Figure 3. Calcineurin supports NGF-mediated axon growth in a transcription-independent manner.

(A–C) NGF does not promote nuclear import of NFAT transcription factors in sympathetic neurons. NFAT immunostaining shows that NGF treatment (100 ng/ml, 30 min) does not induce nuclear localization of NFAT (B, arrow), while NFAT nuclear labeling is evident in neurons expressing CA-CaN (C, arrow). Neurons were also immunostained with β-III Tubulin and DAPI. Scale bar, 10μm. (D) NFAT-luciferase reporter assay shows that NGF and NT-3 do not activate NFAT-dependent transcription in sympathetic neurons. Neurons expressing CA-CaN show activation of NFAT-dependent transcription at 8 and 24 hr post-infection with NFAT-adenovirus, which is blocked by ActD (0.1 μg/ml). **p<0.01 and ***p<0.001. Results are means± SEM from 3 experiments. (E–I) Calcineurin signaling is required for NGF-mediated axon growth in the absence of transcription. NGF and calcineurin inhibitors (CsA+FK506) were added only to distal axons. ActD (0.1 μg/ml) was bath applied. Scale bar, 320μm. (I) Quantification of sympathetic axon growth in compartmentalized cultures over 0–8 hr and 0–24 hr after treatments described in (E–H). *p<0.05, **p<0.01, and ***p<0.001, n=3 experiments. (J) NFAT-luciferase assay shows that NGF does not activate NFAT-dependent transcription in DRG sensory neurons. DRG neurons expressing CA-CaN show robust activation of NFAT-dependent transcription. *p<0.05, **p<0.01, and ***p<0.001, n=5 experiments. (K–N) Calcineurin signaling is required for NGF-mediated axon growth in DRG neurons, in the absence of transcription. Scale bar, 320μm. (O) Quantification of DRG axon growth in compartmentalized cultures. *p<0.05, ** p<0.01, n=4.