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The molecular basis for calcium-dependent axon pathfinding

Key Points

  • For more than 25 years intracellular Ca2+ signalling in growth cones has been recognized to be an important mediator of axon outgrowth and guidance. However, contradictory findings have led to considerable confusion and controversy regarding to the precise functions of Ca2+ in the regulation of growth cone motility. Recent identification of new molecules that function upstream and downstream of Ca2+ has provided new insights into how this ion can exert such diverse effects on growth cone behaviour.

  • Direct experimental manipulation of growth cone Ca2+ concentration shows that Ca2+ signals serve an instructional role in axon guidance. However, the functionally relevant characteristics of local Ca2+ signals are not clear. There is evidence to suggest that the baseline Ca2+ concentration, transient elevations in local Ca2+, and the source of Ca2+ signals may all influence growth cone motility.

  • Growth cones have tight homeostatic control of intracellular Ca2+ concentrations [Ca2+]i. Changes in [Ca2+]i occur in response to environmental factors that alter Ca2+ influx and release from intracellular stores. Growth cones express many different Ca2+-influx and -release channels. The effects of Ca2+ influx and release on growth cone motility probably result from both the combinatorial signals generated (cytosolic) and the specific pathways activated (local).

  • Cytosolic Ca2+ signals with distinct spatiotemporal characteristics can activate specific downstream targets to generate opposing growth cone responses. Some of these targets include kinases and phosphatases that have different affinities for Ca2+, so might serve as decoders of Ca2+ changes of different magnitude. One such pair is Ca2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin, which functions as a bimodal switch to decode local Ca2+ signals of differing magnitude into attraction and repulsion, respectively. Similarly, tyrosine kinase/phosphatase pairs might decode Ca2+ signals, as Src kinase is inhibited by the Ca2+-dependent protease calpain in response to large Ca2+ transients.

  • Cytosolic Ca2+ signals also act through several downstream targets that directly modulate cytoskeletal effectors to influence growth cone motility. For example, cytosolic Ca2+ signals can regulate proteins that activate or inactivate Rho family GTPases. As the Rho GTPases have profound and diverse effects on growth cone motility, crosstalk with this system would allow Ca2+ to influence many aspects of axon pathfinding. Ca2+ signals also interact with other second messengers systems such as cyclic AMP, which is an important modulator of growth cone responses to guidance cues.

  • Future work should seek to better understand the intricate signalling networks that are initiated or modulated by Ca2+ signals. Moreover, determining how these complex signals cooperate to regulate growth cone motility and guidance downstream of guidance cues is necessary for a complete understanding of axon pathfinding. A more complete understanding of the molecular basis of axon pathfinding could provide the necessary basis for developing strategies to enhance axon regeneration and stem cell-based therapies for neurological disorders.

Abstract

Ca2+ signals have profound and varied effects on growth cone motility and guidance. Modulation of Ca2+ influx and release from stores by guidance cues shapes Ca2+ signals, which determine the activation of downstream targets. Although the precise molecular mechanisms that underlie distinct Ca2+-mediated effects on growth cone behaviours remain unclear, recent studies have identified important players in both the regulation and targets of Ca2+ signals in growth cones.

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Figure 1: Measurements of Ca2+ using fluorescent Ca2+ indicators in Xenopus spinal neurons show that Ca2+ signals can vary widely in amplitude, spatial spread and kinetics.
Figure 2: Regulation of growth cone turning by local and global Ca2+ signals.
Figure 3: Local and global changes in growth cone intracellular Ca2+ concentration occur in response to Ca2+ influx through plasma membrane channels and release from intracellular stores.
Figure 4: Ca2+ gradients of various slopes across a growth cone regulate motility by activating different downstream targets.
Figure 5: A local imbalance of kinase and phosphatase activities downstream of Ca2+ signals results in disproportionate effects on growth cone motility, which leads to neurite turning.

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Acknowledgements

The authors thank members of their laboratories for helpful comments on the manuscript. Work on growth cone guidance in the authors' laboratories was supported by grants from the National Institutes of Health, USA, and the National Science Foundation, USA.

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Glossary

Chemotropism

The movement or orientation of an extending axon or cell along a chemical concentration gradient either towards or away from a chemical stimulus.

Antisense nucleotide knockdown

The use of an oligonucleotide with a complimentary sequence to a target mRNA to promote hybridization. When antisense DNA or RNA is added to a cell, it binds to a specific mRNA molecule and prevents translation into protein.

Morpholino

A synthetic oligonucleotide with a modified sugar backbone (morphine ring) that is resistant to degradation by nucleases and therefore forms stable translation-blocking hybrids with endogenous mRNA. This form of oligonucleotide is particularly popular for work with zebrafish and Xenopus systems.

Ca2+ nanodomain

A local Ca2+ signal generated by Ca2+ influx through a single channel. To encode information, Ca2+ sensors must be positioned within 50 nm of the open Ca2+ channel.

Ca2+ microdomain

A local Ca2+ signal generated by integrated Ca2+ influx through a discrete cluster of Ca2+ channels. To encode information, Ca2+ sensors must be positioned <1 μm from the open Ca2+ channels.

Actomyosin

A motor system composed of actin filaments and myosin, which hydrolyse ATP to produce force in processes such as muscle contraction and retrograde actin flow.

Postsynaptic density

An electron-dense complex of proteins located immediately behind the postsynaptic membrane. Proteins in the postsynaptic density have many roles, which include the anchoring and trafficking of neurotransmitter receptors in the plasma membrane, and the clustering of various proteins that modulate receptor function.

Synaptic plasticity

A process in which the efficacy of signal transmission through a synapse is persistently modified. The modification persists beyond the duration of the stimulus and results from post-translational and/or translational changes in the pre- or postsynaptic cell.

Biosensor

A molecule that reports some aspect of cell physiology or molecular function in living cells. Biosensors are often fluorescent molecules, such as fluorescent fusion proteins with green fluorescent protein or its spectral variants. Fluorescent reporters allow investigators to correlate cellular behaviours with spatial and temporal changes in protein localization and function.

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Gomez, T., Zheng, J. The molecular basis for calcium-dependent axon pathfinding. Nat Rev Neurosci 7, 115–125 (2006). https://doi.org/10.1038/nrn1844

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