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

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.