Biomedical subjects

M Popoli

Publications and source records attributed to M Popoli.

At least 19 recordsLinked to original sources

PubMed2002

Association between the ionotropic glutamate receptor kainate 3 (GRIK3) ser310ala polymorphism and schizophrenia.

Schizophrenia is a severe psychiatric illness characterised by disturbance of thought, hallucination and delusions.(1) Several studies have suggested that dysfunctions in the glutamatergic transmission are linked to the pathogenesis of schizophrenia, and in particular an excessive activation of glutamate receptors seems to be related to the disruption of neuronal ionic gradients leading to excitotoxicity.(2-7) Numerous findings suggested that the kainate ionotropic glutamate receptors are primarily involved in this mechanism. Recently it has been demonstrated that the GRIK3 gene encoding for the ionotropic glutamate receptor kainate 3 contains a functional polymorphism (T928G) leading to the substitution of a serine with an alanine in position 310 of the protein sequence.(8-11) We performed an association study between the ser310ala GRIK3polymorphism and schizophrenia in a sample of 99 schizophrenic patients and 116 controls. We found a significant difference in the genotype distribution and in particular considering the ala allele as dominant (P = 0.0105, odds ratio (OR) 2.031, 95% confidence interval (CI) 1.177-3.504). This finding suggests a potential role for GRIK3 for susceptibility to schizophrenia.

Genetic Predisposition to Disease↗

PubMed2001

Long-term treatment with S-adenosylmethionine induces changes in presynaptic CaM kinase II and synapsin I.

BACKGROUND: According to current hypotheses, antidepressant drug action is the result of adaptive changes in neuronal signaling mechanisms rather than a primary effect on neurotransmitter transporters, receptors, or metabolic enzymes. Among the signaling mechanisms involved, protein kinases and phosphorylation have been shown to be modified by drug treatment. Presynaptic signaling (calcium/calmodulin-dependent protein kinase II [CaMKII]) and the protein machinery regulating transmitter release have been implicated in the action of these drugs. METHODS: We investigated the effect of S-adenosylmethionine (SAM), a compound with putative antidepressant activity, on presynaptic CaMKII and its synaptic vesicle substrate synapsin I. The activity of CaMKII was assayed in synaptic subcellular fractions prepared from hippocampus (HI), frontal cortex (FCX), striatum (STR), and parieto-temporal cortex. RESULTS: The kinase activity was increased after SAM treatment in the synaptic vesicle fraction of HI (31.7%), FCX (35.9%), and STR (18.4%). The protein level of CaMKII was also increased in synaptic vesicles of HI (40.4%). The synapsin I level was unchanged in synaptic vesicles but markedly increased in synaptic cytosol of HI (75.8%) and FCX (163.0%). No changes for both CaMKII and synapsin I level were found in homogenates, suggesting that synaptic protein changes are not explained by an increase in total level of proteins, but rather by translocation to nerve terminals. CONCLUSIONS: Similar to typical antidepressant drugs, SAM induces changes in CaMKII activity and increases synapsin I level in HI and FCX nerve terminals, suggesting a modulatory action on transmitter release.

Animals↗

PubMed2001

Effect of reboxetine treatment on brain cAMP- and calcium/calmodulin-dependent protein kinases.

Previous studies showed that the type II Ca(2+)/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA) are affected by long-term antidepressant treatment in presynaptic and somatodendritic compartments, respectively. This study describes the long-term effects of the selective noradrenaline reuptake inhibitor reboxetine on PKA and CaMKII, in both the microtubule and subsynaptosomal fractions of rat brain. Unlike other antidepressants, chronic reboxetine induced in the cerebrocortical soluble and microtubule fractions a decrease in the [(32)P]cAMP binding to the type II PKA regulatory subunit. No change in the cAMP-dependent endogenous phosphorylation of the protein substrate, microtubule-associated protein 2 was observed. In the hippocampal subsynaptosomal fractions (synaptic vesicles and synaptosomal membranes) reboxetine induced a robust increase in the activity but not in the expression of CaMKII. An increase in the calcium/calmodulin-dependent phosphorylation of presynaptic substrates was also detected. These findings showed that reboxetine modulates post-receptor signal transduction systems in rat brain.

Adrenergic Uptake Inhibitors↗

PubMed2001

Serine/threonine kinases as molecular targets of antidepressants: implications for pharmacological treatment and pathophysiology of affective disorders.

It is currently a widely accepted opinion that adaptive, plastic changes in the molecular and cellular components of neuronal signaling systems correlate with the effects on mood and cognition observed after long-term treatment with antidepressant drugs. Protein phosphorylation represents a key step for most signaling systems, and it is involved in the regulation of virtually all cellular functions. Two serine/threonine kinases, Ca2+ /calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, have been shown to be activated in the brain following antidepressant treatment. The changes in kinase activity are mirrored by changes in the phosphorylation of selected protein substrates in subcellular compartments (presynaptic terminals and microtubules), which, in turn, may contribute to the modulation of synaptic transmission observed with antidepressants. The molecular consequences of protein kinase activation may account for some of the alterations in neural function induced by antidepressants, and may suggest novel possible strategies of pharmacological intervention.

Animals↗

PubMed2001

Modifications in brain CaM kinase II after long-term treatment with desmethylimipramine.

The present study investigated the effect of long-term (15 mg/kg for 15 days) and acute (15 mg/kg, single administration) treatment with desmethylimipramine, a tricyclic antidepressant drug, on calcium/calmodulin-dependent protein kinase II (CaMKII), a kinase implicated in the mechanism of antidepressant drug action. Similar to selective and non-selective serotonin reuptake inhibitors, long-term, but not acute, treatment with desmethylimipramine markedly increased the activity of CaMKII in the hippocampal synaptic vesicle fraction (+51.9%). The kinase activity was also increased in the same fraction of frontal cortex (+24.2%) and in the striatum (+45.9%), although in this last area the mechanism appeared to be different because the protein level of the kinase was also markedly increased (+43.7%). However, the effect of treatment was not restricted to the presynaptic kinase, because CaMKII activity was also increased in the total cellular cytosol in cortical areas. The autonomous (calcium-independent) activity of CaMKII was assayed for the first time after antidepressant treatment, and found to be increased in synaptic vesicles of all three areas. These results confirmed the involvement of CaMKII in antidepressant drug action and suggested that modulation of transmitter release is a primary component in the action of psychotropic drugs.

Animals↗

PubMed2000

Modification of presynaptic CaM kinase II affinity for ATP in hippocampus after long term blockade of serotonin reuptake.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is markedly enriched at synapses, where it is involved in the control of synaptic transmission, transmitter release and synaptic plasticity. CaMKII has also been found to be involved in the long-term action of antidepressants on post-receptor signaling mechanisms, because monoamine reuptake inhibitors induced an increase in autophosphorylation and activity of the kinase in nerve terminals of hippocampus. To study whether changes in the amount of enzyme or kinetic changes, due to posttranslational modifications, are responsible for kinase activation in nerve terminals, alpha-CaMKII level and kinetic constants of the autophosphorylation reaction as a function of ATP concentration were measured in presynaptic cytosol from hippocampus. Treatment with two serotonin reuptake inhibitors did not change the level of presynaptic kinase or the Vmax of autophosphorylation reaction. Instead the Km of the kinase for ATP was decreased 2.8-fold with fluvoxamine and 3.5-fold with paroxetine, implying an increase in the affinity for ATP. This result represents the first finding of changes in kinetic constants of a major brain enzyme after treatment with antidepressant drugs.

Adenosine Triphosphate↗

PubMed2000

Second messenger-regulated protein kinases in the brain: their functional role and the action of antidepressant drugs.

Depression has been treated pharmacologically for over three decades, but the views regarding the mechanism of action of antidepressant drugs have registered recently a major change. It was increasingly appreciated that adaptive changes in postreceptor signaling pathways, rather than primary action of drugs on monoamine transporters, metabolic enzymes, and receptors, are connected to therapeutic effect. For some of the various signaling pathways affected by antidepressant treatment, it was shown that protein phosphorylation, which represents an obligate step for most pathways, is markedly affected by long-term treatment. Changes were reported to be induced in the function of protein kinase C, cyclic AMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase. For two of these kinases (cyclic AMP- and calcium/calmodulin-dependent), the changes have been studied in isolated neuronal compartments (microtubules and presynaptic terminals). Antidepressant treatment activates the two kinases and increases the endogenous phosphorylation of selected substrates (microtubule-associated protein 2 and synaptotagmin). These modifications may be partly responsible for the changes induced by antidepressants in neurotransmission. The changes in protein phosphorylation induced by long-term antidepressant treatment may contribute to explain the therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.

Animals↗

PubMed2000

Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation.

The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation-dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin-t-SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation-dephosphorylation balance in regulation of the synaptotagmin-t-SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.

Animals↗

PubMed1998

Modifications in brain cAMP- and calcium/calmodulin-dependent protein kinases induced by treatment with S-adenosylmethionine.

Several lines of evidence suggest that the mechanism of action of antidepressant drugs (AD) involves adaptive changes occurring in intraneuronal post-receptor signal transduction cascades. Protein phosphorylation has a key role in signal transduction and was previously found to be a target in the action of AD (5-HT and/or NA reuptake blockers). Several studies showed that cAMP- and type II Ca2+/calmodulin-dependent protein kinases (PKA and CaMKII) are markedly affected by typical AD in two different and complementary cellular districts, respectively microtubules (a somatodendritic compartment) and synaptic vesicles (a presynaptic terminal compartment). In order to investigate whether the effect on protein kinases may be involved in the therapeutic action of drugs it is interesting to compare the effect of atypical AD with that of typical drugs. In this study the effect of the atypical AD S-adenosylmethionine (SAMe) was tested. Repeated (12 days) SAMe treatment induced in cerebrocortical microtubules an increase in the binding of cAMP to the RII PKA regulatory subunit and an increase in the endogenous phosphorylation of microtubule-associated protein 2, an effect resembling that of typical AD. In synaptic terminals the treatment induced an increase in the activity of CaMKII and in the endogenous phosphorylation of vesicular substrates. However, this modification was found in the cerebral cortex rather than in the hippocampus, where typical AD affect CaMKII. In addition the synapsin I level was decreased in the hippocampus and increased in the cerebral cortex, an effect not detected with typical AD.

Animals↗

PubMed1997

Ca2+/phospholipid-binding and syntaxin-binding of native synaptotagmin I.

Synaptotagmin, a synaptic vesicle protein endowed with multiple properties, is the putative calcium sensor in neuroexocytosis. Ca2+/phospholipid binding and syntaxin binding activity of synaptotagmin were previously investigated using recombinant fusion proteins. In phospholipid binding the EC50 for calcium obtained was different when fusion proteins containing one (C2A) or both (C2A+C2B) binding domains were used. It was alternatively proposed that one or both synaptotagmin binding domains are important for calcium-sensing and triggering of transmitter release. In this study the binding activity of native full-length synaptotagmin, immobilized on beads, was investigated. We found the kinetic parameters of Ca2+/phospholipid binding to be compatible with the role of calcium sensor for synaptotagmin (EC50 for calcium = 72 +/- 7 microM), with the two C2 domains supporting separate and complementary calcium sensing properties. The binding of native syntaxin to synaptotagmin was measurable in the absence of calcium, but was markedly stimulated (2.2-fold) in the presence of mM calcium. It may be speculated that the two domains have a synergistic action in fast synchronous transmitter release, whereas C2B domain alone may support slow asynchronous release, working as a high affinity calcium sensor.

Animals↗

PubMed1997

Long-term blockade of serotonin reuptake affects synaptotagmin phosphorylation in the hippocampus.

Synaptic vesicle trafficking and transmitter release from presynaptic terminals are precisely regulated by a complex array of protein/protein interactions. Several of these proteins are substrates of endogenous protein kinases present in presynaptic terminals. The activity of Ca2+/calmodulin-dependent protein kinase II(CaMKII), one of the kinases involved in the modulation of transmitter release, was previously shown to increase in the hippocampus after long-term blockade of 5-hydroxytryptamine (5-HT) reuptake (a treatment known to elicit an increase in 5-HT release in this area). To investigate the changes induced in presynaptic protein phosphorylation by 5-HT reuptake blockade and concomitant CaMKII up-regulation, we analyzed two major CaMKII presynaptic substrates (synapsin I and synaptotagmin). All 5-HT reuptake blockers that we used, which induce an increase in CaMKII activity and autophosphorylation, also caused a large (2-3-fold) increase in the Ca2+/calmodulin-dependent post hoc phosphorylation of synaptotagmin. Conversely, the phosphorylation of synapsin I is much less affected. The change in synaptotagmin phosphorylation, as determined through immunoprecipitation and quantitative immunoblot analysis after fluvoxamine treatment, is due exclusively to increased phosphate incorporation (presumably caused by the increased kinase activity) and not to a change in the level of substrate protein after the treatment. Thus, drugs known to induce an increase in 5-HT release simultaneously induce an increase in the activity of presynaptic CaMKII and in the phosphate incorporation (post hoc) by a major CaMKII substrate in synaptic vesicles (synaptotagmin). This finding establishes a link between the facilitation of transmitter release induced by antidepressant drugs and the phosphorylation of synaptotagmin by CaMKII.

Animals↗

PubMed1996

Inhibitory effect of lithium on cAMP dependent phosphorylation system.

The aim of the present study was to assess the direct effect of lithium on cAMP dependent phosphorylation. The results show that lithium, but not rubidium, at therapeutic and high concentrations significantly decreases the cAMP stimulated MAP2 endogenous phosphorylation in microtubule fraction. An inhibitory effect of lithium has also been found using purified heat stable microtubule proteins phosphorylated by the catalytic subunit of PKA. These data suggest a direct effect of lithium on the cAMP dependent protein kinase.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Effects of fluvoxamine on the protein phosphorylation system associated with rat neuronal microtubules.

We have studied the phosphorylation system associated with the rat cerebrocortical microtubule fraction after short- and long-term administration (15 mg/kg) of fluvoxamine, a selective serotonin reuptake inhibitor with antidepressant activity. Fluvoxamine administered for 5 days significantly enhanced the 32P incorporation stimulated by cAMP into MAP2, while it failed to produce this effect after 12 and 21 days. Moreover, in the same periods of treatment no changes were observed in basal phosphorylation and in the pattern of microtubule proteins. In conclusion, our results suggest that changes in the protein phosphorylation system associated with the microtubule fraction could represent an early neurochemical modification involved in the action of fluvoxamine.

Animals↗

PubMed1995

Presynaptic Ca2+/calmodulin-dependent protein kinase II: autophosphorylation and activity increase in the hippocampus after long-term blockade of serotonin reuptake.

It is known that long-term treatment with antidepressants induces an enhancement of neurotransmission in the pathway projecting from raphe nuclei to the hippocampus. In the case of selective serotonin (5-HT) reuptake inhibitors, this enhancement is due to a desensitization of presynaptic 5-HT autoreceptors and a concomitant increase in 5-HT release in terminal areas. To investigate whether this effect is accompanied by adaptive changes in the molecular machinery regulating transmitter release at serotonergic terminals, autophosphorylation and activity of Ca2+/calmodulin-dependent protein kinase II were measured in subsynaptosomal fractions from hippocampus and total cortex. Long-term treatment with two selective serotonin reuptake inhibitors (paroxetine and fluvoxamine) and with a nonselective reuptake inhibitor (venlafaxine) induces a large increase of kinase autophosphorylation in synaptic vesicles and synaptic cytosol in the hippocampus but not in synaptosomal membranes. No significant change was detected in total cortex. The change is not reproduced by the direct addition of the drugs to the phosphorylation system and is not elicited by acute treatment of the animals. The increase in autophosphorylation is not accounted for by neosynthesis or translocation of the kinase to synaptic terminals. The change is restricted to the kinase located inside the terminals and is not detected in synaptosomal membranes, containing predominantly postsynaptic kinase, suggesting that only presynaptic kinase is affected. In the same fractions, the kinase activity is increased. These results are in agreement with reports suggesting a presynaptic effect for the SSRIs and disclose a new putative site of action for psychotropic drugs.

Amino Acid Sequence↗

PubMed1995

Dopamine mediated responses in 6-hydroxydopamine lesioned rats involve changes of the signal transduction.

A single dose of the D1 agonist SKF 38393 (3 mg/kg) produces contralateral turning in unilaterally 6-hydroxydopamine lesioned rats only after a previous exposure of the animals to a dopamine agonist. This priming phenomenon is here investigated by studying the phosphorylation of DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein functionally linked to D1 receptors in striatum. Dephospho-form of DARPP-32 in striatal tissue was measured by a back-phosphorylation assay. While the levels of DARPP-32 protein, as measured by quantitative immunoblotting, remained unchanged, a significant decrease of dephospho-DARPP-32 was observed in the denervated striatum of primed rats, indicating an increased phosphorylation in vivo of DARPP-32 in response to the D1 agonist. This study shows that an alteration of the dopamine-dependent signal transduction is related to the behavioral response to dopamine agents, suggesting a possible mechanism involved in the effects of these drugs in parkinsonian patients.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1994

Anti-beta 2 subunit antisense oligonucleotides modulate the surface expression of the alpha 1 subunit of N-type omega-CTX sensitive Ca2+ channels in IMR 32 human neuroblastoma cells.

High voltage activated Ca2+ channels are heteropolymeric complexes in which the alpha 1 subunit forms the channel, while the alpha 2-delta and beta subunits are important for the assembly and regulation of the biophysical properties of the channel. We have tested the role of the beta 2 subunit on the expression and electrophysiological properties of the omega-conotoxin GVIA-sensitive Ca2+ channel expressed in the IMR 32 human neuroblastoma cell line. Anti-beta 2 subunit antisense oligonucleotides supplied to the cells in culture induced a time-dependent increase in the number of [125I]-omega-conotoxin binding sites on the cell surface, which was not paralleled by an increase in current amplitude. We suggest that a reduction in the expression of beta 2 stimulates the transport to the plasma membrane of non-functioning Ca2+ channels and, in particular, of the alpha 1 omega-conotoxin binding subunit.

Amino Acid Sequence↗

PubMed1994

Behavioural sensitization in 6-hydroxydopamine lesioned rats involves the dopamine signal transduction: changes in DARPP-32 phosphorylation.

"Priming" is a phenomenon of behavioural sensitization observed in unilaterally 6-hydroxydopamine lesioned rats following exposure to a dopamine agonist. After priming, a single dose of the D1 agonist SKF 38393 (3 mg/kg) produces contralateral turning, while the same dose is inactive in drug-naive, lesioned animals. The molecular mechanisms of "priming" were investigated here by studying the phosphorylation of dopamine and adenosine 3'-5' monophosphate regulated phosphoprotein (DARPP-32), a dopamine- and cyclic AMP-regulated phosphoprotein functionally linked to D1 receptors in striatum. Dephospho-form of DARPP-32 were measured by a back-phosphorylation assay. All assays were performed in striata from both lesioned and unlesioned sides. A significant decrease of dephospho-DARPP-32 (27%) was observed in the denervated striatum of primed rats, indicating an increased phosphorylation in vivo of DARPP-32 in response to the D1 agonist. The levels of DARPP-32 protein, as measured by quantitative immunoblotting, remained unchanged in all experimental groups. This study shows that priming is expressed as an increased transduction of the D1 receptor message.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1993

Synaptotagmin is endogenously phosphorylated by Ca2+/calmodulin protein kinase II in synaptic vesicles.

The cytoplasmic domain of synaptotagmin (a synaptic vesicle-specific protein) has a high degree of homology with the Ca(2+)-phospholipid binding domain of protein kinase C. The Ca(2+)-phospholipid binding activity of synaptotagmin has been implicated in the docking and fusion of synaptic vesicles with the presynaptic membrane during Ca(2+)-induced exocytosis. The protein sequence contains potential phosphorylation sites for various protein kinases which could modulate its binding activity. At present there is no clear evidence that the protein is endogenously phosphorylated in intact vesicles. Here it is reported that phospho-synaptotagmin was immunoprecipitated from endogenously phosphorylated synaptic vesicles. The conditions used indicate that synaptotagmin, as synapsin I, is phosphorylated by Ca2+/calmodulin-dependent protein kinase II.

Animals↗