In vivo localization and characterization of functional ciliary neurotrophic factor receptors which utilize JAK-STAT signaling.

The ciliary neurotrophic factor receptor is critically involved in embryonic motor neuron development. Postnatally, it may contribute to neuronal maintenance and regeneration. In addition, pharmacological stimulation of the receptor may slow the progression of several neurodegenerative disorders. The widespread nervous system expression of ciliary neurotrophic factor receptor components and the effects of low ciliary neurotrophic factor concentrations on a wide variety of cells in culture combine to suggest that functional ciliary neurotrophic factor receptors are expressed by many classes of neurons in vivo. However, the in vivo signaling properties and distribution of functional ciliary neurotrophic factor receptors have not been directly determined. We developed a novel in vivo assay of functional ciliary neurotrophic factor receptors which revealed that, in the adult nervous system, cranial and spinal motor neurons are very sensitive to ciliary neurotrophic factor and display a rapid, robust increase in phospho-STAT3 in their dendrites, cell bodies and nuclei, which is specifically blocked by the ciliary neurotrophic factor receptor antagonist, AADH-CNTF. In distinct contrast, several other classes of ciliary neurotrophic factor receptor expressing neurons fail to increase phospho-STAT3 levels following ciliary neurotrophic factor treatment, even when ciliary neurotrophic factor is applied at high concentrations. Leukemia inhibitory factor and epidermal growth factor elicit the same cell-type-dependent pattern of phospho-STAT3 increases. Responsive and non-responsive neurons express comparable levels of STAT3.Therefore, in vivo ciliary neurotrophic factor receptor-initiated STAT3 signal transduction is regulated in a very cell-type-dependent manner. The present data suggest that at least some of this regulation occurs at the STAT3 tyrosine phosphorylation step. These unexpected results also suggest that other forms of receptor-initiated STAT3 signal transduction may be similarly regulated.

Regulation of ciliary neurotrophic factor receptor alpha in sciatic motor neurons following axotomy.

Spinal motor neurons are one of the few classes of neurons capable of regenerating axons following axotomy. Injury-induced expression of neurotrophic factors and corresponding receptors may play an important role in this rare ability. A wide variety of indirect data suggests that ciliary neurotrophic factor receptor alpha may critically contribute to the regeneration of injured spinal motor neurons. We used immunohistochemistry, in situ hybridization and retrograde tracing techniques to study the regulation of ciliary neurotrophic factor receptor alpha in axotomized sciatic motor neurons. Ciliary neurotrophic factor receptor alpha immunoreactivity, detected with two independent antisera, is increased in a subpopulation of caudal sciatic motor neuron soma one, two and six weeks after sciatic nerve transection and reattachment, while no changes are detected at one day and 15 weeks post-lesion. Ciliary neurotrophic factor receptor alpha messenger RNA levels are augmented in the same classes of neurons following an identical lesion, suggesting that increased synthesis contributes, at least in part, to the additional ciliary neurotrophic factor receptor alpha protein. Separating the proximal and distal nerve stumps with a plastic barrier does not noticeably affect the injury-induced change in ciliary neurotrophic factor receptor alpha regulation, thereby indicating that this injury response is not dependent on signals distal to the lesion traveling retrogradely through the nerve or signals generated by axonal growth through the distal nerve. The prolonged increases in ciliary neurotrophic factor receptor alpha protein and messenger RNA found in regenerating sciatic motor neurons contrast with the responses of non-regenerating central neurons, which are reported to display, at most, a short-lived increase in ciliary neurotrophic factor receptor alpha messenger RNA expression following injury. The present data are the first to demonstrate, in vivo, neuronal regulation of ciliary neurotrophic factor receptor alpha protein in response to injury. Moreover, they suggest that the ability of a subpopulation of spinal motor neurons to regulate ciliary neurotrophic factor receptor alpha levels in response to injury may play a role in their survival and axonal regeneration. Consistent with such a role, we also find relatively high, and probably elevated, levels of ciliary neurotrophic factor receptor alpha immunoreactivity in regenerating axons.

Antisense studies in PC12 cells suggest a role for H218, a sphingosine 1-phosphate receptor, in growth-factor-induced cell-cell interaction and neurite outgrowth.

Our previous studies of H218, a sphingosine 1-phosphate (S1P) receptor and a member of the G-protein-coupled receptor superfamily, suggest that it may participate in mammalian nervous system development. Thus, brain levels of H218 mRNA are higher during early neurogenesis than postnatally. In addition, embryonic H218 immunoreactivity is preferentially localized in young neuronal cell bodies during their early stages of differentiation and in axons during their extension. This report describes the morphological effects of reducing native H218 levels in PC12 cells. Western blot analyses demonstrated that PC12 cells stably transfected with an expression vector carrying an antisense-oriented H218 cDNA contain less H218 protein than vector-transfected control cells. When differentiated with growth factors, the antisense-H218 cells display more neurite production and form less cell-cell contacts than the control cells. Therefore, these data, along with our previous H218 expression studies and a recent, independent study of H218 overexpression, support the possibility that H218 contributes to developmental processes regulating neuronal interaction and axon growth. The data are also consistent with reports that H218 is a S1P receptor, that S1P is present in serum, like that used in our PC12 cell cultures, and that it causes PC12 cell neurite retraction. Finally, and in agreement with a S1P receptor role for H218, we find that the antisense-H218 cells display less S1P-induced neurite retraction than control cells.