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Homer regulates calcium signalling in growth cone turning.

Gasperini R, Choi-Lundberg D, Thompson MJ, Mitchell CB, Foa L - Neural Dev (2009)

Bottom Line: Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A.In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones.These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Medicine, University of Tasmania, Hobart, 7001, Tasmania, Australia. robert.gasperini@utas.edu.au

ABSTRACT

Background: Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors - such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels - to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance.

Results: Homer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones.

Conclusion: These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.

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Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor (BDNF), Netrin-1 and Sema-3a in an in vitro turning assay. (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). Ti = initial trajectory; Tf = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D). Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: *P < 0.05; **P < 0.005; Mann-Whitney U-test. Error bars indicate standard error of the mean.
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Figure 1: Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor (BDNF), Netrin-1 and Sema-3a in an in vitro turning assay. (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). Ti = initial trajectory; Tf = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D). Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: *P < 0.05; **P < 0.005; Mann-Whitney U-test. Error bars indicate standard error of the mean.

Mentions: Dorsal root ganglia (DRG) sensory neurons are a well-established model for axon guidance and growth cone motility studies [37]. We characterised the behaviour of embryonic rat DRG growth cones in an in vitro growth cone turning assay [38]. Using experimental parameters comparable to those used in other cell types [34,35], DRG growth cones showed reliable responses to attractive and repulsive guidance cues (Figure 1). Isolated DRG growth cones in acute primary culture turned towards micro-gradients of BDNF and Netrin-1, and were repelled by Semaphorin-3a (Sema-3a) when compared to vehicle-only experiments (Figure 1A–C). These effects were specific to turning and did not affect other cytoskeletal events, since axon extensions did not differ significantly between guidance cues (Figure 1D).


Homer regulates calcium signalling in growth cone turning.

Gasperini R, Choi-Lundberg D, Thompson MJ, Mitchell CB, Foa L - Neural Dev (2009)

Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor (BDNF), Netrin-1 and Sema-3a in an in vitro turning assay. (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). Ti = initial trajectory; Tf = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D). Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: *P < 0.05; **P < 0.005; Mann-Whitney U-test. Error bars indicate standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2734570&req=5

Figure 1: Dorsal root ganglia (DRG) growth cone responses to brain derived neurotrophic factor (BDNF), Netrin-1 and Sema-3a in an in vitro turning assay. (A) Representative time-lapse images of DRG growth cones at start (0 minutes) and end (30 minutes). Ti = initial trajectory; Tf = final trajectory; θ = turning angle. Scale bar is 10 μm. (B) growth cone extension/trajectory plots after a 30-minute exposure to gradients of BDNF, vehicle (sensory neuron medium) and Sema-3a. In all cases the micropipette is positioned out of image at upper left quadrant. Quantification of average turning angles (C) and axon extension rates (D). Axon extension rates did not differ significantly after 30 minutes for Netrin-1, BDNF, control or Sema-3a. Positive angles represent attraction; negative angles represent repulsion. Significant differences from control values are marked as: *P < 0.05; **P < 0.005; Mann-Whitney U-test. Error bars indicate standard error of the mean.
Mentions: Dorsal root ganglia (DRG) sensory neurons are a well-established model for axon guidance and growth cone motility studies [37]. We characterised the behaviour of embryonic rat DRG growth cones in an in vitro growth cone turning assay [38]. Using experimental parameters comparable to those used in other cell types [34,35], DRG growth cones showed reliable responses to attractive and repulsive guidance cues (Figure 1). Isolated DRG growth cones in acute primary culture turned towards micro-gradients of BDNF and Netrin-1, and were repelled by Semaphorin-3a (Sema-3a) when compared to vehicle-only experiments (Figure 1A–C). These effects were specific to turning and did not affect other cytoskeletal events, since axon extensions did not differ significantly between guidance cues (Figure 1D).

Bottom Line: Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A.In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones.These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Medicine, University of Tasmania, Hobart, 7001, Tasmania, Australia. robert.gasperini@utas.edu.au

ABSTRACT

Background: Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors - such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels - to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance.

Results: Homer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones.

Conclusion: These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.

Show MeSH
Related in: MedlinePlus