Developmental regulation of beta-thymosins in the rat central nervous system.

HPLC analysis of guanidinium hydrochloride extracts of neonatal and adult rat brain revealed a polypeptide that is present in high concentration in the immature nervous system, but whose levels decline dramatically in the adult. This polypeptide has been isolated and its complete amino acid sequence determined by gas-phase Edman degradation following specific chemical and enzymatic cleavages. The molecule is identified as thymosin beta 10, a member of a multigene family that encodes a structurally conserved series of small acidic polypeptides of uncertain function. Thymosin beta 10 is present in the developing nervous system as early as embryonic day 9. Levels subsequently increase to peak values between embryonic day 15 and postpartum day 3, before falling to adult values (about a 20-fold reduction) by postpartum day 14. The elevated levels of thymosin beta 10 in fetal and neonatal brain correlate with high levels of thymosin beta 10 mRNA, whereas the low values of the polypeptide in the adult and juvenile are mirrored by an approximate 15-fold reduction in specific mRNA. In comparison, the levels of thymosin beta 4 polypeptide, a homologue of thymosin beta 10, only decline by about 20% during the same developmental period. However, the mRNA encoding thymosin beta 4 is elevated in fetal brain, and its levels decrease approximately four-fold to a stable value around the time of birth. The reason for this discrepancy between thymosin beta 4 protein and mRNA levels is unknown. Thymosin beta 10 can also be detected by HPLC in fetal liver, where levels are approximately 5% of those in brain. In liver, thymosin beta 10 also declines following birth. It is concluded that beta-thymosin expression (as measured by steady-state mRNA and polypeptide levels) is both up- and down-regulated during different phases of maturation of the mammalian nervous system.

Retinoic acid regulates thymosin beta 10 levels in rat neuroblastoma cells.

A small acidic polypeptide, termed thymosin beta 10, has been identified and is present in the nervous system of the rat by the ninth day of gestation. Thymosin beta 10 levels rise during the remaining days of life in utero, and then decline to nearly undetectable values between the second and fourth week post partum. The present study investigates the possible developmental signals and mechanisms that might regulate the expression of thymosin beta 10 during neuroembryogenesis. Many cell lines derived from tumors of the central nervous system express thymosin beta 10, as well as its homologue gene product, thymosin beta 4. Because some of these cell lines respond to exogenously applied agents by increasing their apparent state of differentiation, we have determined whether thymosin beta 10 levels are coordinately modulated. In several neuroblastomas, including the B103 and B104 lines, retinoic acid elicits a time- and dose-dependent increase in the content of thymosin beta 10, but not that of thymosin beta 4. The increase in thymosin beta 10 polypeptide is associated with a marked increase in the specific mRNA encoding this molecule. The mRNA for thymosin beta 4 is unaffected by retinoic acid. This is in contrast with the situation in vivo, where the expression of both genes decreases after birth. Other agents that influence the morphology of B104 cells, such as phorbol esters and dibutyryl cyclic AMP, have no influence on beta-thymosin levels. A range of steroids, which like retinoids act upon nuclear receptors, was also inactive. The stimulatory action of retinoic acid is detectable within 4 h, and thymosin beta 10 peptide levels continue to rise for at least 4 days. The influence of the isoprenoid is fully reversible and exhibits structural specificity. We believe that this culture system is mimicking the early rising phase of thymosin beta 10 levels in brain and that endogenous retinoids may be candidate physiological regulators of this gene.

Thymosin beta 10 levels in developing human brain and its regulation by retinoic acid in the HTB-10 neuroblastoma.

Human fetal brain expresses high levels of a polypeptide identified by protein biochemistry and molecular cloning as thymosin beta 10. Within the first 18 months after birth, the thymosin beta 10 content of human brain falls to undetectable levels. In order to establish the molecular basis of this process we screened a number of human tumor cell lines derived from the nervous system for the presence of thymosin beta 10. All of the cell line expressed authentic thymosin beta 10. However, in the HTB-10 neuroblastoma, retinoic acid caused a reduction in the level of thymosin beta 10. This effect of the retinoid was conditional upon its continual presence in the tissue culture medium and was not evident in the other cell lines examined. These results suggest that the thymosin beta 10 gene may be a target for retinoids in the developing nervous system.

Glutamate receptor agonists increase the expression of Fos, Fra, and AP-1 DNA binding activity in the mammalian brain.

Administration of convulsant doses of Metrazole (pentylenetetrazol) and picrotoxin, as well as maximal electroshock, results in a rapid but transient increase in c-fos mRNA in mouse brain. Elevation of c-fos mRNA is followed by the accumulation and subsequent disappearance of Fos, the protein encoded by c-fos. In addition, immunoblots reveal the induction of two additional proteins that are antigenically related to Fos (Fra, Fos-related antigens). Fos and the various Fra appear and disappear in a staggered manner over an 8 hour period, such that at longer times after stimulation the brain contains no Fos but relatively large amounts of Fra (the latter being designated here by their apparent molecular weights, Fra-46K and Fra-35K). Previous studies have established that Fos, as well as several Fra, contribute to transcription factor AP-1 nucleoprotein complexes along with Jun, the product of the jun proto-oncogene. The appearance in brain of Fos and Fra coincides with a protracted increase in total AP-1 DNA binding activity, indicating that all the Fos-like proteins can participate in AP-1 complexes. Furthermore, the molecular composition of these complexes alters with time after stimulation. The induction of c-fos by Metrazole is blocked or attenuated by known anticonvulsants such as diazepam and valproate as well as the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonovaleric acid (APV) and MK-801. This suggests that fos induction might involve stimulation of a glutamate receptor. This conclusion was strengthened by the observations that two glutamate receptor agonists, kainic acid and NMDA, induced c-fos expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of a neuron-specific transcript and its regulation during normal and aberrant cerebellar development.

PEP-19 is a brain-specific polypeptide whose levels increase dramatically during the late maturation of the rodent nervous system. By using immunocytochemistry, PEP-19 is shown to be localized to several regions of the central nervous system, notably cerebellum, thalamus caudate putamen, and olfactory bulb. We have isolated a 0.5-kilobase cDNA clone that encodes the entire PEP-19 protein sequence, making this one of the smallest primary transcripts and translation products ever identified in eukaryotes. The cDNA was used to investigate the developmental expression of PEP-19 in rodent brain. PEP-19 mRNA rises from low levels at embryonic day 17 of gestation in the rat to a plateau value by day 18 postpartum. This mirrored the levels of the protein determined by radioimmunoassay. Since the rise coincided with the formation of synaptic contacts onto Purkinje cells (a major site of PEP-19 expression), the hypothesis was tested that the activity and/or presence of afferent input modulated PEP-19 expression. Parallel fiber innervation was disrupted either by killing granule cells with the cytostatic agent methylazoxymethanol or by examining PEP-19 levels in cerebellar granuloprival mutant mice (reeler and weaver). The influence of climbing fiber input was assessed by either eliminating them with 3-acetylpyridine or stimulating them with harmaline in both neonatal and mature rats. None of the above altered PEP-19 gene expression in cerebellum, leading us to propose that the signals triggering the PEP-19 gene do not emanate from granule cells or neurons in the olivary nucleus. However, preliminary evidence suggests that PEP-19 is under posttranscriptional regulation.

Developmental expression of reactivity to monoclonal antibodies generated against olfactory epithelia.

The developmental expression of immunocytochemical reactivity to 3 monoclonal antibodies (Mabs Neu 4, Neu 5, and Neu 9) that were generated against adult rat olfactory epithelium was examined in olfactory tissues of embryonic rats. Tissues examined included the nasal olfactory epithelium, nerve, and olfactory bulb, as well as vomeronasal epithelium and nerve. Reactivity patterns of these Mabs in adult rats have been described previously (Hempstead and Morgan, 1985a). All 3 Mabs show reactivity on the cell surfaces of neurons, axons, and dendrites of the olfactory epithelium proper. Neu 5 alone shows reactivity on the dendritic knobs, site of transduction of the olfactory stimuli. These reactivities appear early, suggesting developmentally significant roles for the antigens to these Mabs. For Neu 5 and Neu 9 initial reactivity occurs on outgrowing olfactory axons at E13. Dendritic and perikaryal reactivities begin appearing at E14. For Neu 4 initial reactivity occurs simultaneously on olfactory neuronal perikarya, axons, and dendrites at E14. Reactivity also occurs on cells that migrate from the olfactory epithelium and are associated with the olfactory nerves. Within the developing olfactory bulb, Neu 5 behaves as a general cell-surface marker. Neu 4 and Neu 9, however, show enhanced reactivity in the glomerular layer after the onset of synaptogenesis. Reactivity is also seen in the nasal respiratory epithelium and in the vomeronasal epithelia and nerve. Neu 5 and several antibodies to rat neural cell adhesion molecules (N-CAMs) show similar, although not identical, immunohistochemical staining patterns. They also react with the same bands in Western blots of brain membrane preparations. Western blots of Neu 5-reactive material also show developmental and spatial correlations of apparent molecular-weight distributions expected of N-CAM-like components as well.

An immunochemical analysis of the distribution of a brain-specific polypeptide, PEP-19.

Immunochemical and immunohistochemical techniques were used to map the tissue distribution and cellular localization of a rat brain-specific polypeptide, termed PEP-19. PEP-19 was found to be abundant in the cerebellum and olfactory bulbs but was present at much lower levels in other gross brain regions. It was undetectable in all nonneural tissues examined but was present in the cerebellum of several vertebrates, including rat, mouse, guinea pig, monkey, and human. Immunohistochemical analysis revealed that PEP-19 was localized to the soma, axon, and dendritic processes of rat cerebellar Purkinje cells with no demonstrable immunoreactivity in nonneuronal cell types. Furthermore, mutant mice showing degeneration of Purkinje cells exhibit markedly decreased levels of PEP-19. Because PEP-19 appears during the final stages of maturation of Purkinje cells, it may be utilized as a probe to monitor the development of these neurons in vivo.

Cerebellin and related postsynaptic peptides in the brain of normal and neurodevelopmentally mutant vertebrates.

The rat cerebellum was previously shown to contain two polypeptides, a hexadecapeptide termed cerebellin and an apparent metabolite des-Ser1-cerebellin. The cerebellins have a high degree of sequence homology with residues 625-641 of the polyimmunoglobulin (polyIg)-receptor adjacent to its membrane-spanning domain. Since the cerebellins are localized in Purkinje cells and enriched in synaptosomes, this might indicate that cerebellin is a specific proteolytic cleavage fragment of a synaptic protein involved in the transcytosis of an unknown ligand. Using a specific cerebellin radioimmunoassay described here combined with high-performance liquid chromatography, cerebellin immunoreactivity could be demonstrated in the cerebella of all vertebrates examined from man to chicken. Cerebellin immunoreactivity is localized to Purkinje cells in the rat, mouse, and chicken. Furthermore, cerebellin expression is under developmental regulation in both the chicken and mouse. In addition, neurodevelopmental mutations of mice that eliminate granule cells cause a large deficit in cerebellin levels, suggesting some form of transneuronal regulation.

Mapping patterns of c-fos expression in the central nervous system after seizure.

A dramatic and specific induction of c-fos was observed in identifiable neuronal populations in vivo after administration of the convulsant Metrazole. This effect was time- and dose-dependent and was abolished by prior treatment with the anticonvulsant drugs diazepam or pentobarbital. About 60 minutes after administration of Metrazole, c-fos messenger RNA reached a maximum and declined to basal levels after 180 minutes. A further decrease below that in normal brain was observed before a return to basal levels after 16 hours. While Metrazole still elicited seizures during this period, reinduction of c-fos was largely refractory. At 90 minutes, c-fos protein was observed in the nuclei of neurons in the dentate gyrus, and in the pyriform and cingulate cortices. Subsequently, c-fos protein appeared throughout the cortex, hippocampus, and limbic system. Thus, seizure activity results in increased c-fos gene expression in particular subsets of neurons.

Monoclonal antibodies to mammalian carnosine synthetase.

A set of mouse monoclonal antibodies has been generated against rabbit muscle carnosine synthetase. The immunoreactivity of these antibodies has been characterized using an immunoassay that permits the separation and direct measurement of the synthetase activity on a second antibody bead complex. Four IgG monoclonal antibodies bind the carnosine synthetase activity from muscle of all mammals tested (mouse, rat, rabbit, cow, dog, and monkey) but not that from chicken muscle. This indicates the mammalian enzymes share epitopes that are absent from the avian enzyme. In addition, relative tissue levels of synthetase activity can be quantified with this immunoassay. Thus, high levels of carnosine synthetase activity are immunoprecipitated from the olfactory tissues of both rat and rabbit. Synthetase activity is generally lower in other tissues (muscle, brain, heart, liver, and gut). Nevertheless, the cross-reactivity of the synthetase from several tissues (olfactory mucosa, muscle, brain, gut, heart, and liver) of a single species indicates the enzyme protein contains similar epitopes in these tissues. Immunoaffinity purification of this low-abundance, unstable enzyme should now be possible for subsequent studies of structure and regulation.

Cerebellin: a quantifiable marker for Purkinje cell maturation.

The cerebellum-specific hexadecapeptide cerebellin has been localized by immunocytochemical means to the perikarya and dendrites of cerebellar Purkinje cells. Biochemical analysis using ion-pairing HPLC shows cerebellin to first appear 5 days after birth, whereafter levels rise to a maximum at 25 days postpartum, and then decline to stable adult values. This same pattern of development occurs with a lag of approximately 5 days for the major metabolite of cerebellin, des-Ser1-cerebellin. The immunocytochemical picture of cerebellin in developing Purkinje cells mirrors the biochemical data. These results show that cerebellins represent unique quantifiable markers for the investigation of Purkinje cell maturation and lend support to the feasibility of using unique endogenous peptides to chart neurodevelopment.

Monoclonal antibodies reveal novel aspects of the biochemistry and organization of olfactory neurons following unilateral olfactory bulbectomy.

Following unilateral olfactory bulbectomy in rats the ipsilateral olfactory neuroepithelium undergoes degeneration. Subsequently, the receptor neuron complement of the tissue is restored by the proliferation and differentiation of immature neuroblasts. However, as noted by other workers, in the absence of a target organ the dynamics of neuron regeneration is altered such that there is an overall reduction in the number of cells positive for the olfactory marker protein when cellular equilibrium is re-established. Immunocytochemical staining of the olfactory epithelium of unilaterally bulbectomized rats with a series of anti-neuronal monoclonal antibodies reveals an attenuation of binding of some antibodies to the neurons of the ipsilateral epithelium. In contrast, other anti-neuronal monoclonal antibodies show no difference in staining intensity when ipsilateral and control contralateral epithelia are compared. These data suggest that the expression of some neuronal antigens is subject to control by the target olfactory bulb, whereas others are independent of such putative regulation. Besides altering the expression of some antigenic determinants, olfactory bulbectomy also results in certain organizational changes in epithelium. First, bulbectomy produces an increase in the incidence of a cell type that appears to span the neuroepithelium. Although the morphology of these cells is more akin to a sustentacular cell than to a receptor neuron, they are not immunoreactive with antibodies to sustentacular cells. The cells are stained, however, by an anti-neuronal antibody, NEU-9. The second aspect of altered organization is the appearance of novel olfactory marker protein-positive structures in the olfactory mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

A panel of monoclonal antibodies to the rat olfactory epithelium.

BALB/c mice were immunized with dissected olfactory mucosa from young adult Sprague-Dawley-derived, CD, rats and antibody-secreting hybridomas were produced. Supernatants from hybridoma cultures were screened by immunocytochemical methods for their ability to react with specific cell populations in frozen sections of rat olfactory epithelium. Approximately 60 clones were identified that showed various degrees of specific staining. These have been classified on the basis of their particular staining specificities. One group of monoclonal antibodies, designated LUM, reacts with the luminal surface of the epithelium. Closer examination reveals these antibodies to react variously with the apical brush border of sustentacular cells, respiratory cilia, and the luminal membrane of respiratory cells. Another group of monoclonal antibodies reacts primarily with sustentacular cells, as is indicated by the SUS prefix. Some antibodies in this group also react with the membranes of respiratory cells and the cells comprising the acini of Bowman's glands. A larger group of antibodies reacts with olfactory neurons and/or their axons, as denoted by the NEU prefix. This group can be further subdivided by the criteria of whether both the olfactory nerve and vomeronasal nerve react with the same monoclonal antibody. A fourth group of monoclonal antibodies, designated GLA, reacts with Bowman's glands and in some instances with secretory cells present in the respiratory mucosa. Two clones, BCL, stain at the level of the basal cell layer just above the lamina propria. A number of other antibodies react with cells and structures of the epithelium that have not been previously described. One of this group, NIS-1, stains globular structures present in the mucosa of the neuroepithelium.

Isolation and sequencing of two cerebellum-specific peptides.

By comparing the HPLC elution profiles of peptides isolated from different brain regions, two cerebellum-specific species have been identified. The peptides were isolated by sequential chromatography on reverse phase, followed by ion-pairing HPLC using alkane sulfates as pairing reagents. The sequences of both peptides have been determined by gasphase Edman degradation and carboxypeptidase Y analysis and were confirmed by synthesis. The larger peptide, termed cerebellin, is a hexadecamer of primary amino acid sequence NH(2)-Ser-Gly-Ser-Ala-Lys-Val-Ala-Phe-Ser-Ala-Ile-Arg-Ser-Thr-Asn-His- OH. The second peptide is a pentadecamer with a primary sequence identical to residues 2-16 of cerebellin, the nomenclature of which is des-Ser(1)-cerebellin. Subcellular fractionation of cerebellum followed by ion-pairing HPLC analysis shows that both cerebellin and des-Ser(1)-cerebellin are enriched between 2.5- and 4-fold in the P2 crude synaptosomal fraction, suggesting their sequestration in some subcellular particle in vivo.

Monoclonal antibodies to the rat olfactory sustentacular cell.

Immunization of BALB/c mice with rat olfactory epithelium permitted the production of a number of monoclonal antibodies directed towards cell types within the tissue. One of these antibodies, designated SUS-1, reacts specifically with cells located at the luminal surface of the olfactory neuroepithelium which possess a characteristic wine-glass body and a basal process which projects to the basement membrane. Thus these cells have the topographical and morphological properties of olfactory sustentacular cells. The SUS-1-positive cells are not stained by an antibody to the olfactory marker protein and neither does the SUS-1 antibody react with mature olfactory neurons in double labeling experiments. Upon unilateral olfactory bulbectomy there is a marked reduction in olfactory marker protein positive cells while there is no apparent diminution in the labeling of SUS-1 cells. These data show conclusively that the mature olfactory neurons and the SUS-1-positive cells are distinct from one another. SUS-1 staining is absent in all tissues examined except the olfactory epithelium. However, small discrete groups of cells in the lamina propria mucosae (often referred to as the submucosa) do stain in addition to the luminal sustentacular cell layer. The possibility is considered that the mature sustentacular cells may be related to these latter cells.

Production and characterization of antibodies to thymosin beta 4.

Thymosin beta 4 antibodies have been raised in rabbits by conjugating the peptide to keyhole limpet hemocyanin, and a radioimmunoassay for thymosin beta 4 has been established utilizing tritiated thymosin beta 4 as binding ligand. RIA values for thymosin beta 4 in rat tissues reveal levels ranging between 8 and 448 micrograms/g wet weight. Highest levels are observed in spleen, although the peptide is also relatively abundant in thymus, lung, and nasal epithelium. Brain, kidney, liver and testis contain only 10 to 20% the concentration of thymosin beta 4 seen in spleen, whereas heart and muscle contain only 5 to 10% that of spleen. However, thymosin beta 4 probably has a ubiquitous distribution in the rat, being present in all tissues examined. The specificity of the antiserum used in the RIA has been defined. The thymosin beta 4 molecule seemingly bears two major epitopes that lie between amino acid residues 1-8 and 22-32. The antiserum does not cross-react with other unrelated peptides. The antiserum will precipitate biosynthetically labeled beta 4 from cultured rat peritoneal macrophage.

Synthesis of thymosin beta 4 by peritoneal macrophages and adherent spleen cells.

Thymosin beta 4, previously identified as a component of thymosin fraction 5 isolated from calf thymus, is synthesized by peritoneal macrophages derived from rats or mice, including cells from nu/nu mice. Identification of thymosin beta 4 containing [35S]methionine was based on its retention time in reverse-phase HPLC and the recovery of radioactivity in peptides generated by mild acid hydrolysis or by digestion with trypsin. Thymosin beta 4 is also synthesized by rat and mouse spleen cells, and evidence is presented for its release from these cells.

Thymosin beta 4: a ubiquitous peptide in rat and mouse tissues.

Thymosin beta 4, recently isolated from calf thymus, is present in a number of rat and mouse tissues, including spleen, thymus, brain, lung, liver, and heart muscle. High concentrations are found in peritoneal macrophages, suggesting that its occurrence in other tissues may be related to the presence of macrophages or macrophage-like cells in these tissues. The conclusion that "thymosin" beta 4 does not originate solely in the thymus gland is supported by the high concentrations found in tissues of athymic (nu/nu) mice.

Catecholamines in the vasculature of the rat and rabbit: dopamine, norepinephrine, epinephrine.

A highly sensitive radioenzymatic assay for the measurement of catecholamines in small blood vessels was applied to the measurement of the levels of norepinephrine (NE), dopamine (DA) and epinephrine (E). The results showed the presence of NE, E and DA in all segments of rat or rabbit vascular tissue analyzed. The predominant catecholamine in the vasculature from both species was NE, and the contents of E and DA were similar for most vessels. Unexpectedly large concentrations of E were associated with rabbit blood vessels.