Regulation of calcineurin by growth cone calcium waves controls neurite extension.

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.