Intercellular communication involves either direct cell-cell contact or release and uptake of diffusible signals, two strategies mediated by distinct and largely nonoverlapping sets of molecules. Here, we show that the neural cell adhesion molecule NCAM can function as a signaling receptor for members of the GDNF ligand family. Association of NCAM with GFRalpha1, a GPI-anchored receptor for GDNF, downregulates NCAM-mediated cell adhesion and promotes high-affinity binding of GDNF to p140(NCAM), resulting in rapid activation of cytoplasmic protein tyrosine kinases Fyn and FAK in cells lacking RET, a known GDNF signaling receptor. GDNF stimulates Schwann cell migration and axonal growth in hippocampal and cortical neurons via binding to NCAM and activation of Fyn, but independently of RET. These results uncover an unexpected intersection between short- and long-range mechanisms of intercellular communication and reveal a pathway for GDNF signaling that does not require the RET receptor.

Glial cell line-derived neurotrophic factor (GDNF), the most potent trophic factor yet described for both dopaminergic neurons of the substantia nigra and spinal motorneurons, has recently been shown to signal through a multireceptor complex composed of a novel glycosylphosphatidylinositol-anchored GDNF receptor-␣ (GDNFR-␣) and the receptor tyrosine kinase product of the c-ret proto-oncogene (RET). Despite its importance, the individual expression patterns and the relationships between domains of expression of the different components of this trophic system are not understood. We show here by in situ hybridization that GDNF mRNA is expressed in the normal adult rat brain in several targets of substantia nigra neurons, including striatum, nucleus accumbens, thalamic nuclei, olfactory tubercle, hippocampus, cerebellum, and cingulate cortex as well as in the internal granular cell layer of the olfactory bulb. Within the basal ganglia we observe a pronounced segregation of regions expressing GDNF from those expressing GDNF receptors, suggesting that within these structures GDNF is functioning in its anticipated role as a target-derived trophic factor. In addition, the expression of GDNF and both GDNF receptors within the cerebellum, hippocampus, and olfactory bulb may indicate a paracrine mode of action. Importantly, we also see expression of RET mRNA in cellular populations within the cerebellum and the glomerular layer of the olfactory bulb, as well as in the subthalamic nucleus, which lack GDNFR-␣ expression, indicating that RET functions either independently of GDNFR-␣ or with GDNFR-␣ presented in trans. Conversely, GDNFR-␣ is widely expressed in many regions in which RET expression is absent, suggesting that GDNFR-␣ may associate with additional signaling receptors. Finally, RET and GDNFR-␣ show distinct patterns of regulated expression in the brain after kainic acid stimulation and in the sciatic nerve after nerve transection. Taken together these findings indicate that GDNF, RET, and GDNFR-␣ utilize multiple mechanisms to comprise physiologically relevant trophic circuits for different neuronal populations.

Glial-cell-line-derived neutrophic factor (GDNF) promotes the survival and phenotype of central dopaminergic noradrenergic and motor neurons, as well as various subpopulations of peripheral sensory and sympathetic neurons. GDNF is structurally related to members of the transforming growth factor (TGF)-beta superfamily, several members of which have well-characterized receptor systems; however, GDNF receptors still remain undefined. Here we show that GDNF binds to, and induces tyrosine phosphorylation of, the product of the c-ret proto-oncogene, an orphan receptor tyrosine kinase, in a GDNF-responsive motor-neuron cell line. Ret protein could also bind GDNF and mediate survival and growth responses to GDNF upon transfection into naive fibroblasts. Moreover, high levels of c-ret mRNA expression were found in dopaminergic neurons of the adult substantia nigra, where exogenous GDNF protected Ret-positive neurons from 6-hydroxydopamine-induced cell death. Thus the product of the c-ret proto-oncogene encodes a functional receptor for GDNF that may mediate its neurotrophic effects on motor and dopaminergic neurons.

Abstract. Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor- , originally isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons, and more recently shown to be a potent neurotrophic factor for motorneurons. The biological activities and distribution of this molecule outside the central nervous system are presently unknown. We report here on the mRNA expression, biological activities and initial receptor binding characterization of GDNF and a shorter spliced variant termed GDNF[3 in different organs and peripheral neurons of the developing rat. Both GDNF mRNA forms were found to be most highly expressed in developing skin, whisker pad, kidney, stomach and testis. Lower expression was also detected in developing skeletal muscle, ovary, lung, and adrenal gland. Developing spinal cord, superior cervical ganglion (SCG) and dorsal root ganglion (DRG) also expressed low levels of GDNF mRNA. Two days after nerve transection, GDNF mRNA levels increased dramatically in the sciatic nerve. Overall, GDNF mRNA expression was significantly higher in peripheral organs than in neuronal tissues. Expression of either GDNF mRNA isoform in insect cells resulted in the production of indistinguishable mature GDNF polypeptides. Purified recombinant GDNF promoted neurite outgrowth and survival of embryonic chick sympathetic neurons. GDNF produced robust bundle-like, fasciculated outgrowth from chick sympathetic ganglion explants. Although GDNF displayed only low activity on survival of newborn rat SCG neurons, this protein was found to increase the expression of vasoactive intestinal peptide and preprotachykinin-A mRNAs in cultured SCG neurons. GDNF also promoted survival of about half of the neurons in embryonic chick nodose ganglion and a small subpopulation of embryonic sensory neurons in chick dorsal root and rat trigeminal ganglia. Embryonic chick sympathetic neurons expressed receptors for GDNF with Kd 1-5 × 10 -9 M, as measured by saturation and displacement binding assays. Our findings indicate GDNF is a new neurotrophic factor for developing peripheral neurons and suggest possible non-neuronal roles for GDNF in the developing reproductive system. T HE development and maintenance of the vertebrate nervous system requires the activity of a range of polypeptides known as neurotrophic factors. These molecules have been shown to control the generation, survival, differentiation and regeneration of neurons in the peripheral and central nervous systems (Barde, 1989;Thoenen, 1991). They include members of the neurotrophin family, including NGF and related molecules (Thoenen, 1991: Persson andIb~ifiez, 1993), members of the neurokine family, such as ciliary neurotrophic factor (CNTF), 1

Adenosine and dopamine signaling exert opposite effects in the basal ganglia, a brain region involved in sensory-motor integration. Thus, adenosine agonists induce motor depression and adenosine antagonists, such as caffeine, produce motor activation (1). These opposite effects result from specific antagonistic interactions between subtypes of adenosine and dopamine receptors in the striatum, the main input structure of the basal ganglia. In fact, striatal dopamine receptors and, to some extent, adenosine receptors are segregated in the two main populations of ␥-aminobutyric acid (GABA) efferent neurons. EXPERIMENTAL PROCEDURESCell Cultures-Maintenance of SH-SY5Y cells (parental and D 2 Rtransfected cells) as well as the pharmacological characterization and maintenance of D 2 R-and D 1 R-transfected mouse fibroblast Ltk Ϫ cells are described in detail elsewhere (7-9). For primary cultures, striata were removed from 16-day-old Sprague-Dawley rat embryos (B&K Universal) in Ca 2ϩ /Mg 2ϩ -free PBS supplemented with 20 units/ml penicillin and 20 g/ml streptomycin (Invitrogen). The tissue fragments were pooled and mechanically dissociated in SFM Neurobasal serum-free medium (Invitrogen), supplemented with B27 (Invitrogen), glutamine (2 mM; Invitrogen), penicillin/streptomycin (20 units/ml/20 g/ml; Invitrogen), and ␤-mercaptoethanol (25 M) (Invitrogen). Cells were collected by centrifugation at 100 ϫ g for 5 min and resuspended in fresh medium. The resulting single-cell suspension was seeded on 24-well plates coated with gelatin (Sigma) and poly-L-lysine (Sigma), and cells were grown at 37°C in saturation humidity with 5% CO 2 .Immunolabeling Experiments-Neuroblastoma cells were grown on glass coverslips coated with poly-L-lysine (Sigma) and exposed to vari-* This work was

The production of neurotrophin-4 (NT-4) in rat skeletal muscle was found to depend on muscle activity. The amounts of NT-4 messenger RNA present decreased after blockade of neuromuscular transmission with alpha-bungarotoxin and increased during postnatal development and after electrical stimulation in a dose-dependent manner. NT-4 immunoreactivity was detected in slow, type I muscle fibers. Intramuscular administration of NT-4 induced sprouting of intact adult motor nerves. Thus, muscle-derived NT-4 acted as an activity-dependent neurotrophic signal for growth and remodeling of adult motor neuron innervation. NT-4 may thus be partly responsible for the effects of exercise and electrical stimulation on neuromuscular performance.

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