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PubMed1999

A monoclonal antibody, 3A10, recognizes a specific amino acid sequence present on a series of developmentally expressed brain proteins.

Immunoblotting showed that a monoclonal antibody, 3A10, binds to a series of rat brain-specific antigens with molecular masses of 150-, 120-, 118-, 106-, 104-, 79-, and 77-kDa. The expression of 3A10 antigens is dependent on the developmental stage of the brain; only the 106-kDa antigen is detected during embryonic stages of rat brain development, while the expression of the remaining 6 antigens starts after birth and reaches a maximum during postnatal days 15-21. Detection of the 3A10 antigens in cultured neuronal and glial cells derived from cerebral cortices of rat brain at embryonic day 18 showed that the 77-, 79-, 106-, and 150-kDa antigens are specifically expressed in neuronal cells. The 77-kDa antigen was purified and identified as synapsin I by amino acid sequence analyses of the peptide fragments isolated after Achromobacter protease I treatment. During the isolation of 3A10-reactive proteins by immunological screening of cDNA libraries constructed from adult rat brain, we found that all of the 3A10-reactive clones contain nucleotide sequences encoding the unique amino acid sequence TRSP(S, R,G)P. Analyses of 3A10-binding to various synthetic peptides showed that the monoclonal antibody recognizes a specific conformational structure formed by either the TRSPXP sequence or similar amino acid sequences that are expressed on a series of developmentally expressed brain proteins.

Amino Acid Sequence↗
PubMed2006

Inflammation-like glial response in lead-exposed immature rat brain.

Numerous studies on lead (Pb) neurotoxicity have indicated this metal to be a dangerous toxin, particularly during developmental stages of higher organisms. Astrocytes are responsible for sequestration of this metal in brain tissue. Activation of astroglia may often lead to loss of the buffering function and contribute to pathological processes. This phenomenon is accompanied by death of neuronal cells and may be connected with inflammatory events arising from the production of a wide range of cytokines and chemokines. The effects of prolonged exposure to Pb upon glial activation are examined in immature rats to investigate this potential proinflammatory effect. When analyzed at the protein level, glial activation is observed after Pb exposure, as reflected by the increased level of glial fibrillary acidic protein and S-100beta proteins in all parts of the brain examined. These changes are associated with elevation of proinflammatory cytokines. Production of interleukin (IL)-1beta and tumor necrosis factor-alpha is observed in hippocampus, and production of IL-6 is seen in forebrain. The expression of fractalkine is observed in both hippocampus and forebrain but inconsiderably in the cerebellum. In parallel with cytokine expression, signs of synaptic damage in hippocampus are seen after Pb exposure, as indicated by decreased levels of the axonal markers synapsin I and synaptophysin. Obtained results indicate chronic glial activation with coexisting inflammatory and neurodegenerative features as a new mechanism of Pb neurotoxicity in immature rat brain.

Animals↗
PubMed1989

Role of protein phosphorylation in neuronal signal transduction.

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes in the nervous system. Studies in which purified protein kinases or kinase inhibitors have been microinjected into defined cells while a specific response is monitored have demonstrated that protein phosphorylation is both necessary and sufficient to mediate responses of excitable cells to extracellular signals. The precise molecular mechanisms involved in neuronal signal transduction processes can be further elucidated by identification and characterization of the substrate proteins for the various protein kinases. The roles of three such substrate proteins in signal transduction are described in this article: 1) synapsin I, whose phosphorylation increases neurotransmitter release and thereby modulates synaptic transmission presynaptically; 2) the nicotinic acetylcholine receptor, whose phosphorylation increases its rate of desensitization and thereby modulates synaptic transmission postsynaptically; and 3) DARPP-32, whose phosphorylation converts it to a protein phosphatase inhibitor and which thereby may mediate interactions between dopamine and other neurotransmitter systems. The characterization of the large number of additional phosphoproteins that have been found in the nervous system should elucidate many additional molecular mechanisms involved in signal transduction in neurons.

Animals↗
PubMed1992

Tri-iodothyronine regulates survival and differentiation of rat cerebellar granule neurons.

The effects of tri-iodothyronine (T3), which are known to affect cerebellar development, were tested on neuronal survival and differentiation of cultured cerebellar granule neurons. T3 in physiological concentrations increased both granule neuron survival after three days in culture and synaptic vesicle protein formation, as shown by immunostaining with antibodies against synaptophysin. Likewise, T3 increased the mRNA level for synapsin(I), but not that for GAP43 in granule neurons. Antibodies against microtubule associated protein Tau, which is expressed in developing neurites, showed that T3 also enhanced neurite formation.

Animals↗
PubMed1993

Codistribution of choleratoxin binding sites and tyrosine hydroxylase/FGF-2 immunoreactive nigral nerve cells.

By means of light and confocal laser microscopical analysis of choleratoxin (CT) binding sites indicating the localization of the ganglioside GM1, evidence has been obtained for the presence of ganglioside GM1 in discrete nerve terminals, some of them identified by synapsin-1 immunoreactivity (IR), with a focal distribution in the nerve terminal membrane. Double immunolabelling studies demonstrate that GM1 positive nerve terminals are associated with tyrosine hydroxylase/fibroblast growth factor-2 (TH/FGF-2) immunoreactive dopamine (DA) perikarya in the zona compacta of the rat substantia nigra. It is suggested that GM1 may be released from these terminals to become incorporated into the nerve cell membrane of the FGF-2-containing DA nigral nerve cells, where they may enhance the activity of neurotrophic factor receptors such as those for FGF-2.

Animals↗
PubMed1994

Short-term modulation of glutamatergic synapses in adult rat hippocampus by NGF.

Nerve growth factor promotes survival and differentiation of selected groups of neurones through long-term adaptive changes in cell function. While production of this neurotrophin in target cells in hippocampus is regulated in part by afferent glutamate neuronal input, we report now for the first time that nerve growth factor has a direct potentiating effect on spontaneous and depolarization-evoked release of glutamate from hippocampal nerve endings, accompanied by an increase in the excitatory postsynaptic potential of CA1 neurones. This correlated with increased incorporation of [32P]phosphate into synapsin I. Reciprocal positive feedback between production of trophic factors in target cells by neurotransmitter released from afferent neuronal inputs and the modulation of presynaptic neurotransmitter release by target-derived trophic factors provides a novel mechanism for short-term control over synaptic communication.

Animals↗
PubMed1999

Synergistic effects of laminin and thyroid hormones on neuron polarity in culture.

This study is aimed to elucidate whether triiodothyronine (T3) and laminin have additive effects in regulating neural differentiation. We focused our attention on the expression of synapsin I, the 68 kDa component of the neurofilament triplet (NF-68), growth associated protein (GAP)-43 and microtubule associated protein (MAP)-2 as markers of synapses, cytoskeleton, axons and the somatodendritic domain, respectively. The addition of T3 to the medium of differentiating rat cortical neurons cultured on laminin did not have any effect on the concentration of these proteins, but was critical for their subcellular localization, suggesting a synergistic role of thyroid hormones and laminin in the establishment of neural polarity.

Animals↗
PubMed2000

Targeting of tetracycline-regulatable transgene expression specifically to neuronal and glial cell populations using adenoviral vectors.

Targeting regulatable transgene expression specifically to neuronal or glial cell populations would facilitate studies of CNS gene function. We have developed the tetracycline (Tet) regulatable adenoviral system by expressing the Tet-off transactivator (tTA) under the control of the neuronal-specific synapsin I promoter and the well characterized glial-specific glial fibrillary acidic protein (GFAP) promoter. Transfection of primary hippocampal cultures demonstrated that the respective promoters restricted reporter transgene expression exclusively to neuronal or glial populations. Delivery of the vectors into adult rat hippocampus resulted in a similar pattern of cell specific transgene expression. These novel vectors provide a highly effective means of directing regulated, cell-specific, transgene expression and as such are important tools for investigations of neuronal and glial cell function and advancing gene therapy studies.

Adenoviridae↗
PubMed2001

Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

Alleles↗
PubMed1996

Role of zinc-finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human synaptobrevin II gene.

Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1. Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background. Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation.

Base Composition↗
PubMed1997

Protein phosphorylation events in exocytosis and endocytosis.

1. Exocytosis and endocytosis are the continuous outward and inward vesicular transports in a cell that occur constantly for intra- and inter-cellular communications. These events are accomplished with the release and uptake of chemical messages fundamental in a variety of cellular functions, such as neurotransmitter release, hormone secretion and receptor internalization. 2. Although the mechanisms underlying these events have not been fully established, it is widely accepted that they are largely mediated and controlled by a number of effector proteins. These proteins can operate individually and in concert to produce specialized machineries in the sequential steps of exocytotic and endocytic transports. 3. Protein phosphorylation, the most common covalent modification of proteins in cells, has been implicated as playing an important role in the regulation of exocytosis and endocytosis. Many proteins involved in these processes have been identified to be phosphorylated under certain conditions. 4. For instance, synapsin I, myristoylated alanine-rich C kinase substrate and dynamin I undergo dynamic phosphorylation and dephosphorylation cycles during exocytosis and endocytosis, implying that protein phosphorylation regulates the functions of these proteins and, thus, exocytosis and endocytosis.

Animals↗
PubMed2004

Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol.

In order to study estrogen effects on developing human neurons, we have established primary cultures of neurons and glia from 8-13-week human embryo cortex and spinal cord. The neuronal identity of the cultures was verified using the neuronal synaptic vesicle and neuronal endosomal membrane markers synaptotagmin, synapsin and synaptophysin, and the glial contribution to the mixed glial-neuronal cultures was verified using the glial marker glial fibrillary acidic protein (GFAP). We here report expression of estrogen receptor beta (ERbeta) in these cells using RT-PCR and sequencing, RNAse protection assay, immunohistochemistry and immunoblotting. We found that both neuronal and mixed glial-neuronal cultures expressed ERbeta. Treatment with 17beta-estradiol gave an increased expression of ERbeta in both types of cultures. These results suggest that ERbeta is expressed in fetal brain and thus may mediate effects of estrogen in the developing nervous system. Furthermore, the results suggest that expression of ERbeta in fetal brain may be regulated by estrogen.

Calcium-Binding Proteins↗
PubMed2005

Expression of the repressor element-1 silencing transcription factor (REST) is influenced by insulin-like growth factor-I in differentiating human neuroblastoma cells.

The repressor element-1 (RE-1) silencing transcription factor (REST) interacts with an RE-1 cis element and represses the transcription of neuron-specific genes in neuronal progenitors but is down-regulated in post-mitotic neurons. We report that REST expression is modified, in a time-dependent manner, in SH-SY5Y neuroblastoma cells exposed to insulin-like growth factor I (IGF-I), a polypeptide hormone affecting various aspects of neuronal induction and maturation. REST is increased in cells treated with IGF-I for 2 days and then declines in 5-day-treated cells concomitant with a progressive neurite extension. To investigate any role played by REST in neurodifferentiation by IGF-I, we employed an antisense oligonucleotide (AS-ODN) complementary to REST mRNA. In AS-ODN-treated cells, the effects elicited by IGF-I on cell proliferation are not influenced whereas a marked decrease of REST significantly increases neurite elongation without any gross perturbation of neurogenesis. Synapsin I and betaIII-tubulin gene promoters contain an RE-1 motif and their transcription is repressed by REST; both of them are increased in cells exposed to IGF-I for 5 days and further elevated by AS-ODN treatment. A parallel increase of growth cone-associated protein 43, a protein chosen as a neuronal marker not directly regulated by REST, is also observed. Therefore, REST is elevated during early steps of neural induction by IGF-I and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes. These results suggest a model in which differentiating neuroblastoma cells determine their extent of neurite outgrowth on the basis of REST disappearance.

Antibodies↗
PubMed2006

Ca1.2 and CaV1.3 neuronal L-type calcium channels: differential targeting and signaling to pCREB.

Neurons express multiple types of voltage-gated calcium (Ca2+) channels. Two subtypes of neuronal L-type Ca2+ channels are encoded by CaV1.2 and CaV1.3 pore-forming subunits. To compare targeting of CaV1.2 and CaV1.3 L-type Ca2+ channels, we transfected rat hippocampal neuronal cultures with surface-epitope-tagged sHA-CaV1.2 or sHA-CaV1.3a constructs and found that: (i) both sHA-CaV1.2 and sHA-CaV1.3a form clusters on the neuronal plasma membrane surface; (ii) when compared with sHA-CaV1.2 surface clusters, the sHA-CaV1.3a surface clusters were 10% larger and 25% brighter, but 35% less abundant; (iii) 81% of sHA-CaV1.2 surface clusters, but only 48% of sHA-CaV1.3a surface clusters, co-localized with synapsin clusters; (iv) co-expression with GFP-Shank-1B had no significant effect on sHA-CaV1.2 surface clusters, but promoted formation and synaptic localization of sHA-CaV1.3a surface clusters. In experiments with dihydropyridine-resistant CaV1.2 and CaV1.3a mutants we demonstrated that CaV1.3a L-type Ca2+ channels preferentially mediate nuclear pCREB signaling in hippocampal neurons at low, but not at high, levels of stimulation. In experiments with primary neuronal cultures from CaV1.3 knockout mice we discovered that CaV1.3 channels play a more important role in pCREB signaling in striatal medium spiny neurons than in hippocampal neurons. Our results provide novel insights into the function of CaV1.2 and CaV1.3 L-type Ca2+ channels in the brain.

Animals↗
PubMed1985

Blood-brain barrier protein phosphorylation and dephosphorylation.

Capillaries in vertebrate brain have unique permeability properties that make up the blood-brain barrier (BBB). Although it is known that capillaries are innervated by nerve endings of intracerebral origin and that brain capillary function is likely acutely regulated by neuronal inputs, the possible mechanisms of neuronal regulation of capillary function are at present unknown. One possible mode of regulation is via the phosphorylation of brain capillary proteins. The present studies characterize, for the first time, the major phosphoproteins in the bovine brain capillary using both intact bovine brain capillaries and plasma membrane fractions from bovine brain capillaries. The patterns of endogenous phosphorylation of capillary proteins are compared to similar patterns obtained with synaptosomal (P2) fractions from bovine brain. The major findings of this study are: (a) The activity of protein phosphorylation in brain capillaries is localized almost exclusively to the capillary plasma membrane, and is nearly comparable to the activity of protein phosphorylation in synaptosomal membranes. (b) A major phosphoprotein doublet in the capillary fraction comigrates on a sodium dodecyl sulfate gel with a major phosphoprotein doublet of approximate molecular weight of 80K in the synaptosomal fraction, and the latter is presumed to be synapsin I; in dephosphorylation assays the synaptosomal 80K phosphoprotein doublet is not subject to measurable dephosphorylation, whereas the capillary 80K doublet is subject to rapid dephosphorylation, and is essentially completely dephosphorylated within 5 s at 0 degrees C. (c) A prominent triplet of phosphoproteins with molecular weight of 50-55K is present in the capillary fraction, and is not present in the synaptosomal fraction; thus, this 50-55K triplet of phosphoproteins appears specific for brain capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effect of digestion with phospholipase A2 on endogenous protein phosphorylation in particulate fractions from rat brain synaptosomes.

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.

Animals↗
PubMed1989

Endogenous amino acid release from cultured cerebellar neuronal cells: effect of tetanus toxin on glutamate release.

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.

Amino Acids↗
PubMed1989

Introduction of impermeant molecules into synaptosomes using freeze/thaw permeabilization.

Brief freezing as a means of transiently permeabilizing synaptosomes was explored. Rat brain synaptosomes frozen and thawed in the presence of 5% dimethyl sulfoxide, a cryoprotectant, were shown to release, in a calcium-dependent manner, previously accumulated [3H]norepinephrine and [14C]acetylcholine in response to elevated [K+]. In addition, synaptosomes subjected to freeze/thaw were shown to retain their ability to exhibit resting protein phosphorylation, as well as stimulated protein phosphorylation occurring in response to calcium influx. Brief freezing of synaptosomes in the presence of [gamma-32P]ATP and either the catalytic subunit of cyclic AMP-dependent protein kinase or calcium/calmodulin-dependent protein kinase II rendered the synaptosomal interior accessible to these agents, as reflected by the phosphorylation of substrate proteins, such as synapsin I, which reside within the nerve terminal. Inclusion of inhibitors of these protein kinases during freeze/thaw blocked synaptosomal protein phosphorylation, indicating that the inhibitors were also introduced. After freezing, the synaptosomes resealed rapidly and spontaneously, as shown by the inability of any of the agents to elicit an effect on phosphorylation when added at the end of the freezing period. The permeabilization procedure should contribute to an understanding of the functional roles of phosphoproteins, and of their associated protein kinases and protein phosphatases, in nerve terminals.

Acetylcholine↗