Rett syndrome microglia damage dendrites and synapses by the elevated release of glutamate

Abstract

MECP2, an X-linked gene encoding the epigenetic factor methyl-CpG-binding protein-2, is mutated in Rett syndrome (RTT) and aberrantly expressed in autism. Most children affected by RTT are heterozygous Mecp2(-/+) females whose brain function is impaired postnatally due to MeCP2 deficiency. Recent studies suggest a role of glia in causing neuronal dysfunction via a non-cell-autonomous effect in RTT. Here we report a potent neurotoxic activity in the conditioned medium (CM) obtained from Mecp2-null microglia. Hippocampal neurons treated with CM from Mecp2-null microglia showed an abnormal stunted and beaded dendritic morphology, and signs of microtubule disruption and damage of postsynaptic glutamatergic components within 24 h. We identified that the toxic factor in the CM is glutamate, because (1) Mecp2-null microglia released a fivefold higher level of glutamate, (2) blockage of microglial glutamate synthesis by a glutaminase inhibitor abolished the neurotoxic activity, (3) blockage of microglial glutamate release by gap junction hemichannel blockers abolished the neurotoxic activity, and (4) glutamate receptor antagonists blocked the neurotoxicity of the Mecp2-null microglia CM. We further identified that increased levels of glutaminase and connexin 32 in Mecp2-null microglia are responsible for increased glutamate production and release, respectively. In contrast, the CM from highly pure Mecp2-null astrocyte cultures showed no toxic effect. Our results suggest that microglia may influence the onset and progression of RTT and that microglia glutamate synthesis or release could be a therapeutic target for RTT.

Figure 1.

Microglia express MeCP2. A, Cortical sections from the indicated mice of 7 weeks of age were coimmunostained for MeCP2 (red nuclear stain) and the microglia marker CD11b (green cytoplasmic stain). Representative photomicrographs are shown. Filled arrowheads point to wt microglia with clear nuclear MeCP2 immunoreactivity, while arrows point to microglia without MeCP2 immunoreactivity in the null mouse. The brightly MeCP2-immunoreactive nuclei are likely those of neurons (empty arrowheads). B, Microglia derived from male mice of indicated Mecp2 genotypes (wt: Mecp2^+/y; null: Mecp2^−/y) were cultured and RNA was extracted. RT-PCR showed the presence of Mecp2 transcript in wt microglia but not in null microglia. C, Western blot analysis of lysates from indicated cells derived from wt or null mice, analyzed by antibodies to MeCP2, the protein loading control β-actin, microglia marker CD11b, and the astrocytic marker GFAP. D, Microglia cultured from mice of indicated Mecp2 genotypes were doubly immunofluorescently (IF) stained as in A and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

Figure 2.

Conditioned medium from Mecp2-null microglia caused damage to dendrites. wt hippocampal neurons, at least 14 d in vitro, were incubated with 50% conditioned media from wt microglia (wt MCM) or null microglia (null MCM) for 24 h. A, Representative phase contrast images and immunofluorescent images in which the dendrites were demonstrated by immunostaining for MAP2 (red) and Ac-TN (green). B, Sparsely plated neurons immunostained for MAP2. Shown are examples of neurons damaged by null MCM showing stunted dendritic morphology and frequent beaded appearance (arrows). C, NeuN-immunoreactive neurons in each treatment condition were counted and averaged from three independent experiments to demonstrate that neuronal viability was not affected by MCM treatment or the drug treatment shown in Figures 3 and 5. The drugs shown here are glutaminase inhibitor DON, to reduce the glutamate production by microglia, and glutamate receptor antagonists MK801 and NBQX, to block the neuronal action of glutamate.

Figure 3.

Null MCM caused damage to the postsynaptic elements of excitatory synapses. wt hippocampal neurons were cultured and treated as shown in Figure 2. A, Neurons were doubly immunofluorescently stained with antibodies to MAP2 (red) and PSD95 (green). Representative photomicrographs showing MAP2-positive dendrites are presented to illustrate the decreased abundance of PSD95-immunoreactive puncta along the dendrites of null-MCM-treated neurons. B, Western blot analysis of lysates from wt neurons with indicated treatment, analyzed by antibodies to dendritic proteins MAP2 and Ac-TN, postsynaptic protein PSD95 and GRIP1, presynaptic protein synaptophysin, and glutamate receptor subunits NR1, GluR2/3, and GluR6/7. The drugs shown here are glutaminase inhibitor DON and glutamate receptor antagonists MK801 and NBQX (see text). C, The levels of indicated synaptic proteins in synaptosomes prepared from 9-week-old Mecp2-null mice and their male wt littermates (n = 3 in each group) were evaluated by Western blot. D, The band intensities were quantified and expressed as percentages of values obtained from wt. *p < 0.05 and **p < 0.001 compared with the wt values.

Figure 4.

Mecp2-null microglia released a high level of glutamate due to increased production by glutaminase and increased release through Cx32 hemichannels. A, The glutamate concentrations in the 24 h conditioned medium of wt microglia (wt MCM), or Mecp2-null microglia (null MCM) with indicated treatments of microglia were measured. The treatments included culturing in medium lacking glutamine (−GLN) or in the presence of glutaminase inhibitor DON, gap junction blocker CBX, gap junction hemichannel blocker Lan, Cx32-mimetic peptides ³²GAP24 and ³²GAP27, and Cx43-mimetic peptide ⁴³GAP27. The vehicle treatment control was DMSO diluted 500-fold into the medium. n = 3, *p < 0.05 and **p < 0.001 compared with the null-MCM-plus-vehicle group, ^#p < 0.05 compared with the wt MCM group. There was no significant difference between the null-MCM-plus-vehicle and the null-MCM-plus-⁴³GAP27 groups. B, The levels of indicated transcripts were measured by quantitative RT-PCR (see text for results). Shown are representative PCR products in gel. C, Western blot analysis of cell lysates from microglia derived from mice of indicated Mecp2 genotype. See text for quantitative data. D, Microglia derived from mice of indicated Mecp2 genotypes were cultured and immunostained for glutaminase (green) and counterstained with DAPI (blue). E, Sections from the frontal brain region of 7-week-old male littermates of indicated Mecp2 genotypes were coimmunostained for CD11b (green) and glutaminase (red) and counterstained with DAPI (blue). Right, Merged images, in which the CD11b-immunoreactive microglia with high levels of glutaminase are indicated by orange-yellow immunoreactive areas (arrowheads). F, Microglia derived from mice of indicated Mecp2 genotypes were cultured and immunostained for Cx32 (red) and counterstained with DAPI (blue).

Figure 5.

Inhibition of glutamate production/release or of glutamate action on receptors ameliorate the toxicity of null MCM. wt hippocampal neurons were treated with wt MCM or null MCM from indicated treatments as shown in Figure 4A. A–C, Neurons were treated as indicated and immunostained for MAP2 (red) and Ac-TN (green). Representative photomicrographs are shown in A. In B (MAP2) and C (Ac-TN), the immunoreactivities were quantified for each treatment and presented as percentages of control (wt MCM treatment group). n = 3, *p < 0.05 and **p < 0.001 compared with the null-MCM-plus-vehicle group; ^#p < 0.001 compared with the wt MCM group. There was no significant difference between the null-MCM-plus-vehicle and the null-MCM-plus-⁴³GAP27 groups. D, Neurons were doubly immunostained for MAP2 (red) and PSD95 (green). Presented are representative merged images, in which the orange-yellow color represents colocalization of MAP2 and PSD95 immunoreactivities. The quantification of the count of PSD95-immunoreactive puncta per unit (100 μm) length of MAP2-positive dendrite is presented in E. n = 3, *p < 0.05 compared with the null-MCM-plus-vehicle group, ^#p < 0.05 compared with the wt MCM group. There was no significant difference between the null-MCM-plus-vehicle and the null-MCM-plus-⁴³GAP27 groups.