Opinion
GDNF and GFRa: a versatile molecular
complex for developing neurons
Gustavo Paratcha and Fernanda Ledda
Laboratory of Molecular and Cellular Neuroscience, Department of Neuroscience, Karolinska Institute, S-17177 Stockholm, Sweden
The GDNF family ligands (GFLs) signal through the
canonical signaling receptor Ret and a glycosyl-phos-
phatidylinositol-anchored co-receptor, GFRa. In recent
years, signaling by GFLs has been shown to be more
complex than originally assumed. The discrepant
expression between GFRas and Ret has suggested the
existence of additional signal-transducing GDNF recep-
tors, such as NCAM. Here we summarize novel functions
and Ret-independent signaling mechanisms for GDNF
and GFRa, focusing on developing neurons. Emerging
evidence indicates a prominent role of GDNF and GFRa
in the control of neuroblast migration and chemoattrac-
tion and in the formation of neuronal synapses by a new
mechanism of ligand-induced cell adhesion. Therefore,
these data highlight the importance of this versatile
molecular complex for nervous system development,
function and regeneration.
Introduction
Glial cell line-derived neurotrophic factor (GDNF) family
ligands (GFLs) play a crucial role in the development and
function of the nervous system [1,2]. GDNF was originally
characterized in 1993 as a growth factor promoting the
survival of ventral midbrain dopaminergic neurons, those
that degenerate in Parkinson’s disease [3]. Later, it was
found to have a potent survival effect on motor neurons and
other neuronal subpopulations in the central and periph-
eral nervous systems (CNS and PNS, respectively) [1,2,4].
In addition to its role as a survival factor, GDNF is also
essential for proliferation, migration and differentiation of
neuronal cells [2,5–8]. The biological effects summarized in
Table 1 highlight the pleotropic roles played by GDNF
during neuronal development.
The GFLs consist of four members, GDNF, Neurturin
(NRTN), Artemin (ARTN) and Persephin (PSPN), which
are involved in the survival, proliferation and differen-
tiation of several neuronal populations in the CNS and
PNS [2]. A common feature of the receptor complex for the
GFLs is the requirement of two receptor subunits, one
specialized in transmembrane signaling, the receptor tyro-
sine kinase Ret, and the other involved in ligand binding,
the glycosyl-phosphatidylinositol (GPI)-anchored co-recep-
tor GFRa (GDNF family receptor a) [1]. In 1996, the
transmembrane receptor tyrosine kinase Ret was ident-
ified as a GDNF receptor [9,10]. Ret is shared by all of the
GFLs as their common signaling receptor. However, Ret is
unable to bind GDNF or any of the other GFLs in absence
Corresponding authors: Paratcha, G. (gustavo.paratcha@ki.se);
Ledda, F. (fernanda.ledda@ki.se).
384 0166-2236/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2008.05.003 Available online 1 July 2008
of the GFRa co-receptor. The other component of the
GDNF receptor system, GFRa1, was identified in 1996
by expression cloning and screening of GDNF binding
protein [11,12]. The family of GFRa co-receptors includes
four members (GFRa 1–4) which determine ligand speci-
ficity. Thus, GFRa 1–4 are the preferential co-receptors for
GDNF, NRTN, ARTN and PSPN, respectively, but alterna-
tive ligand–co-receptor interactions can also occur [13].
Following homodimeric GFL binding to the cognate GFRa
co-receptor, Ret becomes dimerized and tyrosine phos-
phorylated, and triggers different pathways, including
the Ras-MAPK (mitogen-activated protein kinase), the
phosphatidylinositol-3 kinase (PI3K)-Akt, the PLC-g and
the Src signaling pathways (Figure 1) [13,14].
Several observations have indicated that GDNF
signaling is more complex than originally supposed. In
several regions of the nervous system, GFRas are highly
expressed where Ret is undetectable [2,13]. One possibility
to explain this discordance is that GFRas can activate Ret
signaling in a non-cell-autonomous fashion, presenting
GFLs in trans to Ret-expressing cells (see Figure 1)
[15–17]. Furthermore, the discrepant expression of Ret
and GFRas prompted many groups to intensively explore
for the existence of additional GFL receptors that signal
cooperatively with GFRas but independently of Ret
(Table 1) [18–24]. Thus, in 2003, NCAM was identified
as an alternative signaling receptor for GFLs [21]. This
large body of evidence points out the relevance of GFRa co-
receptors for the versatility of GFL signaling (see Figure 1
and Box 1).
This review summarizes recent advances made in our
understanding of the Ret-dependent and -independent
signaling mechanisms used by the GFL-GFRa complex
and their functional implications for nervous system de-
velopment. Based on the potential therapeutic implica-
tions of GDNF and Ret in multiple neurodegenerative
diseases, we also draw attention to some new mechanisms
through which Ret receptor signaling and biology are
restricted in neuronal cells.
Novel physiological functions of GFRa-Ret signaling
Mice lacking Ret die a few hours after birth owing to
kidney agenesis and show absence of enteric and many
parasympathetic neurons [2]. A similar phenotype was
described for Gdnf- and Gfra1-null mutant mice
[2,25,26]. By contrast, Gfra2-, Gfra3- and Gfra4-mutant
mice are viable and fertile [2]. In light of the known in vitro
survival effects of GDNF/Ret signaling on dopaminergic
and motor neurons, it was surprising that mice lacking
 Opinion Trends in Neurosciences Vol.31 No.8

Table 1. Summary of GDNF effects on neuroblasts and mature neurons

Biological functions Cell type Receptor system Refs
Neuroblast
Proliferation Enteric progenitors Ret/GFRa [6,8]

Migration/chemoattraction Enteric progenitors Ret/GFRa [5,7]

MGE-derived progenitors ?/GFRa [20]
RMS-derived progenitors NCAM/GFRa [18]

Differentiation Enteric progenitors Ret/GFRa [8]

MGE-derived progenitors ?/GFRa [20]

Neuron
Axonal growth Sensory and sympathetic neurons Ret/GFRa* [16]
Motor neurons Ret/GFRa [52]
Hippocampal neurons NCAM/GFRa [21]
Cortical GABAergic neurons ?/GFRa [20]
Axon guidance Sensory and sympathetic neurons Ret/GFRa* [16]
Spinal chord motor neurons Ret/GFRa [29]

Survival VM dopaminergic neurons Ret/GFRa [3]

Spinal chord motor neurons [2,4]
Sympathetic, parasympathetic and sensory neurons [2]
Enteric neurons [8,27]

Synapse formation Midbrain dopaminergic neurons Ret/GFRa [50]

Motor neurons [32,52]
Hippocampal and cortical neurons NCAM/GFRa [19]

The table describes the distinct biological functions of GDNF at different time points during neuronal development. The neuronal type and the receptor system through which
GDNF ligands induce these biological effects are also indicated. Asterisks indicate that the effect was obtained using soluble GFRa presented in trans and ‘‘?’’ indicates that the
signaling receptor is unknown. MGE = medial ganglionic eminence; RMS = rostral migratory stream; VM = ventral midbrain.

Ret, GDNF or GFRa1 show only minor phenotypes in the
CNS during embryonic development. However, the
early lethality of these animals precluded postnatal
analyses of the nervous system. To overcome this obstacle,
recently different groups have generated mice with selec-
tive ablation of Ret or GFRa1 in different neuronal popu-
lations that are compatible with postnatal survival
[27–32]. Here we attempt to summarize the current knowl-
edge regarding the in vivo function of the Ret receptors as
determined from the analysis of tissue-specific Ret-knock-
out and -inducible Gfra1-deficient mice that have been
generated during the last 2 years.
Dopaminergic neurons
Although GDNF was originally characterized as a potent
survival factor for midbrain dopaminergic neurons,
analysis of knockout mice has shown that GFRa1-Ret
signaling is not essential for the embryonic development
of these neurons, illustrating the complex network of sig-
naling pathways and compensatory mechanisms that
regulate neural development. In agreement with this, it
was suggested that the neurotrophic factor CDNF (con-
served dopamine neurotrophic factor) might compensate
GDNF-GFRa1-Ret deficiency during embryonic midbrain
development [33]. Recently, two groups have reported the
generation of different strains of mice with the specific
deletion of Ret in dopaminergic neurons [30,31]. Surpris-
ingly, these animals showed normal development and
maturation of the nigrostriatal system. A detailed study,
including dopaminergic neuron number in substantia
nigra pars compacta (SNpc) and ventral tegmental area
(VTA), fiber density in the striatum and nucleus accum-
bens, and dopamine levels, indicated that Ret is not
required for the maintenance of midbrain dopaminergic
neurons in young adult mice. However, in one of these
studies, Kramer and coworkers found that, only in aged
animals, Ret ablation leads to a progressive and cell-type-
specific loss of SNpc dopaminergic neurons and their
afferents, thereby indicating that Ret is a critical regulator
for the long-term maintenance of the nigrostriatal system
[30].
The significance of Ret signaling for the dopaminergic
system was additionally studied in knockin multiple endo-
crine neoplasia type B (MEN2B) mice by introducing the
Ret-MEN2B mutation (Met919Thr), which renders the
Ret receptor tyrosine kinase constitutively active. In this
work, the authors showed that the constitutive Ret
activity in mice is sufficient to increase brain dopamine

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 Opinion Trends in Neurosciences Vol.31 No.8

Figure 1. Schematic representation of GFL signaling through Ret and p140NCAM receptors. The left side of the figure describes GFL-induced Ret receptor tyrosine kinase
activation and signaling by membrane-bound (cis-signaling) and soluble GFRa (sGFRa, trans-signaling) molecules released from the plasma membrane. During activation
in cis, GFL dimers bind to GPI-anchored GFRa receptors located in lipid rafts. The heterocomplex GFL-GFRa associates with Ret resulting in its dimerization, tyrosine
autophosphorylation and activation of downstream signaling pathways. During activation in trans, GPI-anchored GFRa is released from the membrane of neighboring cells
by the action of unknown phospholipases or proteases. Soluble GFRa molecules bind to and activate Ret inside and outside lipid rafts. For simplicity, the figure only shows
Ret activation by GFRa in trans in raft domains. Only the Ras/MAPK and PI3K/Akt signaling pathways are represented in the scheme. The right side of the figure summarizes
NCAM signaling. In the absence of GFLs, short-range signaling by homophilic interactions between p140NCAM molecules can be disrupted by the presence of GFRa. This is a
GFL-independent mechanism of extracellular signaling crosstalk between GFRa and p140NCAM molecules. In the presence of GFLs, the association of membrane-bound
GFRa and p140NCAM results in the activation of the Fyn-FAK-MAPK signaling pathway and in the ability of p140NCAM to mediate long-range intercellular communication.
Binding affinities of GDNF for Ret and NCAM in the presence of GFRa1 are as follows: DdRet + GFRa1 = 0.27  0.12 nM and DdNCAM + GFRa1 = 1.1  0.32 nM. For simplicity, the
molecules are represented in monomeric form.

concentration and the number of dopaminergic neurons in
the SNpc [34].
Motor neurons
The overexpression of GDNF in skeletal muscle induces
hyperinnervation of the neuromuscular junction (NMJ)
[35], indicating an important role for GDNF in motor
neuron target innervation. Using Ret-null mice and a
mutant mouse strain with a conditional inactivation of
Ret in the embryonic spinal cord, Kramer and coworkers
demonstrated that GDNF-Ret signaling functions as an
instructive guidance cue for certain motor axons toward
dorsal limb muscles. Thus, in these Ret-mutant mice,
motor axons follow an aberrant ventral trajectory away
from the dorsal territory enriched in GDNF. Interestingly,
this phenotype is enhanced in mutant mice lacking both
Ret and Eph receptor tyrosine kinase A4 (EphA4),
suggesting that Ret and EphA4 signals cooperate in the
motor neuron guidance decision [29]. Recently, Baudet and
coworkers have generated another strain of mice with a
specific deletion of Ret in cranial motor neurons. In these
animals, the absence of Ret results in a developmental
deficit in motor neuron maturation and specialization of
presynaptic neuromuscular terminals in specific motor
neuron pools [32].
Sensory neurons
In peripheral neurons of the dorsal root ganglia (DRG), Ret
is expressed by a subpopulation of nonpeptidergic cells
that mediates pain sensation. To analyze the influence of
Ret signaling on postnatal development of sensory
neurons, a new strain of mice with conditional deletion
of Ret in premigratory neural crest cells, including pro-
genitors of DRG neurons, was generated [28]. These
animals survive at birth; however, the majority of them
die by 3 weeks of age. Using these mice, the authors found
that Ret signaling is not required for the viability of non-
peptidergic DRG neurons in vivo but is important for the
acquisition of normal neuronal size, axonal innervation of
the epidermis, postnatal extinction of the NGF receptor
TrkA and expression of nonpeptidergic neuron-specific
genes.
Enteric neurons
Analysis of Ret-null mice embryos revealed that enteric
nervous system (ENS) progenitors are reduced in number
and fail to enter the midgut, precluding the analysis of the
physiological relevance of GDNF signaling in late ENS
development. To study this, Enomoto and coworkers have
engineered mice in which the function of GFRa1 can be
conditionally inactivated in late ENS development. Dis-
ruption of GFRa1 at this late stage resulted in a rapid and
widespread neuronal death in the colon, leading to agan-
glionosis reminiscent of Hirschsprung’s disease [27].
New insights into the negative control of Ret signaling
Despite the critical role played by GDNF and Ret in
orchestrating the development and maintenance of specific
neuronal subpopulations, the molecular mechanisms that
restrict Ret activation and downstream signaling are not
entirely understood. Undoubtedly, the understanding of
the mechanisms that regulate Ret signaling in neuronal
cells could open new therapeutic possibilities for the treat-
ment of both neurodegenerative disorders and nerve

386
Opinion
Box 1. GFL signaling: advantages to having a GFRa
co-receptor
Ret and NCAM signaling receptors require the presence of GFRa co-
receptors for both high-affinity GFL binding and receptor signaling
activation. Therefore, GFRas allow the GFLs to influence many
biological processes by signaling through different transmembrane
receptors (Figure 1) [13].
By virtue of their GPI anchor domain, GFRas are localized in
specialized microenvironments on the cell surface known as lipid rafts
[17,38,40,59]. Rafts are dynamic assemblies of cholesterol and
sphingolipids scattered within the lipid bilayer that have emerged
as membrane platforms specialized in signal transduction events
[60,61]. An important function of GPI-anchored GFRas is to induce a
rapid recruitment of Ret to lipid rafts in response to GFLs (cis
mechanism) [17,59]. As in many other GPI-linked molecules, GPI-
anchored GFRas can function in both membrane-bound and -soluble
forms after release by proteinases and/or phospholipases. Notably, in
cells lacking GFRa, Ret can also be recruited to raft compartments by
simultaneous treatment with a GFL and its cognate GFRa partner in
soluble form (trans mechanism) (Figure 1) [17]. In particular,
recruitment of Ret in trans is delayed and sustained. In this case,
activated Ret can also be detected outside rafts, suggesting the
existence of a dynamic equilibrium in which Ret is partitioning
between raft and nonraft compartments. Indeed, trans stimulation
has the capacity to activate different adaptor signaling molecules
depending on whether Ret is activated outside or inside rafts,
allowing this receptor to transmit different signals depending on its
membrane localization [17]. In addition, released GFRas have the
capacity to influence axonal guidance by creating positional informa-
tion for Ret-positive axons even in the presence of a uniform
concentration of GFLs [16]. Clearly, all of this evidence indicates that
the release of GFRa molecules could represent a general strategy to
diversify and potentiate both signaling and biological functions of
GFLs. The fact that both Schwann cells and injured sciatic nerves
release GFRa1 molecules to the extracellular space [17] could increase
the therapeutic possibilities of GFLs for the treatment of nerve injuries
and neurodegeneration.
Interestingly, GFRa molecules interfere with NCAM function
silencing NCAM homophilic interactions and NCAM-mediated
cell adhesion (Figure 1) [21]. More recent evidence indicates that
GDNF mediates cell adhesion between GFRa1-expressing cells
through a novel mechanism of ligand-induced cell adhesion [19],
revealing the ability of GFRa1 and GDNF to function as a remarkably
versatile molecular complex with capacity to modulate intercellular
communication.
injury. Research over the last 3 years has led to the
identification and characterization of the mechanism of
action of several Ret signaling inhibitors [36–41]. The
picture arising from these studies is that Ret signaling
is tightly regulated through the coordinated action of
different protein inhibitors that function at multiple levels
of the signaling cascade and at different time points after
receptor engagement.
A prevalent mechanism to control Ret is its downregu-
lation by ligand-induced receptor ubiquitination, which
sorts active Ret molecules for intracellular degradation
(irreversible inhibition). Indeed, Ret degradation is accom-
plished via the recruitment and polyubiquitination of the
receptor by Cbl ubiquitin ligases and subsequent degra-
dation by the proteasomal pathway [39,40]. Interestingly,
Pierchala and colleagues uncovered an additional role for
lipid rafts in the control of GDNF signaling. These mem-
brane domains sequester phosphorylated Ret away from
proteasomal degradation, thereby increasing the half-life
of activated Ret and strengthening its downstream sig-
naling [40].
Trends in Neurosciences Vol.31 No.8
Other negative controls include antagonists that
undergo transcriptional upregulation upon GDNF-induced
Ret signaling. These inhibitors function through negative
feedback loops to restrict Ret activation and signaling in a
reversible manner. Examples of this type of Ret inhibitors
include members of the Sprouty family of proteins [36,37]
and the leucine-rich repeat and immunoglobulin-like
domain protein Lrig1 [38].
A genetic study revealed that loss of the Sprouty2 gene
in mice resulted in enteric nerve hyperplasia due to GDNF-
induced hyperactivation of MAPK and Akt in enteric nerve
cells [36]. In agreement with this, Ishida and coworkers
also demonstrated that Sprouty2 is able to regulate growth
and differentiation of human neuroblastoma cells through
the Ret-MAPK pathway [37].
In addition to Sprouty, Lrig1 has also been identified as
an endogenous inhibitor of Ret activation and signaling. In
particular, physical association between Lrig1 and Ret
inhibits GDNF binding, recruitment of Ret to raft domains,
receptor autophosphorylation and MAPK activation in
response to GDNF [38].
NCAM: an alternative signaling receptor for GFLs
The identification of alternative signal-transducing GDNF
receptors has remained an unresolved issue for several
years. Recently, NCAM has been identified as an alterna-
tive signaling receptor for GFLs [21]. Interestingly, high-
affinity GDNF binding and signaling through NCAM
requires the presence of GFRa co-receptors. Indeed, in
cultured Schwann cells and cortical neurons, both of which
express GFRa and p140NCAM isoform, but not Ret, GDNF
treatment activates the cytoplasmic protein tyrosine
kinases Fyn and FAK, two signaling mediators down-
stream of p140NCAM [21]. Structure–function analysis
identified the third immunoglobulin domain of NCAM as
the necessary and sufficient determinant for its interaction
with GDNF [42]. A distinctive characteristic of GFRa is its
ability to act as a negative regulator of the NCAM homo-
philic interaction in the absence of GDNF [21]. Interest-
ingly, the role of GFRa in silencing NCAM–NCAM
interactions but promoting GDNF binding to NCAM
represents a novel mechanism of extracellular crosstalk
between soluble growth factor (long-range) and contact-
mediated (short-range) signaling with the capacity to influ-
ence important biological processes during nervous system
development (Figure 1).
The most distinctive abnormality of the CNS of mice
lacking NCAM is a dramatic reduction in the size of the
olfactory bulb (OB) [43], caused by defects in the migration
of neuronal precursors in the rostral migratory stream
(RMS). The prominent expression of GFRa1 and NCAM,
but absence of Ret, in the developing and adult RMS
indicates that Ret-independent GDNF signaling via
NCAM could play a role in the migration and guidance
of neuroblasts and in the development of the RMS-OB
pathway [21,44]. Consistent with this, Gfra1-deficient
newborn mice have a moderate enlargement in the width
of the developing RMS, a phenotype that is absent in
newborn mice lacking Ret [21]. However, these findings
have been considered inconclusive because the RMS and
OB are only partially developed by postnatal day 0 (P0),
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when GFRa1-mutant mice die. To further address this
issue, Enomoto and colleagues generated mutant mice
lacking GFRa1 expression in cells devoid of Ret, such as
those in the RMS [44]. Although no differences were
observed in either the size or the gross histological organ-
ization of the adult RMS and OB cell layers, it is possible
that some defects in specific neuronal populations might be
found in these structures following a more detailed
analysis. More recently, it has been shown that long-range
NCAM signaling by GDNF is able to evoke chemotropic
effects on neuronal precursors derived from the RMS [18],
indicating that a more detailed analysis of the different
neuronal cell types present in the OB of mice in which Ret-
independent GFRa1 expression is specifically eliminated
deserves further investigation.
In addition to providing guidance of neuroblasts in vitro,
GDNF-GFRa-NCAM signaling stimulates neurite out-
growth of cultured hippocampal and cortical neurons
[21], regulates Schwann cell migration and function
[21,45] and promotes presynaptic maturation and synapse
formation of hippocampal and cortical neurons in vitro and
in vivo [19]. Interestingly, a recent study demonstrating
that NCAM signaling plays a critical role in the analgesic
effect of GDNF in a rat model of neuropathic pain might
support the physiological relevance of this new signaling
pathway [46].
Recently, the possibility that GFRas might also have
cell-autonomous functions independent of Ret or NCAM
has been suggested. Intriguingly, Pozas and Ibáñez
demonstrated that GDNF regulates the differentiation
and tangential migration of cortical GABAergic neurons
via GFRa1 receptors but independently of Ret or NCAM,
suggesting that GFRa1 molecules might associate with as
yet unidentified signaling receptors [20]. In addition, Pop-
sueva and coworkers have shown that in cells expressing
GFRa1, but not Ret, GDNF transactivates Met signaling
through a Src-dependent mechanism [22]. Therefore, the
expression of the hepatocyte growth factor receptor Met in
migrating GABAergic neurons of the forebrain [47] might
be mediating the Ret- and NCAM-independent migratory
activity reported for GDNF and GFRa1 on these cells.
GFRa as a ligand-induced cell-adhesion molecule
Recently, a new role for GFRa proteins as ligand-induced
cell-adhesion molecules (LICAMs) has been described [19].
Unlike other known cell-adhesion molecules (CAMs) which
involve the direct interaction of membrane-associated
adhesion partners in trans, GFRa1-mediated cell adhesion
is dependent on the presence of its ligand, GDNF.
Whereas stable cell interactions are necessary to main-
tain the structural integrity of tissues, dynamic cell-
adhesion mechanisms are involved in the morphogenesis
of developing and regenerating tissues. The majority of the
CAMs known to date are regulated at different levels
including transcription, expression and clustering. There
are also dynamic CAMs that allow for the regulation of
intercellular interactions by conformational changes that
modify their adhesive properties through inside-out sig-
naling in response to external cues (e.g. integrins, cadher-
ins) [48,49]. However, the mechanism of ligand-induced

Figure 2. Model describing the mechanism of ligand-induced cell adhesion by GDNF and GFRa1. (a) Scheme summarizing GDNF-induced trans-homophilic cell adhesion
between GFRa1-expressing cells. (b) Hypothetical model describing trans-synaptic adhesion and presynaptic differentiation induced by GDNF and GPI-anchored GFRa1
molecules in hippocampal neurons. In the presence of GDNF, GFRa1, located at pre- and postsynaptic endings, holds the terminals together triggering presynaptic
maturation. The potential role of GDNF and GFRa1 promoting postsynaptic organization remains to be studied (?). The recruitment of synaptic vesicles (SV) to the
presynaptic terminal is dependent on GFRa1 and partially dependent on p140NCAM, suggesting the involvement of additional transmembrane signaling molecules (X). The
figure only describes the possibility that GDNF binding triggers a conformational change in GFRa1 molecules required for trans-homophilic binding. The alternative option
that GDNF could act as a bridge between the GFRa1 partners remains open and is not included in the scheme. For simplicity, the molecules are represented in monomeric
form. NtR = neurotransmitter receptors; SP = scaffolding proteins.

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Opinion
cell adhesion represents a new way to regulate cell–cell
interaction in which the ligand–receptor system is the
actual mediator of the contact, giving a new level of regu-
lation to the adhesive process (Figure 2a). The role of
GFRa1 as a LICAM predicts that other GPI-linked or
transmembrane receptors and their soluble ligands might
induce cell adhesion working in a similar fashion.
The molecular bases of GDNF-induced trans-homophi-
lic interactions between GFRa1 molecules are not clear
yet. The domains of GFRa1 underlying its LICAM activity
have been studied using GFRa1 deletion mutants. This
study indicated that GFRa1-mediated cell adhesion
requires the presence of an intact GDNF binding domain
in both interacting GFRa1 partners [19]. Because GDNF is
a dimeric molecule with twofold symmetry, it could act as a
bridge between the GFRa1 partners. Another possibility is
that GDNF binding triggers a conformational change in
GFRa1 molecules resulting in the reorientation and expo-
sition of the determinants involved in the trans-homophilic
binding.
The ability of GFRa1 to be released from the plasma
membrane of neuronal and glial cells indicates an
additional way to control cell adhesion mediated by GPI-
GFRa1. In agreement with this, GDNF- and GPI-GFRa-
induced cell adhesion can be disrupted in the presence of
soluble GFRa1 [19]. Thus, ligand-induced trans-homophi-
lic interactions between GFRa1 molecules represent a new
way to regulate intercellular interactions that could have
important implications during the development of the
nervous system, expanding the biological possibilities of
the GDNF–GFRa1 signaling system.
Role of GDNF and GFRa receptors in synapse formation
During the last decade, numerous studies have shown that
GFLs participate in synapse development and plasticity
[2]. Enhancement of transmitter release promoted by
GDNF from midbrain dopaminergic neurons and neuro-
muscular synapses led to the proposition that GDNF
regulates the establishment and functional properties of
synaptic terminals. An increase in the amount of presyn-
aptic vesicle clustering observed in midbrain dopaminergic
neurons indicated a role of GDNF in presynaptic differen-
tiation [50]. Yang and coworkers also demonstrated that
acute GDNF treatment modulates A-type K+ channels and
the excitability of these neurons in culture [51]. Further-
more, using nerve-muscle cocultures, it has been shown
that long-term application of GFLs induces a significant
increase in the number and size of presynaptic vesicle
aggregates as well as acetylcholine receptor (AChR) clus-
ters, indicating that GFLs are able to coordinate the de-
velopment of neuromuscular synapses through both pre-
and postsynaptic mechanisms [52]. The temporal expres-
sion pattern of GDNF and GFRa1 in the developing hippo-
campus and the localization of GFRa1 to pre- and
postsynaptic membranes, together with the ability of this
receptor to function as a LICAM, have suggested that
GDNF-GFRa1 complexes could act as synaptogenic factors
by inducing adhesion between pre- and postsynaptic
specializations (Figure 2b) [19].
Neuronal synapses can be defined as a specialized type
of cell–cell interaction that mediates the communication
Trends in Neurosciences Vol.31 No.8
between neurons and their targets. Trans-synaptic
adhesion molecules influence synapses at different levels
from the establishment of the synaptic contact and differ-
entiation of pre- and postsynaptic specialization to the
modulation of the function of synaptic channels and recep-
tors [53,54]. Recent evidence indicates that, in the presence
of GDNF, a localized source of exogenous GFRa1 that
mimics its postsynaptic localization was able to induce
presynaptic differentiation of hippocampal neurons. This
effect was visualized by the clustering of vesicular proteins
and neurotransmitter transporters and by activity-de-
pendent vesicle recycling. Similar results were observed
in excitatory and inhibitory hippocampal neurons [19].
However, whether this is a common mechanism used by
other neuronal populations remains to be established.
Interestingly, presynaptic differentiation induced by
GDNF was independent of Ret and partially dependent
on NCAM, suggesting that additional effector molecules
are involved in this process. These results are consistent
with learning deficits reported in Gdnf-, Gfra2- and Ncam-
mutant mice [55–57]. In agreement with this evidence,
Gdnf-heterozygous mice showed a transient reduction in
the synaptic localization of presynaptic proteins, indicat-
ing a physiological role for GDNF in hippocampal presyn-
aptic assembly [19]. This transient defect could be
explained by the existence of either compensatory mech-
anisms or the partial reduction in the amount of GDNF
achieved in heterozygous mutants. Recently, Baudet and
coworkers have shown a role of Ret in NMJ synapse
formation, suggesting that GFLs and GFRa1 promote
synaptogenesis functioning through different signal-trans-
ducing receptors along the nervous system [32].
Because of the asymmetric nature of the synapse, the
trans-synaptic interaction between GFRa1 molecules
might activate different signaling responses in pre- and
postsynaptic compartments. However, the contribution of
GDNF and GFRa1 to the postsynaptic density organiz-
ation remains to be explored (Figure 2b).
The function of the nervous system relies on the precise
establishment of neuronal connections, and aberrant con-
nectivity can result in the development of nervous system
disorders. In particular, trans-synaptic adhesion molecules
have been associated with cognitive disorders such as
autism, mental retardation and schizophrenia [53,54].
Thus, elucidating the molecular basis underlying synapse
formation and maturation is critical to the understanding
of the development of nervous system diseases and to
designing therapeutic strategies for them. The ability of
GDNF to induce trans-homophilic interactions between
GFRa1 molecules represents the first example of a new
synaptogenic mechanism that combines soluble and mem-
brane-bound molecules with possible implications in cog-
nitive disorders.
Conclusions and perspectives
Many questions still remain unanswered. The historic
question of why GFRas are more widely distributed than
Ret is perhaps a key issue that remains to be addressed.
Indeed, the physiological contribution of trans-signaling to
nervous system development and axon regeneration still
deserves additional analysis because the genetic models
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Opinion
currently available cannot completely eliminate the poten-
tial compensatory effects of other GFRa molecules. It
would also be of therapeutic value to find out whether
exogenous administration of soluble GFRa1 in combi-
nation with GDNF could promote axonal regeneration
after nerve injury. Furthermore, the identification of the
mechanism by which GPI-anchored GFRas are released
could reveal additional therapeutic possibilities for nerve
regeneration. Animals genetically modified to express
GFRa1 exclusively in Ret-positive cells in GFRa1-null
mice have represented a useful strategy to evaluate only
certain aspects of Ret-independent GFRa1 signaling
during postnatal development [44]. Although the analysis
performed on these mice failed to detect structural
abnormalities in the hippocampus, RMS and OB, it is
possible that functional deficits or morphological changes
in specific neuronal populations could be detected in these
brain areas following a more detailed analysis. Only tissue-
specific or -inducible GFRa1 knockout alone or in combi-
nation with mice deficient in other GFRas will allow us to
unequivocally determine the in vivo relevance of Ret-inde-
pendent signaling during postnatal nervous system de-
velopment. A detailed morphological and functional
analysis of these mice, especially in the forebrain, would
be essential to identify and dissect the physiological con-
tributions of GFLs and GFRa signaling through either
NCAM or still unidentified signaling receptors.
From a therapeutic point of view, mice with specific
deletion of Ret in the nigrostriatal system might be useful
for studying the molecular mechanisms involved in age-
related neurodegeneration, such as Parkinson’s disease
[30]. The physiological requirement of Ret for the mainten-
ance [30] and regeneration [58] of the dopaminergic system
supports additional investigation toward optimizing the
ongoing clinical trials for Parkinson’s disease using activa-
tors or reducing the action of endogenous antagonists of the
Ret signaling pathway. The huge complexity of GDNF-
GFRa signaling also argues for further characterization
and dissection of the molecular mechanisms involved in
protection and/or regeneration of the nervous system.
Acknowledgements
We thank Kimberly Dougherty and Julianna Kele Olovsson for critical
reading of this manuscript, and Sevgi Cakir for secretarial help. Work in
the authors’ laboratory is funded by grants from the Swedish Medical
Research Council, Parkinsonfonden, the Royal Swedish Academy of
Science and the Karolinska Institute. F.L. and G.P. are supported by
assistant researcher positions from the Swedish Medical Research
Council and the Karolinska Institute, respectively. The authors declare
that they have no competing financial interests.
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