Axon Initiation and Growth Cone Turning on Bound Protein Gradients

Abstract

Extracellular gradients of secreted guidance factors are known to guide axon pathfinding and neuronal migration. These factors are likely to bind to cell surfaces or extracellular matrix, but whether and how they may act in bound gradients remains mostly unclear. In this study, we have developed a new technique for rapid production of stable microscopic gradients of substrate-bound proteins by covalent bonding of the proteins with an epoxy-coated glass substrate while they are diffusing in an agarose gel. Using this method, we found that bound gradients of netrin-1 and brain-derived neurotrophic factor (BDNF) can polarize the initiation and turning of axons in cultured hippocampal neurons. Furthermore, bound BDNF gradient caused attractive and repulsive polarizing response on gradients of low-and high-average density of BDNF, respectively. This novel bidirectional response to BDNF depended on the basal level of cAMP in the neuron. Finally, our data showed that the neuron's attractive response to bound BDNF gradient depended on the absolute difference rather than the relative difference in the BDNF density across the neuron, with a minimal effective difference of 1-2 BDNF molecule/m 2 on the substrate surface. Thus, substrate-bound guidance factors are highly effective in polarizing axon initiation and growth, and the diffusive printing technique is useful for studying neuronal responses induced by bound protein gradients.

Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization

“…Next, we generated a gradient of bound laminin using diffusive printing method [33,34] to mimic the potential cortical distribution of laminin [19]. As shown in Figure 2E, the bound laminin gradient was confirmed by immunostaining with anti-laminin antibody.…”

“…Diffusive printing assay was performed to generate bound substrate gradient as described previously [34] with some modifications. Briefly, silicon molds with parallel grooves, each 350 µm in width, 100 µm in depth, and 4 mm apart, were designed and produced by using standard photolithography.…”

More Than Just an OFF-Switch: The Essential Role of Protein Dephosphorylation in the Modulation of BDNF Signaling Events

“…Although BDNF/TrkB signaling has been previously shown to elevate cAMP and calcium levels in neurons (1,42), it is believed to occur and remain local, such as neurite terminals (20). However, it is clear that elevation of intracellular cAMP or calcium facilitate the functional and morphological synaptic responses to BDNF signaling (43,44). Elevation of intracellular calcium activates calcineurin whereas cAMP activates PKA, which in turn phosphorylates and deactivates the upstream CRTC kinase, salt-inducible kinase (SIK).…”

Cyclic nucleotide-dependent switching of mammalian axon guidance depends on gradient steepness

“…In particular, cyclic nucleotide levels can influence the sign of the chemotactic response Song et al, 1997;Song et al, 1998;Wen et al, 2004;Mai et al, 2009;Murray et al, 2009;Tojima et al, 2009). It has been shown that high levels of cAMP relative to cGMP lead to growth cone attraction, while the opposite leads to repulsion (Nishiyama et al, 2003).…”