Role of the CaSR during development of cranial sensory neurons

Virtually all studies on the molecular regulation of axonal growth have been carried out on axotomised neurons that regenerate axons in culture and are dependent on neurotrophic factors not only for survival but also for axonal growth. The technical difficulty of obtaining and culturing early, newly differentiated neurons that initiate axonal growth for the first time in culture has meant that developmentally relevant de novo axonal growth has been almost ignored. Numerous studies have shown that axonal regeneration from embryonic and postnatal neurotrophic factor-dependent neurons is regulated by a variety of signalling pathways that influence the assembly and stability of key components of the cytoskeleton in growth cones. Depending on neuron type, these can include MEK, PI3 kinase, GSK3, NF-kB and calcium signalling. The extent to which these pathways are important for de novo axonal growth, and how their importance changes as axonal growth becomes responsive to neurotrophic factors during early development is not known. I have used the experimentally advantageous placode-derived sensory neurons of the chicken embryo to study the molecular basis of de novo, neurotrophic factor independent axonal growth and to compare this neurotrophic factor-dependent axonal growth at later stages of development. These neurons can be dissected from the earliest stages in their development and cultures can be established in which neurons extend axons for the first time. Previous work has shown that at this stage of development, axonal growth is independent of neurotrophic factors and its rate is correlated with target distance. For example, neurons of the nodose ganglion have the most distant targets, the fastest axonal growth rate and survive longest before becoming dependent on the neurotrophic factor BDNF for survival, whereas neurons of the vestibular ganglion have the nearest targets, slowest axonal growth rate and survive for the shortest time before acquiring BDNF dependence. My initial studies focused on the role of the extracellular calcium-sensing receptor (CaSR), a G protein coupled receptor that has recently been shown to regulate axonal growth from sympathetic neurons during the stage when neurons are innervating their targets. I found that during the stage of development when the earliest axons of placode-derived sensory neurons are growing to their targets, nodose ganglion neurons (which have the fastest axon growth rates) express the highest levels of the CaSR, and vestibular neurons (which have the slowest axon growth rates) express the lowest levels of the CaSR. Experimental manipulation of CaSR activation in cultured nodose neurons at the stage in development when their axons are normally growing to their targets markedly affects axon growth rate (enhancing activation increases growth rate whereas reducing activation has the opposite effect). In contrast, similar manipulations of CaSR activation in cultured vestibular neurons have no effects on axonal growth rate. These findings suggest that the CaSR plays an important role in the regulation of de novo axonal growth rate. Manipulating CaSR activation in older, BDNF-dependent nodose neurons at the stage in development when these neurons are innervating their targets also demonstrated a role for the CaSR in promoting axonal growth at this stage. Having demonstrated a role for the CaSR in promoting axonal growth at these two successive stages of development, I then characterised the intracellular signalling pathways that mediate the effects of the CaSR on axonal growth at these stages. Using Western blot analysis and pharmacological inhibitors of PI3-kinase, GSK3 and MEK1/2, I discovered a clear switch in the signalling pathways that are involved in promoting axon elongation between early BDNF-independent stages of de novo axon growth to later BDNF-dependent stages of axon growth. Whereas PI3-kinase signalling plays a pivotal role in transducing CaSR-enhanced, neurotrophin-independent axon growth, GSK3 signalling plays a major role in transducing the growth enhancing effects of CaSR activation on BDNF-promoted axonal growth from older BDNF-dependent nodose neurons. My findings suggest that PI3-Kinase and GSK3 signalling are not linked in developing nodose neurons, but are regulated independently of each other. Furthermore, Western analysis also suggests the operation of a novel activation mechanism of GSK3 in axon growth in BDNF-dependent nodose neurons that involves tyrosine phosphorylation of GSK3 rather than serine phosphoryation following CaSR activation. In all, my studies have revealed several novel and unexpected aspects of regulation of axonal growth by the CaSR during the early stages of neuronal development.