To our knowledge, this is the first study to systematically compare responses in primary microglia and astrocytes upon treatments with physiologically relevant concentrations of MeHg (Akagi et al., 1998
). Several measurements of responsiveness to MeHg have established that microglia respond to this organometal more rapidly and robustly than astrocytes. For example, MeHg (≤1μM) treatment led to rapid ROS generation and GSH depletion in microglia, commencing as early as 1 min after treatment, well before the 6 hr time point when analogous changes were detected in astrocytes treated with 5 μM MeHg ( and ). Consistent with these observations, comparative studies in cultured cells treated with trimethyltin (TMT) also found significantly higher sensitivity in microglia compared to astrocytes (Monnet-Tschudi et al., 1995
The distinct sensitivities of microglia and astrocytes likely indicate different roles for the two cell types along a temporal axis in response to MeHg. Indeed, Nrf2 expression exhibited distinct upregulation kinetics after MeHg treatment in microglia vs.
astrocytes. Consistent with previous observations (Ni et al., 2010
), the upregulation of microglial Nrf2 occurred as early as 1 min after MeHg treatment (), with Nrf2 undergoing nuclear translocation 10 min post-treatment. In contrast, comparable changes in astrocytes were evident only after treatment with the highest concentration of MeHg (5μM) for the longest time point (6 hr) ( and ). Consistent with the difference in Nrf2 expression kinetics in the two cell types, the temporal expression of genes downstream of Nrf2 was also distinctly regulated. The expression of Ho1, Nqo1
rapidly increased in microglia, but the increase was delayed in astrocytes ( and ). The difference in the kinetics of Nrf2 activation could reflect the temporal difference in the activation of these two cell types. The time scale of the changes is consistent with earlier reports. For example, Long and colleagues reported that microglia responded to titanium dioxide (TiO2
; ≥60ppm) with a rapid (1–5 min) release of H2
(Long et al., 2007
). In contrast, 1mM dibutyryl cAMP and 1μg/ml Lipopolysaccharide (LPS) led to astrocytic activation after 6 hr of treatment (LaDu et al., 2000
). The faster activation of microglia in comparison to astrocytes was also evident in vivo
upon TMT treatment. Reactive microglia in the rat brain were noted as early as 2 days after TMT treatment, while astrocytosis was not evident until 14 days after TMT treatment (Kuhlmann and Guilarte, 2000
From a molecular point of view, the differences between microglial and astrocytic responsiveness to MeHg appear to be related, at least in part, to differences in their respective cellular thiol statuses. This viewpoint is based on three important events: (i) microglia accumulate higher levels of Hg (), which interact with and oxidize thiols; (ii) microglia contain a lower level of GSH (), the major molecule to maintain the normal cellular thiol status; and (iii) Nrf2 activation is regulated by oxidative modifications of its cysteine thiol groups, as well as by thiol oxidation in Keap1 (He and Ma 2009
). The cytoprotective effect of GSH in MeHg-induced toxicity is a well known phenomenon (Mullaney et al., 1994
; Mullaney et al., 1993
). GSH readily binds to MeHg via its sulfhydryl groups, and the conjugated products are actively pumped out of the cells by multi-drug resistance proteins, leading to a decrease in intracellular MeHg and, by inference, its ensuing toxicity (Konig et al., 1999
). In addition, GSH prevents the interaction of Hg with protein thiols (Farina et al., 2009
) and detoxifies ROS generated by MeHg to maintain the redox status in the cell (Das et al., 2006
). As shown in , a greater than four-fold higher basal GSH level was noted in astrocytes compared to microglia. Taking into account the fact that Nrf2 activation is critically regulated by thiol oxidation (He and Ma, 2009
), it is reasonable to assume that the lower GSH content in microglia reflects the more rapid Nrf2 and downstream gene activation in this cell type. The differential thiol status in microglia and astrocytes also explains the observed changes in ROS levels and the GSH/GSSG ratio during MeHg exposure, where microglial GSH was depleted faster given its lower basal levels. Intracellular MeHg has been reported to be exported out of cells as the GSH complex (Heggland et al., 2009
); thus, it is also possible that higher GSH levels in astrocytes facilitate the extrusion of Hg from these cells at a faster rate compared with the microglia (). Notably, the basal level of astrocytic GSH was reported to be 80±10nmol/mg protein (Wang et al., 2002
), which is consistent with our results.
MeHg not only directly activates glial cells, but also alters normal CNS function via molecules released by glial cells. For example, upon activation, microglia produce free radicals ( and ) (Long et al., 2007
) and release proinflammatory cytokines, such as interleukin 6 (IL-6) (Chang, 2007
). IL-6 induces astrogliosis associated with MeHg-induced microglial clusters (Eskes et al., 2002
). Interestingly, as a proinflammatory factor, IL-6 may have also a neuroprotective function. IL-6 co-administered with MeHg has been shown to prevent the MeHg-induced degeneration of the neuronal cytoskeleton (Eskes et al., 2002
). Similar protective effects of IL-6 against the toxic effects of glutamate have also been previously described (Yamada and Hatanaka, 1994
). In contrast, astrocytic dysfunction might mediate MeHg toxicity at later stages of exposure. For example, Yin et al. reported disrupted amino acid homeostasis stemming from astrocyte-mediated MeHg toxicity (Yin et al., 2009
). MeHg inhibits astrocytic glutamate uptake while stimulating glutamate efflux (Aschner et al., 1993
), resulting in an excessive concentration of synaptic glutamate, which ultimately leads to neuronal excitotoxicity and cell death (Mutkus et al., 2006
). Astrocytes also appear to dampen microglial activation under physiological conditions (Thomas, 1992
). Therefore, MeHg-induced astrocytic dysfunction may influence microglial function at later stages of MeHg exposure.
In summary, the results presented herein demonstrate different response kinetics in astrocytes and microglia upon MeHg treatment. Microglia exhibited a faster and more robust response to MeHg compared to astrocytes. The microglial response to MeHg reflects a significantly greater accumulation of Hg in these cells vs. astrocytes, as well as a lower basal GSH pool for detoxifying ROS generated by MeHg and direct binding to MeHg itself. The faster microglia activation of Nrf2 and its downstream genes is likely related to its lower thiol status. Collectively, these studies suggest that microglia and astrocytes assume different roles on a protracted time scale, with microglia representing the early responders and astrocytes taking on a similar role at a later stage following MeHg treatment. A better understanding of the distinctive roles of these cells in mediating MeHg toxicity under in vivo conditions is clearly warranted. Indeed, additional insight gained from future studies offers tremendous promise for the identification and development of potential therapeutic modalities to ameliorate MeHg-induced CNS damage.