- Email: [email protected]
Specification and Survival of Dopaminergic Neurons in the Mammalian Midbrain
908
Arnon Rosenthal
9. Mezey, E., Eisenhofer, G . , Harta, G., Hansson, S., Gould, L., Hunyady, B., and Hoffman, B. J. (1996).A novel nonneuronal catecholaminergic system: Exocrine pancreas synthesizes and releases dopamine. Proc. Nutl. Acud. Sci. U.S.A. 93, 10377-10382. 10. Landis, S. C. (1990).Target regulation of neurotransmitter phenotype. TINS 13,344-350. 11. Habecker, B. A., and Landis, S. C. (1994).Noradrenergic regulation of cholinergic differentiation. Science 264, 1602-1604. 12. Reissmann, E., Ernsberger, U.,Francis-West, P. H., Rueger, D., Brickell, P. M., and Rohrer, H. (1996).Involvement of bone morphogenetic protein-4 and bone morphogenetic protein7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons. Development 122, 2079-2088.
Arnon Rosenthal Department of Neuroscience Genentech, Inc. South San Francisco, California 94080
Specification and Survival of Dopaminergic Neurons in the Mammalian Midbrain Midbrian dopaminergic (DA) neurons, constitute a small group of cells that innervate the striatum, limbic system, and neocortex. These cells play a seminal role in postural reflex, reward-associated behavior, and learning, and their loss or abnormal function has been linked to Parkinson’s disease, schizophrenia, and drug addiction. Despite the physiological and clinical importance of DA neurons, the mechanisms that control their survival and genesis are not well understood. We have shown that several members of the transforming growth factors (TGF) TGFP protein family including, in addition to glial-cell line-derived neurotrophic factor (GDNF), TGFP2 and -p3 can prevent the death of cultured rat embryonic midbrain DA neurons at picomolar concentrations. Furthermore, we found that TGFP2 and -P3 and GDNF are expressed sequentially as local and target-derived trophic factors and that subpopulations of DA neurons projecting to the striatum would have access to GDNF whereas DA neurons projecting to the cortical and limbic system will have access mainly to the TGFs. These findings lead us to propose that multiple members of the TGF protein family may serve as physiological survival factors for distinct subpopulations of DA neurons (1).Surprisingly, given the potent survival activity of TGFp2 and -p3 on DA neurons in uitro, we found that the only member of this protein family that appears to be efficacious in preventing the death of DA neurons in lesioned animal models in viuo was GDNF (2). We, therefore, Advances in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form resewed. 1054-3589198 $25.00
Specification and Survival of DA Neurons in Mammalian Brain
909
undertook to explore the mechanism of action of this protein. We found that physiological responses to GDNF require the presence of a novel glycosylphosphatidyl inositol-linked protein (designated GDNFR) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrated that GDNF promotes the formation of a physical complex between GDNFR and the orphan tyrosine kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF utilizes a multisubunit receptor system in which GDNFR and Ret function as the ligand-binding and signaling components, respectively ( 3 ) . Because GDNF appears to be the most potent and efficacious survival factor for DA neurons known so far, we have explored whether it is indeed the long sought after physiological survival factor for DA neurons during normal development. For this aim, we have generated mice that are deficient in GDNF. Surprisingly, we found that at postnatal day 1, no deficiency in the number of DA neurons in the midbrain or in the density of DA nerve fibers was detected in the GDNF-deficient mice. Furthermore, we have previously demonstrated that GDNF is 75-fold more potent than any other neurotrophic factor in supporting the survival of purified embyronic rat motoneurons in culture and that GDNF mRNA was found in the immediate vicinity of motoneurons during the period of cell death in development. In vivo, GDNF was found to rescue and prevent the atrophy of facial motoneurons that have been deprived of target-derived survival factors by axotomy (4). Despite this, no significant deficit in the number or morphology of motoneurons was found in the GDNFdeficient mice. In addition, we have found that GDNF is a potent survival factor for distinct populations of sensory and sutonomic nervous system neurons (5).Consistent with this finding, GDNF-deficient mice displayed partial deficits in superior cervical sympathetic neurons as well as in subpopulations of dorsal root and nodose sensory ganglia neurons ( 6 ) . Most surprisingly, we find that GDNF-deficient mice completely lack ureters and kidneys as early as embyronic day 10.5. The metanephric kidney develops by reciprocal inductive interactions between the ureteric bud, an evagination of the mesonephric-wolffian duct, which gives rise to the collecting ducts-ureter and the metanephric blastema, a caudal intermediate mesodermal precursor of the renal parenchyma, which gives rise to the renal tubules of the metanephric (definitive) kidney. The pattern of expression of GDNF in the metanephric blastema, combined with the absence of a morphologically defined ureteric bud, raises the possibility that GDNF may participate in the induction of the ureteric bud by the mesonephric duct. Alternatively, GDNF may control the proliferation or survival of ureteric bud cells after they have been specified. In the absence of a ureteric bud, the mesenchyme will not develop into renal tubules, and no kidney will be formed. In addition, these mice fail to develop an enteric nervous system, suggesting that GDNF may be essential for the commitment, migration, or differentiation of ENS neuron precursors ( 6 ) . The discrepancy between the efficacy of GDNF as a survival factor for multiple neuronal populations in culture and the mild neuronal deficient observed in GDNF-deficient mice suggested that in vivo GDNF-like proteins may compensate for its absence. Consistent with this possibility is the recent isolation by the groups of Millbrant and Johnson of a novel protein designated Neurturin, which is structurally related to GDNF. Using survival assays in culture, we
910
Arnon Rosenthal
have demonstrated that Neurturin-like GDNF is a potent survival factor for DA neurons. Moreover, we have identified a multi-component receptor complex for Neurturin composed of a novel, GPI-linked protein designated Neurturin Ra and the tyrosine kinase receptor Ret. In addition to the studies on survival factors for DA neurons, we have been exploring the mechanisms by which these cells acquire their particular cell fate during normal development. We show that DA neurons develop in the midbrain in vicinity to the floor plate, a specialized group of cells that transiently occupy the ventral nerual tube through most of its extant. We further demonstrated, using explant culture in vitro, that the floor plate-produced signals induce the formation of DA neurons in ectopic locations in midbrain explants. In addition, we showed that transgenic mice that harbor a supernumerary floor plate in the dorsal midbrain region develop a duplicated cluster of DA adjacent to the ectopic floor plate (7).Subsequent to these studies, we were able to identify the molecular nature of the floor plate-derived inducer and provided evidence that it is sonic hedgehog (SHH),a secreted protein produced by the floor plate and notochord. Addition of recombinant SHH to midbrain explants led to the formation of DA neurons as well as to the differentiation of floor plate cells, motoneurons, and HNF3P-positive cells. The types of cell that developed in these midbrain explant appeared to be dependent on the concentration of SHH. The concentration of SHH required to induce these different cell types is lowest for motoneurons, about fivefold higher for HNF3@, and higher yet for DA neurons and for FP3/4+ cells. These findings, combined with discoveries from the laboratories of Drs. Jessell and MacMahon, suggest that SHH may function as a morphogene to specify distinct cell types along the dorsoventral axis of the neural tube in a concentration-dependent manner (7). Because SHH appeared to be an important inducer of DA neurons, we undertook the elucidation of its mechanism of action. We provided evidence that SHH mediates its function through a multi-component receptor system composed of 12 transmembrane ligand binding protein patched and a 7 transmembrane signalling component designated smoothened. In sum, the genesis and survival of DA neurons are complex processes regulated by mutliple growth factors that utilize diverse signaling systems.
References 1. Poulsen, K. T., Armanini, M. P., Klein, R. D., Hynes, M. A,, Phillips, H. S . , and Rosenthal, A. (1994). TGFP2 and TGFP3 are potent survival factors for midbrain dopaminergic neurons. Neuron 13, 1245-1252. 2. Beck, K. D., Valverde, J., Alexi, T., Poulsen, K., Moffat, B., Vandlen, R. A., Rosenthal, A., and Hefti, F. (1995). Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature 373, 339-341. 3. Treanor, J., Goodman, L., de Sauvage, F., Stone, D., Poulsen, K., Beck, K., Gray, C., Armanini, M., Pollock, R. A., Hefti, F., Phillips, H., Goddard, A., Davies, A., Asai, N., Takahashi, M., Vandlen, R., Henderson, C., and Rosenthal, A. (1996). Characterization of a multicomponent receptor for GDNF Nature 382, 80-83. 4. Henderson, C. E., Phillips, H. S., Pollock, R. A,, Davies, A. M., Lemeulle, C., Armanini, M., Simmons, L., Moffet, B., Vandlen, R. A., Koliatsos, V. E., and Rosenthal, A. (1994).
Effects of GDNF on Nigrostriatal DA System
9II
GDNF: A potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266,1062-1064. 5 . Buj-Bello, A., Horton, A., Rosenthal, A., and Davies, A. M. (1995).GDNF is an age-specific survival factor for sensory and autonomic neurons. Neuron 15, 821-828. 6. Moore, M. W., Klein, R. D., Farifias, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L. F., Ryan, A. M., Carver-Moore, K., and Rosenthal, A. (1996). Renal and neuronal abnormalities in mice lacking GDNF. Nature 382, 76-79. 7. Hynes, M., Porter, J. A,, Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P. A., Rosenthal, A. (1995).Induction of midbrain dopaminergic neurons by sonic hedgehog. Neuron 15, 35-44.
Don M. Gash,* Greg A. Gerhardt,t and Barry J. Hoffert *Department of Anatomy and Neurobiology and Research Magnetic Resonance Imaging and Spectroscopy Center University of Kentucky College of Medicine Lexington, Kentucky 40536
tDepartments of Pharmacology and Psychiatry Neuroscience Training Program and Rocky Mountain Center for Sensor Technology University of Colorado Health Sciences Center Denver, Colorado 80262
Effects of Glial Cell Line-Derived Neurotrophic Factor on the Nigrostriatal Dopamine System in
Rodents and Nonhuman Primates Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer distantly related to the transforming growth factor+ superfamily, which might have a therapeutic potential for treating Parkinson’s disease (1).While GDNF has been found to play an important role in the survival and development of a variety of cell types throughout the body, our studies have focused on analyzing its effects on the nigrostriatal dopaminergic system in rodents and nonhuman primates. The in uiuo effects of GDNF on midbrain dopamine (DA) neurons in normal adult rats and rhesus monkeys have been shown to be dramatic. In normal Fischer 344 rats, a single intranigral injection of 10 pg of GDNF ( 2 ) , increased nigral DA levels over threefold by 3 wk postinjection (Table I). Striatal DA levels on the injected side were either unchanged (1 wk posttreatment) or significantly lower ( 3 wk posttreatment), while striatal DA turnover was Advances in Pharmacology, Volume 42 Copyrighr 0 1998 by Academic Press. All rlghts of reproduction In any form reserved. 1054-3589/98 $25.00
Arnon Rosenthal
9. Mezey, E., Eisenhofer, G . , Harta, G., Hansson, S., Gould, L., Hunyady, B., and Hoffman, B. J. (1996).A novel nonneuronal catecholaminergic system: Exocrine pancreas synthesizes and releases dopamine. Proc. Nutl. Acud. Sci. U.S.A. 93, 10377-10382. 10. Landis, S. C. (1990).Target regulation of neurotransmitter phenotype. TINS 13,344-350. 11. Habecker, B. A., and Landis, S. C. (1994).Noradrenergic regulation of cholinergic differentiation. Science 264, 1602-1604. 12. Reissmann, E., Ernsberger, U.,Francis-West, P. H., Rueger, D., Brickell, P. M., and Rohrer, H. (1996).Involvement of bone morphogenetic protein-4 and bone morphogenetic protein7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons. Development 122, 2079-2088.
Arnon Rosenthal Department of Neuroscience Genentech, Inc. South San Francisco, California 94080
Specification and Survival of Dopaminergic Neurons in the Mammalian Midbrain Midbrian dopaminergic (DA) neurons, constitute a small group of cells that innervate the striatum, limbic system, and neocortex. These cells play a seminal role in postural reflex, reward-associated behavior, and learning, and their loss or abnormal function has been linked to Parkinson’s disease, schizophrenia, and drug addiction. Despite the physiological and clinical importance of DA neurons, the mechanisms that control their survival and genesis are not well understood. We have shown that several members of the transforming growth factors (TGF) TGFP protein family including, in addition to glial-cell line-derived neurotrophic factor (GDNF), TGFP2 and -p3 can prevent the death of cultured rat embryonic midbrain DA neurons at picomolar concentrations. Furthermore, we found that TGFP2 and -P3 and GDNF are expressed sequentially as local and target-derived trophic factors and that subpopulations of DA neurons projecting to the striatum would have access to GDNF whereas DA neurons projecting to the cortical and limbic system will have access mainly to the TGFs. These findings lead us to propose that multiple members of the TGF protein family may serve as physiological survival factors for distinct subpopulations of DA neurons (1).Surprisingly, given the potent survival activity of TGFp2 and -p3 on DA neurons in uitro, we found that the only member of this protein family that appears to be efficacious in preventing the death of DA neurons in lesioned animal models in viuo was GDNF (2). We, therefore, Advances in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form resewed. 1054-3589198 $25.00
Specification and Survival of DA Neurons in Mammalian Brain
909
undertook to explore the mechanism of action of this protein. We found that physiological responses to GDNF require the presence of a novel glycosylphosphatidyl inositol-linked protein (designated GDNFR) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrated that GDNF promotes the formation of a physical complex between GDNFR and the orphan tyrosine kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF utilizes a multisubunit receptor system in which GDNFR and Ret function as the ligand-binding and signaling components, respectively ( 3 ) . Because GDNF appears to be the most potent and efficacious survival factor for DA neurons known so far, we have explored whether it is indeed the long sought after physiological survival factor for DA neurons during normal development. For this aim, we have generated mice that are deficient in GDNF. Surprisingly, we found that at postnatal day 1, no deficiency in the number of DA neurons in the midbrain or in the density of DA nerve fibers was detected in the GDNF-deficient mice. Furthermore, we have previously demonstrated that GDNF is 75-fold more potent than any other neurotrophic factor in supporting the survival of purified embyronic rat motoneurons in culture and that GDNF mRNA was found in the immediate vicinity of motoneurons during the period of cell death in development. In vivo, GDNF was found to rescue and prevent the atrophy of facial motoneurons that have been deprived of target-derived survival factors by axotomy (4). Despite this, no significant deficit in the number or morphology of motoneurons was found in the GDNFdeficient mice. In addition, we have found that GDNF is a potent survival factor for distinct populations of sensory and sutonomic nervous system neurons (5).Consistent with this finding, GDNF-deficient mice displayed partial deficits in superior cervical sympathetic neurons as well as in subpopulations of dorsal root and nodose sensory ganglia neurons ( 6 ) . Most surprisingly, we find that GDNF-deficient mice completely lack ureters and kidneys as early as embyronic day 10.5. The metanephric kidney develops by reciprocal inductive interactions between the ureteric bud, an evagination of the mesonephric-wolffian duct, which gives rise to the collecting ducts-ureter and the metanephric blastema, a caudal intermediate mesodermal precursor of the renal parenchyma, which gives rise to the renal tubules of the metanephric (definitive) kidney. The pattern of expression of GDNF in the metanephric blastema, combined with the absence of a morphologically defined ureteric bud, raises the possibility that GDNF may participate in the induction of the ureteric bud by the mesonephric duct. Alternatively, GDNF may control the proliferation or survival of ureteric bud cells after they have been specified. In the absence of a ureteric bud, the mesenchyme will not develop into renal tubules, and no kidney will be formed. In addition, these mice fail to develop an enteric nervous system, suggesting that GDNF may be essential for the commitment, migration, or differentiation of ENS neuron precursors ( 6 ) . The discrepancy between the efficacy of GDNF as a survival factor for multiple neuronal populations in culture and the mild neuronal deficient observed in GDNF-deficient mice suggested that in vivo GDNF-like proteins may compensate for its absence. Consistent with this possibility is the recent isolation by the groups of Millbrant and Johnson of a novel protein designated Neurturin, which is structurally related to GDNF. Using survival assays in culture, we
910
Arnon Rosenthal
have demonstrated that Neurturin-like GDNF is a potent survival factor for DA neurons. Moreover, we have identified a multi-component receptor complex for Neurturin composed of a novel, GPI-linked protein designated Neurturin Ra and the tyrosine kinase receptor Ret. In addition to the studies on survival factors for DA neurons, we have been exploring the mechanisms by which these cells acquire their particular cell fate during normal development. We show that DA neurons develop in the midbrain in vicinity to the floor plate, a specialized group of cells that transiently occupy the ventral nerual tube through most of its extant. We further demonstrated, using explant culture in vitro, that the floor plate-produced signals induce the formation of DA neurons in ectopic locations in midbrain explants. In addition, we showed that transgenic mice that harbor a supernumerary floor plate in the dorsal midbrain region develop a duplicated cluster of DA adjacent to the ectopic floor plate (7).Subsequent to these studies, we were able to identify the molecular nature of the floor plate-derived inducer and provided evidence that it is sonic hedgehog (SHH),a secreted protein produced by the floor plate and notochord. Addition of recombinant SHH to midbrain explants led to the formation of DA neurons as well as to the differentiation of floor plate cells, motoneurons, and HNF3P-positive cells. The types of cell that developed in these midbrain explant appeared to be dependent on the concentration of SHH. The concentration of SHH required to induce these different cell types is lowest for motoneurons, about fivefold higher for HNF3@, and higher yet for DA neurons and for FP3/4+ cells. These findings, combined with discoveries from the laboratories of Drs. Jessell and MacMahon, suggest that SHH may function as a morphogene to specify distinct cell types along the dorsoventral axis of the neural tube in a concentration-dependent manner (7). Because SHH appeared to be an important inducer of DA neurons, we undertook the elucidation of its mechanism of action. We provided evidence that SHH mediates its function through a multi-component receptor system composed of 12 transmembrane ligand binding protein patched and a 7 transmembrane signalling component designated smoothened. In sum, the genesis and survival of DA neurons are complex processes regulated by mutliple growth factors that utilize diverse signaling systems.
References 1. Poulsen, K. T., Armanini, M. P., Klein, R. D., Hynes, M. A,, Phillips, H. S . , and Rosenthal, A. (1994). TGFP2 and TGFP3 are potent survival factors for midbrain dopaminergic neurons. Neuron 13, 1245-1252. 2. Beck, K. D., Valverde, J., Alexi, T., Poulsen, K., Moffat, B., Vandlen, R. A., Rosenthal, A., and Hefti, F. (1995). Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature 373, 339-341. 3. Treanor, J., Goodman, L., de Sauvage, F., Stone, D., Poulsen, K., Beck, K., Gray, C., Armanini, M., Pollock, R. A., Hefti, F., Phillips, H., Goddard, A., Davies, A., Asai, N., Takahashi, M., Vandlen, R., Henderson, C., and Rosenthal, A. (1996). Characterization of a multicomponent receptor for GDNF Nature 382, 80-83. 4. Henderson, C. E., Phillips, H. S., Pollock, R. A,, Davies, A. M., Lemeulle, C., Armanini, M., Simmons, L., Moffet, B., Vandlen, R. A., Koliatsos, V. E., and Rosenthal, A. (1994).
Effects of GDNF on Nigrostriatal DA System
9II
GDNF: A potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266,1062-1064. 5 . Buj-Bello, A., Horton, A., Rosenthal, A., and Davies, A. M. (1995).GDNF is an age-specific survival factor for sensory and autonomic neurons. Neuron 15, 821-828. 6. Moore, M. W., Klein, R. D., Farifias, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L. F., Ryan, A. M., Carver-Moore, K., and Rosenthal, A. (1996). Renal and neuronal abnormalities in mice lacking GDNF. Nature 382, 76-79. 7. Hynes, M., Porter, J. A,, Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P. A., Rosenthal, A. (1995).Induction of midbrain dopaminergic neurons by sonic hedgehog. Neuron 15, 35-44.
Don M. Gash,* Greg A. Gerhardt,t and Barry J. Hoffert *Department of Anatomy and Neurobiology and Research Magnetic Resonance Imaging and Spectroscopy Center University of Kentucky College of Medicine Lexington, Kentucky 40536
tDepartments of Pharmacology and Psychiatry Neuroscience Training Program and Rocky Mountain Center for Sensor Technology University of Colorado Health Sciences Center Denver, Colorado 80262
Effects of Glial Cell Line-Derived Neurotrophic Factor on the Nigrostriatal Dopamine System in
Rodents and Nonhuman Primates Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer distantly related to the transforming growth factor+ superfamily, which might have a therapeutic potential for treating Parkinson’s disease (1).While GDNF has been found to play an important role in the survival and development of a variety of cell types throughout the body, our studies have focused on analyzing its effects on the nigrostriatal dopaminergic system in rodents and nonhuman primates. The in uiuo effects of GDNF on midbrain dopamine (DA) neurons in normal adult rats and rhesus monkeys have been shown to be dramatic. In normal Fischer 344 rats, a single intranigral injection of 10 pg of GDNF ( 2 ) , increased nigral DA levels over threefold by 3 wk postinjection (Table I). Striatal DA levels on the injected side were either unchanged (1 wk posttreatment) or significantly lower ( 3 wk posttreatment), while striatal DA turnover was Advances in Pharmacology, Volume 42 Copyrighr 0 1998 by Academic Press. All rlghts of reproduction In any form reserved. 1054-3589/98 $25.00