Alterations of Gene Expression and Glutamate Clearance in Astrocytes Derived from an MeCP2-Null Mouse Model of Rett Syndrome

<div><p>Rett syndrome (RTT) is a neurodevelopmetal disorder associated with mutations in the methyl-CpG–binding protein 2 (MeCP2) gene. MeCP2-deficient mice recapitulate the neurological degeneration observed in RTT patients. Recent studies indicated a role of not only neurons but also glial cells in neuronal dysfunction in RTT. We cultured astrocytes from MeCP2-null mouse brain and examined astroglial gene expression, growth rate, cytotoxic effects, and glutamate (Glu) clearance. Semi-quantitative RT-PCR analysis revealed that expression of astroglial marker genes, including GFAP and S100β, was significantly higher in MeCP2-null astrocytes than in control astrocytes. Loss of MeCP2 did not affect astroglial cell morphology, growth, or cytotoxic effects, but did alter Glu clearance in astrocytes. When high extracellular Glu was added to the astrocyte cultures and incubated, a time-dependent decrease of extracellular Glu concentration occurred due to Glu clearance by astrocytes. Although the shapes of the profiles of Glu concentration versus time for each strain of astrocytes were grossly similar, Glu concentration in the medium of MeCP2-null astrocytes were lower than those of control astrocytes at 12 and 18 h. In addition, MeCP2 deficiency impaired downregulation of excitatory amino acid transporter 1 and 2 (EAAT1/2) transcripts, but not induction of glutamine synthetase (GS) transcripts, upon high Glu exposure. In contrast, GS protein was significantly higher in MeCP2-null astrocytes than in control astrocytes. These findings suggest that MeCP2 affects astroglial genes expression in cultured astrocytes, and that abnormal Glu clearance in MeCP2-deficient astrocytes may influence the onset and progression of RTT.</p> </div

Effect of glutamate on glutamine synthetase and EAAT1 protein expression in MeCP2-null astrocytes.

<p><b>A.</b> Time-dependent expression of GS and EAAT1 proteins in wild-type and MeCP2-deficient astrocyte cultures. Astrocytes were treated with 1.0 mM Glu for 24 h, and subsequently analyzed for expression of GS and EAAT1 by Western blot analysis. Beta-actin protein levels were analyzed in the same way, as an internal control. <b>B.</b> The immunoreactive GS protein bands were quantified by densitometry, normalized against β-actin levels, and expressed as fold change relative to the controls (equals 1.0). Bars represent the means ± SE of samples from three independent experiments (*p<0.05, **p<0.01). Numbers in each column indicate the total number of samples analyzed. <b>C.</b> The immunoreactive EAAT1 protein bands were quantified by densitometry, normalized against β-actin levels, and expressed as % of controls (equals 100%). Bars represent the means ± SE of samples from three independent experiments (**p<0.01). <b>D.</b> Comparison of the effects of Glu on EAAT1 expression in wild-type and MeCP2-null astrocytes. The ratio of EAAT1/β-actin in each treatment group was normalized against that of the non-treated astrocytes from each group. Bars represent the means ± SE of samples from three independent experiments. Numbers in each column indicate the total number of samples analyzed.</p

Cell growth and viability.

<p><b>A.</b> Comparison of cell growth in wild-type and MeCP2-deficient astrocytes. As passage number increased, cell growth rate decreased dramatically in both strains of astrocytes. There was no significant difference in growth rate between the control and MeCP2-null astrocyte cultures. <b>B.</b> Quantification of BrdU-incorporating cells in control and MeCP2-null astrocytes. Astrocytes were cultured for 24 h and incubated with BrdU for 2 h. The graph shows the percentage of BrdU-incorporating cells in the control (white column) and MeCP2-deficient (gray column) astrocytes 2 h after BrdU exposure. The number of BrdU-incorporating cells is expressed as a percentage of the total number of Hoechst-stained cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035354#pone.0035354.s001" target="_blank">Fig. S1</a>). Bars represent the means ± SE of the samples from four independent experiments. The ratio of BrdU-incorporating cells is similar in astrocytes taken from both control and MeCP2-null strains. <b>C–E.</b> Comparison of effects of various neurotoxins (<b>C,</b> H₂O₂; <b>D,</b> NH₄Cl; <b>E,</b> Glutamate) on control and MeCP2-null astrocytes. The graph shows the percentage of viability in the control (white column) and MeCP2-deficient (gray column) astrocytes after neurotoxin treatment at the indicated concentrations. Bars represent the means ± SE of samples from three independent experiments. The glial cultures showed no difference in viability between the control and MeCP2-null strains.</p

Characterization of assay cultures.

<p><b>A.</b> Expression of astroglial genes in primary cultured cortical astrocytes. Semi-quantitative RT-PCR analysis of Mecp2 and astroglial genes was performed in wild-type (white column) and MeCP2-null (gray column) astrocytes. Mecp2 e1 and e2 were detectable in the wild-type astrocytes. The lower graphs show that the GFAP/HPRT or S100β/HPRT expression ratio in each genotype was normalized against the level in control astrocytes. Bars represent the means ± standard errors (SE) of samples from three independent experiments (*p<0.05). The expression of astroglial markers was significantly upregulated by MeCP2 deficiency. <b>B.</b> Expression of MeCP2 in the primary cultured cortical astrocytes. The astrocytes were immunostained with MeCP2 (green) and GFAP (red) as glial-specific astrocytic markers. Scale bars indicate 50 µm.</p

Comparison of glutamate clearance in wild-type and MeCP2-null astrocytes.

<p><b>A.</b> Time-dependent reduction of extracelluar Glu concentration in wild-type (white column) and MeCP2-null (gray column) astrocyte cultures. After treatment with 1.0 mM Glu, culture supernatant was collected at the indicated times for the determination of Glu concentration. The graph shows the concentration of Glu in control and MeCP2-null astrocyte culture medium. Bars represent the means ± SE of samples from three independent experiments (*p<0.05). <b>B–D.</b> Effects of inhibitors of glutamate transporters (<b>B,</b> TBOA; <b>C,</b> DHKA; <b>D,</b> UCPH) on Glu clearance. Astrocytes were exposed to the indicated concentration of Glu transporter inhibitors, and then 0.1 mM Glu was added; culture supernatant was collected for the determination of Glu concentration at 2 h. The graphs show the clearance ratio upon treatment with each inhibitor. The clearance ratio in the indicated concentration groups was expressed by defining the control level (no inhibitor) as 1.0. Bars represent the means ± SE of samples from three independent experiments.</p

Effect of glutamate on glutamine synthetase and glutamate transporter gene expression in MeCP2-null astrocytes.

<p><b>A–C.</b> Effects of Glu on Glu clearance-related genes in wild-type (white column) and MeCP2-null (gray column) astrocytes. Semi-quantitative RT-PCR analysis of Glu clearance-related genes, EAAT1 (<b>A</b>), EAAT2 (<b>B</b>), and GS (<b>C</b>), was performed in the control and MeCP2-null astrocytes 12 or 24 h after treatment with 1.0 mM Glu. The bands corresponding to PCR products were quantified by densitometry, normalized against HPRT levels, and expressed as % of controls (equals 100%). Bars represent the means ± SE of samples from 3–4 independent experiments (*p<0.05, **p<0.01). <b>D–F.</b> Comparison of the effects of Glu on EAAT1, EAAT2 or GS expression in the control and MeCP2-null astrocytes. The ratio of EAAT1/HPRT (<b>D</b>), EAAT2/HPRT (<b>E</b>) or GS/HPRT (<b>F</b>) in each treatment group was normalized against that of the non-treated astrocytes from each group. Bars represent the means ± SE of samples from 3–5 independent experiments (*p<0.05). Numbers in each column indicate the total number of samples analyzed.</p