Abstract
Transcription is essential for neurite and axon outgrowth during development. Recent work points to the involvement of nuclear factor of activated T cells (NFAT) in the regulation of genes important for axon growth and guidance. However, NFAT has not been reported to directly control the transcription of axon outgrowth-related genes. To identify transcriptional targets, we performed an in silico promoter analysis and found a putative NFAT site within the GAP-43 promoter. Using in vitro and in vivo experiments, we demonstrated that NFAT-3 regulates GAP-43, but unexpectedly, does not promote but represses the expression of GAP-43 in neurons and in the developing brain. Specifically, in neuron-like PC-12 cells and in cultured cortical neurons, the overexpression of NFAT-3 represses GAP-43 activation mediated by neurotrophin signaling. Using chromatin immunoprecipitation assays, we also show that prior to neurotrophin activation, endogenous NFAT-3 occupies the GAP-43 promoter in PC-12 cells, in cultured neurons, and in the mouse brain. Finally, we observe that NFAT-3 is required to repress the physiological expression of GAP-43 and other pro-axon outgrowth genes in specific developmental windows in the mouse brain. Taken together, our data reveal an unexpected role for NFAT-3 as a direct transcriptional repressor of GAP-43 expression and suggest a more general role for NFAT-3 in the control of the neuronal outgrowth program.
Highlights
Not surprisingly, axon sprouting and outgrowth are under tight transcriptional control, and the expression of pro-axon growth genes is limited to appropriate spatial and temporal stages of neural development
Transgenic mice containing an nuclear factor of activated T cells (NFAT) reporter showed that NFAT transcriptional activity is highest in the brain [19, 20], and NFAT-3 is expressed in the spinal cord and the brain, with high levels found in the olfactory bulb, cerebellum, and certain regions of the cortex [21,22,23,24]
After 1 h, medium was aspirated off with any dead cells, and Neurobasal medium supplemented with B27, Glutamax, penicillin, and streptomycin was added. (All reagents were from Invitrogen except where indicated.) Embryonic day 13 (E13) mouse brains from wildtype and NFAT-3 null mice were extracted, dissociated, and plated in the presence of Neurobasal medium plus retinoic acid and forskolin to induce neuronal differentiation
Summary
Cell Lines—Pheochromocytoma (PC-12) cell lines were grown in Dulbecco’s modified medium supplemented with 10% horse serum, 5% fetal bovine serum, and 100 units/ml penicillin and 100 mg/ml streptomycin (Invitrogen) in a humidified atmosphere of 5% CO2 in air at 37 °C. The cells were incubated at 37 °C for 20 min, Dulbecco’s modified medium (supplemented with 10% fetal bovine serum) was added, and the cells were dissociated by triturating. After 1 h, medium was aspirated off with any dead cells, and Neurobasal medium supplemented with B27, Glutamax, penicillin, and streptomycin was added. (All reagents were from Invitrogen except where indicated.) Embryonic day 13 (E13) mouse brains from wildtype and NFAT-3 null mice were extracted, dissociated, and plated in the presence of Neurobasal medium plus retinoic acid and forskolin to induce neuronal differentiation After 1 h, medium was aspirated off with any dead cells, and Neurobasal medium supplemented with B27, Glutamax, penicillin, and streptomycin was added. (All reagents were from Invitrogen except where indicated.) Embryonic day 13 (E13) mouse brains from wildtype and NFAT-3 null mice were extracted, dissociated, and plated in the presence of Neurobasal medium plus retinoic acid and forskolin to induce neuronal differentiation
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