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. 2009 Oct 7;29(40):12702-10.
doi: 10.1523/JNEUROSCI.1166-09.2009.

Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

Affiliations

Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

Luis E F Almeida et al. J Neurosci. .

Abstract

cAMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, cAMP response element-binding protein (CREB). However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of l-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca(2+) entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks.

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Figures

Figure 1.
Figure 1.
a, Induction of CREB-dependent gene expression by cAMP requires Ca2+. Stimulation of 4xCRE luciferase activity by forskolin (10 μm) or 8-Br-cAMP (3 mm) was abolished by adding 3 mm Bapta-free acid (Bapta-FA) or by pretreating with Bapta-AM (10 μm). EGTA (3 mm) also blocked stimulation of 4xCRE by cAMP (data not shown). In most experiments, Bapta-FA was used instead of EGTA to chelate extracellular Ca2+ to avoid acidification, which occurs when 3 mm EGTA is added to Ca2+-containing medium. ***Significantly different from forskolin or 8-Br-cAMP alone (p < 0.001). b, Ca2+ is not required for stimulation of cAMP production by forskolin. The forskolin-induced increase in cAMP levels, determined by ELISA, was blocked by the PKA antagonist, H89 (10 μm, data not shown) but was not affected by pretreatment with Bapta-AM (10 μm). ns, Not significantly different from forskolin alone.
Figure 2.
Figure 2.
Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca2+] within a microdomain. One day after transfecting with 4xCRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μm) for 1 h and then stimulated by addition of 10 μm forskolin. Cells were harvested 5 h later for luciferase assays. Bapta completely inhibited forskolin-induced 4xCRE expression, whereas EGTA only inhibited by ∼30%. ***Significantly different from forskolin+Bapta-AM (p < 0.001); ns, not significantly different from untreated.
Figure 3.
Figure 3.
Stimulation of CREB-dependent gene transcription requires activation of NR2B-containing NMDARs and L-type Cavs. a, b, Induction of 4xCRE transcription by 10 μm forskolin (a) or 3 mm 8-Br-cAMP (b) was blocked by the NMDAR antagonists AP5 (100 μm) or ifenprodil (10 μm) and by the L-type Cav antagonists verapamil (100 μm) or nifedipine (100 μm). c, Activation of NMDARs with NMDA (30 μm + 10 μm glycine) induced 4xCRE transcription that was sensitive to AP5 (100 μm) or ifenprodil (10 μm), but not to Cav antagonists. Similarly, NMDA-stimulated 4xCRE expression was blocked by Bapta-free acid (Bapta-FA; 3 mm) or by pretreating neurons with 10 μm Bapta-AM. The L-type Cav antagonist, nifedipine (100 μm), did not reduce NMDA-stimulated 4xCRE transcription. ***Significantly different from agonist only (p < 0.001); ns, not significantly different from NMDA only.
Figure 4.
Figure 4.
Phosphorylation of CREB at serine-133 is not sufficient for 4xCRE transcription. a, Immunoblots were probed for phospho-CREB (top) and then stripped and reprobed for total CREB (bottom) to control for sample variability. Forskolin (F; 10 μm) stimulated CREB phosphorylation when compared with untreated (U) cells. CREB phosphorylation induced by forskolin was insensitive to addition of ifenprodil (F+I) or Bapta-AM (F+B). The effects of forskolin were abolished by incubation with the PKA inhibitor, H89 (10 μm) (data not shown). b, Quantification of CREB phosphorylation (P-CREB/CREB) from 4 experiments; ns, not significantly different from forskolin alone.
Figure 5.
Figure 5.
DA activates CREB-dependent gene transcription. a, Cortical neurons transfected with 4xCRE luciferase were stimulated at 4 DIV with DA (100 μm) or the β-adrenergic agonist, isoproterenol (100 μm), in the absence or presence of 0.1 μm rolipram for 5 h and then harvested for luciferase assay. **Significantly different from untreated neurons, p < 0.005; ***significantly different from DA alone, p < 0.001; ns, not significantly different from untreated control; n = 3 experiments. b, Stimulation by 100 μm DA in the presence of 0.1 μm rolipram was blocked by Bapta-FA (3 mm), AP5 (100 μm), ifenprodil (10 μm), and verapamil (100 μm); this pharmacological profile is identical to that observed with stimulation by cAMP (Fig. 3a,b). The D1 DA receptor antagonist, SCH23390 (30 μm), also completely blocked stimulation by DA+rolipram. ***Significantly different from DA+rolipram, p < 0.001. Ascorbic acid (100 μm) was included to reduce DA oxidation; control experiments demonstrated that ascorbic acid did not stimulate 4xCRE expression (data not shown).
Figure 6.
Figure 6.
Stimulation of mRNA levels of CRE-containing genes by cAMP or D1 DA receptor agonist is mediated by NR2B-containing NMDARs. Cortical neuron cultures were incubated for 24 h in the absence or presence of forskolin (10 μm), the D1 agonist, SKF38393 (100 μm) and/or ifenprodil (10 μm) and cells were harvested for RNA extraction. After reverse transcription, levels of c-fos, and exons I and IV of BDNF were determined by quantitative real-time PCR. Data are expressed relative to untreated cultures. ***Significantly different from corresponding cultures stimulated by forskolin or SKF38393; p < 0.001.
Figure 7.
Figure 7.
EAA uptake inhibition enhances stimulation of CREB-dependent gene transcription by cAMP. The EAA uptake inhibitor, TBOA (10 μm) did not, by itself, stimulate 4xCRE transcription, but potentiated the response induced by forskolin (1 μm) or 8-Br-cAMP (1 mm). ***Significantly different from forskolin or 8-Br-cAMP alone (p < 0.001).
Figure 8.
Figure 8.
l-Aspartate+glutamate scavenger inhibits cAMP-induced 4xCRE transcription but glutamate scavenger does not. a, Addition of a glutamate-only scavenging system (10 mm pyruvate plus 5 U/ml GPT) did not affect forskolin-induced 4xCRE transcription. b, Addition of an l-aspartate+glutamate scavenging system (10 mm pyruvate plus 5 U/ml GPT; 3 mm α-ketoglutarate plus 5 U/ml GOT) completely inhibited the response to forskolin. ns, Not significantly different from forskolin alone; ***significantly different from forskolin alone (p < 0.001).
Figure 9.
Figure 9.
cAMP induces aspartate release from cortical neurons. Four hours after medium replacement, cultures were treated with 100 μm forskolin or vehicle (DMSO) for 30 min. Then, 250 μl of the combined medium from duplicate cultures was removed and subjected to analysis by HPLC as described in Materials and Methods. a, Chromatograms showing aspartate and glutamate peaks; forskolin induced an increase in medium aspartate without significantly affecting glutamate. b, Summary of measurements from 10 pairs of cultures. Bars show means ± SEM; *significantly different from control, p < 0.05 by two-tailed t test; ns, not significantly different from control.
Figure 10.
Figure 10.
Model illustrating the signaling pathways mediating stimulation of CREB-dependent gene transcription by DA and cAMP in mouse cortical neurons prior to synaptogenesis. Like forskolin, activation of the D1/D5 class of DA receptors (D1/5-AC), which are positively linked to adenylyl cyclase, induces a rise in intracellular cAMP, leading to release of l-aspartate (left, dashed arrows). This cAMP can also stimulate CREB phosphorylation via PKA activation (dotted arrow), but CREB-dependent gene transcription requires activation of ifenprodil-sensitive, NR2B-containing NMDA receptors by aspartate and Ca2+ entry (right, solid arrows). Direct activation of NMDARs by NMDA may induce transcription independently of cAMP, because the NMDA-induced rise in Ca2+ is sufficient to activate both CREB and a coactivator (e.g., CBP) via a CaMK. This schema shows aspartate acting via an autocrine mechanism (i.e., aspartate acts on the same cell that releases it); however, our data are also consistent with a paracrine mechanism (if the released aspartate acts on neighboring neurons).

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