Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotrophin-mediated gene expression

Abstract

A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.

Figure 1.

BDNF activates NFAT-dependent transcription. A, BDNF significantly increased NFAT-mediated luciferase expression in a concentration-dependent manner (F = 14.41; p < 0.05 for 1 ng/ml BDNF; p < 0.001 at all other concentrations). B, BDNF induction of NFAT-dependent transcription relies on the activation of calcineurin because pretreatment with FK506 (200 ng/ml) and CsA (1 μg/ml) significantly (F = 37.90; ^**p < 0.001) attenuated luciferase expression. Data represent mean ± SEM.

Figure 2.

BDNF triggers the rapid nuclear translocation of NFATc4. A, Confocal images of immunolabeled NFATc4 (top) and α-tubulin (bottom) in cultured hippocampal neurons. In unstimulated cells (No Stim), the majority of NFATc4 is localized within the cytoplasm. B, Fifteen minutes after BDNF exposure, NFATc4 is observed primarily within the nucleus. C, Pretreatment with FK506 and CsA blocks the nuclear translocation of NFATc4 after BDNF stimulation. D, Quantification of the confocal images demonstrates that BDNF significantly increases the nuclear-to-cytoplasm ratio of immunolabeled NFATc4 (F = 45.00; ^**p < 0.001).

Figure 3.

BDNF activates NFAT-dependent transcription via TrkB receptors. A, Pretreatment with the tyrosine kinase inhibitor K252a (100 nm) abolished the BDNF-induced increase in NFAT-dependent transcription (F = 14.61; ^**p < 0.001). B, Consistent with a TrkB mediated event, BDNF and NT-4/5 (100 ng/ml) were equally efficacious in stimulating NFAT-mediated luciferase expression; NT-3 (100 ng/ml) was less effective (F = 30.94; ^**p < 0.001).

Figure 4.

NMDA receptors and L-type calcium channels are not required for BDNF activation of NFAT-dependent transcription. Pretreatment with the NMDA receptor antagonist AP-5 (25 μm; A) or the L-type calcium channel blocker nifedipine (5 μm; B) had no effect on BDNF-induced NFAT-dependent transcription.

Figure 5.

BDNF activation of NFAT-dependent transcription is dependent on PLC signaling. A, Pretreatment with the PLC inhibitor U73122 (1 μm) significantly (F = 53.21; ^**p < 0.001) attenuated BDNF-induced NFAT-mediated luciferase expression. B, Depletion of calcium from intracellular stores with thapsigargin (1 μm) significantly reduced both basal (F = 28.35; ^*p < 0.05) and BDNF-stimulated (^**p < 0.001) NFAT-dependent transcription. C, BDNF-mediated NFAT-dependent transcription was also significantly attenuated (F = 17.53; ^**p < 0.001) by the PKC inhibitor bisindolylmaleimide (500 nm).

Figure 6.

BDNF and CaN regulation of IP₃R1 expression: a role for NFAT-dependent transcription. A, BDNF increased (F = 15.53; p < 0.001) luciferase expression driven by the IP₃R1 promoter. The effect was significantly (**p < 0.001) attenuated by pretreatment with FK506 and CsA. B, Similar to mRNA expression, BDNF-mediated increases in IP₃R1 protein are partially dependent on CaN. No Stim, Unstimulated cells.

Figure 7.

BDNF expression is regulated through NFAT-dependent transcription. A, Data taken from scRT-PCR experiments. In the control neuron, 40% of the total cellular cDNA template was required to detect BDNF expression. In comparison, in the example neuron expressing a constitutively nuclear NFATc4 construct, amplicons for BDNF were observed when using 40, 20, 10, and 5% of the cDNA template. GAPDH was used as a positive control for RT. B, In neurons where BDNF cDNA was detected (n = 19 of 29), there was a shift (H = 11.27; p < 0.001) in the threshold for detection in cells expressing the constitutively nuclear NFATc4 construct. ND, Not detected. C, BDNF-induced increases in BDNF protein are dependent on CaN, suggestive of an NFAT-mediated event. No Stim, Unstimulated cells.