The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses.

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F(c) chimeric proteins show that constructs containing Ig1-3 or Ig1 domains, but not Ig2-3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.

Transient translocation of protein kinase Cgamma in hippocampal long-term potentiation depends on activation of metabotropic glutamate receptors.

Protein kinase C has been implicated in long-term regulation of cellular functions including induction and maintenance of hippocampal long-term potentiation. In the present study the time-course of long-term potentiation-induced translocation of Ca(2+)-dependent protein kinase C isoenzymes (PKCalpha/beta and PKCgamma) was investigated. Quantitative immunoblot analysis was used to measure translocation of these isoenzymes between cytosolic, membrane-associated and membrane-inserted fraction at 5, 15 and 60 min after induction of long-term potentiation in the dentate gyrus in vivo. To investigate the involvement of metabotropic glutamate receptors in protein kinase C regulation during long-term potentiation induction, additional animals were treated before tetanization with (R,S)-alpha-methyl-4-carboxyphenylglycine, an antagonist of metabotropic glutamate receptors. Brief tetanic stimulation of the perforant path resulted in a 100-150% increase in the population spike amplitude in response to test stimuli 5, 15 or 60 min after stimulation in both untreated and (R,S)-alpha-methyl-4-carboxyphenylglycine-treated animals. Only those rats showing clear potentiation were selected for further biochemical analysis of the potentiated dentate gyrus. Five minutes after high-frequency stimulation the subcellular distribution of all studied protein kinase C isoenzymes was unchanged compared with controls. PKC-gamma translocated into the cytosol 15 min after tetanization and this redistribution was blocked by (R,S)-alpha-methyl-4-carboxyphenylgly-cine pretreatment. By contrast, PKC alpha/beta levels increased in the cytosolic fraction only 60 min after tetanization, but in a (R,S)-alpha-methyl-4-carboxyphenylglycine-independent manner. In an additional set of experiments it was shown that (R,S)-alpha-methyl-4-carboxyphenylglycine alone applied intraventricularly had no effect on the subcellular distribution of the studied isoenzymes. The data suggest that PKCalpha/beta and PKCgamma are activated during different post-tetanic phases and metabotropic glutamate receptor activation might be essential for tetanus-induced translocation of postsynaptic PKCgamma only.

[Genetic polymorphism at FES locus in Chinese and German populations].

OBJECTIVE: The aim of this study on the genetic polymorphism at FES locus was to know whether there is genetic relationship between Chinese and German populations. METHODS: EDTA-blood specimens were collected from 311 healthy unrelated Han individuals in Jilin, Chengdu and Guangzhou in China and from 123 healthy unrelated individuals in Germany. The DNA samples were extracted using Chelex method and amplified by PCR technique. The PAGE horizontal electrophoresis was used for typing the PCR product.DNA sequences were analyzed by ALF. RESULTS: There were nine alleles at FES locus in the Chinese population. The allele frequencies were FES*6,0-0. 0051; FES *8,0-0.0042; FES*9,0.0083-0.0202; FES*10,0.0202-0.0604; FES*11,0.4066-0.5101; FES*12,0.2424-0.3099;FES*13, 0.1860-0.2198; FES*14,0.0041-0.0165; and FES*15,0-0.0050, respectively. In the German population, a variation in 5 flanking region and nine alleles at FES locus were noted. The allele frequencies were FES*8,0.0120; FES*10a,0.02317; FES*10,0.0407; FES*11a,0.0122; FES*11,0.4146; FES*12a, 0.0041; FES*12, 0.2357; FES*13, 0.0447; and FES*14, 0.0041, respectively. The results of test for Hardy-Weinberg equilibrium showed that the genotype distributions observed in the populations were correspondent with the expected. CONCLUSION: There is a remarkable difference in the distribution of allele frequencies at FES locus between Chinese and German populations.

Age-dependent differences in glutamate-induced phosphorylation systems in rat hippocampal slices.

Glutamate receptor induced changes in the activity of different phosphorylation systems were measured in hippocampal slices from 12- and 56-day-old rats, by determining the endogenous phosphorylation of 2.5% perchloric acid (PCA) soluble proteins. We identified among these proteins an 85, 80 kDa and the tau protein as specific substrates for protein kinase A (PKA), MARCKS, and neurogranin as specific substrates for protein kinase C (PKC), and prostaglandin-D-synthase as substrate for casein kinase II (CKII). In addition, a 35 kDa protein was phosphorylated by calcium/calmodulin dependent kinase II and protein kinase C and a 21 kDa protein was a substrate for all investigated kinases. The basal endogenous phosphorylation of 2.5% PCA soluble proteins changed during development qualitatively and quantitatively. Thus, the phosphorylation degree of nearly all proteins declines during maturation. Activation of mGluR induced an increased phosphorylation of PKA, PKC, and CKII substrates in hippocampal slices from 12-day-old rats, but in slices of 56-day-old rats only PKA and to a lower extent PKC substrates were affected. In contrast, stimulation of NMDA receptors led to an enhancement of CKII and PKA dependent phosphorylation only in slices of young animals, whereas the endogenous phosphorylation of some proteins in adult slices was actually decreased. These data showing developmental changes in the coupling of metabotropic and ionotropic glutamate receptors to different phosphorylation systems are discussed in the light of altered physiological properties of the mature hippocampus.

Glycosylation of proteins during a critical time window is necessary for the maintenance of long-term potentiation in the hippocampal CA1 region.

This paper focuses on the role of glycoproteins in activity-dependent synaptic plasticity. The effect of the different inhibitors of protein glycosylation, Tunicamycin, Brefeldin A and Swainsonine, on long-term potentiation was studied in the CA1 region of rat hippocampal slices. Bath application of the inhibitors 60 min before and during tetanization did not interfere with the induction of long-term potentiation of the field excitatory postsynaptic potential. However, the potentiation in inhibitor-treated slices decreased to baseline levels during 90-180 min. Significant differences in the potentiation in non-treated slices were detectable 80 min (Tunicamycin), 60 min (Brefeldin A) and 75 min (Swainsonine) after tetanization, thus indicating the prevention of long-term potentiation maintenance. The application of Swainsonine 120 and 240 min after tetanization did not influence the potentiated field excitatory postsynaptic potential. These data demonstrate the need for undisturbed glycoprotein processing in a time window around long-term potentiation induction to maintain later phases of long-term potentiation and essential functional implications of protein glycosylation in mechanisms underlying synaptic plasticity.

Identification of fucose alpha(1-2) galactose epitope-containing glycoproteins from rat hippocampus.

Fucosylation of terminal galactose residues of brain glycoproteins in the alpha(1-2) position has been shown to be crucial for neuronal plasticity, including phenomena such as long-term potentiation and long-term memory formation. We raised antibodies against the plasticity-relevant fucalpha(1-2)gal epitope and used them to determine the distribution of the epitope in adult rat hippocampus. To identify proteins bearing fucalpha(1-2)gal glycostructures antibodies against known synaptic fucoglycoproteins were used in combination with the fucalpha(1-2)gal antibodies. The NMDA receptor subunit NR1 and fractions of gp65 and cadherin were found to carry the epitope, while fucosylation of NCAM180 and NCAM140 obviously occurs via different linkages to the glycan chains.

Receptor-mediated activation of protein kinase C in hippocampal long-term potentiation: facts, problems and implications.

During the last decade hippocampal long-term potentiation has become one of the most frequently used models to study cellular mechanisms of learning and memory. Receptor-mediated activation of protein kinase C is thought to be involved in LTP stabilisation. In the present review, 1. the molecular structure and activation mechanisms of PKC isoenzymes, 2. the biochemical evidences for PKC activation after induction of LTP using different stimulation paradigms as well as 3. the involvement of metabotropic glutamate receptors in PKC activation after induction of LTP are critically discussed.

Activation of metabotropic glutamate receptors increases endogenous protein kinase C substrate phosphorylation in adult hippocampal slices.

We previously reported (Staak, S., Behnisch, T. and Angenstein, F., Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C (PKC) isoenzymes alpha and beta, Brain Res., 682 (1995) 55-62) that Ca(2+)-dependent PKC isoenzymes alpha/beta and gamma are not translocated between subcellular compartments after stimulation of glutamate receptor subtypes in hippocampal slices. Extending our previous work in this study in situ phosphorylation of endogenous PKC substrates and the translocation of novel PKC isoenzymes delta and epsilon was analysed to detect PKC activation. Two proteins of approximately 94 kDa and 18 kDa were first characterised to be specific PKC substrates. As control of the technique carbachol was shown to increase in situ phosphorylation of the two substrates without any measurable translocation of PKC protein. Activation of metabotropic glutamate receptors by 50 microM DHPG also increased the situ-phosphorylation by 43.9% (94 kDa) and 32.8% (18 kDa) compared to controls but did not induce a measurable subcellular redistribution of conventional and novel PKC isoenzymes. Stimulation by 50 microM trans-ACPD or 0.1 mM quisqualate enhanced the situ phosphorylation in the same range, whereas 0.1 mM NMDA was ineffective. To our knowledge this is the first report showing a direct link between metabotropic glutamate receptor activation and increased endogenous PKC substrate phosphorylation in adult hippocampal slices. This PKC activation was not detectable by a redistribution of enzyme protein between subcellular compartments. We, therefore, conclude, that the failure to detect PKC translocation in physiological experiments is not an indicator for unchanged enzyme activity.

Fucose and fucosyllactose enhance in-vitro hippocampal long-term potentiation.

Bath application of L-fucose and 2-fucosyllactose (2FI) increases the potentiation of the population spike amplitude (POP-spike) and the field excitatory postsynaptic potential (fEPSP) after tetanization of the Schaffer collaterals of the rat hippocampus. The ineffective isomers D-fucose and 3-fucosyllactose (3-FI) have no such effect. Since not only the maintenance of long-term potentiation LTP is influenced but also its induction is drastically improved, an effect of the sugars via the formation of glycoproteins but also via different actions on induction mechanisms is discussed.

Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C isoenzymes alpha and beta.

Using a monoclonal antibody the translocation of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes alpha/beta was studied in hippocampal slices after stimulation of glutamate receptors or induction of long-term potentiation. In submerged slices preincubated for 60 min in a medium usually used in electrophysiological studies, cytosolic PKC was not detectable and the amount of membrane-associated enzyme was increased. The treatment of these slices with 10(-6) M phorbol-12,13-dibutyrate induced a time-dependent translocation of alpha/beta PKC from the membrane-associated into the membrane-inserted state. The glutamatergic agonists N-methyl-D-aspartate, quisqualate and trans-ACPD did not cause a membrane insertion of alpha/beta PKC as observed for the phorbol ester when applied alone or in combination. Furthermore, 2 min and 15 min after induction of LTP in the Schaffer collateral-CA1 pathway the distribution of alpha/beta PKC between the two membrane fractions remained unchanged. An increase in the total amount of PKC immunoreactivity was measured immediately after tetanization (142.6% of controls). The data suggest that a membrane insertion of alpha/beta PKC is not a prerequisite for the LTP-induced increased phosphorylation of PKC substrates and that the enzyme might be recruited from a previously inactive pool.

Fucose and fucose-containing sugar epitopes enhance hippocampal long-term potentiation in the freely moving rat.

Male Wistar rats were intrahippocampally injected with L-fucose and the sugar epitope 2'-fucosyl-lactose prior to induction of long-term potentiation (LTP). Both substances had only a minimal and short-lasting depressive effect on the monosynaptically evoked field potential recorded in the dorsal blade of the dentate gyrus of freely moving rats upon stimulation of the perforant pathway. However, LTP induced by fractionated tetanization of the perforant pathway, which declined within 24 h in control animals injected with Lactose, remained at the initial level even 48 h after tetanization (difference to the control group significant with P < 0.01). The results support earlier findings which have indicated a participation of fucosylated macromolecules in the maintenance of LTP. Different molecular mechanisms concerning the effect of both substances and the significance of the data in elucidation of the relationship between LTP and memory formation are discussed.

Hippocampal long-term potentiation in vivo induces translocation of protein kinase C gamma.

The possible involvement of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes alpha/beta and gamma in mechanisms of long-term potentiation (LTP) was investigated after tetanic stimulation of the perforant path in vivo. Brief tetanic stimulation of the perforant path resulted in a 150% increase in population spike amplitude recorded from the dentate gyrus synapses in response to test stimuli 5 and 10 min after tetanization. Immunoblot analysis of PKC immunoreactivity in cytosolic and membrane fractions revealed a LTP-induced translocation of gamma PKC but not alpha/beta PKC into the cytosol in dentate gyrus but also in the other ipsilateral hippocampal regions. These data suggest different physiological roles of Ca(2+)-dependent PKC isoenzymes in activity-dependent synaptic plasticity.

The maintenance of hippocampal long-term potentiation is paralleled by a dopamine-dependent increase in glycoprotein fucosylation.

Induction of long-term potentiation (LTP) in hippocampal slices of rats caused an increase in both protein synthesis and glycoprotein fucosylation by 38 and 34%, respectively. The enhanced incorporation of [3H]fucose into glycoproteins observed 1 h after tetanization was abolished in the presence of the dopamine D1 receptor antagonist SCH23390 during stimulation whereas the LTP-induced increase of protein synthesis was not influenced by this drug. The enhanced insertion of [3H]fucose into hippocampal glycoproteins 1 h after tetanization was paralleled by an increase in the activity of the fucose metabolizing enzyme, fucokinase. In contrast no changes in protein and glycoprotein synthesis were detectable 5 h after tetanization of the slices. The results provide evidence that in addition to an enhanced protein synthesis a dopamine (D1) mediated increase in glycoprotein fucosylation is necessary for the maintenance of the late stage of LTP.

Phorbol ester-induced changes in rat hippocampal glycoprotein fucosylation.

Studies on glycoprotein fucosylation were carried out using hippocampal slices from rat brain. These slices were incubated in the presence of the protein kinase C (PKC) activating phorbol ester, 4 beta-phorbol-12,13-dibutyrate (PDBu), or an inactive isoform, 4 alpha-phorbol-12,13-didecanoate (PDD), respectively, for 7 min followed by a 60 min pulse of [3H]fucose. PDBu caused an increase in [3H]fucose incorporation into glycoproteins by 29% as well as an activation of the fucokinase enzyme reaction by 21%. The PDBu-induced stimulation of [3H]fucose insertion into hippocampal glycoproteins was abolished by the PKC inhibitors, staurosporine and H7. The importance of a PKC-regulated glycoprotein fucosylation in mechanisms underlying changes in neuronal plasticity is discussed.

Metabolization of a retention-improving dosage of methylglucamine orotate in rat brain.

The present study was carried out to investigate the time course of metabolization in brain and the influence on RNA synthesis of a retention-improving dosage of methylglucamine orotate after intracerebroventricular application. As determined by the HPLC technique, 73% of acid-soluble radioactivity were recovered in unmetabolized orotic acid 30 min after injection of 1 mumole methylglucamine [6-14C]orotate. Two hours later the amount of radioactivity found in this compound was negligible. Analysis of the sequence of labelling of uridine compounds revealed uridine to be the metabolite exhibiting the highest radioactivity at 30 min, whereas UMP- and UDP-sugars attained their maximum 90 min after injection of the precursor. Incorporation of [3H] guanosine into brain RNA was not altered by intraventricular application of 1 mumole methylglucamine orotate as compared to methylglucamine chloride-treated controls. The results are interpreted in the light of behavioural findings in which the pyrimidine nucleotide precursor at the dosage used improved the retention performance of an acquired behaviour in the rat.

Effects of intraperitoneally applied D-galactosamine on uridine and cytidine plasma content and brain activity of uridine kinase in the rat.

As determined by the HPLC technique, injection of 1.85 mmoles/kg D-galactosamine caused strong alterations in the plasma level of cytidime. Thus, 2 h and 4 h after application of the amino sugar only 54% and 61%, respectively, of the normal values were measured, while an increase to 135% of controls was observed 24 h later. In contrast, intraperitoneal application of D-galactosamine had no influence on the plasma level of uridine. The activity of the enzyme, uridine kinase, in the hippocampal area of the rat brain was increased 2 h and 4 h after D-galactosamine treatment. The results are discussed in the light of behavioural findings in which intraperitoneally applied D-galactosamine at this dosage improves the retention performance of an acquired behaviour in the rat.