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. 2010 Apr 1;1(4):e33.
doi: 10.1038/cddis.2010.10.

CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

J Nicolai  1 , S Burbassi, J Rubin, O Meucci
Affiliations

CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival

J Nicolai et al. Cell Death Dis. .

Abstract

Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl-D-aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.

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Figures

Figure 1
Figure 1
CXCL12 pretreatment prevents neuronal death and decreases NMDA-induced intracellular calcium rise. CXCL12 (20 nM) or vehicle was added to the culture media of rat cortical neurons 24 h before NMDA exposure, as described in the ‘Materials and methods' and ‘Results' sections. (a) Cell survival was assayed 24 h after NMDA treatment (20 min, 100 μM). The graph shows NMDA-induced cell death in cultures pretreated with vehicle, or CXCL12 (in the presence or absence of the CXCR4 antagonist AMD3100); basal cell death in untreated neurons was 11.15±1.42, (mean±S.E.M., n=3), as indicated by the line in the graph. AMD3100 (100 ng/ml) was added to the cultures 20 min before CXCL12 (*P<0.01, ANOVA; CXCL12-treated significantly lower than vehicle and AMD3100+CXCL12). (b) CXCL12 pretreatment decreases calcium response stimulated by NMDA (100 μM, 90 s). The effect was blocked by cotreatment with AMD3100 (100 ng/ml). Traces show average ratios (mean±S.E.M.) from at least 60 cells per treatment analyzed individually (*P<0.05, ANOVA; CXCL12 significantly lower than vehicle and AMD3100+CXCL12). (c) Similar responses are observed with lower NMDA concentrations (50 μM). (d) NMDA-induced (50 μM) calcium rise in the absence (first and second peak) and presence (third peak) of the L-type calcium channel inhibitor nifedipine (5 μM)
Figure 2
Figure 2
CXCL12 treatment reduces levels of NR2B protein and mRNA but does not alter other NMDA subunits. (a) Addition of CXCL12 (20 nM, 1–24 h) to neuronal culture media decreases NR2B protein levels in a time-dependent manner. Graph shows data from three independent experiments (*P<0.05 ANOVA; 3, 6, 24 h compared with control) (b) The CXCR4 antagonist AMD3100 (100 ng/ml; added to cultures 20 min before CXCL12), blocked the effect of CXCL12 (20 nM) on NR2B protein levels. Bar graph reports data from four independent experiments (*P<0.05 ANOVA; CXCL12 compared with all other groups). (c) mRNAs isolated from neuronal cultures treated with CXCL12 (20 nM, 6 h) were analyzed by RT-PCR for NR2B, NR2A, or NR1 receptor levels. Separate gels were run for NR2A and AldoA because of close fragment lengths. (d) qPCR studies were performed on control and CXCL12-treated neurons. The graph indicates relative changes in NR2B and NR1 levels normalized to the housekeeping gene enolase 1 (*P<0.05 ANOVA; 15 h compared with control)
Figure 3
Figure 3
CXCL12 reduces global histone H3 acetylation in neurons, and histone deacetylase (HDAC) inhibitors prevent the effects of CXCL12 on the NR2B. (a) Global H3 acetylation levels were measured through a colorimetric acetylation assay as indicated in the ‘Materials and methods' section. Reduced levels of histone acetylation were found in CXCL12-treated (20 nM) neurons compared with control; this effect is blocked by cotreatment with TSA (100 nM) (*P<0.05 ANOVA; CXCL12 compared with all other groups) (b) Changes in NR2B protein levels in CXCL12- and/or TSA-treated neurons were analyzed by western blot. CXCL12 (20 nM) and/or TSA (100 nM) were added to neuronal culture media for 24 h; the bar graph reports the band density normalized to actin from three independent experiments (*P<0.05 ANOVA; CXCL12 compared with all other groups). (c) Fura-2 studies indicate that TSA (100 nM) treatment abolished the effect of CXCL12 (20 nM) on NMDA-induced (100 μM) calcium responses. Data in the bar graph are reported as changes in the 340/380 ratios (i.e., from baseline to peak); at least 20 cells per treatment were analyzed from three independent experiments (*P<0.05 ANOVA; CXCL12 compared with all other groups)
Figure 4
Figure 4
In vivo AMD3100 administration increases NR2B protein levels in the rat cortex. (a) AMD3100 treatment decreases CXCR4 phosphorylation in brain slices of treated animals as detected through immunohistochemistry, using phospho-specific antibodies against ligand-activated CXCR4. Three animals per group were analyzed and no changes were observed in total levels of CXCR4. (b) Studies in homogenized tissue samples (cerebral cortex and hippocampus) also show a reduction in phosphorylated levels of CXCR4 compared with total CXCR4 (*P<0.05 Student's t-test). The graph reports averaged data obtained from eight animals per group (representative immunoblot on the top). (c) AMD3100 pretreatment in vivo decreases [35S] GTPγS incorporation after stimulation of brain slices with CXCL12. Analysis was performed in different brain areas as previously reported: medial cortex (MC) and lateral cortex (LC). Three animals per treatment group were analyzed (*P<0.05 Student's t-test; AMD3100 compared with control in each brain region). (d) In vivo AMD3100 (1.25 mg/kg) treatment decreases NR2B protein levels in brain tissue (cerebral cortex and hippocampus) collected from P4 pups and analyzed by western blot. In all, 11 animals per treatment group were analyzed (*P<0.05 Student's t-test)
Figure 5
Figure 5
Effect of CXCL12 and RO-256981 on neuronal survival and NMDA-induced calcium changes. (a) CXCL12 (20 nM, 24 h) and the NMDA antagonist RO-256981 (10 μM, 24 h) independently protect neurons from NMDA treatment (*P<0.05 ANOVA; NMDA-treated compared with basal and all other treatment groups). (b) Calcium imaging studies show that RO-256981 markedly reduces NMDA-induced intracellular calcium rise both in control and CXCL12-treated neurons. The graph shows average calcium responses (mean±S.E.M.; ∼20 cells per group)
Figure 6
Figure 6
CXCL12 selectively decreases calcium responses induced by stimulation of NMDA extrasynaptic receptors. (a) Control neurons and CXCL12 pretreated neurons were stimulated with bicuculline (50 μM) and 4-AP (2.5 mM) to evoke a synaptic response, followed by a coapplication of bicuculline, 4-AP, and MK801 (10 μM) to specifically and irreversibly block the synaptic NMDA receptors response. The following NMDA stimulation represents the extrasynaptic response induced by a bath application of NMDA (50 μM). These experiments indicate a dramatic reduction in extrasynaptic response (graph b) and modest stimulation in synaptic responses (graphs c and d). Data obtained from three or more independent experiments; a total of at least 45 neurons per group were studied (*P<0.05)
Figure 7
Figure 7
CXCL12 treatment enhances ERK activation stimulated by synaptic NMDA receptor activation. (a) Effect of CXCL12 on ERK activation after treatment with bicuculline and 4-AP, as reported above. Phospho-ERK immunoreactivity was normalized to total ERK, and the bar graph represents band density as a percentage of control from four independent experiments. (*P<0.05 ANOVA; 5 and 15 min Bic/4AP-treated in CXCL12 group compared with control). (b) Selective activation of synaptic glutamate receptors by application of bicuculline and 4-AP for the indicated time increases CXCR4 protein levels in a time-dependent manner. Bar graph reports average data from three independent experiments (*P<0.05 ANOVA; 4 and 6 h versus control)
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