of the central nervous system (CNS), but its toxic mechanisms have yet to be fully recognized. In the present study, we tested the hypothesis that MeHg induces neurotoxicity via oxidative stress mechanisms, and that these effects are attenuated by the antioxidant, ebselen. Rat neonatal primary cortical astrocytes were pretreated with or without 10 μM ebselen for 2 hours followed by MeHg (0, 1, 5, and 10 μM) treatments. MeHg-induced changes in astrocytic [ 3 H]-glutamine uptake were assessed along with changes in mitochondrial membrane potential (ΔΨ m ), using the potentiometric dye tetramethylrhodamine ethyl ester (TMRE). Western blot analysis was used to detect MeHginduced ERK (extracellular-signal related kinase) phosphorylation and caspase-3 activation. MeHg treatment significantly decreased (p<0.05) astrocytic [ 3 H]-glutamine uptake at all time points and concentrations. Ebselen fully reversed MeHg's (1 μM) effect on [ 3 H]-glutamine uptake at 1 min. At higher MeHg concentrations, ebselen partially reversed the MeHg-induced astrocytic inhibition of [ 3 H]-glutamine uptake [at 1 min (5 and 10 μM) (p<0.05); 5 min (1, 5 and 10 μM) (p<0.05)]. MeHg treatment (1 hour) significantly (p<0.05) dissipated the ΔΨ m in astrocytes as evidenced by a decrease in mitochondrial TMRE fluorescence. Ebselen fully reversed the effect of 1 μM MeHg treatment for 1 hour on astrocytic ΔΨ m and partially reversed the effect of 5 and 10 μM MeHg treatments for 1 hour on ΔΨ m . In addition, ebselen inhibited MeHg-induced phosphorylation of ERK (p<0.05) and blocked MeHg-induced activation of caspase-3 (p<0.05 to 0.01). These results are consistent with the hypothesis that MeHg exerts its toxic effects via oxidative stress and that the phosphorylation of ERK and the dissipation of the astrocytic mitochondrial membrane potential are involved in MeHg toxicity. In addition, the protective effects elicited by ebselen reinforce the idea that organic selenocompounds represent promising strategies to counteract MeHg-induced neurotoxicity.

Methylmercury (MeHg) is a global environmental pollutant with significant adverse effects on human health. As the major target of MeHg, the central nervous system (CNS) exhibits the most recognizable poisoning symptoms. The role of the two major nonneuronal cell types, astrocytes and microglia, in response to MeHg exposure was recently compared. These two cell types share several common features in MeHg toxicity, but interestingly, these cells types also exhibit distinct response kinetics, indicating a cell-specific role in mediating MeHg-induced neurotoxicity. The aim of this study was to review the most recent literature and summarize key features of glial responses to this organometal.

Laparoscopic gastric resection for selected cases of gastrointestinal stromal tumors of the stomach appears to be safe, feasible, and effective.

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