Abstract
BackgroundHomer 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.ResultsHomer1 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.ConclusionThese 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.
Highlights
Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis
Homer1 expression is crucial for growth cone turning Dorsal root ganglia (DRG) sensory neurons are a wellestablished model for axon guidance and growth cone motility studies [37]
The data presented here support the hypothesis that the post-synaptic scaffolding protein Homer1 acts pre-synaptically to mediate the activity of store-operated channels in growth cones to regulate crucial aspects of calcium signalling in response to guidance cue receptor activation
Summary
Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. Changes in spatial growth cone [Ca++]i gradients mediate the activation of calcium-calmodulin dependent protein kinase II (CaMKII) and calcineurin phosphatase (CaN) in a molecular switch-like mechanism that controls calcium-dependent growth cone turning [8]. This molecular switch is consistent with the [Ca++]i set point hypothesis [1,9,10] that predicts baseline [Ca++]i and/or frequency of transients is maintained at a low level, in order for discrete and/or global changes in [Ca++]i to be instructional to growth cone extension [11]. Calcium is a promiscuous second messenger and the complete molecular repertoire that regulates basal [Ca++]i and guidancecue-induced changes in [Ca++]i within growth cones is yet to be fully resolved
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