Sphingosine-1-Phosphate Receptors Modulators Decrease Signs of Neuroinflammation and Prevent Parkinson's Disease Symptoms in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Mouse Model.

Sphingosine-1-phosphate (S1P) is a potent bioactive lipid mediator that acts as a natural ligand upon binding to five different receptors that are located in astrocytes, oligodendrocytes, microglial and neuronal cells. Recently, global activation of these receptors by FTY720 (fingolimod) has been suggested to provide neuroprotection in animal model of Parkinson's disease (PD). Among S1P receptors, the subtype 1 (S1P1R) has been linked to features of neuroprotection and, using the selective agonist SEW2871, the present investigation assessed potential benefits (and mechanisms) of this receptor subtype in an established animal model of PD. We demonstrated that oral treatments with SEW2871 are able to provide protection to the same levels as FTY720 against loss of dopaminergic neurons and motor deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg, i.p., 5 days) mouse model of PD. At the molecular level, we observed that the beneficial effects of both S1PR agonists were not associated with alterations in ERK and Akt levels, two markers of molecular adaptations in the striatum neurons. However, these compounds have the capacity to prevent signs of neuroinflammation such as the activation of astrocytes and glial cells, as well as MPTP-induced reduction of BDNF levels in key regions of the brain implicated in motor functions. These findings suggest that selective S1P1R modulation has the ability to provide neuroprotection in response to MPTP neurotoxicity. Targeting S1P1R in PD therapy may represent a prominent candidate for treatment of this neurodegenerative conditions.

Motor learning deficits and striatal GSK-3 hyperactivity in Akt3 knockout mice.

Akt protein family (Akt1, Akt2 and Akt3) of serine/threonine kinases, also known as protein kinase B, are enzymes implicated in many physiological and pathological processes in the central nervous system. A striking feature of these enzymes is their ability to interact with several molecular targets such as the glycogen synthase kinase 3 (GSK-3). Among Akt isoforms, the Akt3 is significantly more expressed in the brain and the present investigation was designed to determine whether the Akt3/GSK-3 pathway plays a role in the learning of a complex motor skill. Using the accelerating rotarod task, known to reproduce different motor learning phases, we demonstrated in mouse models that genetic deletion of GSK-3α or GSK-3ß had no effect on rotarod performances. However, Akt3 deletion robustly compromised rotarod learning when compared with wild-type animals. Biochemical analysis in the striatum revealed modifications in the levels of both phosphorylated GSK-3 and tau in Akt3-deficient mice, which are reminiscent of enhanced GSK-3 activity. In this line, we observed that both biochemical and motor learning impairments were prevented in Akt3-deficent mice by chronic treatments with lithium, a well-known GSK-3 inhibitor. Altogether, our findings raised the interesting possibility that interconnection between Akt3 and GSK-3 kinases is required in the learning of new complex motor tasks. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Genetic Deletion of Akt3 Induces an Endophenotype Reminiscent of Psychiatric Manifestations in Mice.

The protein kinase B (PKB/Akt), found in three distinctive isoforms (PKBα/Akt1, PKBß/Akt2, PKBγ/Akt3), is implicated in a variety of cellular processes such as cell development, growth and survival. Although Akt3 is the most expressed isoform in the brain, its role in cerebral functions is still unclear. In the present study, we investigated the behavioral, electrophysiological and biochemical consequences of Akt3 deletion in mice. Motor abilities, spatial navigation, recognition memory and LTP are intact in the Akt3 knockout (KO) mice. However, the prepulse inhibition, three-chamber social, forced swim, tail suspension, open field, elevated plus maze and light-dark transition tests revealed an endophenotype reminiscent of psychiatric manifestations such as schizophrenia, anxiety and depression. Biochemical investigations revealed that Akt3 deletion was associated with reduced levels of phosphorylated GSK3α/ß at serine 21/9 in several brain regions, although Akt1 and Akt2 levels were unaffected. Notably, chronic administration of lithium, a mood stabilizer, restored the decreased phosphorylated GSK3α/ß levels and rescued the depressive and anxiety-like behaviors in the Akt3 KO mice. Collectively, our data suggest that Akt3 might be a critical molecule underlying psychiatric-related behaviors in mice.

The sphingosine-1-phosphate receptor 1 agonist SEW2871 reduces Tau-Ser262 phosphorylation in rat hippocampal slices.

Recent studies indicate that Tau phosphorylation can be modulated by the compound FTY720-P, a global sphingosine-1-phosphate receptor (S1PR) agonist. The present work compared the effects of more selective S1PR agonists on Tau properties, using rat hippocampal slices as model system. Whereas Tau phosphorylation was not modified by the S1PR3 agonist CYM5541, Tau-Ser262 phosphorylation was significantly decreased by treatment with the S1PR1 agonist SEW2871. This effect appears to be quite restricted, as no changes in phosphorylation were elicited by the agonist on Tau-Ser199/202, Tau-Ser396 and Tau-Ser404 residues. In terms of molecular mechanisms, it is proposed that SEW2871-induced reduction of Tau-Ser262 phosphorylation depends on AMP-activated protein kinase alpha (AMPKα) inactivation via a pathway requiring AMPKα dephosphorylation at Thr172 by the protein phosphatase 2A (PP2A).

GluN2B-containing NMDA receptors are upregulated in plasma membranes by the sphingosine-1-phosphate analog FTY720P.

Sphingosine-1-phosphate (S1P) is a ceramide derivative serving not only as a regulator of immune properties but also as a modulator of brain functions. To better understand the mechanism underlying the effects of S1P on brain functions, we investigated the potential impact of S1P receptor (S1PR) activation on NMDA receptor subunits. We used acute rat hippocampal slices as a model system, and determined the effects of the active phosphorylated S1P analog, fingolimod (FTY720P) on various NMDA receptors. Treatment with FTY720P significantly increased phosphorylation of GluN2B-containing NMDA receptors at Tyr1472. This effect appears rather specific, as treatment with FTY720P did not modify GluN2B-Tyr1336, GluN2B-Ser1480, GluN2A-Tyr1325 or GluN1-Ser897 phosphorylation. Pre-treatment of hippocampal slices with the compounds W146 and PP1 indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 epitopes was dependent on activation of S1PR subunit 1 (S1PR1) and Src/Fyn kinase, respectively. Cell surface biotinylation experiments indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 was also associated with increased levels of GluN1 and GluN2B subunits on membrane surface, whereas no change was observed for GluN2A subunits. We finally demonstrate that FTY720P is inclined to favor Tau and Fyn accumulation on plasma membranes. These results suggest that activation of S1PR1 by FTY720P enhances GluN2B receptor phosphorylation in rat hippocampal slices, resulting in increased levels of GluN1 and GluN2B receptor subunits in neuronal membranes through a mechanism probably involving Fyn and Tau.

Blockade of lysosomal acid ceramidase induces GluN2B-dependent Tau phosphorylation in rat hippocampal slices.

The lysosomal acid ceramidase, an enzyme known to limit intracellular ceramide accumulation, has been reported to be defective in neurodegenerative disorders. We show here that rat hippocampal slices, preincubated with the acid ceramidase inhibitor (ACI) d-NMAPPD, exhibit increased N-methyl-D-aspartate (NMDA) receptor-mediated field excitatory postsynaptic potentials (fEPSPs) in CA1 synapses. The ACI by itself did not interfere with either paired pulse facilitation or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-mediated fEPSPs, indicating that its influence on synaptic transmission is postsynaptic in origin and specific to the NMDA subtype of glutamate receptors. From a biochemical perspective, we observed that Tau phosphorylation at the Ser262 epitope was highly increased in hippocampal slices preincubated with the ACI, an effect totally prevented by the global NMDA receptor antagonist D/L(-)-2-amino-5-phosphonovaleric acid (AP-5), the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and the GluN2B (but not the GluN2A) receptor antagonist RO25-6981. On the other hand, preincubation of hippocampal slices with the compound KN-62, an inhibitor known to interfere with calcium/calmodulin-dependent protein kinase II (CaMKII), totally abolished the effect of ACI on Tau phosphorylation at Ser262 epitopes. Collectively, these results provide experimental evidence that ceramides play an important role in regulating Tau phosphorylation in the hippocampus via a mechanism dependent on GluN2B receptor subunits and CaMKII activation.

mTOR signaling contributes to motor skill learning in mice.

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of mRNA translation and is suspected to be involved in various long-lasting forms of synaptic and behavioral plasticity. However, its role in motor learning and control has never been examined. This study investigated, in mice, the implication of mTOR in the learning processes associated with the accelerating rotarod task. We first observed that the rotarod learning did not alter the levels of total mTOR in the striatum, hippocampus, cerebellum, and anterior cortex of trained mice. However, it increased the levels of phosphorylated mTOR in the striatum and hippocampus exclusively during the first session of training; no change was observed at the second and third sessions. In order to further investigate the potential role of mTOR during motor skill learning, we performed systemic and intrastriatal inhibitions of mTOR using the pharmacological inhibitor rapamycin, as well as a genetic knockdown of striatal mTOR using intrastriatal infusion of mTOR siRNA. These three independent approaches were all associated with a significant reduction in rotarod performances that were reminiscent of impaired consolidation processes. Notably, these treatments did not affect the capacity of mice to execute the pole test, suggesting that mTOR activity was mainly controlling motor learning rather than motor abilities. Moreover, all treatments decreased the levels of phosphorylated 4EBP1 and P70S6K, two molecular downstream targets of mTORC1. Our findings demonstrate that striatal mTOR kinase, via the phosphorylation of 4EBP1 and P70S6K, plays an important role in the cellular and molecular processes involved in motor skill learning.

Regulation of tyrosine phosphatase STEP61 by protein kinase A during motor skill learning in mice.

Recently, striatal-enriched protein tyrosine phosphatase (STEP) and its upstream regulator protein kinase A (PKA) have been suspected to play a role in the intracellular mechanisms of fear conditioning and spatial memory. However, whether they contribute to the learning and memory of motor skills is totally unknown. In this study, we have investigated the role of STEP and PKA activities during motor skill learning associated with the accelerating rotarod task. We observed that learning the rotarod task differentially modulated the levels of phosphorylated STEP61 at serine 221, a site directly regulated by PKA, in the hippocampus, motor cortex and striatum. In a second set of experiments, we have pharmacologically inhibited PKA by the injection of Rp-cAMPS directly into the dorsal striatum of mice before rotarod trainings. PKA phosphorylation of STEP prevents the dephosphorylation of STEP substrates, whereas inhibition of PKA promotes STEP activity. Striatal PKA inhibitions dose-dependently impaired mice performances on the accelerating rotarod task. General motor abilities testing revealed an intact motor control in mice treated with 5 and 20 µg of Rp-cAMPS, but not at the highest dose of 40 µg. This suggested that motor learning was selectively affected by PKA inhibition at lower doses. Most notably, striatal inhibition of PKA reduced the levels of phosphorylated STEP61 at serine 221. Our data support that inactivation of STEP61 by the PKA activity is part of the molecular process associated with motor skill learning.

NMDA reduces Tau phosphorylation in rat hippocampal slices by targeting NR2A receptors, GSK3ß, and PKC activities.

The molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. In the present study, pharmacological inhibitors were deployed to investigate potential processes by which the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors modulates Tau phosphorylation in rat hippocampal slices. Our results demonstrated that Tau phosphorylation at Ser199-202 residues was decreased in NMDA-treated hippocampal slices, an effect that was not reproduced at Ser262 and Ser404 epitopes. NMDA-induced reduction of Tau phosphorylation at Ser199-202 was further promoted when NR2A-containing receptors were pharmacologically isolated and were completely abrogated by the NR2A receptor antagonist NVP-AAM077. Compared with nontreated slices, we observed that NMDA receptor activation was reflected in high Ser9 and low Tyr216 phosphorylation of glycogen synthase kinase-3 beta (GSK3ß), suggesting that NMDA receptor activation might diminish Tau phosphorylation via a pathway involving GSK3ß inhibition. Accordingly, we found that GSK3ß inactivation by a protein kinase C- (PKC-) dependent mechanism is involved in the NMDA-induced reduction of Tau phosphorylation at Ser199-202 epitopes. Taken together, these data indicate that NR2A receptor activation may be important in limiting Tau phosphorylation by a PKC/GSK3ß pathway and strengthen the idea that these receptors might act as an important molecular device counteracting neuronal cell death mechanisms in various pathological conditions.

Maintenance of synaptic stability requires calcium-independent phospholipase A2 activity.

Phospholipases A2 (PLA2s) represent one of the largest groups of lipid-modifying enzymes. Over the years, significant advances have been made in understanding their potential physiological and pathological functions. Depending on their calcium requirement for activation, PLA2s are classified into calcium dependent and independent. This paper mainly focuses on brain calcium-independent PLA2 (iPLA2) and on the mechanisms by which they influence neuronal function and regulate synaptic plasticity. Particular attention will be given to the iPLA2γ isoform and its role in the regulation of synaptic glutamate receptors. In particular, the paper discusses the possibility that brain iPLA2γ deficiencies could destabilise normal synaptic operation and might contribute to the aetiology of some brain disorders. In this line, the paper presents new data indicating that iPLA2γ deficiencies accentuate AMPA receptor destabilization and tau phosphorylation, which suggests that this iPLA2 isoform should be considered as a potential target for the treatment of Tau-related disorders.

Sphingomyelinase selectively reduces M1 muscarinic receptors in rat hippocampal membranes.

Although there is evidence that nicotinic acetylcholine (Ach) receptors are influenced by ceramides, we do not currently know whether or not these sphingolipids can also regulate the muscarinic subtypes of Ach receptors. Using the whole-cell patch technique, we demonstrated that the effectiveness of the muscarinic receptor agonist pilocarpine, in enhancing spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, was completely abolished in hippocampal slices pre-exposed to the ceramide-generating enzyme sphingomyelinase (SMase). Western blot experiments, performed with biotinylated hippocampal membranes, showed that this electrophysiological defect possibly relies on the loss of M1 muscarinic Ach receptors at the cell surface. However, the effect appears to be relatively specific as the cell-surface expression of M4 muscarinic receptors was not found to be impacted by SMase treatment. Interestingly, we observed that G protein-coupled receptor kinases 2 and ß-arrestin1/2 interactions with M1-immunoprecipitated proteins were substantially augmented in SMase-treated slices and that the reduction of cell-surface M1 muscarinic receptor expression generated was completely suppressed by the muscarinic antagonist atropine. Collectively, our data suggest that selective internalization of M1 muscarinic receptors can be accentuated in neurons subjected to high ceramide levels. The potential physiopathological implications of this finding are presented.

Blockade of NR2A-containing NMDA receptors induces Tau phosphorylation in rat hippocampal slices.

Physiological activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been proposed to play a key role in both neuronal cell function and dysfunction. In the present study, we used selective NMDA receptor antagonists to investigate the involvement of NR2A and NR2B subunits in the modulatory effect of basal NMDA receptor activity on the phosphorylation of Tau proteins. We observed, in acute hippocampal slice preparations, that blockade of NR2A-containing NMDA receptors by the NR2A antagonist NVP-AAM077 provoked the hyperphosphorylation of a residue located in the proline-rich domain of Tau (i.e., Ser199). This effect seemed to be Ser199 specific as there was no increase in phosphorylation at Ser262 and Ser409 residues located in the microtubule-binding and C-terminal domains of Tau proteins, respectively. From a mechanistic perspective, our study revealed that blockade of NR2A-containing receptors influences Tau phosphorylation probably by increasing calcium influx into neurons, which seems to rely on accumulation of new NR1/NR2B receptors in neuronal membranes and could involve the cyclin-dependent kinase 5 pathway.

Dopamine D1 receptor activation induces tau phosphorylation via cdk5 and GSK3 signaling pathways.

Increasing evidence is demonstrating that drugs affecting dopamine levels in the brain induce cytoskeletal modifications. These evolving changes may impact neuronal synaptic plasticity as cytoskeletal constituents are involved in the maintenance of dendritic processes, and any alterations in their stability could influence major cellular compartments of neurons, such as dendrites, spines and synapses. Here, we describe a molecular chain of events that links dopamine D1 receptor activation to hyperphosphorylation of the microtubule-associated protein tau, which is normally involved in microtubules stabilization. We show, in SK-N-MC cells and rat striatal sections, that phosphorylation of tau at serines 199-202 and 214 appears to be mediated through activation of calcium-dependent intracellular mechanism, subsequent to D1 receptor-induced cAMP-dependent protein kinase A (PKA). We demonstrate, using pharmacological tools, that PKA activation causes increase of calcium levels, leading to cyclin-dependent kinase 5 activation by calpain proteolysis of p35 to p25 and glycogen synthase kinase 3beta activation by its phosphorylation at tyrosine 216. The D2 receptor agonism or lowering cAMP levels has no effect in our experimental settings. Moreover, we do not observe any association between phosphorylated tau and cellular damage. These data unravel novel mechanisms of tau hyperphosphorylation during G-protein-coupled receptor activation and are the first to show that stimulation of D1 receptors could have a profound influence on the neuronal cytoskeletal constituent tau.

AMPA receptor-mediated cell death is reduced by docosahexaenoic acid but not by eicosapentaenoic acid in area CA1 of hippocampal slice cultures.

Over the last decade, several studies have shown that high levels of polyunsaturated fatty acids (PUFAs) in the brain might limit neuronal damage in various pathological conditions. For example, in animal models of Alzheimer's disease, omega-3 type PUFAs such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids have been proposed to decrease both the cognitive and the cellular manifestations of premature ageing. The mechanisms by which they promote brain integrity remain to be established, and the experiments on cultured hippocampal slices described here examine the possibility that omega-3 fatty acids can modulate brain cell viability by interacting with glutamate receptors. We observed, by lactate dehydrogenase release and propidium iodide (PI) uptake, that excitotoxicity triggered by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist was markedly reduced in hippocampal slices treated with DHA. PI uptake experiments also revealed that neuroprotection by DHA was restricted to the hippocampal CA1 region and could not be reproduced by EPA or arachidonic acid, an omega-6 PUFA. Moreover, the beneficial effect of DHA was specific to AMPA receptor stimulation, insofar as the toxicity induced by N-methyl-D-aspartate or kainate receptor agonists was not diminished by DHA preincubation. Biotinylation experiments finally indicated that the neuroprotective actions of DHA could result from down-regulation of AMPA receptors in hippocampal membranes. This investigation thus provides the first indication that a beneficial outcome of DHA on the brain could derive from specific modulation of AMPA-mediated toxicity, reinforcing the notion that dietary DHA uptake might be useful in preventing neurodegenerative diseases.

CRMP3 is required for hippocampal CA1 dendritic organization and plasticity.

In vitro studies have pointed to the collapsin response mediator proteins (CRMPs) as key regulators of neurite outgrowth and axonal differentiation. CRMP3 is expressed mostly in the nervous system during development but remains at high levels in the hippocampus of adults. To explore CRMP3 function in vivo, we generated mice with targeted disruption of the CRMP3 gene. Immunohistochemistry and Golgi staining of CA1 showed abnormal dendrite and spine morphogenesis in the hippocampus of CRMP3-deficient mice. Apical dendrites displayed an increase in undulation and a reduction in length and branching points. Basal dendrites also exhibited a reduction in length with an alteration in soma stem distribution and an increased number of thick dendrites localized in stratum oriens (SO). Long-term potentiation (LTP) was impaired in this area. These data indicate an important role for CRMP3 in dendrite arborization, guide-posts navigation, and neuronal plasticity.

Calcium-independent phospholipase A(2) influences AMPA-mediated toxicity of hippocampal slices by regulating the GluR1 subunit in synaptic membranes.

We have recently documented that phosphorylation of the GluR1 subunit of alpha-amino-3-hydroxy-5-methylisoxazole-propionate (AMPA) glutamate receptors is influenced by calcium-independent forms of phospholipase A(2) (iPLA(2)) activity in the brain. Given the importance of GluR1 subunit phosphorylation in the control of AMPA receptor delivery to synaptic membranes, we tested the influence of iPLA(2) activity on AMPA receptor distribution between neuronal compartments, using organotypic cultured hippocampal slices. In agreement with earlier reports, the iPLA(2) inhibitor bromoenol lactone (BEL) markedly enhanced the phosphorylation of the GluR1 subunit at both Ser831 and Ser845 residues. GluR1 subunit phosphorylation levels were selectively increased by (R)-BEL, an enantio-selective inhibitor of iPLA(2)gamma, but not by (S)-BEL, an iPLA(2)beta inhibitor. The iPLA(2)gamma inhibitor R-BEL also promoted the insertion of new GluR1 subunits into synaptic membranes and exacerbated AMPA-mediated cell death in the CA1 region of the hippocampus. The latter effect was selectively abolished by IEM 1460 and philanthotoxin-433, two antagonists specific for AMPA receptors lacking GluR2 subunits. These results provide evidence that iPLA(2)gamma-related regulation of AMPA receptor GluR1 subunit phosphorylation could represent an important mechanism modulating hippocampal cell death induced by AMPA receptor overstimulation.

A novel role for calcium-independent phospholipase A in alpha-amino-3-hydroxy-5-methylisoxazole-propionate receptor regulation during long-term potentiation.

A considerable body of evidence indicates that phospholipase A(2) (PLA(2)) enzymes participate in long-term potentiation (LTP) of excitatory synaptic transmission. In the present study, we have undertaken experiments to identify which calcium-independent isoform of PLA(2) is involved in synaptic plasticity and to determine whether calcium-independent PLA(2) (iPLA(2)) contributes to post-synaptic processes of LTP. Using field recordings from rat CA1 hippocampal slices, we found that theta-burst stimulation (TBS)-induced LTP of field excitatory post-synaptic potentials (fEPSPs) was abolished by the iPLA(2) inhibitor bromoenol lactone (BEL) but not by the Ca(2+)-dependent PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)). The ionic currents generated during TBS were not affected during iPLA(2) inhibition as BEL by itself had no effect on the magnitude of facilitation during burst responses. In addition, (R)-BEL, an enantioselective inhibitor of iPLA(2)gamma, precluded TBS-induced LTP, an action that was not replicated by the iPLA(2)beta inhibitors (S)-BEL and methyl arachidonyl fluorophosphonate. (R)-BEL was, however, ineffective on pre-established LTP. Finally, BEL also prevented the potentiation of fEPSPs elicited by brief exposure to 50 microM N-methyl-d-aspartate, as well as the associated up-regulation of alpha-amino-3-hydroxy-5-methylisoxazole-propionate (AMPA) receptor GluR1 subunit levels and the increase of (3)H-AMPA binding in crude synaptic fractions. Collectively, these results unravel a new role for iPLA(2)gamma in LTP, which appears to favor the insertion of AMPA receptors at post-synaptic membranes.

The rapid onset of hyperglycaemia in ZDF rats was associated with a widespread alteration of metabolic proteins implicated in glucose metabolism in the heart.

The present study tested the hypothesis that the phosphorylation and regulation of metabolic proteins implicated in glucose homeostasis were impaired in the heart of the type 2 diabetic Zucker-diabetic-fatty (ZDF) rat model. The onset of hyperglycaemia in ZDF rats was not uniform, instead it either progressed rapidly (3-4 weeks) or was delayed (6-8 weeks). In both the early and late onset hyperglycaemic ZDF rats, AMPKalpha Thr172 phosphorylation in the heart was significantly decreased. In the early onset hyperglycaemic ZDF rats, PKB Ser473 phosphorylation was reduced, whereas Thr308 phosphorylation was significantly increased. In the late onset hyperglycaemic ZDF rats, PKB Ser473 phosphorylation was unchanged, but Thr308 phosphorylation remained elevated. Cardiac GLUT4 protein and mRNA expression were significantly reduced in the early onset hyperglycaemic ZDF rats, whereas increased protein expression was observed in the late onset hyperglycaemic ZDF rats. In conclusion, the present study has demonstrated that following a more rapid onset of hyperglycaemia, the type 2 diabetic heart is more prone to alterations in the signaling proteins implicated in glucose metabolism.

Phosphorylation of AMPA receptor subunits is differentially regulated by phospholipase A2 inhibitors.

Our laboratory recently discovered that the phosphorylation of subunits forming the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of glutamate receptors is regulated by constitutive phospholipase A2 (PLA2) activity in rat brain sections. In the present investigation, arachidonyl trifluoromethyl ketone (AACOCF3) and bromoenol lactone (BEL) were used to compare the influence of calcium-dependent (cPLA2) and calcium-independent (iPLA2) enzymes on phosphorylation of AMPA and N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors. Incubation of rat brain sections with 3 microM BEL enhanced phosphorylation on the serine (Ser) 831 residue of the AMPA receptor GluR1 subunit in synaptosomal P2 fractions, whereas AACOCF3 at the same concentration resulted in increased phosphorylation on residues Ser880/891 of GluR2/3 subunits. These effects were restricted to the AMPA receptor subtype as no changes in phosphorylation were elicited on the NMDA receptor NR1 subunit. The effects of BEL and AACOCF3 were not occluded during blockade of protein phosphatases since AMPA receptor phosphorylation was still apparent in the presence of okadaic acid, indicating that the PLA2 inhibitor-induced increase in AMPA receptor phosphorylation does not rely on a decrease in dephosphorylation reactions. However, pretreatment of rat brain sections with a cell-permeable protein kinase C (PKC) inhibitor prevented BEL- and AACOCF3-induced phosphorylation on the Ser831 and Ser880/891 sites of GluR1 and GluR2/3 subunits, respectively. These results suggest that constitutive cPLA2 and iPLA2 systems may differentially influence AMPA receptor properties and function in the rat brain through mechanisms involving PKC activity.