GAP43 phosphorylation is critical for growth and branching of retinotectal arbors in zebrafish

Abstract

Visual activity acts via NMDA Receptors to refine developing retinotectal maps by shaping retinal arbors. Retinal axons add and delete transient branches, and the dynamic rates increase when MK801 blocks NMDARs, as if this prevents release of a stabilizing signal. Ca(++) entry through NMDARs activates phospholipase A2 (cPLA2) to release arachidonic acid (AA), which taps into a presynaptic growth control mechanism. NCAM, L1, N-cadherin, and FGF all stimulate axon growth via AA activation of protein kinase C to phosphorylate GAP43 and polymerize/stabilize F-actin. Our previous results show that blocking cPLA2 mimics NMDAR blockers, whereas exogenous AA reverses the increased dynamics, and PKC inhibitors also arrest growth. To test whether this activity-driven F-actin control mechanism shapes retinotectal arbors in zebrafish, we used the alpha-1-tubulin promoter to express GAP43-GFP fusion proteins in retinal ganglion cells, and imaged arbors in time-lapse to test for effects of GAP43 levels and its phosphorylation. Overexpressing wildtype GAP43 gave faster growth and larger arbors (#branches, spatial extent, total length of branches) at three days and especially four days. Surprisingly, the N-terminal 20 amino acid segment alone caused the same increase in branching, but no increase in growth. Earlier studies implicate this region in activating G(o) resulting in collapse of growth cones, which is now known to precede branch initiation. In contrast, GAP43 with ser41 mutated to ala (S41A) to prevent phosphorylation did not increase either branching or growth but resulted in immature, elongated arbors even at four to five days. In support of this atrophic effect, only half of brain/spinal neurons expressing S41A successfully initiated axonal outgrowth (vs. nearly 100% for wtGAP43). These results suggest that the region around the ser41 phosphorylation site, which binds CaM and PIP2, promotes growth only when phosphorylated, and also activates the branching control region in the first 10-20 amino acids. Whereas phosphorylation introduces a bulky negative charge group, mutation of serine to arginine introduces a bulky positive charge. But this also produced the same growth and branching as phosphorylation, suggesting that the effect of phosphorylation is through hydrophilic bulk rather than negative charge, in agreement with other IQ motifs. The results implicate the cPLA2-AA-PKC-GAP43 pathway as part of an F-actin based mechanism that both stabilizes new synapses and initiates new branches near effective synapses.

An α7 nicotinic receptor-G protein pathway complex regulates neurite growth in neural cells*

“…As illustrated in Pathway B, CaMKII phosphorylation of its target protein GAP-43 functions as an activator of Ga i/o signaling within the cell. CaMKII phosphorylation of GAP-43 has also been shown to promote its dissociation from CaM (Denny, 2006;Leu et al, 2010), a finding consistent with our crosslinking experiments showing an effect of Bgtx on GAP-43/CaM dissociation within the cell (Fig. 2).…”

“…The phosphorylation of GAP-43 by protein kinase C (PKC) (Esdar et al, 1999) and CaMKII (Denny, 2006;Leu et al, 2010) or dephosphorylation by calcineurin (PP2B) (Liu and Storm, 1989;Madsen et al, 1998) has been shown to regulate CaM association and neurite growth. In its unphosphorylated form, GAP-43 binds CaM leading to an inhibition in neurite growth (Skene, 1990;Slemmon et al, 1996).…”

In vivo imaging of the zebrafish retinotectal map

“…Transfecting a permanently phosphorylated GAP43 into cells increases branching and outgrowth. The axons arborize with several focal areas instead of just one, disrupting the retinotectal map by stabilizing incorrect connections [186]. These known factors interact with many additional molecules, activity and visual experience, and downstream factors to shape the arbors.…”

“…GAP43 is usually targeted to the end of the axon, where it first causes growth cone collapse and then initiates branching from the growth cone remnants [186]. When overexpressed, GAP43 causes larger RGC arbors through faster growth, increasing the number of branches, the area covered, and total branch length.…”

“…(neuromodulin) is membrane-linked and when phosphorylated it binds to, and stabilizes, actin [186]. GAP43 is usually targeted to the end of the axon, where it first causes growth cone collapse and then initiates branching from the growth cone remnants [186].…”