Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion.

Glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor beta 3 (TGF-beta 3) are members of the TGF-beta superfamily with high neurotrophic activity on cultured nigral dopamine neurons. We investigated the effects of intracerebral administration of GDNF and TGF-beta 3 on the delayed cell death of the dopamine neurons in the rat substantia nigra following 6-hydroxydopamine lesions of dopaminergic terminals in the striatum. Fluorescent retrograde tracer injections and tyrosine hydroxylase immunocytochemistry demonstrated nigral degeneration with an onset 1 week after lesion, leading to extensive death of nigral neurons 4 weeks postlesion. Administration of recombinant human GDNF for 4 weeks over the substantia nigra at a cumulative dose of 140 micrograms, starting on the day of lesion, completely prevented nigral cell death and atrophy, while a single injection of 10 micrograms 1 week postlesion had a partially protective effect. Continuous administration of TGF-beta 3, starting on the day of lesion surgery, did not affect nigral cell death or atrophy. These findings support the notion that GDNF, but not TGF-beta 3, is a potent neurotrophic factor for nigral dopamine neurons in vivo.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.

Interleukin 4 signals through two related pathways.

The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.

Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells.

Enteropathogenic Escherichia coli (EPEC), a major cause of pediatric diarrhea, adheres to epithelial cells and activates host cell signal transduction pathways. We have identified five proteins that are secreted by EPEC and show that this secretion process is critical for triggering signal transduction events in epithelial cells. Protein secretion occurs via two pathways: one secretes a 110-kDa protein and the other mediates export of the four remaining proteins. Secretion of all five proteins was regulated by temperature and the perA locus, two factors which regulate expression of other known EPEC virulence factors. Amino-terminal sequence analysis of the secreted polypeptides identified one protein (37 kDa) as the product of the eaeB gene, a genetic locus previously shown to be necessary for signal transduction. A second protein (39 kDa) showed significant homology with glyceraldehyde-3-phosphate dehydrogenase, while the other three proteins (110, 40, and 25 kDa) were unique. The secreted proteins associated with epithelial cells, and EaeB became resistant to protease digestion upon association, suggesting that intimate interactions are required for transducing signals.

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.

A role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury.

Within the central nervous system (CNS) ciliary neurotrophic factor (CNTF) is expressed by astrocytes where it remains stored as an intracellular protein; its release and function as an extracellular ligand are thought to occur in the event of cellular injury. We find that overexpression of CNTF in transgenic mice recapitulates the glial response to CNS lesion, as does its injection into the uninjured brain. These results demonstrate that CNTF functions as an inducer of reactive gliosis, a condition associated with a number of neurological diseases of the CNS.

Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.

Signaling by interferon gamma (IFN-gamma) requires two structurally related cell surface proteins: a ligand-binding polypeptide, known as the IFN-gamma receptor (IFN-gamma R), and an accessory factor. However, it is not known whether IFN-gamma forms a ternary complex with the IFN-gamma R and accessory factor to initiate signaling. Here we demonstrate complex formation between IFN-gamma and the two proteins, both in solution and at the cell surface. We observe complexes containing ligand, two molecules of IFN-gamma R (designated the IFN-gamma R alpha chain), and one or two molecules of accessory factor (designated the IFN-gamma R beta chain). Transfected cells expressing both IFN-gamma R chains bind IFN-gamma with higher affinity than do cells expressing alpha chain alone. Anti-beta-chain antibodies prevent the beta chain from participating in the ligand-receptor complex, reduce the affinity for IFN-gamma, and block signaling. Soluble alpha- or beta-chain extracellular domains also inhibit function. These results demonstrate that IFN-gamma signals via a high-affinity multisubunit complex that contains two types of receptor chain and suggest a potential approach to inhibiting specific actions of IFN-gamma by blocking the association of receptor subunits.

Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway.

Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants.

Chemical and physical signals have been reported to mediate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Among the chemical signals, phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and the peptide systemin represent the best characterized systems. Furthermore, electrical and hydraulic mechanisms have also been postulated as putative Pin2-inducing systemic signals. Most of the chemical agents are able to induce Pin2 gene expression without any mechanical wounding. Thus, ABA, JA, and systemin initiate Pin2 mRNA accumulation in the directly treated leaves and in the nontreated leaves (systemic) that are located distal to the treated ones. ABA-deficient tomato and potato plants do not respond to wounding by accumulation of Pin2 mRNA, therefore providing a suitable model system for analysis of the signal transduction pathway involved in wound-induced gene activation. It was demonstrated that the site of action of JA is located downstream to the site of action of ABA. Moreover, systemin represents one of the initial steps in the signal transduction pathway regulating the wound response. Recently, it was reported that heat treatment and mechanical injury generate electrical signals, which propagate throughout the plant. These signals are capable of inducing Pin2 gene expression in the nontreated leaves of wounded plants. Furthermore, electrical current application to tomato leaves leads to an accumulation of Pin2 mRNA in local and systemic tissues. Examination of photosynthetic parameters (assimilation and transpiration rate) on several types of stimuli suggests that heat-induced Pin2 gene expression is regulated by an alternative pathway from that mediating the electrical current and mechanical wound response.

Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro.

Oligogalacturonides are plant cell wall-derived regulatory molecules which stimulate defense gene expression during pathogenesis. In vitro, these compounds enhance the phosphorylation of an approximately 34-kDa protein (pp34) in purified plasma membranes from potato and tomato leaves. We now show that polygalacturonate-enhanced phosphorylation of pp34 occurs in plasma membranes purified from tomato roots, hypocotyls, and stems and from undifferentiated potato cells. Furthermore, a similar phosphorylation is detected in leaf plasma membranes from soybean, a plant distantly related to tomato. Purified oligogalacturonides 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro. This stimulation pattern differs from the induction of many known defense responses in vivo, where a narrower range of smaller fragments, between approximately 10 and 15 residues long, are active. On the basis of these differences we suggest that observed effects of applied exogenous oligogalacturonides on defense responses may not necessarily reflect the situation during pathogenesis. The cell wall could act as a barrier to many exogenous oligo- and polygalacturonides as well as other large regulatory ligands.

Redundant 3' end-forming signals for the yeast CYC1 mRNA.

The cyc1-512 mutation is a 38-bp deletion in the 3' untranslated region of the CYC1 gene, which encodes iso-1-cytochrome c in Saccharomyces cerevisiae. This deletion caused a 90% reduction in the levels of the CYC1 mRNA and protein because of the absence of the normal 3' end-forming signal. Although the 3' end-forming signal was not defined by previous analyses, we report that concomitant alteration by base-pair substitution of three 3' end-forming signals within and adjacent to the 38-bp region produced the same phenotype as the cyc1-512 mutation. Furthermore, these signals appear to be related to the previously identified 3' end-forming signal TATATA. A computer analysis revealed that TATATA and related sequences were present in the majority of 3' untranslated regions of yeast genes. Although TATATA may be the strongest and most frequently used signal in yeast genes, the CYC1+ gene concomitantly employed the weaker signals TT-TATA, TATGTT, and TATTTA, resulting in a strong signal.

Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent. Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional in promoting the active nuclear transport of MyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells. Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor. In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.

Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Residue replacements were made at five positions (Arg-73, Asp-76, Tyr-87, Asp-106, and Asp-201) in the Halobacterium salinarium phototaxis receptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The sites were chosen for their correspondence in position to residues of functional importance in the homologous light-driven proton pump bacteriorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling function: Asp-201. The effect of the substitution with the isosteric asparagine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substitution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signaling. Wild-type two-photon repellent signaling by the receptor is intact in the Asp-201 mutant, genetically separating the wild-type attractant and repellent signal generation processes. A possible explanation and implications of the inverted signaling are discussed. Results of neutral residue substitution for Asp-76 confirm our previous evidence that proton transfer reactions involving this residue are not important to phototaxis but that Asp-76 functions as the Schiff base proton acceptor in proton translocation by transducer-free SR-I.

Control of somite patterning by signals from the lateral plate.

The body musculature of higher vertebrates is composed of the epaxial muscles, associated with the vertebral column, and of the hypaxial muscles of the limbs and ventro-lateral body wall. Both sets of muscles arise from different cell populations within the dermomyotomal component of the somite. Myogenesis first occurs in the medial somitic cells that will form the epaxial muscles and starts with a significant delay in cells derived from the lateral somitic moiety that migrate to yield the hypaxial muscles. The newly formed somite is mostly composed of unspecified cells, and the determination of somitic compartments toward specific lineages is controlled by environmental cues. In this report, we show that determinant signals for lateral somite specification are provided by the lateral plate. They result in a blockade of the myogenic program, which maintains the lateral somitic cells as undifferentiated muscle progenitors expressing the Pax-3 gene, and represses the activation of the MyoD family genes. In vivo, this mechanism could account for the delay observed in the onset of myogenesis between muscles of the epaxial and hypaxial domains.