Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Synapsins”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed1999

A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes.

Cultured astrocytes can release a variety of messenger substances via receptor-mediated mechanisms, implicating their potential for regulated exocytosis and the participation of proteins of the SNARE complex. Here we demonstrate the astrocytic expression and organellar association of a large variety of synaptic proteins (synaptobrevin II, synaptotagmin I, synaptophysin, rab3a, synapsin I, SNAP-25, and syntaxin I) and also of the ubiquitous cellubrevin. As revealed by immunoblotting the expression of synaptic proteins was highest within the first few days after plating. Synaptophysin and SNAP-25 showed the most significant decline with prolonged culture time. Rab3a and synaptobrevin II were retained at a high level and synaptotagmin I, synapsin I, and syntaxin I at a lower level until 20 DIV. The immunoreaction for cellubrevin was low at the beginning and increased with prolonged culture time. As revealed by light microscopical immunocytochemistry the proteins are expressed by GFAP-positive astrocytes and associated with organelles of varying size. Immunoelectron microscopical analysis allocates synaptobrevin II and synaptophysin to the membranes of vesicular organelles. Double labeling experiments for pairs of synaptic proteins reveal that individual synaptic proteins can be entirely colocalized or partly reside on different organelles. Subcellular fractionation of astrocyte cultures by sucrose density gradient centrifugation after 2, 6, 13, and 20 DIV showed that the proteins sediment with ATP containing organelles of a broad density range. Our data suggest that messenger substances may be released from cultured astrocytes via receptor-mediated, Ca2+-dependent exocytosis.

Adenosine Triphosphate↗
PubMed1993

Synaptophysin--a common constituent of presumptive secretory microvesicles in the mammalian pinealocyte: a study of rat and gerbil pineal glands.

Recent studies have established that pinealocytes of the mammalian pineal gland contain marker molecules of neuroendocrine cells or paraneurons like the synaptic vesicle-associated protein synaptophysin (p38). The objective of this study was to identify the subcellular synaptophysin-positive compartment and to characterize in detail the intracellular distribution of this protein in rat and gerbil pinealocytes. An analysis of serial semithin sections of plastic-embedded pineals immunostained for synaptophysin, including computer-assisted optical density measurements of synaptophysin immunoreactivities, demonstrated unequivocally that synaptophysin was highly concentrated in dilated process terminals of the pinealocytes. More than 75% of these process terminals were found to border or lie within the pericapillary space. At the ultrastructural level, they contained accumulations of small clear vesicles of variable size that turned out to be the site of synaptophysin immunoreactivity when immunogold staining was performed. In addition, microvesicles surrounding synaptic ribbons were also immunolabeled. Hence, the pinealocyte is the first neuroendocrine cell type that has now been shown to concentrate synaptophysin-positive microvesicles in perivascular process endings. This observation lends strong support to the hypothesis that small clear vesicles in neuroendocrine cells in general, and in pinealocytes in particular, serve secretory functions. The quantitative analysis of completely sectioned process endings revealed that the microvesicles outnumber by far the amount of dense core vesicles and therefore cannot arise by endocytosis of dense core vesicle membranes. Thus, small synaptic-like vesicles probably constitute an independent secretory pathway of the paraneuronal pinealocytes. In the present study, we could also establish the absence of immunoreactivity for synapsin I (belonging to a family of neuron-specific nerve terminal phosphoproteins) from pinealocytes. Synapsin I immunoreactivity was only detectable in intrapineal nerve terminals and varicosities. Taken together, the immunostaining patterns of the pineal gland obtained with antibodies directed against synaptic vesicle-associated proteins render the mammalian pinealocyte a very special type of neuroendocrine cell or paraneuron rather than a "classic" neuron.

Animals↗
PubMed2002

Hydroxyurea-induced partial mushroom body ablation in the honeybee Apis mellifera: volumetric analysis and quantitative protein determination.

Hydroxyurea (HU) treatment of first instar honeybee larvae was previously shown to cause mushroom body (MB) ablations. Predominantly, either one or both median MB subunits were ablated. This prompted us to analyze the effects of asymmetrical or symmetrical HU-induced MB ablation on both the morphology of the brain and on the level of three proteins (synapsin, PKA RII, and PKC), which are considered to play a role in synaptic plasticity, learning, and memory. In brains with one median MB subunit missing the volume of the overall MB calyx neuropil in the lesioned side was diminished by 35%. This strong reduction occurred although the remaining lateral MB calyx of the lesioned brain side was found to be significantly larger than that of the intact side. Accordingly, in brains with both median MB subunits missing the size of the remaining lateral calyces increased. The various types of MB ablation differentially affected the amounts of synapsin, PKA RII, and PKC expressed in the central brain. In animals with bilateral and thus symmetrical MB ablation (both median calyces ablated) the protein amount was found to be similar to that in control animals. However, unilateral MB ablation causes an increase in the amounts of the tested proteins in the intact brain side, while the levels in the ablated side were the same as in control animals. These findings not only show that HU-induced ablation of MB subunits is accompanied by volume changes and by changes in protein expression, but also suggest that these processes are highly regulated between the brain sides. The latter is of general importance in understanding the potential contribution of the MB subunits to learning and memory and their interaction between the brain sides.

Animals↗
PubMed1987

Regulation of the phosphorylation and dephosphorylation of a 96,000 dalton phosphoprotein (P96) in intact synaptosomes.

When intact rat brain synaptosomes are depolarized there is a significant increase in the phosphorylation of many proteins, and a rapid dephosphorylation of a 96,000 dalton protein termed P96. The mechanisms governing dephosphorylation are shown to be distinct from the mechanisms leading to increased phosphorylation of proteins such as synapsin I. Depolarization-dependent P96 dephosphorylation was found to be rapid (preceding the phosphorylation of synapsin I) and fully reversible, and required both depolarization and calcium entry. The phosphorylation of P96 was specifically increased by fluphenazine and by the calcium channel agonist (BAY K 8644) and antagonist (verapamil) by unknown mechanisms. Phosphorylation was also increased in the presence of dibutyryl cAMP indicating some role for cAMP-dependent protein kinase in P96 labeling. Preliminary evidence also raises the possibility of a role for protein kinase C. The characteristics of this unique synaptosomal protein suggest that it may play an important role in nerve terminal function.

Animals↗
PubMed1995

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording.

Low-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosities, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5-7 days in vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeks in vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortex in vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.

Animals↗
PubMed1993

Light- and electron-microscopic study of synaptic connections in the paracervical ganglion of the female rat: special reference to calcitonin gene-related peptide-, galanin- and tachykinin (substance P and neurokinin A)-immunoreactive nerve fibers and terminals.

Nerve fibers and varicosities in the pelvic paracervical ganglia (PG) are immunoreactive for the neuropeptides calcitonin gene-related peptide, galanin, and the tachykinins substance P and neurokinin A. Many of these fibers and varicosities are capsaicin-sensitive, originate in dorsal root ganglia and, thus, are considered to be primary afferent fibers. Numerous immunoreactive varicosities are pericellular to principal neurons in the PG. The present study examines the ultrastructure of calcitonin gene-related peptide-, galanin-, substance P-, and neurokinin A-immunoreactive nerve fibers and varicosities in the ganglia to determine their relationships to principal neurons and their synaptic connectivity. Paracervical ganglia of female rats were processed for light-microscopic immunohistochemistry using antisera against synapsin I, as a nerve terminal marker, and microtubule-associated protein-2 to define soma and dendrites. The rationale for performing this co-immunohistochemical analysis was to reveal the relationship between nerve endings and principal neurons. Synapsin I endings were predominantly axosomatic with fewer being axodendritic. Other ganglia were processed for electron-microscopic immunohistochemistry using both standard immunogold and peroxidase-anti-peroxidase procedures. Unmyelinated fibers and varicosities immunoreactive for calcitonin gene-related peptide, galanin, and the tachykinins were routinely observed in the interstitium between neuron somas. Numerous immunoreactive axon profiles were present in small groups that were ensheathed by Schwann cells. Immunoreactive fibers and varicosities were also observed within the satellite-cell sheath of the neuron soma and often intimately associated with the membrane of the soma, somal protrusions, or with the proximal part of a dendrite. Membrane specializations, indicative of synaptic contacts, between the fibers and the principal neurons were observed. It is suggested that these peptide-immunoreactive sensory fibers and varicosities are involved in regulation of activity in the PG.

Animals↗
PubMed2004

Autoantibody synthesis in primary progressive multiple sclerosis patients treated with interferon beta-1b.

We conducted an open-labeled clinical trial of interferon beta-1b (IFNB) treatment in 20 patients with primary progressive multiple sclerosis (PPMS) and longitudinally monitored autoantibodies against double-stranded DNA (dsDNA), thyroid peroxidase (TPO),myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), synapsin and S-100B. Before treatment, one patient had elevated TPO antibodies, four patients had elevated antibodies against S-100B, two patients against MOG or synapsin and one patient against MBP. In two patients we observed a continuous increase of dsDNA or TPO antibodies above the normal range. This rise paralleled IFNB treatment. In addition, 11 of 20 patients developed neutralizing antibodies against IFNB. There was no increase of autoantibodies directed against central nervous system antigens. Like patients with relapsing remitting or secondary progressive multiple sclerosis, PPMS patients may be at risk of an autoimmune response during IFNB treatment.

Adjuvants, Immunologic↗
PubMed1986

A rapid method for isolation of synaptosomes on Percoll gradients.

A new rapid method for fractionation of crude synaptosomes (postmitochondrial pellet, P2) on a discontinuous 4-step Percoll gradient is described. The homogeneity and integrity of the 5 major subcellular fractions were determined by analysis of the distribution of protein, lactate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, synapsin I (a synaptic vesicle marker) and the myelin basic proteins. The biochemical results were substantiated by quantitative electron microscopy. Fractions 3, 4 and 5 were enriched in synaptosomes and contained 19.7, 40.6 and 19.5% of the intact, identifiable synaptosomes in P2, respectively. Fraction 1 was enriched in membranous material, fraction 2 in myelin and fraction 5 in extrasynaptosomal mitochondria. The synaptosomes in fractions 3, 4 and 5 differed in their size, and their content of mitochondria, synapsin I and neurotransmitters. These results suggest that partial separation of different pools of synaptosomes has been achieved. The synaptosomes in fractions 3, 4 and 5 are viable, as they take up calcium, phosphate and noradrenaline; they are metabolically normal as judged by their ability to perform protein phosphorylation and they respond normally to depolarization by increasing calcium uptake, protein phosphorylation and neurotransmitter release. The synaptosomes in fraction 4 are relatively homogeneous and appear to be free of contamination from lysed synaptosomes and synaptic plasma membranes. This constitutes a major advantage of the Percoll method over traditional procedures which involve centrifugation to equilibrium. We have therefore confirmed (J. Neurochem., 43 (1984) 1114-1123) the advantages of Percoll use over traditional procedures, while further reducing the time taken, and extended our analysis to show that the present procedure provides a fractionation of synaptosomes into different pools of viable synaptosomes.

Animals↗
PubMed1988

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions.

A method for preparation of synaptosomes from rat cerebral cortex, on a discontinuous Percoll gradient, was previously developed for use with a P2 pellet (Brain Research, 372 (1986) 115-129). Here the Percoll method has been adapted for use with an S1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S1-Percoll method was 1.4-fold greater than with the P2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1-3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 micron mean diameter) only 8% of which contained mitochondria, while fractions 2-4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 micron mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1-5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S1-Percoll procedure.

Animals↗
PubMed1988

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions.

The metabolic and functional viability of synaptosomes was examined in 5 subcellular fractions obtained after centrifugation of an S1 fraction from rat cerebral cortex on a discontinuous Percoll gradient (Brain Research, this volume, 1987). Fraction 4 was the most enriched for viable synaptosomes since, although it accounted for only 11.8% of the total protein recovered from the gradient, this fraction contained 23.7% of the basal synapsin I phosphorylation activity, the greatest degree of depolarisation-stimulated increase in synapsin I phosphorylation, 36.1% of the total [3H]noradrenaline uptake capacity and 46.9% of the total [3H]noradrenaline release capacity. Noradrenaline release from fraction 4 was consistent with a neuronal mechanism as it was increased with increasing K+ concentrations and was dependent on calcium. Fractions 1 and 2 contained few viable synaptosomes as judged by their capacity for noradrenaline uptake and release, yet these fractions accounted for some 62.6% of the endogenous content of noradrenaline. In part their lack of viability was due to a low content of intrasynaptosomal mitochondria, while their high content of endogenous noradrenaline was due to the presence of synaptic vesicles released from damaged nerve terminals. The synaptosomes in fraction 3 were metabolically and functionally viable, but their capacity for uptake and release of noradrenaline was lower than for fraction 4. The synaptosomes in fraction 5 showed only a small depolarisation-stimulated release of noradrenaline, suggesting a lack of viability. Part of the capacity for uptake of [3H]noradrenaline into fraction 5 was attributed to the presence of extrasynaptosomal mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity.

Activation of microglia is a well-documented phenomenon associated with diverse pathological conditions of the central nervous system. In order to investigate the involvement of microglial cells in the neurotoxic action of the heavy metal compound trimethyltin, three-dimensional brain cell cultures were treated during an early developmental period, using concentrations at or below the limit of cytotoxicity. Microglial cells were studied by cytochemical staining, using horseradish peroxidase-conjugated B4 isolectin (GSI-B4). In parallel, neurotoxic effects were assessed by determining the content of synaptophysin and synapsin I, both in the total homogenates and in the synaptosomal fraction of the cultures. Changes in the content of the specific growth cone protein, GAP-43, were also analyzed. It was found that low, non-cytotoxic concentrations of TMT (10(-9) to 10(-8) M) caused a significant increase in the number and/or the clustering of microglial cells. A decrease in the synaptic protein (synapsin I, synaptophysin) content was detected at 10(-8) M of TMT in synaptosomal fractions, whereas in the total homogenates, changes in synaptic proteins and GAP-43 were observed only at the cytotoxic TMT concentration (10(-6) M). Although it remains to be shown whether the microglial response is caused by direct or indirect action of TMT, the present findings show that microglial responsiveness can be detected prior to any sign of neuronal degeneration, and may serve as a sensitive indicator for heavy metal neurotoxicity in the brain.

Animals↗
PubMed1984

Comparison of calmodulin-dependent glycogen synthase kinase from skeletal muscle and calmodulin-dependent protein kinase-II from brain.

Calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle and calmodulin-dependent protein kinase-II from rat brain were found to have remarkably similar substrate specificities. Both protein kinases phosphorylated synapsin -I, glycogen synthase, smooth muscle myosin light chains, histone H1 and acetyl-CoA carboxylase at the same relative rates. Site-2 of glycogen synthase was preferentially phosphorylated by both enzymes, followed by a slower phosphorylation of site-1b. Each protein kinase catalysed a 2-fold activation of tryptophan 5-monooxygenase. Calmodulin-dependent protein kinase-II and glycogen synthase kinase exhibited similar immunological cross-reactivity in the presence of Ca2+ and calmodulin, using monoclonal antibody raised against the rat brain enzyme. In the absence of Ca2+ and calmodulin, cross-reactivity of glycogen synthase kinase was decreased, whereas that of calmodulin-dependent protein kinase-II was not. The two enzymes appear to represent different isoenzymes of a multifunctional calmodulin-dependent protein kinase that may mediate many of the actions of Ca2+ in mammalian tissues. The results demonstrate that calmodulin-dependent protein kinase-II is identical to calmodulin-dependent synapsin -I kinase-II, previously shown to be very similar to calmodulin-dependent glycogen synthase kinase [(1983) FEBS Lett. 163, 329-334].

Animals↗
PubMed1990

Inhibition of phosphorylation of rat synaptosomal proteins by snake venom phospholipase A2 neurotoxins (beta-bungarotoxin, notexin) and enzymes (Naja naja atra, Naja nigricollis).

Some snake venom neurotoxins, such as beta-bungarotoxin (beta-BuTX) and notexin, which inhibit the release of neurotransmitter at both peripheral and central presynaptic terminals possess phospholipase A2 activity. In contrast, most snake venom phospholipase A2 enzymes such as those isolated from Naja naja atra and Naja nigricollis are structurally homologous to these neutrotoxins but do not have any specific or potent presynaptic action although they have higher enzymatic activities than the neurotoxins. In order to investigate the mechanisms of presynaptic action of the snake venom neurotoxins, we studied their effects on phosphorylation of rat brain synaptosomal proteins. It is known that phosphorylation of synapsin I, a neuron specific and synaptic vesicle associated phosphoprotein, increases neurotransmitter release. Incubation of cerebral cortical synaptosomes with 32P-orthophosphate at 37 degrees C for 30 min, caused significant phosphorylation of a wide mol. wt range of proteins including most markedly those proteins in the mol. wt range (81,000-86,000) of synapsin I. Both snake venom phospholipase A2 neurotoxins and enzymes (5, 15 and 50 nM) inhibited phosphorylation in a Ca2(+)-dependent manner with the following order of potencies: beta-BuTX greater than N.n. atra phospholipase A2 greater than or equal to notexin greater than N. nigricollis phospholipase A2. Five nanomoles of beta-BuTX, which has the lowest phospholipase A2 activity, inhibited phosphorylation of a wide range of mol. wt proteins (51,000-188,000) by 42-58%. At the same concentration, N.n. atra phospholipase A2 (which possesses the highest enzymatic activity), notexin and N. nigricollis phospholipase A2 caused less inhibition than beta-BuTX, ranging from 0-40% depending on the agent used. These results indicate that there is no correlation between their potencies in inhibiting phosphorylation and the levels of their phospholipase A2 activities. An inhibitory activity on phosphorylation may be at least partially responsible for a presynaptically-induced block of neurotransmitter release.

Animals↗
PubMedDate not supplied

The central nervous system of Bulla gouldiana: peptide localization.

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.

Animals↗
PubMed1996

CaM kinase II in long-term potentiation.

The observation that autophosphorylation converts CaM kinase II from the Ca(2+)-dependent form to the Ca(2+)-independent form has led to speculation that the formation of the Ca(2+)-independent form of the enzyme could encode frequency of synaptic usage and serve as a molecular explanation of "memory". In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of CaM kinase II through autophosphorylation, and this response was blocked by an NMDA receptor antagonist, D-2-amino-5-phosphonopentanoate (AP5). In addition, we confirmed that high, but not low frequency stimulation, applied to two groups of CA1 afferents in the rat hippocampus, resulted in LTP induction with concomitant long-lasting increases in Ca(2+)-independent and total activities of CaM kinase II. In experiments with 32P-labeled hippocampal slices, the LTP induction in the CA1 region was associated with increases in autophosphorylation of both alpha and beta subunits of CaM kinase II 1 h after LTP induction. Significant increases in phosphorylation of endogenous CaM kinase II substrates, synapsin I and microtubule-associated protein 2 (MAP2), which are originally located in presynaptic and postsynaptic regions, respectively, were also observed in the same slice. All these changes were prevented when high frequency stimulation was applied in the presence of AP5 or a calmodulin antagonist, calmidazolium. Furthermore, in vitro phosphorylation of the AMPA receptor by CaM kinase II was reported in the postsynaptic density and infusion of the constitutively active CaM kinase II into the hippocampal neurons enhanced kainate-induced response. These results support the idea that CaM kinase II contributes to the induction of hippocampal LTP in both postsynaptic and presynaptic regions through phosphorylation of target proteins such as the AMPA receptor, MAP2 and synapsin I.

2-Amino-5-phosphonovalerate↗
PubMed2005

Brain-derived neurotrophic factor-induced potentiation of glutamate and GABA release: different dependency on signaling pathways and neuronal activity.

The mechanisms underlying BDNF-modulated neurotransmitter release remain elusive. Here, we found that 24-h exposure of postnatal cortical neurons to BDNF potentiated depolarization-evoked glutamate and GABA release in a protein synthesis-dependent manner. BDNF-potentiated glutamate release occurred through the PLC-gamma and MAPK pathways. The expression of synapsin I, synaptotagmin, and synaptophysin, but not of syntaxin or SNAP25, increased through the PLC-gamma and MAPK pathways. In contrast, BDNF-up-regulated GABA release and GAD65/67 expression depended on MAPK. Furthermore, neuronal activity was necessary for the up-regulation of glutamate release and synapsin I, synaptotagmin, and synaptophysin expression, but not of GABA or GAD65/67. PLC-gamma inhibitor attenuated BDNF-stimulated long-lasting MAPK activation. As BDNF rapidly potentiates glutamatergic transmission through PLC-gamma (J. Biol. Chem. 277, (2002) 6520-6529), PLC-gamma-mediated neuronal activity might sustain MAPK activation, resulting in BDNF-potentiated glutamate release. In conclusion, BDNF potentiates the excitatory and inhibitory system separately, which may be important for the regulation of synaptic plasticity.

Animals↗
PubMed2006

Activity-related redistribution of presynaptic proteins at the active zone.

Immunogold labeling distributions of seven presynaptic proteins were quantitatively analyzed under control conditions and after high K+ depolarization in excitatory synapses from dissociated rat hippocampal cultures. Three parallel zones in presynaptic terminals were sampled: zones I and II, each about one synaptic vesicle wide extending from the active zone; and zone III, containing a distal pool of vesicles up to 200 nm from the presynaptic membrane. The distributions of SV2 and synaptophysin, two synaptic vesicle integral membrane proteins, generally followed the distribution of synaptic vesicles, which were typically evenly distributed under control conditions and had a notable depletion in zone III after stimulation. Labels of synapsin I and synuclein, two synaptic vesicle-associated proteins, were similar to each other; both were particularly sparse in zone I under control conditions but showed a prominent enrichment toward the active zone, after stimulation. Labels of Bassoon, Piccolo and RIM 1, three active zone proteins, had very different distribution profiles from one another under control conditions. Bassoon was enriched in zone II, Piccolo and RIM 1 in zone I. After stimulation, Bassoon and Piccolo remained relatively unchanged, but RIM 1 redistributed with a significant decrease in zone I, and increases in zones II and III. These results demonstrate that Bassoon and Piccolo are stable components of the active zone while RIM 1, synapsin I and synuclein undergo dynamic redistribution with synaptic activity.

Animals↗
PubMed2006

GAL4-NF-kappaB fusion protein augments transgene expression from neuronal promoters in the rat brain.

Targeted gene expression mediated by a mammalian cellular promoter is desirable for gene therapy in the brain, where there are a variety of different neuronal phenotypes, several types of supportive cells, and blood vessels. However, this approach can be hampered by weak activity of some cellular promoters. In view of the potency of the transcription factor NF-kappaB in regulating neuronal gene expression, we have assessed whether it can be used to enhance the strength of neuron-specific promoters. Our approach was to use a neuronal promoter to drive expression of a chimeric transactivator, which consisted of a part of the transcriptional activation domain of the NF-kappaB p65 protein fused to the DNA-binding domain of GAL4 protein from yeast. The second copy of the neuronal promoter was modified by introducing the unique GAL4 binding sequences at its 5' end and used to drive the expression of a transgene. Binding of the chimeric transcriptional activator upstream of the second promoter was expected to potentiate its transcriptional activity. In this study, the approach was applied to the platelet-derived growth factor beta chain and synapsin-1 neuron-specific promoters and tested in vitro and in vivo using plasmid, lentiviral, and baculoviral vectors. We observed up to a 100-fold improvement in reporter gene expression in cultured neurons and 20-fold improvement in the rat brain in vivo. Moreover, the cell-type specificity of the two tested promoters was well preserved and restricted to neurons. Finally, the expression driven by the new lentiviral vectors with the p65-potentiated synapsin-1 promoter showed no signs of decline or cell damage 4 weeks after injection. This approach should be suitable for constructing powerful and stable gene expression systems based on weak cell-specific promoters in neuronal phenotypes.

Animals↗