GDNF Family Neurotrophic Factor Signaling: Four Masters, One Servant?

Molecular and Cellular Neuroscience 13, 313–325 (1999) Article ID mcne.1999.0754, available online at http://www.idealibrary.com on

MCN

REVIEW GDNF Family Neurotrophic Factor Signaling: Four Masters, One Servant? Matti S. Airaksinen, Alexey Titievsky, and Mart Saarma1 Program of Molecular Neurobiology, Institute of Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, FIN-00014 Helsinki, Finland

GDNF FAMILY NEUROTROPHIC FACTORS Glial cell line-derived neurotrophic factor (GDNF), and related factors neurturin (NTN), artemin (ART), and persephin (PSP), are members of the GDNF family of neurotrophic factors (Fig. 1). They form a new subgroup in the transforming growth factor-␤ (TGF-␤) superfamily. These factors are basic, dimeric, secretory proteins with a cysteine knot structure (for a review see Iba´n˜ez, 1998; Saarma and Aruma¨e, 1999). GDNF, found as a trophic factor for midbrain dopaminergic neurons (Lin et al., 1993), has received much attention as a potential therapeutic agent for the treatment of neurodegenerative diseases (reviewed by Lapchak et al., 1997; Olson et al., 1997; Grondin and Gash, 1998). It promotes survival of many types of neurons including subpopulations of peripheral autonomic and sensory, as well as central motor, dopamine, and noradrenaline neurons (Lin et al., 1993; Henderson et al., 1994; Arenas et al., 1995; Buj-Bello et al., 1995; Trupp et al., 1995; Hearn et al., 1998; Heuckeroth et al., 1998). The three GDNF-related proteins, NTN, ART, and PSP, were discovered by research groups led by Drs. J. Milbrandt and E. M. Johnson. NTN was purified as a survival factor for sympathetic neurons (Kotzbauer et al., 1996), after which ART and PSP could be identified on the basis of sequence homology. NTN and ART have many neurotrophic effects similar to GDNF; they all support the survival of peripheral sympathetic and sensory neurons as well as midbrain dopamine neurons (Kotzbauer et al., 1996; Baloh et al., 1998b; Horger et al., 1 To whom correspondence should be addressed. E-mail: saarma@ operoni.helsinki.fi. All three authors contributed equally.

1044-7431/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

1998). PSP is expressed at low levels in most tissues and supports CNS dopamine and motor neurons, but not peripheral neurons (Milbrandt et al., 1998).

GDNF FAMILY RECEPTORS The receptor complex for GDNF (Fig. 2) was discovered simultaneously by several groups (for a review see Lindsay and Yancopoulos, 1996; Mason, 1996; Olson, 1997; Saarma and Aruma¨e, 1999). It consists of the transmembrane Ret receptor tyrosine kinase (Durbec et al., 1996b; Trupp et al., 1996; Worby et al., 1996) and a ligand-binding component, the GDNF-family receptor ␣1 (GFR␣1) (Jing et al., 1996; Treanor et al., 1996). Ret was originally described as an oncogene (REarranged in Transformation; Takahashi et al., 1985). Activating mutations of RET are found in human thyroid carcinomas (PTC and FMTC) and in multiple endocrine neoplasia type 2 (MEN2) cancer syndromes (for reviews see Eng, 1996; Pasini et al., 1996; Edery et al., 1997). Inactivating mutations of Ret, in contrast, cause Hirschsprung’s disease (aganglionic megacolon). Mutations in the extracellular part of Ret found in MEN2A lead to the formation of constitutively active homodimers. Intracellular mutations found in MEN2B produce a mutant Ret kinase with changed substrate specificity, but whose activity can be modulated by GDNF family ligands (Bongarzone et al., 1998; Carlomagno et al., 1998). GFR␣1 was discovered by expression cloning and screening of GDNF binding proteins (Jing et al., 1996; Treanor et al., 1996). It is anchored to the outer plasma membrane by a glycosyl phosphatidylinositol (GPI) link. In the model proposed by Jing et al. (1996), a dimeric GDNF first binds to either monomeric or di-

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314 meric GFR␣1 and the GDNF/GFR␣1 complex can then interact with Ret and induce its homodimerization. All the four known GDNF family ligands have their own preferred coreceptors (Fig. 2). NTN signals preferentially via GFR␣2 receptor (Baloh et al., 1997; Buj-Bello et al., 1997; Jing et al., 1997; Klein et al., 1997; Sanicola et al., 1997; Suvanto et al., 1997) while ART signals through GFR␣3 (Baloh et al., 1998b), a more distantly related GFR␣ receptor (Jing et al., 1997; Baloh et al., 1998a; Masure et al., 1998; Naveilhan et al., 1998; Nomoto et al., 1998; Trupp et al., 1998; Worby et al., 1998). The PSP receptor, GFR␣4 (Enokido et al., 1998), has been found so far only in chicken (Thompson et al., 1998); the sequence of GRF␣4 shows more similarity to GFR␣1 and GFR␣2 than to GFR␣3. A putative mouse Gfra4 gene has recently been identified (Gunn et al., 1999).

A PUTATIVE DOMAIN STRUCTURE OF GFR␣ MOLECULES A comparison of GFR␣1 and GFR␣2 amino acid sequences revealed internal homologies within the conserved cysteine-rich sequences and suggested a putative domain structure for these receptors (Fig. 3; modified from Suvanto, 1997). In this model, based on secondary structure analysis, the three Cys-rich domains are joined together by less conserved adapter sequences. Although all four GFR␣ receptors conform to this model, the amino acid sequence of GFR␣3 shows the largest deviation from the consensus sequence; this may lead to differences in the 3D structure of GFR␣3 relative to the other GFR␣ molecules. However, its ligand ART does not show more deviation from the GDNF family consensus sequence than the other members.

GDNF FAMILY SIGNALING GDNF-Dependent Ret Activation Triggers Various Intracellular Signaling Pathways Since, for a long time, Ret was an orphan oncogenic receptor tyrosine kinase, extensive studies on Ret signaling have been performed using chimeric and/or oncogenic forms of Ret. The intracellular domain of Ret consists of 14 tyrosine residues (a shorter isoform of Ret lacks 2 tyrosine residues at the C-terminus). Interactions of Ret with a variety of downstream targets have been correlated with clinical syndromes (MEN2A, MEN2B, FMTC, Hirschsprung’s disease; Fig. 4). Phosphorylated tyrosine residues Tyr905, Tyr1015, Tyr1062, and Tyr1096 were

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identified as docking sites for the adaptor proteins Grb7/Grb10, phospholipase C␥ (PLC␥), Shc/Enigma, and Grb2, respectively (Asai et al., 1996; Borrello et al., 1996; Durick et al., 1996; Liu et al., 1996; Arighi et al., 1997; Lorenzo et al., 1997; Ohiwa et al., 1997; Alberti et al., 1998; Xing et al., 1998). The specific phosphotyrosine residues of Ret required for the mitogenic and transforming activity depend on the cell type and the oncogenic form of Ret that is expressed. For example, Tyr905 is essential for the transforming activity of MEN2A-Ret mutant protein, whereas Tyr1062 regulates the activity of both MEN2A-Ret and MEN2B-Ret (Takahashi et al., 1998). Ret has been shown to activate several pathways typical for receptor tyrosine kinase signaling (Fig. 4). These include the Ras–MAPK (Santoro et al., 1994; Worby et al., 1996) and phosphoinositol-3-kinase (PI3K; van Weering et al., 1997) as well as Jun N-terminal kinase (JNK) (Chiariello et al., 1998; Xing et al., 1998) and PLC␥ (Borrello et al., 1996) dependent pathways. Although some intracellular events following GDNFdependent Ret activation are relatively well studied, specific signaling pathways leading to proliferation, survival, neurite outgrowth, and other differentiation effects of different GDNF family ligands in neurons are still poorly understood, compared to the neurotrophins (Kaplan and Miller, 1997, for review). It is also not known whether Ret activation by each of the GDNF family ligands and coreceptors will stimulate a distinct profile of signaling pathways, as was suggested for the activation of TrkB by its two ligands BDNF and NT-4 (Minichello et al., 1998). Recently, the effects of the GDNF family ligands in normal Ret signaling pathways have been studied in cell lines or in primary neurons. The Ras–MAPK pathway appears to be necessary for the survival and neurite growthstimulating actions of GDNF and NTN (Creedon et al., 1997; van Weering et al., 1997; Xing et al., 1998). PI3K signaling (possibly independent of its downstream substrate, the Ser/Thr-kinase Akt) is required for GDNF-induced formation of large lamellipodia, which are implicated in neuritogenesis (van Weering et al., 1997, 1998) and differentiation of cultured dopamine neurons (Pong et al., 1998). GDNF can activate the JNK pathway via Rho/Rac-related small GTPases, such as Cdc42 (Chiariello et al., 1998). Both PI3KAkt and JNK pathways are key regulators of neurotrophindependent neuronal survival (Kaplan and Miller, 1997), suggesting that they may also play key roles in mediating the trophic effects to the GDNF family ligands. Abrogation of the PLC␥-dependent signaling blocks the oncogenic activity of Ret (Borrello et al., 1996). We have shown that GDNF can trigger potent PLC␥-dependent Ca2⫹ release (Titievsky et al., submitted) and changes in intracellular free Ca2⫹ concentration [Ca2⫹]i are known to be

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important in the action of neurotrophic factors by activating many signal transduction cascades, for example, those that regulate gene expression (Finkbeiner and Greenberg, 1998). The role of the Zn-finger protein Enigma in neurons remains enigmatic. Based on its homology with proteins that link receptors to postsynaptic densities, Enigma may be required for the correct localization of Ret in specific signaling zones (Durick et al., 1998).

The Expression Patterns of GDNF Family Receptors Suggest Novel Mechanisms of Action In vivo, GDNF family ligands are thought to be released in limiting amounts by distinct target tissues in which they bind their specific coreceptor on the innervating nerve endings and activate Ret. Ret is normally coexpressed with one or several of the Gfras in different tissues, which is often complementary to the expression of their ligand mRNAs (Luukko et al., 1997, 1998; Trupp et al., 1997, 1998; Widenfalk et al., 1997; Naveilhan et al., 1998; Yu et al., 1998). In many cells, however, the Gfras are expressed without Ret (e.g., in Schwann cells and many areas of adult brain). This suggests that the GFR␣ receptors may also interact with Ret in soluble forms (Naveilhan et al., 1997) or in trans (Trupp et al., 1997) or that Ret is not necessarily required for GDNF family signaling. Although bead-immobilized GFR␣1/GDNF complex is able to activate Ret in trans (Yu et al., 1998), as yet there is no evidence of a trans action in vivo. Alternatively, other Ret-like or unrelated molecules may be involved in GFR␣ signaling, or the GFR␣ receptors may signal on their own (see below).

Ret-Independent Signaling of GDNF via GFR␣ Molecules According to current understanding, GPI-anchored proteins, transmembrane tyrosine kinase proteins, and acylated tyrosine kinases of the Src family all associate with so-called lipid rafts. These rafts are structures of sphingolipids and cholesterol packed into moving platforms within the lipid bilayer (Sargiacomo, 1993; for reviews see Simons and Ikonen, 1997; Brown and London, 1998). At least in the immune system, GPI-linked proteins are able to mediate intracellular signaling events, such as the activation of the Src family kinases and elevation of [Ca2⫹ ] i (Green et al., 1997; Xavier et al., 1998). The existence of microdomains of GPI-anchored proteins was recently shown also in living cells (Varma and Mayor, 1998; Friedrichson and Kurzchalia, 1998). GDNF promotes the survival of postnatal rat cochlear neurons, which express Gfra1 but lack detectable Ret

(Ylikoski et al., 1998). In sensory neurons from Ret⫺/⫺ mice, 10–100 ng/ml GDNF can induce a rapid activation of Src-type kinases and PLC␥ followed by long-lasting [Ca2⫹ ] i elevation. In addition, Src kinases can be coimmunoprecipitated with GFR␣1 in Triton-insoluble fractions (Titievsky et al., submitted). Thus, we propose a model (Fig. 5), in which GFR␣1 receptor, located in the raft, recruits and activates Src-type kinases upon GDNF binding. Src kinases, in turn, phosphorylate phospholipase C␥, leading to the production of IP3 and release of Ca2⫹ from intracellular pools. Depending on the cell type, ligand-induced clustering of GPI-anchored proteins may activate different signaling pathways. In support of our model, GDNF promotes survival and Fos activation in cell lines expressing GFR␣1 but lacking Ret (Trupp, 1998). The mechanism of GFR␣1-mediated Src activation, as well as the in vivo relevance of this signaling are unclear. Src family kinases have been shown to promote proliferation and neurite outgrowth (Thomas and Brugge, 1997). For example, the Ret-independent signaling by GDNF may play a role postnatally in brain plasticity or in Schwann cells after nerve lesion as an autocrine signaling to promote regeneration. It is also not known whether other GFR␣ receptors can signal independent of Ret or how, in a neuron that expresses Ret and GFR␣1, the two signaling systems are related to each other.

Cross-talk between the Ret Signaling Components in Vitro Although each GDNF family member binds one preferred GFR␣ receptor in vitro, the coreceptors show a degree of promiscuity in their ligand specificities and differences in how they activate Ret (Fig. 2; Baloh et al., 1997, 1998b; Buj-Bello et al., 1997; Creedon et al., 1997; Jing et al., 1997; Klein et al., 1997; Sanicola et al., 1997; Suvanto et al., 1997; Trupp et al., 1998). In a cell-free system without Ret, in which equilibrium binding of ligands was compared in solution, soluble GFR␣1 showed high specificity for GDNF over NTN (Klein et al., 1997). In another study, in which the plates were coated with the ligands, soluble GFR␣1 could bind equally well to immobilized GDNF and NTN (Baloh et al., 1998b). In both studies, GFR␣2 showed high binding specificity to NTN when tested alone (Klein et al., 1997; Baloh et al., 1998b); but together with soluble Ret, GFR␣2 was also able to bind GDNF (Sanicola et al., 1997). In cell lines, GDNF and NTN appear almost equally effective in causing Ret phosphorylation through GFR␣1 (Baloh et al., 1997; Creedon et al., 1997; Jing et al., 1997), whereas NTN is 30–100 times more potent than GDNF for Ret activation via GFR␣2 (Baloh et al., 1997; Jing et al.,

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FIG. 1. Schematic structure of glial-derived neurotrophic factor (GDNF) family ligands showing the relative lengths (and number of amino acids) of the mature molecules (green) and their pre- (pink) and pro- (peach) domains, as well as relative positions of the seven conserved cysteine residues (yellow). Neurturin (NTN), artemin (ART), and persephin (PSP). FIG. 2. The four masters and a servant. All the GDNF family ligands (GDNF, NTN, ART, and PSP) activate the transmembrane Ret tyrosine kinase via different glycosyl phosphatidylinositol-linked GFR␣ receptors. Thick lines represent preferred functional binding, whereas interactions shown by dotted lines may not be physiologically significant in vivo. Mammalian GFR␣4 has not yet been found. It is thought that GDNF first binds to GFR␣1 and then the complex can interact with Ret and promote its dimerization (Jing et al., 1996). In contrast, GDNF may be able to bind GFR␣2 only if GFR␣2 is already bound to Ret (Sanicola et al., 1997).

1997). Interestingly, the binding of GDNF to both GFR␣1 and GFR␣2 can be competed by both GDNF and NTN, but that of NTN can only be competed by NTN (Jing et al., 1997). This suggests the presence of two distinct

binding sites in GFR␣ molecules: one that binds only NTN and another that can harbor both GDNF and NTN. In cultured, microinjected sympathetic neurons, GDNF at high concentrations was able to promote survival

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FIG. 3. (Right) Alignment of cysteines (yellow) and predicted secondary structures of mouse GFR␣1, GFR␣2, and GFR␣3 and chicken GFR␣4. Amino acids of GFR␣3 that are different from the consensus sequence and secondary structure are highlighted (red). (Left) The proposed domain structure is modified, by permission of the publisher, from Suvanto (1997). Globular Cys-rich domains linked with flexible hinge domains, as well as putative N-glycosylation sites (Y) are shown. GFR␣2 has an additional cysteine-rich domain in its C-terminus (Suvanto, 1997). GFR␣ proteins are attached to the plasma membrane through a GPI anchor. FIG. 4. Known or proposed intracellular Ret-binding proteins (red) and signaling pathways. The phosphorylated tyrosine residues (-(P)Y) in Ret used for docking are shown. Enigma binding to Tyr1062 is phosphorylation independent. The most common activating mutation sites of Ret in the cancer syndromes multiple endocrine neoplasia 2 (MEN2) and familial medullary thyroid cancer (FMTC) are indicated on the right side (light

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component for PSP, shows some cross-talk in vitro, because the Ret–GFR␣4 receptor complex is capable of mediating modest survival responses to high concentrations of NTN (Enokido et al., 1998). GFR␣3 seems to be the least promiscuous coreceptor—of the four ligands, only ART can activate Ret and induce any biological activity via GFR␣3 (Fig. 2; Baloh et al., 1998b).

Other Trophic Factors Required for Ret-Dependent GDNF Family Signaling Recently, TGF-␤ was shown to be a necessary cofactor for the trophic effects of GDNF in a variety of neurons in vitro (Kriegelstein et al., 1998) and also in vivo (Schober et al., 1999). However, the mechanism of action and the in vivo physiological relevance of TGF-␤ in Ret signaling are unknown. Interestingly, promotion of survival of axotomized corticospinal neurons by GDNF appears to require endogenous BDNF. This suggests cross-talk between GDNF and BDNF signaling in regeneration and possibly in other types of neuronal plasticity (Giehl et al., 1998a). FIG. 5. Model of Ret-independent signaling of GFR␣1. GDNF induces GFR␣1 clustering in rafts and phosphorylation (-P) of Srcfamily kinases anchored to rafts. Src kinases activate PLC␥, which leads to IP3 production and long-lasting Ca2⫹ release from intracellular stores and subsequently activation of downstream targets such as transcription factors. Other signaling pathways are also possible.

through GFR␣2, but NTN could not promote survival through GFR␣1 (Buj-Bello et al., 1997). In contrast, midbrain dopamine neurons, which express GFR␣1 but not GFR␣2, appear to survive equally well with both GDNF and NTN both in vitro and in vivo (Horger et al., 1998) and respond to neither factor in the absence of GFR␣1 (Cacalano et al., 1998). It is still unclear whether NTN signals directly through GFR␣1 in vivo or if the effect of NTN is indirect. In an indirect effect, GFR␣2 expressed by cells in the vicinity of the dopamine neurons could activate Ret in trans (Horger et al., 1998) or the cells could produce GDNF after NTN stimulation. GFR␣1 seems to be the more promiscuous coreceptor, because it can also bind and mediate the survival effects of ART (Baloh et al., 1998b) in addition to NTN and its preferred partner GDNF. Also GFR␣4, the preferred ␣

PHENOTYPES OF MICE LACKING GDNF FAMILY LIGANDS AND THEIR RECEPTORS Mice lacking Ret (Schuchardt et al., 1994), GDNF (Moore et al., 1996; Pichel et al., 1996; Sa´nchez et al., 1996), or GFR␣1 (Cacalano et al., 1998; Enomoto et al., 1998) all die soon after birth and share a similar phenotype of kidney agenesis and absence of enteric neurons below the stomach. This suggests a tight coupling of GDNF and GFR␣1 in Ret signaling in vivo. There are some differences, however, most notably in the superior cervical sympathetic ganglia (Table 1 and below). In contrast to the GDNF and GFR␣1 knockout mice, mice lacking NTN (Heuckeroth et al., 1999) or GFR␣2 (Rossi et al., 1999) are viable and fertile. They share a similar eye phenotype and deficits in the enteric and parasympathetic nervous system (Table 1), which indicates a tight pairing also of NTN and GFR␣2 in vivo. The cholinergic innervation of the lacrimal and salivary glands is almost absent and is severely reduced in the

blue). Inactivating mutations found in Hirschsprung’s disease are dispersed throughout the sequence and are not shown. The extracellular part of Ret is not in scale. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; PLC, phospholipase-C.

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TABLE 1 Phenotypes of GDNF-Family Ligand- and Receptor-Deficient Mice Gene knockout:

Ret

Gdnf

Gfra1

Ntn

Gfra2

Gross phenotype

Lethal P0

Lethal P0

Lethal P0

Droopy eyelids with discharge, normal growth, fertile

Dry eyes, blinking eyelids, poor growth after weaning, fertile

Sensory Trigeminal

ND

n.s.

Fewer Gfra2 neurons

ND ND

n.s., fewer Gfra1 neurons 40% loss 23% loss

n.s. n.s.

n.s. Fewer Gfra2 neurons

Fewer ‘‘Gfra2’’ neurons a,b n.s. ND

100% loss Fewer neurons b

35% loss ND

n.s. ND

No neurons in bowel, few in stomach

No neurons in bowel, few in stomach

No neurons in bowel, few in stomach

n.s. 45% loss (P0), no fibers in lacrimal gland (adult) AChE fibers 40% loss in small intestine

n.s. 80% loss (adult), no fibers in lacrimal gland (adult) AChE fibers 45% loss in small intestine

ND

Spinal 22–31% loss

n.s. (oculomotor)

n.s. (oculomotor)

No gross defects No kidneys Schuchardt et al., 1994; Durbec et al., 1996

n.s. (LC less TH) No kidneys Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996; Granholm et al., 1997; Naveilhan et al., 1998

Spinal 24% loss1 or n.s.2 n.s. (SN and LC) No kidneys 1Cacalano et al., 1998; 2Enomoto et al., 1998

No gross defects No gross defects Heuckeroth et al., 1999

No gross defects No gross defects Rossi et al., 1999

Nodose DRG Autonomic Sympathetic (SCG) Parasympathetic (SMG) Enteric CNS Motor Brain Other Reference

Note. DRG, dorsal root ganglia; LC, locus coeruleus; ND, not determined; Nodose, nodose–petrosal complex; n.s., not significantly different; SCG, superior cervical ganglion; SMG, submandibular ganglion; SN, substantia nigra; TH, tyrosine hydroxylase. aA short form of Gfra2 mRNA is still expressed in Gfra2⫺/⫺ brain (with pattern similar to the full-length wild-type mRNA) but not in the sensory ganglia. bM.S.A. et al., unpublished data.

small bowel. In contrast to the dramatic growth retardation in Gfra2⫺/⫺ mice, the Ntn⫺/⫺ mice are reported to grow well. This suggests NTN-independent signaling of GFR␣2 in vivo. The reason for the growth retardation of Gfra2⫺/⫺ mice is unclear at present. Our hypothesis is that malnutrition caused by salivary gland and/or small intestinal dysfunction may be more severe in GFR␣2- than in NTN-deficient mice. However, other possibilities exist, for example, high expression of Gfra2, and Ret, in the hypothalamus, and the side effect of GDNF infusion into ventricles on body weight may indicate CNS involvement (Giehl et al., 1998b). To resolve this issue, it will be important to compare the neuronal defects and growth of Gfra2⫺/⫺ and Ntn⫺/⫺ mice under identical conditions. In the mice lacking different GDNF family members or receptors, only minor phenotypes have been found in the CNS so far (Table 1). This is in agreement with the idea of trophic redundancy for central neurons (Snider,

1994). Below we discuss in more detail the phenotypes in autonomic, sensory, and motor neurons, which show the clearest neuronal phenotypes in these mice. Because knockouts of ART/GFR␣3 or PSP/GFR␣4 are not available, it is difficult to do full comparison.

Sympathetic Neurons There are the normal numbers of SCG neurons in Gfra1⫺/⫺ (Cacalano et al., 1998; Enomoto et al., 1998), Gfra2⫺/⫺ (Rossi et al., 1999), and Ntn⫺/⫺ (Heuckeroth et al., 1999) mice and only 30% loss in Gdnf⫺/⫺ mice (Moore et al., 1996). This contrasts with the complete lack of SCG in Ret⫺/⫺ mice (Durbec et al., 1996a). Even if Gfra1 and Gfra2 were coexpressed in most SCG neurons, complete redundancy between GDNF and NTN in Ret activation via these coreceptors appears unlikely. Supporting this notion, the SCG is clearly present in Gdnf⫺/⫺/Gfra2⫺/⫺ double-mutant mice at birth (M.S.A. et al., unpublished

320 findings). This strongly suggests a role for other GFR␣ molecules and their ligands during SCG formation in sympathetic neuroblasts. A good candidate is Gfra3, which is strongly expressed in embryonic and newborn mouse SCG neurons. These neurons do not respond to GDNF or NTN (our unpublished observations) but should respond to the GFR␣3 ligand ART (Baloh et al., 1998b).

Enteric Neurons Enteric neurons and glial cells are derived from the vagal and sacral neural crest (Gershon 1997). These cells are missing below stomach in mice with inactivating mutations in GDNF/Ret or endothelin system (reviewed by Pasini et al., 1996). GDNF promotes proliferation of enteric precursor cells, migrating throughout the gut, and promotes their differentiation into neurons (Heuckeroth et al., 1998; Natarajan et al., 1999). Endothelin-3 modulates GDNF action, inhibiting the neuronal differentiation (Hearn et al., 1998). Accordingly, mutations affecting GDNF or endothelin signaling in humans cause aganglionic megacolon either through inadequate proliferation or through precocious differentiation, respectively. In mice lacking NTN or GFR␣2, the phenotype in enteric neurons is more mild than in GDNF- or GFR␣1mutant mice (Table 1). This correlates with the delayed expression of Gfra2 in the gut (Widenfalk et al., 1997, Rossi et al., 1999). The loss of acetylcholinesterase and substance P-positive fibers is more pronounced than the loss of NADPH-diaphorase and VIP-positive fibers in these mice. This is consistent with the idea that NTN/ GFR␣2 signaling is required for the development and maintenance of the excitatory cholinergic subpopulation of myenteric neurons (Costa et al., 1996). Interestingly, GDNF and NTN and their receptor components are strongly expressed, with a distinct expression pattern, also in the adult gut (our unpublished data). These results suggest that Ret signaling could also be required by adult enteric neurons. Thus, Ret signaling may be involved in pathogenesis of functional bowel diseases (for example, recurrent abdominal pain and irritable bowel syndrome) and may represent a target for therapy.

Parasympathetic Neurons Defects in the parasympathetic nervous system have been found in mice lacking either NTN or GFR␣2 (Table 1). Their phenotypes show that NTN signaling via GFR␣2 is essential for the development of specific postganglionic parasympathetic neurons and suggest that it could be involved in human disorders such as dry

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eye and dry mouth syndromes. As expected, the parasympathetic ganglia, which innervate the lacrimal and salivary glands and are affected in Gfra2⫺/⫺ mice, express Ret and Gfra2 (Rossi et al., 1999). Interestingly, low GDNF levels support early chick ciliary neurons in vitro (Buj-Bello et al., 1995) but not mouse submandibular neurons. The latter respond to low NTN concentrations and do not require GFR␣1 (Cacalano et al., 1998). Ciliary neurons appear largely unaffected in Gfra2⫺/⫺ mice, which suggests that they do not depend on a single GDNF or other family ligand in vivo. Future studies should compare in more detail the development and target innervation of different parasympathetic ganglia of mice lacking different GDNF family ligands and receptors.

Sensory Neurons In the dorsal root ganglia, Ret is expressed by a subpopulation of mainly small unmyelinated, IB4 lectinpositive, TrkA- and neuropeptide-negative neurons which mediate pain sensation (Molliver et al., 1997; Bennett et al., 1998). The neurons, many of which are supported by GDNF, terminate in the inner part of lamina II of the spinal cord. Exogenous GDNF prevents several axotomy-induced changes in these neurons and also prevents the A fiber sprouting into lamina II. This suggests that GDNF may be useful in the treatment of peripheral neuropathies (Bennett et al., 1998; Snider and McMahon, 1998). Interestingly, GDNF-like immunoreactivity is found in the neuropeptidepositive, Ret-negative neurons terminating in lamina I and outer lamina II (Holstege et al., 1998). Thus, endogenous GDNF signaling may also play a role in pain sensation. During development, Gfra1, Gfra2, and Gfra3 are expressed in distinct but partially overlapping subpopulations of trigeminal ganglion neurons (Bennett et al., 1998; Naveilhan et al., 1998), while GDNF and NTN are expressed by their target areas (Luukko et al., 1997, 1998; Widenfalk et al., 1997). In vitro, GDNF, NTN, and ART support a subpopulation of sensory neurons (Baloh et al., 1998b), and NTN, but not GDNF, fails to support Gfra2⫺/⫺ trigeminal neurons (Rossi et al., 1999). In trigeminal ganglion, Gfra1- but not Gfra2- or Gfra3expressing neurons are reduced in Gdnf⫺/⫺ mice (Naveilhan et al., 1998), whereas Gfra2- but not Gfra1-expressing neurons are reduced in Ntn⫺/⫺ and Gfra2⫺/⫺ mice (Heuckeroth et al., 1999; M.S.A. et al., unpublished data). This suggests that most of the responsive neurons are missing. However, because direct evidence of neuronal death is lacking, it remains possible that the neurons survive and merely down-regulate Gfra mRNA levels. Taken together, these results suggest that GDNF, NTN, and ART support the survival and/or differentiation of

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distinct subpopulations of primary sensory neurons in vivo. The functional identity of these neurons, as well as the in vivo roles of GDNF family factors, and the stages at which they are critical for development are unknown.

Motor Neurons Consistent with a role for GDNF as an in vitro motoneuron survival factor (Henderson et al., 1994; Acre et al., 1998), there is about a 20–30% loss of motoneurons in both Gdnf⫺/⫺ and Gfra1⫺/⫺ mice (Table 1). However, no motoneuron loss has been reported in Ret⫺/⫺ mice. In vitro, GDNF, NTN, and PSP are trophic factors for specific subpopulations of motoneurons and act in synergy with other factors such as the neurokine cardiotrophin-1 (Arce et al., 1998). After peripheral axotomy, Gdnf is up-regulated in the denervated muscle, Gdnf and Gfra1 in the Schwann cells of the distal nerve stump, and Ret and Gfra1 in the motoneurons. These observations suggest an important trophic role for GDNF in regenerating motor axons in adults (Naveilhan et al., 1997; Trupp et al., 1997). Overexpression of GDNF in muscle produced hyperinnervation of neuromuscular junctions in neonatal mice probably by preventing multiple synapse elimination (Nguyen et al., 1998). Normal locomotor behavior and a lack of gross motoneuron defects in Ntn⫺/⫺ or Gfra2⫺/⫺ mice suggest that NTN/GFR␣2 signaling is not essential for motoneuron development.

The GDNF Family and Activity-Dependent Neuronal Plasticity in the Brain In addition to their well-established survival and neurite outgrowth effects, GDNF family members may have other effects on neuronal phenotype such as regulating neurotransmitter content (e.g., Beck et al., 1996). mRNAs of GDNF family ligand and receptors are differentially expressed in specific neuronal populations and differentially regulated after brain insults (Humpel et al., 1994; Schmidt-Kastner et al., 1994; Trupp et al., 1997; Reeben et al., 1998; Kokaia et al., 1999). It is conceivable that the GDNF family members, in a manner similar to the neurotrophins (see Thoenen, 1995, for a review), may play a role in neuronal regeneration and in activity-dependent synaptic plasticity. For example, the dynamic changes in GDNF family ligand and receptor mRNAs following recurrent seizures could contribute to subsequent development of abnormal excitability in epilepsy (Kokaia et al., 1999, and unpublished data). GDNF can affect neuronal transmission via potentiation of neurotransmitter release. Quantal release of

dopamine recorded directly from axonal varicosities of midbrain dopamine neurons increased severalfold after GDNF exposure (Pothos et al., 1998). In neonatal mouse neuromuscular junction, GDNF produced a twofold increase in transmitter release (Ribchester et al., 1998). The mechanisms and regulation of neuronal GDNF synthesis and release are poorly known. Many factors (e.g., fibroblast growth factors) and multiple signaling pathways and secondary messenger systems (e.g., cAMP) can promote GDNF secretion in cell lines (Verity et al., 1998). In response to growth factors, cytokines, and pharmacophores, the synthesis/release of GDNF appears to be differentially regulated in cells of glial and neuronal origin (Verity et al., 1999).

CONCLUSIONS Is the GDNF family complete? All four masters of the GDNF family have their coreceptor partners, and all have been shown to be served by Ret. All neurons and tissues affected in Gfra1 and Gfra2 knockout mice also express Ret. However, additional masters (ligands with their coreceptors) with restricted or postnatal expression may still exist. They may need more than the one servant signaling receptor or may signal themselves without the service of Ret. It appears that each GDNF family factor with its coreceptor is crucial for specific subtypes of peripheral neurons. Double knockouts may reveal new functions of various Ret-signaling components in development, and conditional knockouts will permit us to explore their functions in the adult CNS. The distinct expression of ART and GFR␣3 suggests a role for this pair in the development of sympathetic and of specific sensory neurons in vivo. The weak and widespread expression of PSP and the lack of information on mammalian GFR␣4 make it difficult to predict an in vivo role for PSP/ GFR␣4. GDNF appears to be a unique member of the family in that it can act as a kidney morphogen (for a review see Sariola and Sainio, 1997), but other family members may also exhibit nonneuronal effects via Ret postnatally. Mutations in different GDNF family ligands and receptors could be involved in the etiology of other human diseases, in addition to Hirschsprung’s disease and MEN2 cancer syndromes. For example, rare mutations in the GDNF pathway have been found in the congenital hypoventilation syndrome (Ondine’s curse; Amiel et al., 1998), and Ret⫺/⫺ mice show a similar phenotype (Burton et al., 1998). In addition, specific stimulation or inhibition of Ret-signaling pathways may be useful drug targets for therapy.

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ACKNOWLEDGMENTS We thank U. Aruma¨e, D. Rice, H. Sariola, and A. Vieira for critical comments about the manuscript. This work was supported by the Academy of Finland, Biocentrum Helsinki, Cephalon, Inc., EU Biomed (Grant BMH4-97-2157), and the Sigrid Juselius Foundation.

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