Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic polypeptide, distantly related to transforming growth factor-beta (TGF-beta), 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 beta 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 x 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.

Muscle-derived neurotrophin-4 as an activity-dependent trophic signal for adult motor neurons.

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.

CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor.

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary.

Evolutionary conservation of members of the NGF family in vertebrates was studied by DNA sequence analysis of PCR fragments for NGF, BDNF, and NT-3 from human, rat, chicken, viper, Xenopus, salmon, and ray. The results showed that the three factors are highly conserved from fishes to mammals. Phylogenetic trees reflecting the evolution and speciation of the members of the NGF family were constructed. In addition, the gene for a fourth member of the family, neurotrophin-4 (NT-4), was isolated from Xenopus and viper. The NT-4 gene encodes a precursor protein of 236 amino acids, which is processed into a 123 amino acid mature NT-4 protein with 50%-60% amino acid identity to NGF, BDNF, and NT-3. The NT-4 protein was shown to interact with the low affinity NGF receptor and elicited neurite outgrowth from explanted dorsal root ganglia with no and lower activity in sympathetic and nodose ganglia, respectively. Northern blot analysis of different tissues from Xenopus showed NT-4 mRNA only in ovary, where it was present at levels over 100-fold higher than those of NGF mRNA in heart.

GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo.

The locus coeruleus (LC), the main noradrenergic center in the brain, participates in many neural functions, as diverse as memory and motor output, and is severely affected in several neurodegenerative disorders of the CNS. GDNF, a neurotrophic factor initially identified as dopaminotrophic, was found to be expressed in several targets of central noradrenergic neurons in the adult rat brain. Grafting of genetically engineered fibroblasts expressing high levels of GDNF prevented > 80% of the 6-hydroxydopamine-induced degeneration of noradrenergic neurons in the LC in vivo. Moreover, GDNF induced a fasciculated sprouting and increased by 2.5-fold both tyrosine hydroxylase levels and the soma size of lesioned LC neurons. These findings reveal a novel and potent neurotrophic activity of GDNF that may have therapeutic applications in neurodegenerative disorders affecting central noradrenergic neurons, such as Alzheimer's, Parkinson's, and Huntington's diseases.

Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts.

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.

BDNF regulates reelin expression and Cajal-Retzius cell development in the cerebral cortex.

Cajal-Retzius (CR) cells of the cerebral cortex express receptors for the neurotrophin brain-derived neurotrophic factor (BDNF) and downregulate expression of the extracellular matrix protein Reelin during early postnatal development, coincident with the onset of cortical BDNF expression. During this period, mice lacking BDNF have elevated levels of Reelin in CR cells. Acute BDNF stimulation of cortical neuron cultures and overexpression of BDNF in the developing brain of transgenic mice prior to the onset of endogenous production causes a profound, dose-dependent reduction of Reelin expression in CR cells. In addition, overexpression of BDNF produces gaps and heterotopias in the marginal zone and disorganization and aggregation of cortical CR cells and induces several other malformations, including aberrant cortical lamination, similar to the phenotype of reeler mutant mice, which lack Reelin. These results demonstrate a role for BDNF on cortical CR cells and identify Reelin as a direct effector of this neurotrophin during brain development.

p75LNGFR regulates Trk signal transduction and NGF-induced neuronal differentiation in MAH cells.

We have examined NGF-induced signal transduction events and neuronal differentiation in MAH cells, a neuronal progenitor cell line, in which the expression of the two NGF receptors, p140trk (Trk) and p75LNGFR (p75), has been independently manipulated. Coexpression of a large molar excess of p75 substantially enhances the NGF-induced tyrosine autophosphorylation of Trk, compared with cells expressing Trk alone. MAH cells expressing both Trk and p75 stop dividing and acquire a mature neuronal morphology more rapidly and with greater efficiency than MAH cells expressing Trk alone. These biochemical and biological influences of p75 are not observed using a mutant form of NGF that binds Trk but not p75. These data provide evidence that p75 can modulate signal transduction through Trk in a neuronal progenitor cell context and that such modulation has functional consequences for the neuronal differentiation pathway induced by NGF.

Self-association of the transmembrane domain of RET underlies oncogenic activation by MEN2A mutations.

In patients with medullary thyroid carcinoma (MTC) and type 2A multiple endocrine neoplasia (MEN2A), mutations of cysteine residues in the extracellular juxtamembrane region of the RET receptor tyrosine kinase cause the formation of covalent receptor dimers linked by intermolecular disulfide bonds between unpaired cysteines, followed by oncogenic activation of the RET kinase. The close proximity to the plasma membrane of the affected cysteine residues prompted us to investigate the possible role of the transmembrane (TM) domain of RET (RET-TM) in receptor-receptor interactions underlying dimer formation. Strong self-association of the RET-TM was observed in a biological membrane. Mutagenesis studies indicated the involvement of the evolutionary conserved residues Ser-649 and Ser-653 in RET-TM oligomerization. Unexpectedly, RET-TM interactions were also abrogated in the A639G/A641R double mutant, first identified in a sporadic case of MTC. In agreement with this, no transforming activity could be detected in full-length RET carrying the A639G and A641R mutations, which remained fully responsive to glial cell-line-derived neurotrophic factor (GDNF) stimulation. When introduced in the context of C634R - a cysteine replacement that is prevalent in MEN2A cases - the A639G/A641R mutations significantly reduced dimer formation and transforming activity in this otherwise highly oncogenic RET variant. These data suggest that a strong propensity to self-association in the RET-TM underlies - and may be required for - dimer formation and oncogenic activation of juxtamembrane cysteine mutants of RET, and explains the close proximity to the plasma membrane of cysteine residues implicated in MEN2A and MTC syndromes.

Emerging themes in structural biology of neurotrophic factors.

Neurotrophic factors control the survival, differentiation and maintenance of neurons in the peripheral and central nervous systems. Their discovery and characterization have been instrumental to our understanding of a wide range of phenomena in the development, plasticity and repair of the nervous system. Their potential importance in the development of therapeutic agents against neurodegenerative disorders and nerve injury has led to a flurry of activity towards understanding their structure, function and signaling mechanisms. This knowledge has increased dramatically in recent years, in particular due to the elucidation of three-dimensional structures, the discovery of families of structurally related neurotrophic factors and the characterization of receptors and downstream signaling components. Common themes are emerging from these recent studies that allow us to make new insights and predictions as to the function and possible clinical utility of these molecules.

Expression of neurotrophin-4 mRNA during oogenesis in Xenopus laevis.

Neurotrophin-4 (NT-4), a recently discovered novel member of the family of neurotrophic factors structurally related to nerve growth factor (NGF), is abundantly expressed in the Xenopus laevis ovary. In this study we have localized NT-4 mRNA expressing cells in the Xenopus ovary by in situ hybridization and have used this technique together with Northern blot analyses to quantify NT-4 mRNA expression during oogenesis in Xenopus. In situ hybridization of sections through the Xenopus ovary using an alpha-[35S]-dATP labeled Xenopus NT-4 mRNA specific probe showed an intense labeling over the cytoplasm of oocytes with a diameter of 50-200 microns corresponding to stage I according to Dumont (1972). Labeling was also seen over the cytoplasm of stages II to IV although with a lower intensity than over stage I oocytes. No labeling was seen over more mature oocytes of stages V and VI. NT-4 mRNA could not be detected in the early embryo from the onset of cleavage division to the neurula stage suggesting that the NT-4 gene is not expressed during Xenopus early embryogenesis. The confinement of NT-4 mRNA in the Xenopus ovary to immature oocytes suggests that NT-4 mRNA expression is strictly regulated during oogenesis and that the NT-4 protein could play a role as a maturation factor for immature oocytes.

Neurotrophic factors: from structure-function studies to designing effective therapeutics.

The development and maintenance of the vertebrate nervous system requires the continuous supply of a number of polypeptide hormones known as neurotrophic factors. The ability of neurotrophic factors to promote the survival of peripheral and central neurones during development and after neuronal damage, has stimulated an interest in these molecules as potential therapeutic agents for the treatment of nerve injuries and neurodegenerative diseases. Understanding the molecular basis of the biological specificity of neurotrophic polypeptides has provided an insight into their mechanisms of action, and allowed the design of derivatives and analogues with specific pharmacological properties.

Nerve growth factor: structure/function relationships.

Nerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted beta-hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2 cell-surface receptor types expressed in a variety of neuronal and non-neuronal cells. Residues involved in these interactions and in the maintenance of tertiary and quaternary structure have been identified by chemical modification and site-directed mutagenesis, and this information can be related to their location in the 3-dimensional structure. For example, interactions between aromatic residues contribute to the stability of the NGF dimer, and specific surface lysine residues participate in receptor contacts. The conclusion from these studies is that receptor interactions involve broad surface regions, which may be composed of residues from both promoters in the dimer.

Neurotrophin-7: a novel member of the neurotrophin family from the zebrafish.

A novel member of the neurotrophin family, zebrafish neurotrophin-7 (zNT-7), was isolated from the zebrafish Danio rerio. The amino acid sequence of zNT-7 is more closely related to that of fish nerve growth factor (NGF) and neurotrophin-6 (NT-6) than to that of any other neurotrophin. zNT-7 is, however, equally related to fish NGF and NT-6 (65% and 63% amino acid sequence identity, respectively) indicating that it represents a distinct neurotrophin sequence. zNT-7 contains a 15 amino acid residue insertion in a beta-turn region in the middle of the mature protein. Recombinant zNT-7 was able to bind to the human p75 neurotrophin receptor and to induce tyrosine phosphorylation of the rat TrkA receptor tyrosine kinase, albeit less efficiently than rat NGF. zNT-7 did not interact with rat TrkB or TrkC, indicating a similar receptor specificity as NGF. We propose that a diversification of the NGF subfamily in the neurotrophin evolutionary tree occurred during the evolution of teleost fishes which resulted in the appearance of several additional members, such as zNT-7 and NT-6, structurally and functionally related to NGF.

Coexpression of mRNA for the full-length neurotrophin receptor trk-C and trk-A in favourable neuroblastoma.

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may sometimes regress spontaneously in infants, or progress to a poor clinical outcome despite intensive therapy. Neuroblastomas express neurotrophin receptors and high levels of mRNA for trk-A correlates with favourable outcome, whereas trk-B mRNA is expressed by more unfavourable tumours. Using a sensitive RNase protection assay, mRNA expression for the neurotrophin receptor trk-C was investigated in 50 tumour samples from 45 children at different stages including metastatic and relapsing tumour tissue, out of which 22 were also investigated for trk-A mRNA. Thirty-seven of 43 primary tumours (86%) showed trk-C mRNA with more than 300-fold difference between the highest and the lowest values. A higher trk-C index (trk-C mRNA/GAPDH mRNA) was associated with favourable features such as younger age (P = 0.009-0.003), favourable tumour stage (1, 2 or 4S; P < 0.001) and favourable prognosis (P = 0.044). Better survival probability was shown in children with intermediate or high trk-C index compared with patients with low or undetectable levels (P = 0.031). All localised tumours co-expressed mRNA for trk-A and trk-C receptors. RT-PCR analysis detected mRNA encoding the cytoplasmic trk-C tyrosine kinase region only in favourable neuroblastomas. We conclude that favourable neuroblastoma may express the full-length trk-C receptor while unfavourable tumours, especially those with MYCN amplification, seem to either express no trk-C or truncated trk-C receptors with unknown biological function. Trk-C and possibly its preferred ligand NT-3 may be involved in the biology of favourable neuroblastomas showing apoptosis or differentiation.

Antigenic determinants of Trypanosoma cruzi defined by cloning of parasite DNA.

A genomic DNA library from Trypanosoma cruzi, the agent of Chagas' disease, was constructed in the gt11 lambda vector and was screened with serum from a Chagasic patient. Out of 53 positive clones, 23 plaques were purified to homogeneity and 10 different groups were defined by cross-hybridization experiments and by reaction of antibodies selected with products from each recombinant clone. Native T. cruzi proteins of molecular mass ranging from 85 to larger than 205 kDa that share antigenic determinants with products of the recombinant clones were observed in Western blots of parasite extracts. Some of the native proteins were detected in the trypomastigote stage of the parasite, while others were present in epimastigotes as well. The latter result was confirmed for some recombinant clones by hybridization of the cloned DNA with Northern blots of parasite RNA. Clones from each group reacted differently with nine sera from rabbits infected with several T. cruzi strains as well as with eight sera from human patients. Clone 7 was detected by all rabbit sera but not by three human sera. Conversely, clones 1, 2 and 30 were detected by all human sera but failed to be detected by most rabbit sera. We conclude that several proteins from T. cruzi are antigenically active during infection and that some of them differ in their ability to generate antibodies in rabbit or human infections.

Multiple Trypanosoma cruzi antigens containing tandemly repeated amino acid sequence motifs.

Chromosomal DNA from Trypanosoma cruzi, the agent of the American trypanosomiasis (Chagas' disease), was used for construction of a DNA library, employing the expression vector lambda gt11. Nine clones encoding different parasite antigens were isolated from this library by screening with an antiserum from a Chagasic patient. Nucleotide sequence analysis showed that seven out of the nine isolated clones code for antigens which contain tandemly repeated amino acid sequence motifs. Each of the seven antigens contains a unique repeat, ranging in length between 5 and 68 amino acids. The length of the repeats is highly conserved within each clone. Fusion proteins, expressed from two of the clones, reacted with a large proportion of sera collected from Chagasic patients in Argentina, Brazil and Chile. These clones appear thus to encode antigens which are shared between different strains of T. cruzi. Immunofluorescence experiments with live parasites showed that three of the antigens were detectable on the surface of trypanosomes.

Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease.

A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.

On the role of the low-affinity neurotrophin receptor p75LNTR in nerve growth factor induction of differentiation and AP 1 binding activity in PC12 cells.

Three clones of PC12 cells that differ with respect to their nerve growth factor (NGF) receptors were examined: wild-type PC12 cells that have both trkA and p75LNTR receptors; the MR-1 clone that possesses a normal trkA receptor and a truncated form of p75LNTR without the extracellular NGF-binding part; and a new PC12 variant, called v-clone, that is partly characterized here. The v-clone had no demonstrable binding to trkA, but displayed binding to p75LNTR as assessed by chemical crosslinking. NGF did not induce any change in the tyrosine phosphorylation of phosphatidy-3'-kinase in the v-clone. NGF induced neurite extension in wild-type cells, induced it more rapidly in mR-1, but not at all in v-clone cells. The v-clone lacked the b-form of protein kinase C, but transfection with this enzyme did not restore responsiveness to NGF. Neurite extension in response to staurosporine and basic fibroblast growth factor was equal in wild-type and v-clone cells. All three clones responded to forskolin, with the mR-1 clone the most responsive. NGF stimulated AP 1 binding activity in all clones. The response was transient in the MR-1 clone but prolonged in the wild-type and v-clone cells. In the wild-type and MR-1 clone cells, AP 1 binding activity was reduced by a tyrphostin analog, whereas in the v-clone cells it was inhibited by staurosporine. NGF increased inositol (1,4,5)-trisphosphate (InsP3) formation in all clones. In the wild-type and v-clone cells the InsP3 responses were followed by [Ca2+]i increases. It is concluded that although trkA is required for differentiation in response to NGF in PC12 cells, the concomitant stimulation, by NGF, of p75LNTR may affect phospholipase C and AP 1. This may be important for the reported ability of p75LNTR to modify the phenotypic changes induced in PC12 cells by NGF.

[Nerve growth factors in future therapies. Protein mutation broadens the range of therapeutic possibilities]. / Nervtillväxtfaktorer i framtida terapier. Muterade proteiner vidgar behanlingsmöjligheterna.

The development and maintenance of the vertebrate nervous system requires a continuous supply of a number of polypeptide hormones known as neurotrophic factors. The ability of neurotrophic factors to promote the survival of peripheral and central neurons during development and after neuronal damage has aroused interest in these molecules as potential therapeutic agents for the treatment of nervous lesions and neurodegenerative diseases. Understanding of the molecular basis of the biological specificity of neurotrophic polypeptides has provided insight into their mechanisms of action and allowed strategies to be developed for the rational design of derivatives and analogues with specific pharmacological properties.