Zhaobao Yin

Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes

The neurotoxicity of high levels of methylmercury (MeHg) is well established both in humans and experimental animals. Astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS). Although the precise mechanisms of MeHg neurotoxicity are ill-defined, oxidative stress and altered mitochondrial and cell membrane permeability appear to be critical factors in its pathogenesis. The present study examined the effects of MeHg treatment on oxidative injury, mitochondrial inner membrane potential, glutamine uptake and expression of glutamine transporters in primary astrocyte cultures. MeHg caused a significant increase in F(2)-isoprostanes (F(2)-IsoPs), lipid peroxidation biomarkers of oxidative damage, in astrocyte cultures treated with 5 or 10 microM MeHg for 1 or 6 h. Consistent with this observation, MeHg induced a concentration-dependant reduction in the inner mitochondrial membrane potential (DeltaPsi(m)), as assessed by the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE). Our results demonstrate that DeltaPsi(m) is a very sensitive endpoint for MeHg toxicity, since significant reductions were observed after only 1 h exposure to concentrations of MeHg as low as 1 microM. MeHg pretreatment (1, 5 and 10 microM) for 30 min also inhibited the net uptake of glutamine ((3)H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and ASCT2, was inhibited only at the highest (10 microM) MeHg concentration, suggesting that the reduction in glutamine uptake observed after 30 min treatment with lower concentrations of MeHg (1 and 5 microM) was not due to inhibition of transcription. Taken together, these studies demonstrate that MeHg exposure is associated with increased mitochondrial membrane permeability, alterations in glutamine/glutamate cycling, increased ROS formation and consequent oxidative injury. Ultimately, MeHg initiates multiple additive or synergistic disruptive mechanisms that lead to cellular dysfunction and cell death.

Ferroportin is a manganese-responsive protein that decreases manganese cytotoxicity and accumulation.

J. Neurochem. (2010) 112, 1190–1198.

The methylmercury-L-cysteine conjugate is a substrate for the L-type large neutral amino acid transporter

Methylmercury (MeHg) is a potent neurotoxin. The mechanism(s) that governs MeHg transport across the blood‐brain barrier and other biological membranes remains unclear. This study addressed the role of the L‐type large neutral amino acid transporter, LAT1, in MeHg transport. Studies were carried out in CHO‐k1 cells. Over‐expression of LAT1 in these cells was associated with enhanced uptake of [14C]‐MeHg when treated with l‐cysteine, but not with the d‐cysteine conjugate. In the presence of excess l‐methionine, a substrate for LAT1, l‐cysteine‐conjugated [14C]‐MeHg uptake was significantly attenuated. Treatment of LAT‐1 over‐expressing CHO‐k1 cells with l‐cysteine‐conjugated MeHg was also associated with increased leakage of lactate dehydrogenase into the media as well as reduced cell viability measured by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction assay. In contrast, knock‐down of LAT1 decreased the uptake of l‐cysteine‐conjugated MeHg and attenuated the effects of MeHg on lactate dehydrogenase leakage and CHO‐k1 cell viability. These results indicate that the MeHg‐l‐cysteine conjugate is a substrate for the neutral amino acid transporter, LAT1, which actively transports MeHg across membranes.

Estrogen and tamoxifen reverse manganese‐induced glutamate transporter impairment in astrocytes

Chronic exposure to manganese (Mn) can cause manganism, a neurodegenerative disorder similar to Parkinson’s disease. The toxicity of Mn includes impairment of astrocytic glutamate transporters. 17β‐Estradiol (E2) has been shown to be neuroprotective in various neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease, and some selective estrogen receptor modulators, including tamoxifen (TX), also possess neuroprotective properties. We have tested our hypothesis that E2 and TX reverse Mn‐induced glutamate transporter impairment in astrocytes. The results established that E2 and TX increased glutamate transporter function and reversed Mn‐induced glutamate uptake inhibition, primarily via the up‐regulation of glutamate/aspartate transporter (GLAST). E2 and TX also increased astrocytic GLAST mRNA levels and attenuated the Mn‐induced inhibition of GLAST mRNA expression. In addition, E2 and TX effectively increased the expression of transforming growth factor β1, a potential modulator of the stimulatory effects of E2/TX on glutamate transporter function. This effect was mediated by the activation of MAPK/extracellular signal‐regulated kinase (ERK) and phosphoinositide 3‐kinase (PI3K)/Akt signaling pathways. These novel findings suggest, for the first time, that E2 and TX enhance astrocytic glutamate transporter expression via increased transforming growth factor β1 expression. Furthermore, the present study is the first to show that both E2 and TX effectively reverse Mn‐induced glutamate transport inhibition by restoring its expression and activity, thus offering a potential therapeutic modality in neurodegenerative disorders characterized by altered glutamate homeostasis.

Methylmercury induces acute oxidative stress, altering Nrf2 protein level in primary microglial cells

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.

Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity

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 with 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-induced alterations in astrocyte functions are attenuated by ebselen

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 2h 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 MeHg-induced 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 MeHgs (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 (1h) 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 1h on astrocytic ΔΨ(m) and partially reversed the effect of 5 and 10 μM MeHg treatments for 1h on ΔΨ(m). In addition, ebselen inhibited MeHg-induced phosphorylation of ERK (p<0.05) and blocked MeHg-induced activation of caspase-3 (p<0.05-0.01). 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 toxicity and Nrf2-dependent detoxification in astrocytes.

Methylmercury (MeHg) is a potent neurotoxicant and preferentially induces oxidative injury in astrocytes. In neuronal tissues, nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor determining the protective antioxidant response against various environmental toxicants. Nrf2 is subjected to regulation by many other signaling pathways. The purpose of this study is to characterize its interaction with the phosphatidylinositol 3 (PI3) kinase in cultured rat neonatal primary astrocytes. The results showed that at pathologically relevant concentrations, exposure of primary astrocytes to MeHg led to Nrf2 activation and upregulation of its downstream antioxidant genes. Inhibition of the PI3 kinase resulted in decreased Nrf2 activity, decreased cellular glutathione, and increased cell death to high-dose MeHg. The functional interaction between the two signaling pathways underlined an important mechanism for astrocyte protection against MeHg toxicity. Modulation of Nrf2 by pharmacological modalities should afford a treatment to attenuate MeHg-induced neurotoxicity.

Transforming growth factor-α mediates estrogen-induced upregulation of glutamate transporter GLT-1 in rat primary astrocytes.

Glutamate transporter‐1 (GLT‐1) plays a central role in preventing excitotoxicity by removing excess glutamate from the synaptic clefts. 17β‐Estradiol (E2) and tamoxifen (TX), a selective estrogen receptor (ER) modulator, afford neuroprotection in a range of experimental models. However, the mechanisms that mediate E2 and TX neuroprotection have yet to be elucidated. We tested the hypothesis that E2 and TX enhance GLT‐1 function by increasing transforming growth factor (TGF)‐α expression and, thus, attenuate manganese (Mn)‐induced impairment in astrocytic GLT‐1 expression and glutamate uptake in rat neonatal primary astrocytes. The results showed that E2 (10 nM) and TX (1 μM) increased GLT‐1 expression and reversed the Mn‐induced reduction in GLT‐1, both at the mRNA and protein levels. E2/TX also concomitantly reversed the Mn‐induced inhibition of astrocytic glutamate uptake. E2/TX activated the GLT‐1 promoter and attenuated the Mn‐induced repression of the GLT‐1 promoter in astrocytes. TGF‐α knockdown (siRNA) abolished the E2/TX effect on GLT‐1 expression, and inhibition of epidermal growth factor receptor (TGF‐α receptor) suppressed the effect of E2/TX on GLT‐1 expression and GLT‐1 promoter activity. E2/TX also increased TGF‐α mRNA and protein levels with a concomitant increase in astrocytic glutamate uptake. All ERs (ER‐α, ER‐β, and G protein‐coupled receptor 30) were involved in mediating E2 effects on the regulation of TGF‐α, GLT‐1, and glutamate uptake. These results indicate that E2/TX increases GLT‐1 expression in astrocytes via TGF‐α signaling, thus offering an important putative target for the development of novel therapeutics for neurological disorders.

Rat brain endothelial cells are a target of manganese toxicity

Manganese (Mn) is an essential trace metal; however, exposure to high Mn levels can result in neurodegenerative changes resembling Parkinsons disease (PD). Information on Mns effects on endothelial cells of the blood-brain barrier (BBB) is lacking. Accordingly, we tested the hypothesis that BBB endothelial cells are a primary target for Mn-induced neurotoxicity. The studies were conducted in an in vitro BBB model of immortalized rat brain endothelial (RBE4) cells. ROS production was determined by F(2)-isoprostane (F(2)-IsoPs) measurement. The relationship between Mn toxicity and redox status was investigated upon intracellular glutathione (GSH) depletion with diethylmaleate (DEM) or L-buthionine sulfoximine (BSO). Mn exposure (200 or 800 microM MnCl(2) or MnSO(4)) for 4 or 24h led to significant decrease in cell viability vs. controls. DEM or BSO pre-treatment led to further enhancement in cytotoxicity vs. exposure to Mn alone, with more pronounced cell death after 24-h DEM pre-treatment. F(2)-IsoPs levels in cells exposed to MnCl(2) (200 or 800 microM) were significantly increased after 4h and remained elevated 24h after exposure compared with controls. Consistent with the effects on cell viability and F(2)-IsoPs, treatment with MnCl(2) (200 or 800 microM) was also associated with a significant decrease in membrane potential. This effect was more pronounced in cells exposed to DEM plus MnCl(2) vs. cells exposed to Mn alone. We conclude that Mn induces direct injury to mitochondria in RBE4 cells. The ensuing impairment in energy metabolism and redox status may modify the restrictive properties of the BBB compromising its function.