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Homer, TRP channels and calcium : the signalling triad of growth cone motility

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Gasperini, Robert John (2008) Homer, TRP channels and calcium : the signalling triad of growth cone motility. PhD thesis, University of Tasmania.

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Abstract

The nervous system is an elaborate network of intricate circuits linking, monitoring and
controlling all functions in the body. This circuitry, established early in development, is
defined by a process known as axon guidance. The precision and accuracy of this
circuitry is ultimately a correlate of the navigational capabilities of specialised structures
at the distal tips of extending axons, the growth cones.
Growth cones are equipped with an array of fine antennal projections, or filipodia,
sensitive to a variety of attractive or repulsive signals. These guidance cues are
detected and interpreted by intracellular signal transduction mechanisms that mediate
cytoskeletal rearrangements within the growth cone, ultimately providing directional
control of growth cone trajectories. Guidance cues may be diffusible molecules from
distant target tissues or components of contacting cells, however, the complete
repertoire of molecules that transduce these extracellular signals to the cytosolic
cytoskeletal machinery are yet to be fully understood.
Neurons have evolved a variety of important intracellular signal transduction pathways,
many of which rely on calcium as a key second messenger molecule. Many crucial pre- and
post-synaptic functions in neurons are mediated by changes in intracellular calcium
concentration ([Ca2+]i,) including filipodial protrusion and neurite elongation. Indeed,
spatial [Ca2+]i gradients within the growth cone are crucial for the appropriate recognition
and motile responses to the key guidance molecules netrin-1 and brain derived
neurotrophic factor (BDNF). Cytosolic calcium is highly regulated with the key calcium
buffering organelle being the endoplasmic reticulum (ER). The mechanisms regulating
the transduction of extracellular guidance signals to changes in ER mediated calcium release, however, are still to be determined. This thesis describes work focusing on the
elucidation of a molecular correlate of such a mechanism.
Homer proteins are best known as facilitators of receptor clustering and signalling at the
post synaptic density. Long form Homer (H1b/c) forms dimers via C-terminal coiled-coil
domains, cross-linking multiple signalling partners through N-terminal, enabled-VASP
homology (EVH1) domains. This molecular motif enables Homer proteins to couple cell-surface
receptors such as metabotropic glutamate receptors (mGluR) and transient
receptor potential cation channels (TRPC) to intracellular calcium stores via inositol
triphosphate (IP3 R) and ryanodine (RyR) receptors. Homer is necessary for axon
pathfinding in the amphibian visual system in vivo, in a mechanism that to date, has
remained elusive. The unique binding characteristics of this synaptic molecule, the
subcellular location and physiological relevance of its binding partners makes Homer a
good candidate molecule to facilitate the coupling of extracellular guidance cues to
changes in [Ca++]i.
This study addresses the following questions: What is the biochemical nature of Homer
function in axon guidance? Does Homer facilitate the transduction of extracellular
guidance cues to the cellular machinery required to adjust motility and guidance? Is
Homer required for calcium signalling in the growth cone?
The study describes the development and characterisation of a growth cone turning
assay using a relevant developmental system, i.e. primary cultures of embryonic rat
dorsal root ganglion sensory neurons (DRG). Combining this assay with a targeted
morpholino knockdown approach, the study shows that a crucial level of H1b/c is
necessary for calcium dependant motile responses to netrin-1 and BDNF, with Homer morphant DRG showing a reversal of motile responses from attraction to repulsion
(control morphants +15.8 and +18.7 degrees for netrin and BDNF respectively while for
Homer morphants -19.7 and -18.6 degrees for netrin and BDNF). Furthermore,
pharmacological experiments suggest that Homer functions through the activational
state of a CaMKII/calcineurin molecular switch. Such a molecular switch has recently
been found to be crucial in other axon guidance model systems and is sensitive to the
depth of growth cone calcium gradients, lending support to a role for Homer in the "setpoint
hypothesis" of growth cone motility.
On the basis of these experiments, it was hypothesised that perturbation of growth cone
calcium dynamics would be a feature of Homer knockdown. Indeed, single wavelength
calcium imaging experiments showed that Homer morphant DRGs exhibited altered
calcium responses to BDNF microgradients and a higher frequency of TRPC-mediated
calcium transients, or spike events (control morphants 0.3 events/min and Homer
morphants 1.5 events/min). These results describe a crucial role for Homer in growth
cone calcium homeostasis.
The relevance and importance of Homer in sensory systems is further demonstrated
through an examination of the ontogeny of a putative Homer1b/c homologue in the
developing zebrafish embryo. Significantly, Homer protein is prominent in the
developing sensory architecture of the zebrafish larva at important developmental,
behavioural and synaptogenic timepoints, supporting previous experimental data
showing a crucial role for Homer in the developing amphibian visual system.
In summary, the work demonstrates that Homer acts as a facilitator of calcium signalling
and thus motile events in the growth cone. These findings, therefore, may ultimately have implications for the design and implementation of pharmacological interventions in
neurological diseases and clinical conditions such as spinal cord injury.

Item Type: Thesis (PhD)
Keywords: TRP channels, Proteins, Molecular neurobiology
Copyright Holders: The Author
Copyright Information:

Copyright 2008 the Author – The University is continuing to endeavour to trace the copyright owner(s) and in the meantime this item has been reproduced here in good faith. We would be pleased to hear from the copyright owner(s).

Additional Information:

Available for library use only and copying in accordance with the Copyright Act 1968, as amended. Thesis (PhD)--University of Tasmania, 2008. Includes bibliographical references

Date Deposited: 09 Dec 2014 00:12
Last Modified: 09 Jun 2016 01:24
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