Antonio Caputi

Calcium/Calmodulin‐Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca2+/calmodulin‐dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2‐3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either 32P‐autophosphorylated CaMKII or 32P‐NR2A/B and by evaluating the formation of a CaMKII‐NR2A/B complex by means of the cross‐linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.

Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses

A double methylazoxymethanol (MAM) intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. Thirty adult rats from 8 different progenies were investigated with a combined electrophysiological and neuroanatomical analysis. The offspring of treated dams was characterized by extensive cortical layering abnormalities, subpial bands of heterotopic neurons in layer I, and subcortical nodules of heterotopic neurons extending from the periventricular region to the hippocampus and neocortex. The phenotype of cell subpopulations within the heterotopic structures was analyzed by means of antibodies raised against glial and neuronal markers, calcium binding proteins, GABA, and AMPA glutamate receptors. Neurons within the subcortical heterotopic nodules were characterized by abnormal firing properties, with sustained repetitive bursts of action potentials. The subcortical nodules were surrounded by cell clusters with ultrastructural features of young migrating neurons. The immunocytochemical data suggested, moreover, that the subcortical heterotopia were formed by neurons originally committed to the neocortex and characterized by morphological features similar to those found in human periventricular nodular heterotopia. The present study demonstrates that double MAM treatment at gestational day 15 induces in rats developmental brain abnormalities whose anatomical and physiological features bear resemblance to those observed in human brain dysgeneses associated with intractable epilepsy. Therefore, MAM treated rats could be considered as useful tools in investigating the pathogenic mechanisms involved in human developmental brain dysgeneses.

Increased Secretion of the Amino‐Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up‐Regulation of Protein Kinase C

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APPs) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol‐treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane‐bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up‐regulated, with a concomitant increase in APPs production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up‐regulated. In addition, phorbol esters or H‐7 modulate APPs release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.

CaMKII-dependent phosphorylation of NR2A and NR2B is decreased in animals characterized by hippocampal damage and impaired LTP.

The calcium‐calmodulin‐dependent protein kinase II (CaMKII) subserves activity‐dependent plasticity in central neurons. To examine in vivo the implication of CaMKII activity in synaptic plasticity, we used an animal model characterized by developmentally induced targeted neuronal ablation within the cortex and the hippocampus, and showing, at presynaptic level, molecular alterations leading to facilitation of glutamate release in hippocampal synapses (methylazoxymethanol‐treated rats, MAM‐rats). We report here that at the postsynaptic side, the activity of CaMKII is markedly decreased in MAM‐rats when compared to controls, although the concentration of the enzyme in Post Synaptic Density (PSD) is not altered. This effect is confined to PSD‐associated CaMKII, as enzyme activity tested in the soluble fraction is unchanged in MAM‐rats. In addition, the decreased activity is not due to inhibition by autophosphorylation in specific sites within the calmodulin‐binding domain, as preincubation with purified phosphatases 1 and 2A failed to restore CaMKII activity in PSD of MAM‐rats. The CaMKII‐dependent phosphorylation of NR2A/B subunits of NMDA receptor is lower in MAM‐rats when compared to controls (51.77 ± 7.39% of controls level), as revealed in back‐phosphorylation experiments. In addition, a treatment able to restore long‐term potentiation (LTP) in hippocampal slices from MAM‐rats, e.g. exposure to d‐serine, is able to restore CaMKII activity to the control value.

Changes in Protein Kinase C and its Presynaptic Substrate B-SO/GAP-43 after Intrauterine Exposure to Methylazoxy-methanol, a Treatment Inducing Cortical and Hippocampal Damage and Cognitive Deficit in Rats

The involvement of protein kinase C (PKC)‐dependent processes in adaptive and plastic changes underlying neuronal plasticity was tested in an in vivo animal model characterized by targeted cellular ablation of cortical and hippocampal neurons, cognitive impairment and lack of induction of long‐term potentiation. [3H]Phorbol ester binding performed on brain slices revealed a 67.4 and 35.0% increase in membrane‐bound protein kinase C in the cortex and hippocampus respectively of rats treated with methylazoxy‐methanol acetate compared with saline‐treated control rats, and there was no modification in the expression of mRNAs of different protein kinase C isozymes. In situ phosphorylation experiments performed with 32Pi‐labelled synaptosomes from the affected areas demonstrated that the phosphorylation of the nervous tissue‐specific presynaptic membrane‐associated protein kinase C substrate B‐50lGAP‐43 was increased by 51.4 and 44.8% in cortex and hippocampus respectively. Western blot analysis of protein kinase C in synaptosomal cytosol and membrane fractions prepared from cortex and hippocampus showed an increased proportion of protein kinase C in the membrane compartment in treated animals, but no change in the total synaptosomal protein kinase C activity. Our data are consistent with increased activity of presynaptic protein kinase C and predict a sustained increase in glutamate release in methylazoxy‐methanol‐treated rats.

Increased Presynaptic Protein Kinase C Activity and Glutamate Release in Rats with a Prenatally Induced Hippocampal Lesion

We have previously shown that protein kinase C (PKC) activity is up‐regulated in nerve terminals of animals that have been subjected to targeted cellular ablation of cortical and hippocampal neurons by treatment with methylazoxymethanol (MAM), which results in impaired long‐term potentiation (LTP) and cognitive deficit. In this study we investigated the consequences of increased membrane‐bound PKC in the regulation of release of glutamate, the major excitatory transmitter involved in LTP. We show that nerve terminals of MAM‐treated rats show higher PKC activity, as monitored by the in situ phosphorylation of B‐50/GAP‐43, in both basal and phorbol ester‐stimulated conditions. In these animals, hippocampal nerve endings release a greater amount of glutamate than those of controls, both in basal conditions and when synaptosomes are stimulated with KCI or 3,4–diaminopyridine. The potentiation observed in MAM‐treated rats was counteracted by the PKC blocker H‐7 and the clostridial tetanus toxin. On the contrary, GABA release was not significantly up‐regulated, either in basal or in depolarization‐evoked conditions. Therefore our data show that the increase in synaptosomal PKC activity is paralleled by increased glutamate but not GABA release in this animal model. Whether this reflects specific up‐regulation of membrane PKC activity in glutamatergic terminals or an alteration in the regulation of glutamate release remains to be determined.

Determination of the endogenous phosphorylation state of B-50/GAP-43 and neurogranin in different brain regions by electrospray mass spectrometry

Electrospray mass spectrometry coupled to liquid chromatography was utilized to measure two PKC neuronal substrates, B‐50/GAP‐43 and neurogranin, in single rat brain areas. Aliquots of perchloric acid extracts were directly injected and mass spectra recorded. At elution times of 14.2 and 27.0 min two molecular species of MW 7450 and 23 602 Da were observed. These values are in excellent agreement for the expected MW for rat neurogranin and B‐50/GAP‐43. The presence of molecular species shifted by 80 mass units in both cases indicates that these proteins are present in phosphorylated forms in cortical and hippocampal extracts.

Differential translocation of protein kinase C isozymes in rats characterized by a chronic lack of LTP induction and cognitive impairment

The translocation of protein kinase C isozymes was investigated in an animal model of cognitive deficit and lack of induction of long‐term potentiation (LTP). In MAM rats, presynaptic α, β, ϵ PKC showed enhanced translocation, while postsynaptic γ PKC displayed decreased translocation when compared to control levels. This imbalance of PKC isozyme translocation between the pre‐ and post‐synaptic compartment might therefore represent a possible molecular cause for the lack of synaptic plasticity observed in these animals.

Cysteinyl-leukotriene receptors and transduction mechanisms in airway cells

A variety of inflammatory cells synthesize cys-leukotrienes (cys-LTs) C4, D4 and E4 in response to biological and non-biological stimuli1: eosinophils, basophils and mast cells2,3 are able to synthesize cys-LTs from arachidonic acid but cys-LTs can also be produced through transcellular metabolism from neutrophil-derived LTA4, by vascular endothelial cells4-6 and platelets7.