Localization and Expression of GFRα2 Receptors in Neonatal and Adolescent Mouse Heart.

Neurturin (NRTN), a member of the glial cell-line derived neurotrophic factor(GDNF) family, is required in the development of parasympathetic cholinergic neurons. It signals through binding to a glycosyl-phophatidyl inositol (GPI)-linked receptor, GDNF family receptor α2 (GFRα2), which couples to Ret tyrosine kinase. Studies of NRTN knockout mice have shown that NRTN is essential for normal cholinergic innervation of the heart, but the precise role of NRTN remains unknown. For NRTN to evoke a biological response, GFRα2 must be localized to the surface of target neurons. The aim of this study was to determine the expression and localization of GFRα2 at two developmental time points in the atria of mouse hearts, postnatal day (P)1 and P18. Atria were used because of their extensive cholinergic innervation, particularly at the sinoatrial node and the right atrium. By P21, neurons are adult sized and substantial growth of cholinergic nerve fibers has occurred; therefore, it was hypothesized that GFRα2 signaling would be higher in P1 neuronal fibers than in P18 fibers. Because NRTN only activates surface receptors, and once activated, internalization occurs, we further hypothesized that GFRα2 would be cytoplasm localized when treated with NRTN. Atria were analyzed for GFRα2 expression in cholinergic neurons by immunohistochemistry with and without Triton X-100, a cell membrane permeabilizing detergent, to visualize cytoplasmic localization in one group and cell membrane localization in the other. In an additional group, excised P1 atria were cultured in petri dishes with and without NRTN (400 ng/ml, 3h) to determine if GFRα2 was internalized in response to NRTN treatment. Stained atria were viewed using a fluorescence microscope, and digital images were collected using a confocal microscopy system. Within each age group, Triton X-100 treated tissues exhibited cytoplasmic localization within ganglia; however, P1 neurons had distinct membrane staining, whereas in the P18 model, the majority of the GFRα2 was localized to the cytoplasm. NRTN-treated P1 ganglia showed a substantial decrease in membrane localization in central cell bodies and an increase in localization in perimeter cell bodies compared to the non-NRTN group. The results from this study show that GFRα2 is extensively localized to the cell membrane in P1 cholinergic neurons and is primarily localized to the cytoplasm in P18 cholinergic neurons. NRTN treatments lead to internalization of GFRα2 and may lead to a better understanding of GFRα2 trafficking. These findings suggest that GFRα2 cellular localization may be increased in periods of elevated nerve fiber growth and may serve as a regulator of responsiveness to NRTN