Patricia A. Osborne

Expression of Neurturin, GDNF, and GDNF Family-Receptor mRNA in the Developing and Mature Mouse

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.

Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury

SummaryThe reaction of dorsal root ganglia (DRG) neurons to axotomy and its alteration by locally supplied nerve growth factor (NGF) were examined in adult rats. Surgically implanted silicone chambers attached to the severed tip of the sciatic nerve acted as reservoirs capable of providing prolonged access of NGF to the site of injury. The time course of NGF activity within the chambers was determined by using the standard NGF chick DRG bioassay. The fluid from chambers filled with the NGF-saline solution maintained NGF activity for periods up to 6 weeks after implantation. By 9 weeks, however, the fluid from most chambers failed to show any NGF activity in the bioassay.Experiments were designed to compare the response in adult rats to injury of DRG neurons receiving chambers filled with either NGF-saline or with only saline. The total neuronal counts in the lumbar fourth and fifth DRG at 3 weeks and 6 weeks after sciatic nerve section showed 22% and 16% cell death, respectively, in those injured neurons receiving saline-filled chamber implants. The animals that received chamber implants which contained an NGF-saline solution showed no cell death in the ipsilateral DRG at either 3 or 6 weeks after injury. Morphometric analysis of injured DRG neurons showed evidence of atrophy in the injured neurons which did not receive NGF. The degree of atrophy among all cell sizes was significantly decreased in those injured neurons receiving NGF. At 3 weeks after section the mean volume of injured neurons not treated with NGF was decreased by 28% as compared with only a 13% decrease in neurons treated with NGF. Similar findings were noted in the 6-week studies. NGF treatment did not alter the incidence of classic changes of chromatolysis in the groups of injured neurons receiving either NGF-saline-filled chambers or only saline-filled chambers.

Selective dependence of mammalian dorsal root ganglion neurons on nerve growth factor during embryonic development

We have investigated the NGF dependence of dorsal root ganglion (DRG) neurons in mammals using a paradigm of multiple in utero injections of a high titer anti-NGF antiserum. We have determined the specificity of our antiserum in relation to other members of the NGF neurotrophin family and found no cross-reactivity with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). To identify various classes of DRG neurons, we have stained their characteristic central projections with Dil. We show here that the NGF dependence of DRG neurons is strikingly selective. Although a majority of DRG neurons are lost after NGF deprivation during embryonic life, these are almost exclusively small diameter neurons that project to laminae I and II of the dorsal horn and presumably subserve nociception and thermoreception. Larger neurons that project to more ventral spinal laminae and subserve other sensory modalities do not require NGF for survival. These NGF-independent DRG neurons likely require one of the more recently identified neurotrophins, BDNF or NT-3.

Expression of neurturin, GDNF, and their receptors in the adult mouse CNS

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 (GFRα‐1 and GFRα‐2). We have used in situ hybridization to study the mRNA expression of NTN, GDNF, RET, GFRα‐1, and GFRα‐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. J. Comp. Neurol. 398:139–150, 1998.

Neuronal Heterotopias in the Developing Cerebral Cortex Produced by Neurotrophin-4

The marginal zone (MZ) of embryonic neocortex is crucial to its normal development. We report that neurotrophin-4 (but not NT3 or NGF), applied to embryonic rodent cortex in vitro or in vivo, produces heterotopic accumulations of neurons in the MZ. Although heterotopia production is TrkB mediated, BDNF is >10-fold less effective than NT4. Heterotopic neurons have the same birth date and phenotype as normal MZ neurons; they are not the result of NT4-induced proliferation or rescue from apoptosis. We suggest that NT4 causes excess neurons to migrate into the MZ and thus may play a role in normal MZ formation as well as in the pathogenesis of certain human cortical dysplasias.

Characterization of the effects of autoimmune nerve growth factor deprivation in the developing guinea-pig

Abstract We have previously reported that female guinea-pigs immunized with mouse nerve growth factor (NGF) make antibodies which cross-react with guinea-pig NGF. Offspring born to the immunized female guinea-pig were born with marked decreases in numbers of sympathetic and dorsal root ganglion sensory neurons. In this paper, we describe the degree and permanence of the destruction of these components in the nervous system of the offspring. Considerable variability is seen in the immunological response to NGF. Only those animals that generate a high titer antibody against mouse NGF which cross-reacts well with guinea-pig NGF produce offspring with deleterious effects on the developing nervous system. Female guinea-pigs with titers of approximately 4000 against guinea-pig NGF produce ‘severely’ affected offspring with gross sensory deficits which were easily observed as a complete lack of response to several painful stimuli. These ‘severely’ affected animals showed a virtual absence of unmyelinated axons in the sciatic nerve. These animals did not survive beyond a few days of age. Animals born to females with titers of 1000–2000 were ‘moderately’ affected; responses to painful stimuli were blunted but not absent. ‘Moderately’ affected animals survived and grew normally; maternal antibody in their circulation disappeared by six weeks of age. None of the moderately affected animals exhibited obvious deficits in proprioception and general coordination. Analysis of catecholamine levels showed that in severely and moderately affected animals norepinephrine levels were permanently reduced to almost non-detectable levels in heart and spleen. Less severe permanent effects were observed in small intestine and transient effects were seen in the sympathetic innervation of the male urogenital system. No adverse effects were seen upon light-microscopic examination of the adrenal medulla, nor was there any decrease in tyrosine hydroxylase activity in this tissue; thus, we found no evidence for an adverse effect of anti-NGF on the developing adrenal medulla. We conclude that varying degrees of destruction of the sympathetic and sensory nervous systems are produced by in utero exposure to maternal anti-NGF in the guinea-pig. The sources of the variability include differences in immunological responses among animals and varying degrees of susceptibility to NGF deprivation among neuronal types.

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclude that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.

Destruction of sympathetic and sensory neurons in the developing rat by a monoclonal antibody against the nerve growth factor (NGF) receptor.

The ability of the monoclonal antibody, 192-IgG, directed against the rat nerve growth factor (NGF) receptor to mimic or inhibit the actions of NGF was examined in vitro and in vivo. 192-IgG had no effect on morphology, survival, or protein synthesis rates of sympathetic neuronal cultures. When injected into newborn rats, destruction of sympathetic, but not sensory, neurons was produced. Injection prenatally produced more dramatic destruction of sympathetic neurons and, in addition, destruction of neural crest-derived sensory neurons. Therefore, although 192-IgG had no discernible effects in vitro, it produced a pattern of neuronal destruction in vivo qualitatively similar to that produced by antibodies to NGF itself.

NGF deprivation and neuronal degeneration trigger altered β-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro

In order to study the expression of beta-amyloid precursor protein (APP) isoforms during neuronal degeneration we have used the rat superior cervical ganglia (SCG) as an experimental model. In the neonate these sympathetic ganglia are nerve growth factor (NGF) dependent and in vivo administration of anti-NGF antiserum results in exaggerated neuronal degeneration. Analysis of APP mRNA transcripts in the SCG, following NGF deprivation, revealed a coincident decrease in APP695 and augmentation of APP751/770. These changes were specific to the SCG and were not seen in sensory ganglia. Subsequent in vitro studies, using primary dissociated cultures of sympathetic or cortical neurones, confirmed these changes in APP gene expression during neuronal degeneration. These observations may have important implications for the generation of beta-amyloid in Alzheimers disease.

Expression and function of GDNF family ligands and receptors in the carotid body

The carotid body is a neural crest-derived neuroendocrine organ that detects the oxygen level in blood and regulates ventilation. Unlike many other neural crest derivatives, the trophic factors mediating survival and differentiation of neuroendocrine cells of the carotid body are unknown. Given that many neural crest derivatives rely on the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) for survival and function, we undertook an analysis of the carotid body as a potential site of GFL action. RET and GDNF family receptor alphas (GFRalpha) 1-3 are expressed in the developing carotid body as detected by RT-PCR and immunocytochemistry. mRNA for GDNF, and artemin (ARTN) were also present. In vitro, treatment with GDNF, neurturin (NRTN), or ARTN, individually or in combination, produced an increase in the number and length of processes of the Type-I glomus cells of the carotid body [embryonic day-17 (E17) rats]. However, GFLs alone or in combination had no effect on glomus cell survival in either postnatal day-1 (P1) or E17 carotid body cultures. These results suggest that one or more GFLs may have a role in carotid body function. In addition, the results of this study suggest that endogenous or exogenous GFLs may enhance target innervation by carotid body transplants.