Neurturin signalling via GFRα2 is essential for innervation of glandular but not muscle targets of sacral parasympathetic ganglion neurons

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Abstract

Neurturin, a member of the glial cell-derived neurotrophic factor familys of ligands, is important for development of many cranial parasympathetic ganglion neurons. We have investigated the sacral component of the parasympathetic nervous system in mice with gene deletions for neurturin or its preferred receptor, GFRα2. Disruption of neurturin signalling decreased cholinergic VIP innervation to the mucosa of the reproductive organs, but not to the smooth muscle layers of these organs or to the urinary bladder. Thus, neurturin and its receptor are involved in parasympathetic innervation of a select group of pelvic visceral tissues. In contrast, noradrenergic innervation was not affected by the gene ablations. The epithelium of reproductive organs from knockout animals was atrophied, indicating that cholinergic innervation may be important for the maintenance of normal structure. Cholinergic neurons express GFRα2 on their terminals and somata, indicating they can respond to neurotrophic support, and their somata are smaller when neurturin signalling is disrupted. Colocalisation studies showed that many peripheral glia express GFRα2 although its role in these cells is yet to be determined. Our results indicate that neurturin, acting through GFRα2, is essential for parasympathetic innervation of the mucosae of reproductive organs, as well as for maintenance of a broader group of sacral parasympathetic neurons.

Introduction

Neurturin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) which also includes GDNF, artemin and persephin (Kotzbauer et al., 1996). The GFLs are growth factors for several types of central and peripheral neurons such as sensory, autonomic, motor and midbrain dopaminergic neurons Baloh et al., 1998, Baudet et al., 2000, Creedon et al., 1997, Garces et al., 2001, Henderson et al., 1994, Heuckeroth et al., 1999, Horger et al., 1998, Kotzbauer et al., 1996, Lin et al., 1993, Milbrandt et al., 1998, Rossi et al., 1999. These growth factors activate the Ret tyrosine kinase in the presence of a glycosylphosphatidylinositol (GPI)-linked coreceptor belonging to the GFRα family of receptors: GDNF binds preferably to GFRα1, neurturin to GFRα2, artemin to GFRα3 and persephin to GFRα4 Baloh et al., 1997, Baloh et al., 1998, Durbec et al., 1996, Jing et al., 1996, Masure et al., 2000, Thompson et al., 1998, Treanor et al., 1996, Trupp et al., 1996.

The distributions of neurturin and its receptor, GFRα2, have been described in detail for developing and adult rats and mice Golden et al., 1999, Laurikainen et al., 2000, Widenfalk et al., 1997, Xian et al., 1999. During development, GFRα2 is found in most cranial parasympathetic neurons and remains highly expressed in adults, whereas in the sympathetic chain and superior cervical ganglia, GFRα2 is found at moderate levels in embryos but its expression decreases in adults Golden et al., 1999, Rossi et al., 2000. Evidence from gene ablation studies has further shown that neurturin plays a critical role in the development of some parts of the peripheral nervous system. Mice lacking neurturin or GFRα2 have defects in their enteric, sensory and cranial parasympathetic nervous systems Heuckeroth et al., 1999, Rossi et al., 1999. In particular, cranial parasympathetic innervation and cholinergic innervation of the small intestine and heart are reduced Heuckeroth et al., 1999, Hiltunen et al., 2000, Rossi et al., 1999. The somata of the surviving cranial parasympathetic and enteric neurons are smaller in neurturin and GFRα2 knockout mice than in their wild-type littermates (Heuckeroth et al., 1999).

The role of neurturin and GFRα2 in the second major component of the parasympathetic nervous system, the sacral system supplying pelvic organs, is less well understood. In rodents, the paired major pelvic ganglia provide the majority of the sacral autonomic innervation to the lower urinary tract and reproductive organs, and part of the extrinsic motor innervation of the lower bowel (Keast, 1999). The pelvic ganglia are quite unique and difficult to study as they contain both parasympathetic and sympathetic neurons which receive synaptic inputs from preganglionic neurons in the sacral and lumbar cord, respectively (Keast, 1999). These neuronal types can be distinguished by chemical markers. In mouse pelvic ganglia, sympathetic neurons express the enzymes dopamine-β-hydroxylase (DBH) and tyrosine hydroxylase (TH), as well as expressing neuropeptide Y (NPY) (Wanigasekara et al., 2003). Parasympathetic pelvic ganglion neurons express choline acetyltransferase (ChAT) but do not express either DBH or TH. Some of these parasympathetic neurons also express vasoactive intestinal peptide (VIP) and/or NPY (Wanigasekara et al., 2003). Conversely, all of the pelvic ganglion VIP neurons are ChAT-positive and fail to express TH. In rats, many of the parasympathetic VIP neurons also express nitric oxide synthase (NOS) (Dun et al., 1996). Although Laurikainen et al. (2000) demonstrated that some major pelvic ganglion cells express GFRα2, this study did not indicate which of this heterogenous group of neurons are influenced by neurturin. However, mice lacking GFRα2 have fewer parasympathetic axons innervating the penis, suggesting that neurturin may be a target-derived survival or neuritogenic factor for these neurons (Laurikainen et al., 2000).

The aim of this study was therefore to determine the role of neurturin in various functionally distinct classes of the sacral parasympathetic ganglion neurons using mice lacking either the neurturin or the GFRα2 gene. Comparison of the structure of the pelvic organs of neurturin and GFRα2 knockout and wild-type mice was performed to determine neurturin's role in these tissues. Immunohistochemical studies of axons in the pelvic organs and of neurons in the major pelvic ganglion of wild-type and knockout animals were conducted to determine which neurons expressed GFRα2 and were dependent on neurturin for innervation of target organs. GFRα2 expression in pelvic ganglia was also studied in different chemical classes of neurons in mice and adult rats. Finally, we employed retrograde labelling from the vas deferens and the bladder to determine whether neurturin was involved in other neurotrophic activities such as the maintenance of neuronal soma size.

Section snippets

Structure of the reproductive tissues is abnormal in neurturin and GFRα2 knockout mice

Gross comparison between neurturin and GFRα2/Bl6 knockout mice and their respective wild-type littermates was conducted to confirm previous reports that although the GFRα2 knockout mice were smaller that their wild-type littermates, there was no size difference between neurturin knockout and wild-type mice Heuckeroth et al., 1999, Rossi et al., 1999. In these reports, the two mice colonies were housed in different institutions, and differences in housing or diet may have contributed to growth

Discussion

The importance of neurturin for the development and maintenance of the cranial parasympathetic nervous system has been established and its actions are complex Heuckeroth et al., 1999, Rossi et al., 1999, Rossi et al., 2000. Neurturin is not a neurotrophic factor for all cranial parasympathetic neurons and its neurotrophic action also varies between the cranial ganglia. Here, we have investigated the sacral parasympathetic nervous system by studying the pelvic ganglion and its targets. This

Animals

The generation of neurturin and GFRα2 knockout mice has been described elsewhere Heuckeroth et al., 1999, Rossi et al., 1999. The neurturin knockout mice used in this study were back-crossed to the F4 generation with C57BL/6 mice. The GFRα2 mutation was maintained by backcrossing on two inbred mouse strains (129S2/SvHsd and C57BL/6JolaHad, both from Harlan) for eight generations to investigate whether the genetic background of the mouse influences the phenotype of the gene ablation. All

Acknowledgements

This work was funded by the National Health and Medical Research Council (grant 102447) and Australian Research Council (grant A00104654). The anti-GFAP antibody was a generous gift from Glenda Halliday and the anti-NOS antibody was a generous gift from Piers Emson. We are grateful to Heather Campbell for expert technical assistance with genotyping.

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