RESUMO
The GDNF family ligands (GFLs: GDNF, neurturin, persephin, and artemin) signal through RET and a gly-cosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRalpha1-alpha4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRalpha1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRalpha1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.
Assuntos
Proteínas de Drosophila , Lipídeos/análise , Fatores de Crescimento Neural , Neurônios/química , Neurônios/enzimologia , Proteínas Proto-Oncogênicas/análise , Receptores Proteína Tirosina Quinases/análise , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Animais , Diferenciação Celular/fisiologia , Fracionamento Celular , Membrana Celular/química , Sobrevivência Celular/fisiologia , Cerebelo/citologia , Detergentes , Teste de Complementação Genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Ligantes , Metabolismo dos Lipídeos , Proteínas Luminescentes , Proteínas do Tecido Nervoso/farmacologia , Neuroblastoma , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkA/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Células Tumorais CultivadasRESUMO
Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.
Assuntos
Proteínas de Drosophila , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células 3T3/química , Células 3T3/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Sequência de Bases , Northern Blotting , Química Encefálica/fisiologia , Mapeamento Cromossômico , Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hibridização in Situ Fluorescente , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurturina , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Ratos , Nervo Isquiático/química , Nervo Isquiático/citologia , Nervo Isquiático/lesões , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Testículo/química , Regulação para Cima/fisiologiaRESUMO
The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRalpha1-GFRalpha4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRalpha3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRalpha1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRalpha1-RET) and NTN (GFRalpha2-RET), Artemin has a preferred receptor (GFRalpha3-RET) but that alternative receptor interactions also occur.
Assuntos
Proteínas de Drosophila , Mesencéfalo/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/fisiologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sítios de Ligação , Sobrevivência Celular , Células Cultivadas , Clonagem Molecular , Embrião de Mamíferos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Ligantes , Mesencéfalo/citologia , Camundongos , Modelos Químicos , Dados de Sequência Molecular , Fatores de Crescimento Neural/química , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Gânglio Cervical Superior/citologiaRESUMO
A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.
Assuntos
Neurônios Motores/química , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/metabolismo , Animais , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Gânglios Espinais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Mesencéfalo/citologia , Camundongos , Dados de Sequência Molecular , Neurônios Motores/fisiologia , Neurturina , Gânglio Nodoso/citologia , Reação em Cadeia da Polimerase/métodos , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento/fisiologia , Receptores do Ácido Retinoico/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/citologia , Transfecção , Gânglio Trigeminal/citologia , Ureter/citologia , Ureter/embriologiaRESUMO
The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system.
Assuntos
Proteínas de Drosophila , Família Multigênica , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/embriologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Ligantes , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Sistema Nervoso Parassimpático/citologia , Sistema Nervoso Parassimpático/embriologia , Sistema Nervoso Parassimpático/crescimento & desenvolvimento , Sistema Nervoso Periférico/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/embriologia , Sistema Nervoso Simpático/crescimento & desenvolvimentoRESUMO
Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.
Assuntos
Esclerose Lateral Amiotrófica/imunologia , Demência Frontotemporal/imunologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Macrófagos/imunologia , Microglia/imunologia , Células Mieloides/imunologia , Proteínas/fisiologia , Envelhecimento/imunologia , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72 , Demência Frontotemporal/genética , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Heterozigoto , Humanos , Doenças Linfáticas/genética , Doenças Linfáticas/imunologia , Camundongos , Camundongos Knockout , Proteínas/genética , Ratos , Esplenomegalia/genética , Esplenomegalia/imunologiaAssuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Doença dos Neurônios Motores/genética , Proteinopatias TDP-43/genética , Proteinopatias TDP-43/patologia , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Autopsia , Encéfalo/patologia , DNA/genética , Éxons/genética , Evolução Fatal , Humanos , Masculino , Mutação/fisiologia , Mutação de Sentido Incorreto/genética , Medula Espinal/patologiaRESUMO
Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF) are the first two members of the GDNF family (GF) of neurotrophic factors. These two proteins are potent survival factors for several populations of central and peripheral neurons in mature and developing rodents. The receptor for these factors is a multicomponent complex that includes the RET (rearranged during transfection) tyrosine kinase receptor and one of two glycosyl phosphatidylinositol (GPI)-linked ligand-binding components called GDNF family receptor alphas (GFRalpha-1 and GFRalpha-2). We have used in situ hybridization to study the mRNA expression of NTN, GDNF, RET, GFRalpha-1, and GFRalpha-2 in the central nervous system (CNS) of adult mice. GF receptors are expressed in several areas in which neuronal populations known to respond to NTN and GDNF are located, including the ventral horn of the spinal cord and the compacta region of the substantia nigra. In addition, we have demonstrated receptor expression in other areas of the brain including the thalamus and hypothalamus. Neurons in these areas express GF receptors, and therefore, may respond to NTN or GDNF. NTN and GDNF are expressed in targets of neurons that express GF receptors. The pattern of GF factor and receptor expression in the adult brain suggests a role for these factors in maintaining neuronal circuits in the mature CNS.
Assuntos
Proteínas de Drosophila , Camundongos Endogâmicos ICR/fisiologia , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fatores Etários , Animais , Química Encefálica/fisiologia , Tronco Encefálico/química , Tronco Encefálico/citologia , Cerebelo/química , Cerebelo/citologia , Feminino , Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hipotálamo/química , Hipotálamo/citologia , Hibridização In Situ , Mesencéfalo/química , Mesencéfalo/citologia , Camundongos , Neurturina , Bulbo Olfatório/química , Bulbo Olfatório/citologia , Prosencéfalo/química , Prosencéfalo/citologia , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/análise , Medula Espinal/química , Medula Espinal/citologia , Tálamo/química , Tálamo/citologiaRESUMO
OBJECTIVE: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600). METHODS: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed. RESULTS: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state-a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function. CONCLUSIONS: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.
Assuntos
Cromossomos Humanos Par 14 , Dineínas do Citoplasma/genética , Genes Dominantes , Extremidade Inferior , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Atrofias Musculares Espinais da Infância/genética , Pré-Escolar , Cromossomos Humanos Par 14/genética , Dineínas do Citoplasma/metabolismo , Feminino , Genes Dominantes/genética , Humanos , Lactente , Masculino , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVE: Spinal muscular atrophies (SMAs) are hereditary disorders characterized by weakness from degeneration of spinal motor neurons. Although most SMA cases with proximal weakness are recessively inherited, rare families with dominant inheritance have been reported. We aimed to clinically, pathologically, and genetically characterize a large North American family with an autosomal dominant proximal SMA. METHODS: Affected family members underwent clinical and electrophysiologic evaluation. Twenty family members were genotyped on high-density genome-wide SNP arrays and linkage analysis was performed. RESULTS: Ten affected individuals (ages 7-58 years) showed prominent quadriceps atrophy, moderate to severe weakness of quadriceps and hip abductors, and milder degrees of weakness in other leg muscles. Upper extremity strength and sensation was normal. Leg weakness was evident from early childhood and was static or very slowly progressive. Electrophysiology and muscle biopsies were consistent with chronic denervation. SNP-based linkage analysis showed a maximum 2-point lod score of 5.10 (theta = 0.00) at rs17679127 on 14q32. A disease-associated haplotype spanning from 114 cM to the 14q telomere was identified. A single recombination narrowed the minimal genomic interval to Chr14: 100,220,765-106,368,585. No segregating copy number variations were found within the disease interval. CONCLUSIONS: We describe a family with an early onset, autosomal dominant, proximal SMA with a distinctive phenotype: symptoms are limited to the legs and there is notable selectivity for the quadriceps. We demonstrate linkage to a 6.1-Mb interval on 14q32 and propose calling this disorder spinal muscular atrophy-lower extremity, dominant.
Assuntos
Cromossomos Humanos Par 14/genética , Genes Dominantes , Ligação Genética/genética , Extremidade Inferior , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Adulto , Criança , Feminino , Humanos , Extremidade Inferior/fisiopatologia , Masculino , Pessoa de Meia-Idade , Atrofia Muscular Espinal/fisiopatologia , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Adulto JovemRESUMO
Multiple-frequency rotatory testing was performed in a series of 12 normal subjects and 27 patients who had significant labyrinthine paralysis. Fourteen of the patients had total loss of labyrinthine function. Phase leads of the responses were much greater in the lower-frequency measurements and measured gains of the responses to ampullopetal stimulation were qualitatively greater than those of the responses to ampullofugal stimulation. However, many patients' responses continued to fall within the normal range because of the large variability of the responses of normal subjects. The value of rotatory testing in clinical diagnosis is discussed.
Assuntos
Doenças do Labirinto/diagnóstico , Testes de Função Vestibular , Testes Calóricos , Feminino , Humanos , Doenças do Labirinto/fisiopatologia , Masculino , Doença de Meniere/diagnóstico , Pessoa de Meia-Idade , ReflexoRESUMO
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified sites along the second finger of GDNF critical for activating the GFRalpha1.RET and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRalpha1. RET, but additional determinants are required for interaction with the other GFRalphas. This difference in the molecular basis of GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific agonists and provides a foundation for understanding of GFL-GFRalpha.RET signaling at the molecular level.
Assuntos
Proteínas de Drosophila , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Ligantes , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
GDNF, neurturin, and persephin are transforming growth factor beta-related neurotrophic factors known collectively as the GDNF family (GF). GDNF and neurturin signal through a multicomponent receptor complex containing a signaling component (the Ret receptor tyrosine kinase) and either of two glycosyl-phosphatidylinositol-linked binding components (GDNF family receptor alpha components 1 and 2, GFRalpha1 or GFRalpha2), whereas the receptor for persephin is unknown. Herein we describe a third member of the GF coreceptor family called GFRalpha3 that is encoded by a gene located on human chromosome 5q31.2-32. GFRalpha3 is not expressed in the central nervous system of the developing or adult animal but is highly expressed in several developing and adult sensory and sympathetic ganglia of the peripheral nervous system. GFRalpha3 is also expressed at high levels in developing, but not adult, peripheral nerve. GFRalpha3 is a glycoprotein that is glycosyl-phosphatidylinositol-linked to the cell surface like GFRalpha1 and GFRalpha2. Fibroblasts expressing Ret and GFRalpha3 do not respond to any of the known members of the GDNF family, suggesting that GFRalpha3 interacts with an unknown ligand or requires a different or additional signaling protein to function.
Assuntos
Glicoproteínas de Membrana/isolamento & purificação , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Fator de Crescimento Neural , Células 3T3 , Adulto , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Fibroblastos/metabolismo , Gânglios/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Hibridização In Situ , Glicoproteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Neurturina , Receptores de Superfície Celular/química , Alinhamento de Sequência , Transdução de Sinais , TransfecçãoRESUMO
Neurturin (NTN) is a neurotrophic factor that shares homology with glial cell line-derived neurotrophic factor (GDNF). Recently, a receptor complex has been identified for GDNF that includes the Ret tyrosine kinase receptor and a glycosylphosphatidylinositol-linked protein termed "GDNFRalpha." However, differences in the phenotype of Ret and GDNF knockout animals suggest that Ret has at least one additional ligand. In this report, we demonstrate that NTN induces Ret phosphorylation in primary cultures of rat superior cervical ganglion (SCG) neurons. NTN also caused Ret phosphorylation in fibroblasts that were transfected stably with Ret and GDNFRalpha but not in cells expressing Ret alone. A glycosylphosphatidylinositol-linked protein also was important for NTN and GDNF signaling in SCG neurons; phosphatidylinositol-specific phospholipase C treatment of SCG cultures reduced the ability of NTN to phosphorylate Ret and the ability of NTN or GDNF to activate the mitogen-activated protein kinase pathway. NTN and GDNF also caused sustained activation of Ret and the mitogen-activated protein kinase pathway in SCG neurons. Finally, both NTN and GDNF activated the phosphatidylinositol 3-kinase pathway in SCG neurons, which may be important for the ability of NTN and GDNF to promote neuronal survival. These data indicate that NTN is a physiologically relevant ligand for the Ret receptor and suggest that NTN may have a critical role in the development of many neuronal populations.