[Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential].

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson's disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.

Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.

Vascular endothelial growth factor C acts as a neurotrophic factor for dopamine neurons in vitro and in vivo.

Neurotrophic factors regulate the development and maintenance of the nervous system and protect and repair dopaminergic neurons in animal models of Parkinson's disease (PD). Vascular endothelial growth factors A (VEGF-A) and B have also neurotrophic effects on various types of neurons, including dopaminergic neurons. We examined the ability of the key lymphangiogenic factor VEGF-C to protect dopaminergic cells in vitro and in vivo. The study was initiated by a finding from microarray profiling of Neuro2A-20 cells which revealed up-regulation of VEGF-C by glial cell-line-derived neurotrophic factor (GDNF). Next, we observed that VEGF-C can rescue embryonic dopaminergic neurons and activate the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway in vivo. VEGF receptors 1-2 and co-receptors, neuropilins 1-2, were expressed both in mouse embryonic cultures and adult midbrains. In vivo, VEGF-C had a robust functional effect in the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD and there was a small additive effect on the survival of tyrosine hydroxylase (TH)-positive cells with GDNF. The neuroprotective effect of VEGF-C is most likely due to a combination of direct and indirect neurotrophic effects because, VEGF-C, unlike GDNF, induced also angiogenesis in the striatum following 6-OHDA insult as it did in human umbilical vein endothelial cells (HUVEC). However, we detected activation of astroglia and microglia as well as blood-brain barrier disruption after intracerebral delivery of VEGF-C, raising a concern of its safe usage as a therapeutic molecule. Our results provide evidence of VEGF-C as a neurotrophic factor that influences the dopaminergic system through multiple mechanisms.

RET(MEN 2B) is active in the endoplasmic reticulum before reaching the cell surface.

MEN 2B (multiple endocrine neoplasia type 2B) is an autosomal dominant cancer syndrome caused by an oncogenic form of the receptor tyrosine kinase REarranged during transfection (RET). The MEN 2B syndrome is associated with an abnormal autophosphorylation of the mutated receptor even without ligand-stimulation. Here, we characterize the activation of a RET(MEN 2B) variant carrying the point mutation Met918Thr, and show that the 150 kDa precursor of RET(MEN 2B) becomes phosphorylated already during synthesis in the endoplasmic reticulum (ER). At least three different tyrosine residues (Tyr905, Tyr1062, Tyr1096) of the RET(MEN 2B) precursor are phosphorylated before the oncogenic receptor reaches the cell surface. We also demonstrate that the precursor of RET(MEN 2B) interacts with both growth factor receptor-bound protein and Src homology 2 domain-containing already in the ER, and that this interaction is dependent on the kinase activity of RET. With the aid of two RET mutants (RET(MEN 2B/S32L) and RET(MEN 2B/F393L)), which accumulate in the ER, we show that the oncogenic precursor of the receptor has the capacity to activate AKT, extracellular signal-regulated kinase and signal transducer and activator of transcription 3 from the ER. Taken together, our data demonstrate that the oncogenic precursor of RET(MEN 2B) is phosphorylated, interacts with adapter proteins and induces downstream signalling from the ER.

The mouse soluble GFRalpha4 receptor activates RET independently of its ligand persephin.

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) all signal through the transmembrane receptor tyrosine kinase RET. The signalling complex consists of GFLs, GPI-anchored ligand binding GDNF family receptor alphas (GFRalphas) and RET. Signalling via RET is required for the development of the nervous system and the kidney, as well as for spermatogenesis. However, constitutive activation of RET is implicated as a cause in several diseases. Mutations of the RET proto-oncogene cause the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). Recently, it has been suggested that mutations in the persephin binding GFRalpha4 receptor may have a potentially modifying role in MEN 2. Several naturally occurring, different splice variants of the mammalian GFRalpha4 have been reported. A 7 bp insertion-mutation in the human GFRalpha4 gene causes a shift of reading frame and thereby changes the balance between the transcripts encoding GPI-anchored and soluble GFRalpha4 receptors. We report here that the mammalian soluble GFRalpha4 can activate RET independently of its preferential ligand, persephin. Our data show that soluble GFRalpha4 can associate with, and induce, phosphorylation of RET. In addition, our data show that this isoform of GFRalpha4 can induce downstream signalling, as well as neuronal survival and differentiation, in the absence of persephin. These results suggest that, in line with the previous report, GFRalpha4 may be a candidate gene for, or modifier of, the MEN 2 diseases.

Effects of repeated morphine on locomotion, place preference and dopamine in heterozygous glial cell line-derived neurotrophic factor knockout mice.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be involved in the maintenance of striatal dopaminergic neurons. Neurotrophic factors are crucial for the plasticity of central nervous system and may be involved in long-term responses to drug exposure. To study the effects of reduced GDNF on dopaminergic behaviour related to addiction, we compared the effects of morphine on locomotor activity, conditioned place preference (CPP) and extracellular accumbal dopamine in heterozygous GDNF knockout mice (GDNF+/-) with those in their wild-type (Wt) littermates. When morphine 30 mg/kg was administered daily for 4 days, tolerance developed towards its locomotor stimulatory action only in the GDNF+/- mice. A morphine 5 mg/kg challenge dose stimulated locomotor activity only in the GDNF+/- mice withdrawn for 96 h from repeated morphine treatment, whereas clear and similar sensitization of the locomotor response was seen after a 10 mg/kg challenge dose in mice of both genotypes. Morphine-induced CPP developed initially similarly in Wt and GDNF+/- mice, but it lasted longer in the Wt mice. The small challenge dose of morphine increased accumbal dopamine output slightly more in the GDNF+/- mice than in the Wt mice, but doubling the challenge dose caused a dose-dependent response only in the Wt mice. In addition, repeated morphine treatment counteracted the increase in the accumbal extracellular dopamine concentration we previously found in drug-naive GDNF+/- mice. Thus, reduced endogenous GDNF level alters the dopaminergic behavioural effects to repeatedly administered morphine, emphasizing the involvement of GDNF in the neuroplastic changes related to long-term effects of drugs of abuse.

Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons.

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.

Stroke induces widespread changes of gene expression for glial cell line-derived neurotrophic factor family receptors in the adult rat brain.

Gene expression for glial cell line-derived neurotrophic factor (GDNF) family ligands and receptors was analyzed with in situ hybridization after two focal ischemic insults of different severities. Focal ischemia was induced in rats by either 30 min or 2 h of middle cerebral artery occlusion (MCAO), causing damage to the striatum only, or involving also the parietal cortex, respectively. We found modest, transient elevation of GDNF mRNA in the dentate granule cell layer. In addition, the number of GDNF mRNA-expressing cells increased in the cortex and striatum after 2 h or 30 min of MCAO, respectively. No changes of neurturin or persephin mRNA expression were detected. Both c-Ret and GFRalpha1 mRNA levels were markedly increased in the ipsilateral cortex outside the ischemic lesion at 6-24 h after the 2-h insult, whereas GFRalpha2 expression was decreased in cortical areas both within and outside the lesion. Similar increases of c-Ret and GFRalpha1 mRNA levels were detected in the striatum, and to a lesser extent, in the cortex following 30 min of MCAO. The 2-h insult also gave rise to transient increases of c-Ret and GFRalpha1 mRNA in hippocampal subregions. Thirty minutes and 2 h of MCAO lead to elevated c-Ret, and GFRalpha1 or GFRalpha2 mRNA expression, respectively, in the ipsilateral ventroposterolateral thalamic nucleus. Both insults induced increased levels of GFRalpha1 mRNA in the subventricular zone of the lateral ventricle. Our data indicate major changes of GDNF family signaling in the forebrain, regulated mainly through altered receptor levels, in the post-ischemic phase. These changes could enhance neuroprotective and neuroregenerative responses both to endogenous and exogenous GDNF ligands.

GDNF recruits the signaling crew into lipid rafts.

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL) are potent survival factors and regulators for central and peripheral neurons. GFLs bind to specific glycosyl phosphatidylinositol (GPI)-anchored co-receptors (GFRalpha1-alpha4), but signal through a common c-Ret receptor. Both GPI-anchored and soluble GFRalpha1 recruit c-Ret to lipid rafts following GDNF stimulation, where c-Ret interacts with different proteins than outside the rafts. Soluble GFRalpha1 mobilizes c-Ret to rafts by a different mechanism compared with GPI-anchored GFRalpha1.

Nerve growth factor and brain-derived neurotrophic factor mRNAs are regulated in distinct cell populations of rat heart after ischaemia and reperfusion.

Neurotrophins play a crucial role in the development of the peripheral nervous system and their mRNAs are often regulated after several types of tissue injury. This study has investigated the regulation of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNAs 30 min after myocardial ischaemia followed by reperfusion, by northern blotting, and in situ hybridization in a rat model. Between 2 and 120 h of reperfusion, Ngf mRNA levels showed two- to four-fold up-regulation compared with sham-operated hearts. Scattered Ngf-expressing cells, probably pericytes, were detected in the viable border zone of the myocardium in close association with capillaries, venules, and arterioles. In addition, diffuse Ngf expression was seen in the infarct area after 120 h of reperfusion. Bdnf mRNA showed transient up-regulation after 2 and 5 h of reperfusion and remained at control levels thereafter. Bdnf was expressed in the myocytes of the viable border zone. Nt-3 expression showed no significant changes compared with sham-operated hearts. These results suggest a role for NGF and/or BDNF in the pathogenesis of reperfusion injury or in the alterations of cardiac sensory and sympathetic neuronal function after myocardial ischaemia and reperfusion.

[The GDNF family: from neurotrophic factors to oncogenesis]. / Semeistvo GDNF: ot neirotrofnykh faktorov k onkogenezu.

The structure and in vivo functions of the glial cell-derived neurotrophic factor (GDNF) family ligands (GFLs) and their high-affinity receptors are considered. These proteins play an important role in the development of the nervous system, morphogenesis of the kidneys, and in regulation of spermatogenesis. Tyrosine kinase Ret is a receptor component common for all GFLs. Its role in multiple endocrine neoplasia type 2 (MEN2) is discussed.

Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis.

We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have been found. By several histological, molecular, and histochemical characteristics, the GDNF-induced tumors mimic classic seminomas in men, representing a useful experimental model for testicular germ-cell tumors. The data also show that a deregulated stimulation of a normal proto-oncogene by its ligand can be an initiative event in carcinogenesis.

Neuron-specific Bcl-2 homology 3 domain-only splice variant of Bak is anti-apoptotic in neurons, but pro-apoptotic in non-neuronal cells.

We have identified and characterized N-Bak, a neuron-specific isoform of the pro-apoptotic Bcl-2 family member Bak. N-Bak is generated by neuron-specific splicing of a novel 20-base pair exon, which changes the previously described Bak, containing Bcl-2 homology (BH) domains BH1, BH2, and BH3, into a shorter BH3-only protein. As demonstrated by reverse transcription-polymerase chain reaction and RNase protection assay, N-Bak transcripts are expressed only in central and peripheral neurons, but not in other cells, whereas the previously described Bak is expressed ubiquitously, but not in neurons. Neonatal sympathetic neurons microinjected with N-Bak resisted apoptotic death caused by nerve growth factor (NGF) removal, whereas microinjected Bak accelerated NGF deprivation-induced death. Overexpressed Bak killed sympathetic neurons in the presence of NGF, whereas N-Bak did not. N-Bak was, however, still death-promoting when overexpressed in non-neuronal cells. Thus, N-Bak is an anti-apoptotic BH3-only protein, but only in the appropriate cellular environment. This is the first example of a neuron-specific Bcl-2 family member.

Functional characterization of two splice variants of rat bad and their interaction with Bcl-w in sympathetic neurons.

Neuronal cell death is in many cases regulated by competitive interactions between pro- and antiapoptotic proteins of the Bcl-2 family. In this study we have identified two splice variants of the rat proapoptotic molecule Bad, which differ in their carboxy-terminal regions. Both splice variants of Bad interacted with the antiapoptotic molecule Bcl-w as shown by yeast two-hybrid assay and by co-immunoprecipitation experiments from transfected cells. mRNA expression for the two variants of bad were detected in all neonatal and adult rat tissues tested. Overexpression of either of the two isoforms of Bad in nerve growth factor (NGF)-maintained sympathetic neurons by microinjection induced the cell death of these neurons, which was neutralized by co-expression of Bcl-w. Overexpression of Bcl-w in sympathetic neurons also counteracted death induced by NGF deprivation, which was not reduced by co-expression of either of the two Bad variants. The results suggest that Bcl-w, Bad-alpha, and Bad-beta may participate in the regulation of apoptosis in the sympathetic nervous system.

Identification of the genome-linked protein in virions of Potato virus A, with comparison to other members in genus Potyvirus.

Viruses of the genus Potyvirus, the largest genus of plant-infecting viruses, have a messenger-polarity ssRNA genome encapsidated by approximately 2000 units of the viral coat protein (CP), resulting in filamentous virions. Only few studies have examined potyvirus virions for the presence of other structural proteins. A protein linked covalently to the 5'-end of the genome has been identified in Tobacco vein mottling virus (TVMV) and Tobacco etch virus (TEV). In TEV, it is either the viral NIa protein or only its N-terminal domain (VPg) separated autocatalytically from the C-terminal proteinase domain (NIa-Pro). Virions of TVMV carry only the VPg. We examined virions of Potato virus A (PVA) for the genome-linked protein using immunoblotting or iodination and immunoprecipitation. The VPg ( approximately 25 kDa) only, and not the unprocessed NIa, was detected. Another signal corresponding to approximately 49 kDa was detected in disrupted, RNase-treated virions with anti-VPg antibodies but not with antibodies to NIa-Pro. Since it possibly represented a dimeric form of the VPg, self-interaction of the VPg was tested using the yeast two-hybrid system, which showed that the VPg self-interacts in the absence of viral RNA.

Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus A.

Plant viruses encode movement proteins (MPs) to facilitate transport of their genomes from infected into neighboring healthy cells through plasmodesmata. Growing evidence suggests that specific phosphorylation events can regulate MP functions. The coat protein (CP) of potato virus A (PVA; genus Potyvirus) is a multifunctional protein involved both in virion assembly and virus movement. Labeling of PVA-infected tobacco leaves with [(33)P]orthophosphate demonstrated that PVA CP is phosphorylated in vivo. Competition assays established that PVA CP and the well characterized 30-kDa MP of tobacco mosaic virus (genus Tobamovirus) are phosphorylated in vitro by the same Ser/Thr kinase activity from tobacco leaves. This activity exhibits a strong preference for Mn(2+) over Mg(2+), can be inhibited by micromolar concentrations of Zn(2+) and Cd(2+), and is not Ca(2+)-dependent. Tryptic phosphopeptide mapping revealed that PVA CP was phosphorylated by this protein kinase activity on multiple sites. In contrast, PVA CP was not phosphorylated when packaged into virions, suggesting that the phosphorylation sites are located within the RNA binding domain and not exposed on the surface of the virion. Furthermore, two independent experimental approaches demonstrated that the RNA binding function of PVA CP is strongly inhibited by phosphorylation. From these findings, we suggest that protein phosphorylation represents a possible mechanism regulating formation and/or stability of viral ribonucleoproteins in planta.

Human glial cell line-derived neurotrophic factor receptor alpha 4 is the receptor for persephin and is predominantly expressed in normal and malignant thyroid medullary cells.

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.

GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.

Distinct roles for GFRalpha1 and GFRalpha2 signalling in different cranial parasympathetic ganglia in vivo.

Neurturin (NRTN), signalling via the GDNF family receptor alpha2 (GFRalpha2) and Ret tyrosine kinase, has recently been identified as an essential target-derived factor for many parasympathetic neurons. NRTN is expressed in salivary and lacrimal glands, while GFRalpha2 and Ret are expressed in the corresponding submandibular, otic and sphenopalatine ganglia. Here, we have characterized in more detail the role of GDNF and NRTN signalling in the development of cranial parasympathetic neurons and their target innervation. Gfra1 mRNA was expressed at E12 but not in newborn cranial parasympathetic ganglia, while Gfra2 mRNA and protein were strongly expressed in newborn and adult cranial parasympathetic neurons and their projections, respectively. In newborn GFRalpha1- or Ret-deficient mice, where many submandibular ganglion neurons were still present, the otic and sphenopalatine ganglia were completely missing. In contrast, in newborn GFRalpha2-deficient mice, most neurons in all these ganglia were present. In these mice, the loss and atrophy of the submandibular and otic neurons were amplified postnatally, accompanied by complete loss of innervation in some target regions and preservation in others. Surprisingly, GFRalpha2-deficient sphenopalatine neurons, whose targets were completely uninnervated, were not reduced in number and only slightly atrophied. Thus, GDNF signalling via GFRalpha1/Ret is essential in the early gangliogenesis of some, but not all, cranial parasympathetic neurons, whereas NRTN signalling through GFRalpha2/Ret is essential for the development and maintenance of parasympathetic target innervation. These results indicate that GDNF and NRTN have distinct functions in developing parasympathetic neurons, and suggest heterogeneity among and within different parasympathetic ganglia.

Development and persistence of kindling epilepsy are impaired in mice lacking glial cell line-derived neurotrophic factor family receptor alpha 2.

Seizure activity regulates gene expression for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and their receptor components, the transmembrane c-Ret tyrosine kinase and the glycosylphosphatidylinositol-anchored GDNF family receptor (GFR) alpha 1 and alpha 2 in limbic structures. We demonstrate here that epileptogenesis, as assessed in the hippocampal kindling model, is markedly suppressed in mice lacking GFR alpha 2. Moreover, at 6 to 8 wk after having reached the epileptic state, the hyperexcitability is lower in GFR alpha 2 knock-out mice as compared with wild-type mice. These results provide evidence that signaling through GFR alpha 2 is involved in mechanisms regulating the development and persistence of kindling epilepsy. Our data suggest that GDNF and NRTN may modulate seizure susceptibility by altering the function of hilar neuropeptide Y-containing interneurons and entorhinal cortical afferents at dentate granule cell synapses.