doi: 10.1523/JNEUROSCI.4873-09.2009.
Nuclear factor of activated T-cells isoform c4 (NFATc4/NFAT3) as a mediator of antiapoptotic transcription in NMDA receptor-stimulated cortical neurons
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- PMID:19955386
- PMCID: PMC2836809
- DOI: 10.1523/JNEUROSCI.4873-09.2009
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Nuclear factor of activated T-cells isoform c4 (NFATc4/NFAT3) as a mediator of antiapoptotic transcription in NMDA receptor-stimulated cortical neurons
Aruna Vashishta et al. J Neurosci..
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Abstract
During cortical development, when NR2B subunit is the major component of the NMDA glutamate receptors (NMDARs), moderate NMDAR activity supports neuronal survival at least in part by regulating gene transcription. We report that, in cultured cortical neurons from newborn rats, the NMDARs activated the calcium-responsive transcription regulator nuclear factor of activated T cells (NFAT). Moreover, in developing rat cortex, the NFAT isoforms c3 and c4 (NFATc3 and NFATc4) were expressed at relatively higher levels at postnatal day 7 (P7) than P21, overlapping with the period of NMDAR-dependent survival. In cultured cortical neurons, NFATc3 and NFATc4 were regulated at least in part by the NR2B NMDAR. Conversely, knockdown of NFATc4 but not NFATc3 induced cortical neuron apoptosis. Likewise, NFATc4 inhibition prevented antiapoptotic neuroprotection in response to exogenous NMDA. Expression of the brain-derived neurotrophic factor (BDNF) was reduced by NFATc4 inhibition. NFATc4 regulated transcription by the NMDAR-responsive bdnf promoter IV. In addition, NMDAR blockers including NR2B-selective once reduced BDNF expression in P7 cortex and cultured cortical neurons. Finally, exogenous BDNF rescued from the proapoptotic effects of NFATc4 inhibition. These results identify bdnf as one of the target genes for the antiapoptotic signaling by NMDAR-NFATc4. Thus, the previously unrecognized NMDAR-NFATc4-BDNF pathway contributes to the survival signaling network that supports cortical development.
Figures
Regulation of NFAT-driven transcription by NMDAR.A, At DIV4, rat cortical neurons were transfected with the indicated reporter plasmids and EF1αLacZ (0.2 + 0.125 μg of plasmid DNA/5 × 105 cells, respectively). Forty-eight hours after transfection, cells were treated for 6 h with 10 μm ifenprodil (IFN), 10 μm MK801, or vehicle (Veh, 0.2% DMSO). Cortical neurons from newborn transgenic mice carrying an NFAT-driven luciferase reporter gene underwent the same pharmacological treatment at DIV7. A specific decrease of NFAT-driven transcription after ifenprodil or MK801 treatments suggests NFAT dependence on the basal NR2B NMDAR activity.B, Rat cortical neurons were transfected as inA. To enhance synaptic activity, bicuculline/4AP (50 μm + 2.5 mm , Bicc/4AP) was applied for 16 h at DIV6. After blocking neuronal electrical activity with 1 mm TTX, bicuculline/4AP failed to activate NFAT-driven transcription. In addition, the synaptic activity-induced NFAT-driven transcription was reduced after blocking NR2B NMDAR. InA, data represent means ± SEM of triplicate determinations from at least three independent experiments; inB, triplicate determinations from sister cultures ± SD are depicted. **p < 0.01; ***p < 0.001. Results similar to those inB were obtained in at least two independent experiments.
NFAT isoform expression in cultured rat cortical neurons and in developing rat cortex.A, Reverse transcription-PCR analysis of NFAT isoform mRNA expression in cultured rat cortical at DIV7. GAPDH mRNA levels were also determined.B,C, The qRT-PCR was used to determine the NFAT isoform mRNA levels in developing rat neocortex at P1, P7, and P21. The 18S rRNA levels were used for normalizations. Note that expression of NFATc3 and NFATc4 peaks at P7 and declines at P21, overlapping with the maximum expression of the NR2B NMDAR and the survival dependence of cortical neurons on NMDAR (Sheng et al., 1994; Ikonomidou et al., 1999). InB andC, each data point represents six to seven animals ± SEM. *p < 0.05; ***p < 0.001.
Regulation of NFATc3/c4 by the NR2B NMDAR.A, The DIV4 cortical neurons were cotransfected with NFAT–luciferase reporter plasmid, EF1αLacZ, and wild-type NFATc3 or NFATc4 (0.2 + 0.125 + 0.2 μg of plasmid DNA/5 × 105 cells, respectively). Forty-eight hours after transfection, cells were treated with NMDA for 6 h in the presence of the NMDAR inhibitors: 10 μm ifenprodil (IFN), 0.5 μm Ro-25-6981 (Ro), 100 μm APV, or 10 μm MK801. Vehicle control was 0.2% DMSO (Veh). NFATc3/c4 activation by exogenous NMDA required NR2B.B, The DIV4 cortical neurons were cotransfected with the expression plasmid for wild-type NFATc4 that was tagged with GFP (0.4 μg of plasmid DNA/5 × 105 cells). Forty-eight hours after transfection, cells were treated with NMDA in the presence of the PP2B inhibitor FK506 or the ERK1/2 pathway blocker U0126 for 6 h. Thez-stack confocal images of GFP–NFATc4 revealed NMDAR-mediated nuclear translocation of NFATc4. This translocation was blocked by PP2B but not extracellular signal-regulated kinase inhibition. Similar results were obtained in three independent experiments using either GFP–NFATc3 or GFP–NFATc4.C, Forty-eight hours after transfection that was performed as inA, cells were treated with NMDA in the presence of FK506 or U0126 for 20 h as indicated. NMDAR-mediated activation of NFATc3/c4 was antagonized by blocking either PP2B or the ERK1/2 pathway. InA andC, averages ± SD of triplicate determinations from sister cultures are shown. Similar results were obtained in at least two independent experiments.
The shRNA-mediated inhibition of NFATc3/c4 reduces activation of NFAT-driven transcription in synaptically stimulated cortical neurons.A,B, DIV4 cortical neurons were cotransfected with expression plasmids for the GFP-tagged NFATc3 or the HA-tagged NFATc4 together with the NFAT–luciferase reporter, the β-gal expression plasmid EF1αLacZ, and the shNFATc3 or shNFATc4-N (0.2 + 0.2 + 0.125 + 1.1 μg of plasmid DNAs/5 × 105 cells, respectively). Control shRNAs included shRNA expression plasmids targeting SRF coactivator MKL1 (control shRNA for shNFATc3) or GFP (shGFP, used as a control for shNFATc4-N). Two days after transfection, cells were stimulated with 10 μm NMDA for 20 h. The shNFATc3 or shNFATc4 inhibited NMDAR-mediated activation of their respective targets. These results together with data from additional validation studies (supplemental Fig. 1, available atwww.jneurosci.org as supplemental material) suggest that shNFATc3 or shNFATc4 specifically inhibited NFATc3 or NFATc4, respectively.C, Neurons were transfected with shRNAs as described inA andB except omission of the NFATc3/c4 expression plasmids. At 72 h after transfection, cells were stimulated with bicuculline/4AP (50 μm + 2.5 mm , Bic/4AP) for 8 h. The synaptic activity-mediated increase of NFAT-driven transcription was reduced by shNFATc3 or shNFATc4-N. Because synaptic activity-mediated stimulation of NFAT is NMDAR dependent, these results indicate a role for NMDAR in regulating endogenous NFATc3/c4 in rat cortical neurons. InA andB, averages ± SD of triplicate determinations from sister cultures are depicted. Similar results were obtained in at least two independent experiments. InC, data represent averages ± SEM from six sister cultures in two independent experiments. **p < 0.01; ***p < 0.001.
Cortical neuron apoptosis in response to NFATc4 knockdown. The DIV4 cortical neurons were cotransfected with control shRNA (shGFP), shNFATc4-N, or shNFATc4-C together with the β-gal expression plasmid pON260 (1.0 + 0.2 μg of plasmid DNA/5 × 105 cells, respectively). The shNFATc3 was used as an additional control.A, Representative micrographs of shNFATc4-N-transfetced neurons at 96 h after transfection. Immunofluorescence for β-gal was used to identify transfected neurons. Counterstaining with Hoechst 33258 revealed the noncondensed chromatin of live cells (arrow) or chromatin condensation accompanying apoptosis (arrowhead).B,C, The shNFATc4-N or the shNFATc4-C but not shNFATc3 induced cortical neuron apoptosis. Data represent duplicate determinations from at least three independent experiments ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001;nsp > 0.05.
Requirement of NFATc4 for the NMDA-induced neuroprotection. DIV4 cortical neurons were cotransfected with control shRNA (shGFP) or shNFATc4-N together with a β-gal expression plasmid pON260 (1.0 + 0.2 μg of plasmid DNA/5 × 105 cells, respectively). Forty-eight hours later, cells were exposed to 30 μm PI3K inhibitor LY294002 (LY) or vehicle (Veh, 0.2% DMSO). In addition, cells were treated with 10 μm NMDA as indicated. After 24 h, LY294002 increased apoptosis in shGFP- or shNFATc4-transfected neurons. In shGFP-transfected neurons, NMDA suppressed the apoptotic response to LY294002. After NFATc4 knockdown, NMDA-mediated neuroprotection was removed. Data represent four independent experiments ± SEM; *p < 0.05;nsp > 0.05.
NFATc4 as a regulator of BDNF expression. Freshly isolated cortical neurons were electroporated with control shRNA (shGFP) or shNFATc4-N together with DN-p53 that was added to reduce electroporation toxicity (2.5 + 0.5 μg of plasmid DNA/8 × 106 cells, respectively). Four days later, RNA was isolated and expression of NFATc4 as well as its NMDAR-responsive candidate target genes includingbdnf,alivin, andl1 was determined using qRT-PCR. rRNA levels (18S) were used for normalizations. The shNFATc4-N significantly reduced NFATc4 and BDNF mRNA levels. Average of three independent experiments are presented. **p < 0.01; ***p < 0.001;nsp > 0.05. Error bars are SEM.
NFATc4 regulation of the NMDA-responsive BDNF promoter IV.A, DIV4 cortical neurons were cotransfected with the β-gal expression plasmid EF1αLacZ and either the wild-type (WT) or mutant (MT) form of human BDNF promoter IV–luciferase reporter construct (0.125 + 0.2 μg of plasmid DNAs/5 × 105 cells, respectively). The mutation consisted of four substitutions inactivating the composite binding sites for NFAT/MEF2 that is fully conserved between primates and rodents (positions 140–156 from the major transcription start site of exon 4). Two days after transfection, cells were stimulated with 10 μm NMDA for 20 h. Disruption of the NFAT/MEF site inhibited NMDA-induced activation of the BDNF promoter IV. Data ± SD are averages of triplicate sister cultures; similar trends were observed in three independent experiments.B, DIV4 cortical neurons were cotransfected with expression plasmids for NFATc4 or shNFATc4-N together with the rat BDNF promoter IV–CAT reporter construct and the β-gal expression plasmid EF1αLacZ (0.2 or 1.1 + 0.2 + 0.125 μg of plasmid DNAs/5 × 105 cells, respectively). The control plasmids included the empty NFATc4 expression vector pBJ5 (vector) and the shNFATc4-N control shGFP. NMDA stimulation was as inA. InB, data from a representative experiment are shown. Analysis of five independent experiments revealed that the wtNFATc4 enhanced NMDA-mediated promoter IV activation by 40 ± 9.5% and the shNFATc4 reduced the activation by 25 ± 3.65% (p < 0.001).
Role of the NR2B in maintenance of BDNF mRNA expression.A, P7 rat pups received single subcutaneous injections of 0.9% NaCl (vehicle control), 10 mg/kg MK801, or 20 mg/kg ifenprodil (IFN). After 3 h, expression of the candidate NMDAR-regulated NFAT target genes was analyzed by qRT-PCR. MK801 or IFN reduced BDNF expression, whereas alivin and l1 declined only in response to MK801. Hence, NR2B appears critical for maintenance of BDNF expression in developing rat cortex.B, In DIV7 cortical neurons, 24 h treatments with 10 μm MK801 (MK), 10 μm ifenprodil (IFN), or 0.5 μm Ro-25-6981 (RO) lowered BDNF mRNA levels. Veh, Vehicle. InA, three animals were treated for each data point; inB, data represent three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars are SEM.
Rescue of shNFATc4-transfected neurons by exogenous BDNF.A, Cortical neurons were transfected as in Figure 5. After 48 h, cells were treated with BDNF for the next 24 h. The shNFATc4-induced apoptosis was suppressed by BDNF. Data from three independent experiments ± SEM are depicted. **p < 0.01; ***p < 0.001.B, Our results support a model of NR2B-activated survival networks, in which BDNF maps downstream of NFATc4.
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