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Biomedical subjects

P N de Graan

Publications and source records attributed to P N de Graan.

At least 19 recordsLinked to original sources
PubMed2001

A grading system for hippocampal sclerosis based on the degree of hippocampal mossy fiber sprouting.

In patients suffering from temporal lobe epilepsy (TLE) a highly variable degree of hippocampal sclerosis (HS) can be observed. For standard neuropathological evaluation after hippocampal resection, neuronal cell loss in the hippocampal subareas is assessed (Wyler score 0-4) [Wyler et al. (1992) J Epilepsy 5: 220-225]. Other marked morphological changes in the sclerotic hippocampus are gliosis and loss of mossy fibers in the hilus and mossy fiber sprouting in the supragranular layer. In this study we quantified changes in mossy fiber density using Timm's stain in resected hippocampal tissue from patients with various degrees of sclerosis. We found that tissue specimens from patients without sclerosis (W0) show almost no mossy fiber sprouting. Patients with moderate sclerosis show sprouting without fiber loss in the hilus, whereas specimens from patients with severe sclerosis show sprouting as well as fiber loss in the hilus. Thus, analysis of mossy fiber abundance in hilus and supragranular layer by the rapid and simple Timm's stain is a sensitive measure for hippocampal sclerosis. It provides a reliable rapid tool for neuropathological evaluation, even if the tissue only contains dentate gyrus due to the sectioning procedure.

Axons↗
PubMed2000

Long term depression in the CA1 field is associated with a transient decrease in pre- and postsynaptic PKC substrate phosphorylation.

Induction of homosynaptic long term depression (LTD) in the CA1 field of the hippocampus is thought to require activation of N-methyl-d-aspartate receptors, an elevation of postsynaptic Ca(2+) levels, and a subsequent increase in phosphatase activity. To investigate the spatial and temporal changes in protein phosphatase activity following LTD induction, we determined the in situ phosphorylation state of a pre- (GAP-43/B-50) and postsynaptic (RC3) protein kinase C substrate during N-methyl-d-aspartate receptor-dependent LTD in the CA1 field of rat hippocampal slices. We show that LTD is associated with a transient (<30 min) and D-AP5-sensitive reduction in GAP-43/B-50 and RC3 phosphorylation and that LTD is prevented by the phosphatase inhibitors okadaic acid and cyclosporin A. Our data provide strong evidence for a transient increase in pre- and postsynaptic phosphatase activity during LTD. Since the in situ phosphorylation of the calmodulin-binding proteins GAP-43/B-50 and RC3 changes during both LTD and long term potentiation, these proteins may form part of the link between the Ca(2+) signal and Ca(2+)/calmodulin-dependent processes implicated in long term potentiation and LTD.

Animals↗
PubMed2000

Glutamate and gamma-aminobutyric acid content and release of synaptosomes from temporal lobe epilepsy patients.

During surgical intervention in medically refractory temporal lobe epilepsy (TLE) patients, diagnosed with either mesial temporal lobe sclerosis (MTS)- or tumor (T)-associated TLE, biopsies were taken from the anterior temporal neocortex and the hippocampal region. Synaptosomes, isolated from these biopsies were used to study intrasynaptosomal Ca(2+) levels ([Ca(2+)](i)), and glutamate and gamma-aminobutyric acid (GABA) contents and release. All synaptosomal preparations demonstrated a basal [Ca(2+)](i) of about 200 nM, except neocortical synaptosomes from MTS-associated TLE patients (420 nM). K(+)-induced depolarization resulted in a robust increase of the basal [Ca(2+)](i) in all preparations. Neocortical synaptosomes from TLE patients contained 22.9 +/- 3.0 nmol glutamate and 4.6 +/- 0.5 nmol GABA per milligram synaptosomal protein, whereas rat cortical synaptosomes contained twice as much glutamate and four times as much GABA. Hippocampal synaptosomes from MTS-associated TLE patients, unlike those from T-associated TLE patients, contained about 70% less glutamate and 55% less GABA than neocortical synaptosomes. Expressed as percentage of total synaptosomal content, synaptosomes from MTS-associated TLE patients exhibited an increased basal and a reduced K(+)-induced glutamate and GABA release compared to rat cortical synaptosomes. In MTS-associated TLE patients, only GABA release from neocortical synaptosomes was partially Ca(2+)-dependent. Control experiments in rat synaptosomes demonstrated that at least part of the reduction in K(+)-induced release can be ascribed to resection-induced hypoxia in biopsies. Thus, synaptosomes from MTS-associated TLE patients exhibit a significant K(+)-induced increase in [Ca(2+)](i), but the consequent release of glutamate and GABA is severely impaired. Our data show that at least part of the differences in glutamate and GABA content and release between human biopsy material and fresh rat tissue is due to the resection time.

Animals↗
PubMed2000

Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals.

Munc18-1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18-1 pool by endogenous, presynaptic PKC-isoforms. In isolated rat brain nerve terminals, munc18-1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+-evoked depolarization also increased munc18-1 phosphorylation, by 50% within 5 s in a Ca2+-dependent manner. Munc18-1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18-1 with its binding partner syntaxin-1A by 50%. Munc18-1 antisera precipitated 80% of native, brain-derived munc18-1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin-1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four-fold lower than the total pool. We conclude that the synaptic munc18-1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin-1A make munc18-1 a prominent candidate to account for PKC-dependent enhancement of secretion.

Animals↗
PubMed2000

Immunohistochemical characterization of mossy fibre sprouting in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy.

Hippocampal sclerosis (HS) is a common derangement in many patients with temporal lobe epilepsy. As a result of neuronal cell loss in the hilar region of the hippocampus, it is proposed that mossy fibres sprout and re-innervate new regions of the dentate gyrus. This sprouting may cause recurrent excitation that may lead to the generation of seizures. Here, we determined neuronal density, and synaptophysin and glial fibrillary acidic protein (GFAP) immunoreactivity in hippocampal specimens from patients with pharmaco-resistant temporal lobe epilepsy. Patients were classified into two groups: those with severe and those with no HS. Non-epileptic autopsy tissue served as controls. Mossy fibre sprouting was investigated in these two groups of epilepsy patients using Timm's staining and an immunohistochemical staining of the presynaptic growth-associated protein B-50 (also known as GAP-43, neuromodulin, F1). B-50 immunoreactivity in the different sub-areas of the hippocampus was quantified by image analysis. Our results show the following: (i) in both groups of temporal lobe epilepsy patients, there was a significant loss in cell number in all major hippocampal sub-areas compared with autopsy control tissue; (ii) in HS patients, when compared with non-HS patients, there was a further decline in the number of principal cells in all hippocampal sub-areas analysed, which was associated with an increase in GFAP immunoreactivity; (iii) the decline in cell density was accompanied by a reduced number of synaptic terminals; (iv) in the HS group, there were sprouted mossy fibres in the supragranular layer (SGL) of the dentate gyrus; (v) there was an increase in synaptophysin immunostaining in the SGL indicating that functionally active nerve terminals were formed; and (vi) B-50 immunoreactivity was also increased in the SGL in the HS group compared with the non-HS and control groups. These data showed that all temporal lobe epilepsy hippocampi investigated had severe neuronal cell loss which was most dramatic in the HS group, where it was accompanied by a severe loss of synapses. In the HS group, mossy fibre sprouting into the SGL was found. The increase in B-50 immunoreactivity in the SGL indicated that there was still active sprouting. This sprouting was accompanied by an increased density of synapses, indicating that mossy fibre terminals are not only anatomically present, but probably also functional. Thus, functional glutamatergic mossy fibre terminals are in the right position to synapse on to the dendrites of granule cells and thus may contribute to the onset of seizures.

Adolescent↗
PubMed1999

Characterization of neocortical and hippocampal synaptosomes from temporal lobe epilepsy patients.

To investigate epilepsy-associated changes in the presynaptic terminal, we isolated and characterized synaptosomes from biopsies resected during surgical treatment of drug-resistant temporal lobe epilepsy (TLE) patients. Our main findings are: (1) The yield of synaptosomal protein from biopsies of epilepsy patients was about 25% of that from rat brain. Synaptosomal preparations were essentially free of glial contaminations. (2) Synaptosomes from TLE patients and naive rat brain, quickly responded to K(+)-depolarization with a 70% increase in intrasynaptosomal Ca(2+) ([Ca(2+)](i)), and a 40% increase in B-50/GAP-43 phosphorylation. (3) Neocortical and hippocampal synaptosomes from TLE patients contained 20-50% of the glutamate and gamma-aminobutyric acid (GABA) contents of rat cortical synaptosomes. (4) Although the absolute amount of glutamate and GABA released under basal conditions from neocortical synaptosomes of TLE patients was lower than from rat synaptosomes, basal release expressed as percentage of total content was higher (16.4% and 17.3%, respectively) than in rat (11.5% and 9. 9%, respectively). (5) Depolarization-induced glutamate and GABA release from neocortical synaptosomes from TLE patients was smaller than from rat synaptosomes (3.9% and 13.0% vs. 21.9% and 25.0%, respectively). (6) Analysis of breakdown of glial fibrillary acid protein (GFAP) indicates that resection time (anoxic period during the operation) is a critical parameter for the quality of the synaptosomes. We conclude that highly pure and viable synaptosomes can be isolated even from highly sclerotic human epileptic tissue. Our data show that in studies on human synaptosomes it is of critical importance to distinguish methodological (i.e., resection time) from pathology-related abnormalities.

Adult↗
PubMed1999

Substrate phosphorylation in the protein kinase Cgamma knockout mouse.

The phosphorylation state of three identified neural-specific protein kinase C substrates (RC3, GAP-43/B-50, and MARCKS) was monitored in hippocampal slices of mice lacking the gamma-subtype of protein kinase C and wild-type controls by quantitative immunoprecipitation following 32Pi labeling. Depolarization with potassium, activation of glutamate receptors with glutamate, or direct stimulation of protein kinase C with a phorbol ester increased RC3 phosphorylation in wild-type animals but failed to affect RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C. Our results suggests the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates the gamma-subtype of protein kinase C, which in turn phosphorylates RC3. The inability to increase RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C by membrane depolarization or glutamate receptor activation may contribute to the spatial learning deficits and impaired hippocampal LTP observed in these mice.

Animals↗
PubMed1995

4-Aminopyridine differentially affects the spontaneous release of radiolabelled transmitters from rat brain slices in vitro.

4-Aminopyridine increased the release of [3H]noradrenaline from dorsal hippocampus slices in vitro in a concentration-dependent manner. When the slices were exposed to 4-aminopyridine for 5 min, the overflow of radioactivity returned to pre-exposure values within 20-25 min. When the exposure of the slices was continued, a sustained enhancement of the release of [3H]noradrenaline was observed for the duration of the exposure. 4-Aminopyridine, 10(-4) M, had an effect of similar magnitude, or an even more pronounced effect, on the release of [3H]catecholamine from cortex, septum, periaqueductal gray and striatum slices. The effects of the compound on the release of [3H]5-hydroxytryptamine and [14C]acetylcholine were less pronounced. At this concentration 4-aminopyridine had no effect on the release of [3H]D-aspartate from hippocampus or septum slices, whereas the effect on the release of this transmitter in striatal slices was marginal. The effect of 4-aminopyridine on the release of [3H]noradrenaline in hippocampus slices was largely dependent on the presence of Ca2+ in the superfusion medium. This was also the case for the effect on the release of [3H]noradrenaline from preloaded dorsal hippocampus synaptosomes. In the presence of nitrendipine the effect of 4-aminopyridine was dose-dependently reduced, but the maximal reduction, at a nitrendipine concentration of 10(-4) M, was only 40%. Cd2+ completely abolished the effect of 4-aminopyridine on the release of [3H]noradrenaline. These results confirm that the enhancing effect of 4-aminopyridine on the release of [3H]noradrenaline depends on the entry of extracellular Ca2+ into the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗
PubMed1995

Long-term potentiation and synaptic protein phosphorylation.

Long-term potentiation (LTP) is a well known experimental model for studying the activity-dependent enhancement of synaptic plasticity, and because of its long duration and its associative properties, it has been proposed as a system to investigate the molecular mechanisms of memory formation. At present, there are several lines of evidence that indicate that pre- and postsynaptic kinases and their specific substrates are involved in molecular mechanisms underlying LTP. Many studies focus on the involvement of protein kinase C (PKC). One way to investigate the role of PKC in long-term potentiation is to determine the degree of phosphorylation of its substrates after in situ phosphorylation in hippocampal slices. Two possible targets are the presynaptic membrane-associated protein B-50 (a.k.a. GAP 43, neuromodulin and F1), which has been implicated in different forms of synaptical plasticity in the brain such as neurite outgrowth, hippocampal LTP and neurotransmitter release, and the postsynaptic protein neurogranin (a.k.a. RC3, BICKS and p17) which function remains to be determined. This review will focus on the protein kinase C activity in pre- and postsynaptic compartment during the early phase of LTP and the possible involvement of its substrates B-50 and neurogranin.

Animals↗
PubMed1993

Effects of ACTH-(1-24) on dopamine and noradrenaline release, B-50 phosphorylation and calmodulin binding to B-50 in vitro.

ACTH-(1-24), 1 microM, enhanced the Ca(2+)-dependent release of [3H]dopamine ([3H]DA) from intact septal synaptosomes by approximately 30%, but had no effect on the release of [3H]noradrenaline ([3H]NA) from intact cortical synaptosomes. Since a strong correlation has been reported between B-50 (phosphorylation) and [3H]NA release from intact or streptolysin-O- (SL-O-) permeated cortical synaptosomes, we investigated whether the effects of ACTH-(1-24) on the release of radiolabelled transmitters are mediated by B-50. We observed that the increment in the release of [3H]DA from SL-O-permeated septal synaptosomes as a result of exposure to a high Ca2+ concentration was much less pronounced than that of the release of [3H]NA from SL-O permeated septal and cortical synaptosomes. ACTH-(1-24) concentration-dependently inhibited [3H]NA release from SL-O-permeated cortical synaptosomes (IC50 value of approximately 10 microM) when ACTH-(1-24) was added 150 s prior to the Ca2+ trigger. Simultaneous addition of ACTH-(1-24), SL-O and Ca(2+)-buffers to cortical synaptosomes did not lead to a change in [3H]NA release at any of the ACTH-(1-24) concentrations tested. ACTH-(1-24) had no effect on B-50 phosphorylation in intact synaptosomes, whereas it concentration-dependently inhibited B-50 phosphorylation in permeated cortical synaptosomes (IC50 value of 100 microM). ACTH-(1-24) inhibited (IC50 value of 10 microM) B-50/calmodulin binding in vitro. We conclude that the effects of high concentrations of ACTH-(1-24) on various biochemical B-50 related parameters are not likely to represent the mechanisms underlying the action of ACTH-(1-24) on neurotransmitter release.

Adenosine Triphosphate↗
PubMed1992

Measurement of relative amounts of phospho- and dephospho-B-50(GAP-43) peptides by fast atom bombardment-mass spectrometry.

The biological role of phosphoproteins depends upon their degree of phosphorylation in vivo. Methods currently available to measure the degree of phosphorylation of a protein involve indirect procedures to detect the 32P-phosphate incorporation. We report here a direct method to measure relative amounts of phospho- and dephospho-forms of peptides based upon a mass spectrometric technique. The intensities of the molecular ions corresponding to the two forms of the peptides are proportional to their relative amounts. This is demonstrated for a peptide fragment of the protein B-50(GAP-43) and for kemptide, respectively substrates for protein kinases C and A, and demonstrates the applicability of fast atom bombardment-mass spectrometry to quantitate peptides bearing post-translational modifications.

Amino Acid Sequence↗
PubMed1987

4-Aminopyridine inhibits synaptosomal plasma membrane protein phosphorylation in vitro: effect of the selective NMDA-antagonist 2-amino-5-phosphonovalerate.

Phosphorylation of synaptosomal plasma membranes from rat hippocampus in the presence of the convulsant drug 4-aminopyridine resulted in the inhibition of the phosphorylation of the nervous tissue specific protein kinase C substrate protein B-50 (48 kDa) and the alpha-subunit of calcium/calmodulin-dependent protein kinase II (50 kDa). Preincubation of SPM with 2-amino-5-phosphonovalerate prevents the inhibition of B-50 phosphorylation by 4-aminopyridine, but had no effect on the inhibition of 50 kDa phosphorylation. 2-Amino-5-phosphonovalerate is known to be a specific N-methyl-D-aspartate antagonist and has anti-epileptic activity in vitro and in vivo. Several other anti-epileptic drugs tested did not influence the 4-aminopyridine-induced inhibition of protein phosphorylation.

2-Amino-5-phosphonovalerate↗
PubMed1987

alpha-MSH-induced changes in protein phosphorylation of Cloudman S91 mouse melanoma cells.

The role of protein phosphorylation in MSH-induced melanogenesis was investigated with an in vivo phosphorylation assay using intact cultured Cloudman S91 mouse melanoma cells preincubated with [32P]orthophosphate. Exposure of the cells to alpha-MSH increased the extent of labelling of two protein bands on SDS gel electrophoresis with estimated molecular weights of 43 and 34 kDa, respectively. The 32P incorporation was concentration-dependent and reached a maximal value at 10(-8) M alpha-MSH for the 43 kDa band (156% of controls) and at 10(-5) M alpha-MSH for the 34 kDa band (250% of controls). The corresponding ED50s were 5 X 10(-10) M (43 kDa) and 3 X 10(-8) M (34 kDa). The 32P incorporation into the 34 kDa band reached a maximum after a 5 min exposure to alpha-MSH whereas 43 kDa phosphorylation was maximal after a 30-60 min incubation with hormone. The effect was completely reversible after removal of the hormone and specific for melanotropic peptides. Dibutyryl cAMP (10(-3) M) and forskolin (10(-4) M) together with isobutylmethylxanthine (10(-4) M) mimicked the effect of alpha-MSH, pointing to an involvement of adenylate cyclase activation in the phosphorylation of both the 34 kDa and the 43 kDa protein. Preliminary observations showed that the 34 kDa protein is membrane-bound whereas the 43 kDa protein is of mitochondrial or melanosomal origin.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Kindling induces a long-lasting change in the activity of a hippocampal membrane calmodulin-dependent protein kinase system.

Septal kindling has been shown to produce a long-lasting decrease in endogenous calcium/calmodulin-dependent phosphorylation of hippocampal synaptic plasma membrane proteins, including two major bands of approximately 50,000 and 60,000 Daltons. These two proteins differ from the B-50 protein and tubulin, as evidenced by differences in migration in SDS-PAGE gels and by lack of cross-immunoreactivity with specific antibodies. Identity of these two proteins with the rho and sigma subunits of purified calmodulin-dependent kinase (CaM Kinase II) is suggested by similar migration in SDS-PAGE and two-dimensional gels, by similar calmodulin binding in two-dimensional gels, and similar 125I-peptide mapping of the 50,000 Dalton protein. These results demonstrate that septal kindling is associated with changes in the activity of a major Ca2+/calmodulin-dependent kinase system in hippocampal synaptic plasma membrane. This long-lasting modulation of kinase activity may provide a molecular insight into some aspects of neuronal plasticity.

Animals↗
PubMed1986

Dioctanoylglycerol and phorbol diesters enhance phosphorylation of phosphoprotein B-50 in native synaptic plasma membranes.

The short chain diacylglycerol, 1,2-dioctanoylglycerol, at concentrations of 100-300 microM stimulated phosphorylation of the nervous system-specific membrane protein B-50 (Mr 48 kDa, IEP 4.5) in isolated synaptic plasma membranes both in the presence and absence of exogenous protein kinase C. Comparable enhancement of histone phosphorylation by purified protein kinase C was achieved with 1 microM neutral lipid. Phorbol dibutyrate was 100 times more potent than the diacylglycerol in stimulating endogenous B-50 kinase in the membranes, whereas 4-alpha-phorbol was without effect. These results further confirm that B-50 is phosphorylated physiologically by a C kinase. Our data are consistent with a negative feedback mechanism in which generation of 1,2-diacylglycerol by enhanced phosphatidylinositol-4,5-bisphosphate hydrolysis could stimulate B-50 phosphorylation, thereby diminishing phosphatidylinositol-4-phosphate kinase activity and decreasing phosphatidylinositol-4,5-bisphosphate biosynthesis.

Animals↗
PubMed1986

Comparison of a 52-kDa phosphoprotein from synaptic plasma membranes related to long-term potentiation and the major coated vesicle phosphoprotein.

In the in vitro hippocampal slice preparation a short tetanus induces long-term potentiation (LTP) and an increase in the post hoc phosphorylation of a 52-kDa protein in synaptosomal plasma membranes (SPM) prepared from these slices. This 52-kDa SPM phosphoprotein closely resembles the predominant phosphoprotein in coated vesicles, pp50, with respect to the insensitivity of its phosphorylation to Ca2+/calmodulin and cyclic AMP. This resemblance prompted us to compare in rat brain the 52-kDa SPM protein with pp50 in isolated coated vesicles. Both proteins appear to be very similar on basis of the following criteria: relative molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, phospho-amino acid content, and isoelectric point. Since coated vesicles are thought to be involved in receptor-mediated endocytosis and membrane recycling, our data suggest that LTP-correlated changes in 52-kDa phosphorylation may reflect increased coated vesicle activity.

Action Potentials↗