Cell-Autonomous Enhancement of Glutamate-Uptake by Female Astrocytes

Abstract

Since gonadal female hormones act on and protect neurons, it is well known that the female brain is less vulnerable to stroke or other brain insults than the male brain. Although glial functions have been shown to affect the vulnerability of the brain, little is known if such a sex difference exists in glia, much less the mechanism that might cause gender-dependent differences in glial functions. In this study, we show that in vitro astrocytes obtained from either female or male pups show a gonadal hormone-independent phenotype that could explain the gender-dependent vulnerability of the brain. Female spinal astrocytes cleared more glutamate by GLAST than male ones. In addition, motoneurons seeded on female spinal astrocytes were less vulnerable to glutamate than those seeded on male ones. It is suggested that female astrocytes uptake more glutamate and reveal a stronger neuroprotective effect against glutamate than male ones. It should be noted that such an effect was independent of gonadal female hormones, suggesting that astrocytes have cell-autonomous regulatory mechanisms by which they transform themselves into appropriate phenotypes.

Introduction

Astrocytes control synaptic transmission by releasing gliotransmitters such as ATP and glutamate or by uptaking excess neurotransmitters (Haydon 2001; Koizumi et al. 2003). As for the clearance of neurotransmitters, astrocytes express excitatory amino acid transporter 1 (EAAT1; GLAST) or EAAT2 (GLT-1), by which they control the extracellular glutamate concentrations and excitatory neurotransmission. Thus, the functions of these transporters are highly involved in glutamate-dependent excitotoxicity or various neuronal diseases.

It is well known clinically and experimentally that female brain is more resistant to various brain insults or neurodegenerative diseases than male brain, and such sex differences have been historically attributed to the protective effect of gonadal female hormones such as estrogen. Studies by Sato et al. (2003) and Pawlak et al. (2005) have already shown that, exogenously applied 17β-estradiol (E2), the most potent mammalian estrogen, affects the activity of glutamate uptake in astrocytes, which may in part explain the gender difference in brain vulnerability. However, sexual dimorphism generally persists well beyond menopause (Sacco et al. 1998), suggesting that sex differences in brain injury may not be entirely related to the influence of gonadal female hormones. In this study, we demonstrate that female astrocytes in vitro are a high glutamate-uptake phenotype that removes more glutamate and protects motoneurons more against glutamate than male ones. We also demonstrate that such a difference in astrocytic function does not depend on gonadal female hormones but depends rather on a local cell-autonomous mechanism (s).

Materials and Methods

All of the animals used in this study were obtained, housed, cared for, and used in accordance with the guidelines of the Universities of Yamanashi and Hiroshima.

Cell Culture

The culture of spinal astrocytes was prepared as described previously (Shibata et al. 2011) with minor modifications. The spinal cord was removed from neonatal male or female Wistar rats. Male and female rat pups were distinguished by the larger genital papilla and longer ano-genital distance in male versus female pups. To remove serum-derived hormones, charcoal-stripped fetal bovine serum was used. The culture of rat motoneurons was prepared as described previously (Nishijima et al. 2001).

Measurement of Glutamate Uptake

Glutamate clearance was measured as previously described (Sato et al. 2003).

Ca²⁺ Imaging in Single Motor Neurons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]i) were measured by the fura-2 method as previously described (Koizumi et al. 2003). The amplitude of the high K⁺-evoked [Ca²⁺]i elevation in motoneurons seeded on either male or female astrocytes was used as an index of neuronal function (Koizumi et al. 1994).

Chemicals

DL-threo-β-Benzyloxyaspartic acid (TBOA) and Dihydrokainate (DHK) were purchased from TOCRIS Bioscience (Bristol, UK). Anti-neurofilament H non-phosphorylated (SMI-32) antibody was from COVANCE Japan Co. Ltd (Tokyo, Japan). All other reagents were from Sigma-Aldrich Japan (Tokyo, Japan).

Statistical Analysis

Experimental results are expressed as means ± S.E.M. Statistical analysis was performed using Student’s t test. One way analyses of variance (ANOVA) followed by Tukey test were applied for multiple comparisons. The differences between means were considered to be significant when the p values were less than 5 %.

Results

Female astrocytes (grey columns) cleared significantly larger amounts of glutamate than male ones (open columns) both at 30 and 60 min (Fig. 1). At 30 min, female astrocytes showed two times higher glutamate uptake activity. When extracellular Na⁺ was removed, the glutamate clearance disappeared almost completely (data not shown), indicating that the extracellular glutamate was uptaken by Na⁺-dependent glutamate transporter(s). To identify the predominant glutamate transporter(s) of cultured astrocytes, we co-applied 0.3 mM TBOA, an inhibitor of both GLAST and GLT-1 (Shimamoto et al. 2004) or 1 mM DHK, a selective inhibitor of GLT-1 (Johnston et al. 1974) with glutamate. TBOA dramatically inhibited the glutamate uptake in both male and female astrocytes, whereas DHK showed only slight inhibition or no effect, suggesting that GLAST was dominant in both cultures.

Fig. 1.

Differences in glutamate (Glu) clearance in female and male astrocytes. The uptake activity of female (grey columns) and male (open columns) astrocytes, 30 and 60 min after incubation with glutamate in the absence (Ctr) and the presence (TBOA or DHK) of inhibitors of glutamate transporters. Female astrocytes uptook higher amounts of glutamate than male ones at both time periods (Ctr). DHK had no or only a slight effect on the glutamate uptake, but TBOA significantly decreased the glutamate clearance both in male and female astrocytes

We then investigated whether such sex-dependent differences in glutamate uptake might affect neuronal damage/death induced by exogenously applied glutamate. Since the glutamate clearance by spinal astrocytes greatly affects the survival of motoneurons (Jimonet et al. 1999), we used motoneurons cultured on either male or female spinal astrocytes. Figure 2a shows phase-contrast images of motoneurons, showing the effects of glutamate-treatment. Healthy motoneurons show phase bright morphology (left panel), but when damaged, they show a dark, flattened shape. Motoneurons were stimulated with glutamate (100 μM) for 30 min, and then washed-out and further incubated with glutamate-free medium for 24 h. The fraction of motoneurons with phase bright morphology was dramatically decreased by the glutamate-treatment. For quantitative analysis, we employed a high K⁺-evoked increase in [Ca²⁺]i in neurons (Koizumi et al. 1994). The treatment with glutamate (100 μM, 30 min, and then 24 h washout) significantly decreased the high K⁺-evoked responses in motoneurons on male astrocytes, whereas it had almost no effect on the [Ca²⁺]i responses in motoneurons on female astrocytes (Fig. 2c). After the Ca²⁺ imaging experiments, cells were stained with anti-SMI-32 antibody to confirm that the cells of interest were motoneurons (Fig. 2b, right).

Fig. 2.

Potent protection of motoneurons by female astrocytes. a Phase-contrast images of motoneurons seeded on male astrocytes, showing the effects of glutamate-treatment. Left panel, healthy motoneurons show phase bright morphology; right panel, motoneurons damaged by glutamate seeded on male astrocytes, show dark, flattened shape. b Fura-2 fluorescent images and immunostaining by SMI-32 antibody. Left panel, fura-2 fluorescence; right panel, immunocytochemical images of anti-SMI32 antibody of the motoneurons after Ca²⁺ imaging experiments. c The high K⁺-evoked increase in [Ca²⁺]i in motoneurons after treatment of cells with glutamate (100 μM, 30 min, and then 24 h washout). Changes in [Ca²⁺]i in cells were expressed as ∆ ratio of F340/F380. The high K⁺-evoked increase in [Ca²⁺]i in motoneurons seeded on male astrocytes was decreased by glutamate but not in those seeded on female astrocytes

Discussion

In this study, we demonstrated that (1) female astrocytes cleared more glutamate by GLAST than male ones; (2) spinal female astrocytes showed stronger protective action against glutamate-evoked neuronal damage in motoneurons than male ones; and most importantly, (3) these characteristic features of female astrocytes were not necessarily dependent on gonadal female hormones, since astrocytes were obtained separately from either female or male pups and cultured in the absence of sex hormones. Although differences in the vulnerability to several types of brain insults between female and male brains are often explained by the fact that gonadal female hormones act on and protect neurons, it is unlikely that such differences are entirely related to the hormonal effects on neurons. Thus, our present results could be novel and important as that we have shown that such sex differences could be explained by (i) functional differences in astrocytes but not neurons, and that (ii) these differences in astrocytic functions do not totally depend on gonadal sex hormones but, presumably, depend on the property of XX versus XY chromosomes, by which each astrocyte is transformed into a distinct phenotype in a cell-autonomous mechanism, although we must await further studies to clarify the detail molecular mechanisms.

As for the peripheral sex hormone-independent mechanisms, extragonadal production of E2 may be involved. E2 can be synthesized locally from testosterone by the aromatase cytochrome P450 in the CNS. In an experimental stroke model, mice with targeted deletion of cyp19, which codes for aromatase P450, showed more severe brain injury than wild-type litter mates (McCullough et al. 2003), suggesting that aromatase and extragonadal E2 play an important role in protection of the brain. It should be noted that astrocytes express aromatase P450, and more importantly, that the expression of P450 is higher in female astrocytes than that in male astrocytes (Liu et al. 2007). These findings suggest that female astrocytes locally produce more estrogen than male, thereby leading to higher expression of glutamate-transporters. The higher capacity to clear glutamate causes the female astrocytes to have higher neuroprotection against glutamate. However, further study is required to clarify this issue.

Spinal astrocytes in vivo express more GLT-1 than GLAST. It is well known that, similar to cultured astrocytes obtained from the hippocampus or cortex, GLT-1 expression becomes less dominant by cultivation. If cultured with neurons, or in the presence of several factors such as cAMP-forming reagents or β-lactam antibiotics, GLT-1 expression is increased (Rothstein et al. 2005). When spinal astrocytes were co-cultured with motoneurons, it is possible that GLT-1 was upregulated contributing to the clearance of glutamate in female astrocytes. Thus, we do not exclude the involvement of GLT-1 in the higher uptake of glutamate in female astrocytes.

Taken together, we demonstrated that spinal astrocytes obtained from female pups showed higher glutamate uptake activity and more intensive neuroprotection against glutamate than those obtained from males. The effect was independent of gonadal female hormones, suggesting that astrocytes have cell-autonomous regulatory mechanisms by which they transform themselves into less vulnerable phenotypes.