Deletion of adenosine A2A receptors from astrocytes disrupts glutamate homeostasis leading to psychomotor and cognitive impairment: relevance to schizophrenia

Abstract

Background: Adenosine A2A receptors (A2AR) modulate dopamine and glutamate signaling and thereby may influence some of the psychomotor and cognitive processes associated with schizophrenia. Because astroglial A2AR regulate the availability of glutamate, we hypothesized that they might play an unprecedented role in some of the processes leading to the development of schizophrenia, which we investigated using a mouse line with a selective deletion of A2AR in astrocytes (Gfa2-A2AR knockout [KO] mice].

Methods: We examined Gfa2-A2AR KO mice for behaviors thought to recapitulate some features of schizophrenia, namely enhanced MK-801 psychomotor response (positive symptoms) and decreased working memory (cognitive symptoms). In addition, we probed for neurochemical alterations in the glutamatergic circuitry, evaluating glutamate uptake and release and the levels of key proteins defining glutamatergic signaling (glutamate transporter-I [GLT-I], N-methyl-D-aspartate receptors [NMDA-R] and α-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors [AMPA-R]) to provide a mechanistic understanding of the phenotype encountered.

Results: We show that Gfa2-A2AR KO mice exhibited enhanced MK-801 psychomotor response and decreased working memory; this was accompanied by a disruption of glutamate homeostasis characterized by aberrant GLT-I activity, increased presynaptic glutamate release, NMDA-R 2B subunit upregulation, and increased internalization of AMPA-R. Accordingly, selective GLT-I inhibition or blockade of GluR1/2 endocytosis prevented the psychomotor and cognitive phenotypes in Gfa2-A2AR KO mice, namely in the nucleus accumbens.

Conclusions: These results show that the dysfunction of astrocytic A2AR, by controlling GLT-I activity, triggers an astrocyte-to-neuron wave of communication resulting in disrupted glutamate homeostasis, thought to underlie several endophenotypes relevant to schizophrenia.

Keywords: A(2A)R; Adenosine; Astrocytes; GLT-I; NMDA-R; Schizophrenia.

Figure 1

Selective deletion of A_2AR in astrocytes triggered psychomotor and cognitive endophenotypes of schizophrenia. (A) Whole-brain astrocytes (GFAP+ cells) and neurons (β-tubulin III1 cells, Tub1) from Gfa2-A_2AR KO mice [Cre(+)] and corresponding WT littermates [Cre(−)] were separated by flow cytometry, followed by PCR analysis of genomic DNA of the sorted cells. PCR analysis demonstrated the accuracy of the recombination, showing that the band corresponding to the recombined (“Rec”) allele was present only in astrocytes but not in neurons from Gfa2-A_2AR KO mice. (B) Representative images show upregulation of A_2AR density in astrocytic-like cells in the cerebral cortex of WT but not Gfa2-A_2AR KO mice in response to MPTP treatment. Following MPTP treatment, both GFAP (red staining) and A_2AR (green staining) levels were specifically upregulated in cortical astrocytes of WT mice (shown in the first panel from the right and second panel from the left, respectively). Conversely, the Gfa2-A_2AR KO mice challenged with MPTP displayed no A_2AR immunoreactivity in cortical astrocytes (second panel from the right), despite the increased GFAP immunoreactivity (first panel from the left). (B) Bottom row are 15× higher magnification sections of the boxes in the top row. These findings confirmed the selective deletion of A_2AR in astrocytes of MPTP-intoxicated Gfa2-A_2AR KO mice. Bar = 500 μm. (C) When mice were injected with MK-801 (.5 mg/kg, IP) and activity was recorded for another 3 hours, Gfa2-A_2AR KO mice showed a 15× higher peak response than WT mice; in contrast, MK-801 caused a lower increase of locomotor activity in CaMKIIα-A_2AR KO than in WT mice. (D) Graph shows the grouped analysis of the total locomotor activity in the MK-801 post-injection period (75–200 minutes), confirming that the selective deletion of astrocytic A_2AR versus neuronal A_2AR produced opposite effects on the MK-801-induced hyperlocomotor response. (E) Gfa2-A_2AR KO mice displayed a spatial working memory deficit in a classical Y-maze paradigm. (F,G) Working memory performance was consistently impaired in Gfa2-A_2AR KO mice compared to WT mice, in an 8-baited radial arms test, as evident by increased working memory errors over 6 consecutive trials (F) or by total number of errors for the entire 6 trials (G). Values are means 6 SEM, and statistical differences were assessed with two-way ANOVA and Bonferroni post-hoc analysis with n = 12 mice per group. ^*p < .05, ^**p < .01, GFAP-A_2AR WT versus GFAP-A_2A R KO mice. ^#p < .05, ^## p < .01, CaMKIIα-A_2AR- WT versus CaMKIIα-A_2AR KO. DHK, dihydrokainate; KO, knockout; R, receptor; WT, wild type.

Figure 2

Selective deletion of astrocytic A_2AR upregulated glutamate uptake activity in Gfa2-A_2AR KO mice. Gliosomes and synaptosomes from Gfa2-A_2AR KO, CaMKIIα-A_2AR KO mice, and corresponding WT mice were prepared after acute (30-minute) IP administration of vehicle or SCH58261 (SCH, .1 mg/kg, IP). Then, glutamate uptake was estimated by assaying D-[³H]aspartate (Asp) uptake for 10 minutes. Glutamate uptake was selectively enhanced in gliosomes (A) but not synaptosomes of Gfa2-A_2AR KO mice (B) and was not modified in either gliosomes (B) or in synaptosomes (D) from CaMKIIα-A_2AR KO mice. Furthermore, the A_2AR antagonist SCH58261 enhanced glutamate uptake only in gliosomes from CaMKIIα-A_2AR KO mice (B) but not from Gfa2-A_2AR KO mice (B) and was devoid of effect in synaptosomes from both types of mice (C,D). Accordingly, GLT-I density (E) was also significantly increased in total membranes from the cerebral cortex of Gfa2-A_2AR KO mice (black columns) compared to those in WT mice (white columns), with no differences in the densities of GLAST (F) or GFAP (G). (E–G) Bars represent the relative immunoreactivity obtained with each primary antibody compared to β-actin immunoreactivity (reference), expressed as percentages of the immunoreactivity found in WT littermates. Data are means 6 SEM from at least six independent mice or experiments. ^*p < .05, ^***p < .001; one-way ANOVA Tukey’s post hoc test was used to compare results with those from naive WT mice. DHK, dihydrokainate; KO, knockout; R, receptor; WT, wild type.

Figure 3

Selective inhibition of the glutamate transporter GLT-I reverts psychomotor and cognitive abnormalities in Gfa2-A_2AR KO mice. (A) Mice were habituated and then treated with dihydrokainate (DHK, 10 mg/kg, IP) 30 minutes before MK-801 (.5 mg/kg, IP), and activity was recorded for another 3 hours. DHK blunted the exacerbated response of Gfa2-A_2AR KO mice to MK-801, whereas it was devoid of effects in WT mice. Statistical differences were assessed using Tukey’s post hoc test, applied after one-way ANOVA. (B) Grouped analysis of the total locomotor activity in the MK-801 post-injection period (75–200 minutes) shows DHK blockade of the enhanced psychomotor response to MK-801 in Gfa2-A_2AR KO mice. (C) DHK treatment abrogated the impairment of working memory performance displayed by Gfa2-A_2AR KO mice, assessed as the spontaneous alternation in a Y-maze test. Data are means ± SEM of the percentages of alternations of total possible alternations. Statistical differences, ^*p < .05 and ^**p < .01, comparing the indicated columns, were assessed using Tukey’s post-hoc test applied after one-way ANOVA with n = 8 to 12 mice per group. DHK, dihydrokainate; KO, knockout; MK, N-Methyl-D-aspartate receptor antagonist; R, receptor; WT, wild type.

Figure 4

Gfa2-A_2AR KO mice displayed adaptive changes in glutamatergic synapses, with increased evoked release of glutamate from nerve terminals, enhanced density of NR2B, and decreased membrane surface levels of GluR1/GluR2. Release of basal or depolarization Ca²¹-dependent–evoked release of L-[³H]glutamate from cerebral cortical gliosomes (A) or synaptosomes (B) was evaluated in the absence or presence of the A_2AR agonist CGS21680 (CGS, 50 nM). Depolarizing conditions triggered an evoked release from both the gliosomes (A) and the synaptosomes (B), which was larger in synaptosomes from Gfa2-A_2AR KO mice, the only preparation where CGS21680 exacerbated the evoked release of glutamate. Comparison of Western blots of the densities of NMDA-R-NR1 (C), NMDA-R-NR2A (D), and NMDA-R-NR2B (E) in synaptosomal membranes and those of AMPA-R-GluR1 (F) and AMPA-R-GluR2 (G) in crude membranes and biotinylated extracts from synaptosomes from the cortex of Gfa2-A_2AR KO and WT mice. Density ratios for AMPA-R subunits were determined by dividing the surface intensity by the total intensity. Data are means ± SEM from six independent mice or experiments performed in triplicate. ^*p < .05, ^**p < .01, compared with basal conditions. ^#p < .05, ^##p < .01 compared with WT mice, one-way ANOVA, Tukey’s post-hoc test. DHK, dihydrokainate; KO, knockout; R, receptor; WT, wild type.

Figure 5

Blockade of the regulated endocytosis of AMPA-R GluR2 subunits with Tat-Glur2_3Y reversed the working memory impairment and blunted the exacerbation of MK-801-induced psychomotor activity in Gfa2-A_2AR KO mice, namely in the nucleus accumbens (NAc). (A,B) Tat-GluR2_3Y and the scrambled Tat-GluR2_3S control peptide were injected IP in Gfa2-A_2AR KO and WT mice 60 minutes before they were sacrificed for preparation of cerebral cortical synaptosomes, followed by biotinylation and Western blot analysis. Decrease of the plasma membrane levels of GluR1 (A) and GluR2 (B) in Gfa2-A_2AR KO mice was reverted by Tat-Glur2_3Y but unaffected by Tat-GluR2_3S. (C–F) Tat-GluR2_3Y and Tat-GluR2_3S were IP injected in Gfa2-A_2AR KO and WT mice 90 minutes before MK-801 administration or 60 minutes before Y-maze testing. (C) Gfa2-A_2AR KO mice treated with Tat-GluR2_3Y did not display the exacerbated response to MK-801, observed in the presence of Tat-GluR2_3S. (D) Grouped analysis of the total locomotion in the MK-801 post-injection period confirmed that Tat-Glur2_3Y prevented the enhanced MK-801 response in Gfa2-A_2AR KO mice. (E) Gfa2-A_2AR KO mice bilaterally injected in the NAc with Tat-GluR2_3Y did not display the significantly increased response to MK-801 observed with intra-NAc injection of Tat-GluR2_3S. (F) Data show that the impairment of working memory performance observed in Gfa2-A_2AR KO mice subject to a conventional Y-maze test was reverted by Tat-Glur2_3Y but not by Tat-GluR2_3S. Values are the means ± SEM of n = 8 to 12 mice per group. ^*p < .05, ^**p < .01, ^***p < .001, comparing the indicated columns in the bar graphs (A,B,D,F) and ^*p < .05, ^**p < .01 comparing Gfa2-A_2AR KO versus WT mice in the time course graphs (C,E), using two-way ANOVA followed by Tukey’s post hoc test. DHK, dihydrokainate; KO, knockout; MK, N-Methyl-D-aspartate receptor antagonist; R, receptor; WT, wild type.