Protein kinase C activation modulates alpha-calmodulin kinase II binding to NR2A subunit of N-methyl-D-aspartate receptor complex

The N-methyl-d-aspartate (NMDA) receptor subunits NR2 possess extended intracellular C-terminal domains by which they can directly interact with a large number of postsynaptic density (PSD) proteins involved in synaptic clustering and signaling. We have previously shown that PSD-associated alpha-calmodulin kinase II (alphaCaMKII) binds with high affinity to the C-terminal domain of the NR2A subunit. Here, we show that residues 1412-1419 of the cytosolic tail of NR2A are critical for alphaCaMKII binding, and we identify, by site directed mutagenesis, PKC-dependent phosphorylation of NR2A(Ser(1416)) as a key mechanism in inhibiting alphaCaMKII-binding and promoting dissociation of alphaCaMKII.NR2A complex. In addition, we show that stimulation of PKC activity in hippocampal slices either with phorbol esters or with the mGluRs specific agonist trans-1-amino-1,3- cyclopentanedicarboxylic acid (t-ACPD) decreases alphaCaMKII binding to NMDA receptor complex. Thus, our data provide clues on understanding the molecular basis of a direct cross-talk between alphaCaMKII and PKC pathways in the postsynaptic compartment.     

Calcium/calmodulin-dependent protein kinase II phosphorylation drives synapse-associated protein 97 into spines

Synapse-associated protein 97 (SAP97) has been involved in the correct delivery and clustering of glutamate ionotropic receptors to the postsynaptic compartment. Here we demonstrate that synaptic trafficking of SAP97 itself was modulated by calcium/calmodulin-dependent protein kinase II (CaMKII) in cultured hippocampal neurons. CaMKII activation led to increased targeting of SAP97 into dendritic spines, whereas CaMKII inhibition was responsible for SAP97 high colocalization in the cell soma with the endoplasmic reticulum protein disulfide-isomerase. No effect was detected for other members of the membrane-associated guanylate kinase protein family, such as SAP102 and PSD-95. Transfection of activated alphaCaMKII T286D dramatically increased concentration of both endogenous and transfected SAP97 at postsynaptic terminals. In vitro CaMKII phosphorylation of the SAP97 N-terminal fusion protein and metabolic labeling of transfected COS7 cells indicated SAP97-Ser-39 as a CaMKII phosphosite in the SAP97 protein sequence. Moreover, transfection in hippocampal neurons of SAP97 mutants that blocked or mimicked Ser-39 phosphorylation had effects similar to those observed upon inhibiting or constitutively activating CaMKII. Further, CaMKII-dependent SAP97-Ser-39 phosphorylation determined a redistribution of the glutamate receptor subunit (GluR1) of the AMPA receptor. In conclusion, our data show that CaMKII-dependent SAP97-Ser-39 phosphorylation regulates the association of SAP97 with the postsynaptic complex, thus providing a fine molecular mechanism responsible for the synaptic delivery of SAP97 interacting proteins, i.e. ionotropic glutamate receptor subunits.     

CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction

Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.     

Induction of COX-2 and reactive gliosis by P2Y receptors in rat cortical astrocytes is dependent on ERK1/2 but independent of calcium signalling

The present study has been aimed at characterizing the ATP/P2 receptor (and transductional pathways) responsible for the morphological changes induced in vitro by alphabetamethyleneATP on rat astrocytes obtained from cerebral cortex, a brain area highly involved in neurodegenerative diseases. Exposure of cells to this purine analogue resulted in elongation of cellular processes, an event reproducing in vitro a major hallmark of in vivo reactive gliosis. alphabetamethyleneATP-induced gliosis was prevented by the P2X/P2Y blocker pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid, but not by the selective P2X antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, ruling out a role for ligand-gated P2X receptors. Conversely, the Gi/Go protein inactivator pertussis toxin completely prevented alphabetamethyleneATP-induced effects. No effects were induced by alphabetamethyleneATP on intracellular calcium concentrations. RT-PCR and western blot analysis showed that alphabetamethyleneATP-induced gliosis involves up-regulation of cyclooxygenase-2 (but not lipooxygenase). Also this effect was fully prevented by pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid. Experiments with inhibitors of mitogen-activated protein kinases (MAPK) suggest that extracellular signal regulated protein kinases (ERK)1/2 mediate both cyclooxygenase-2 induction and the associated in vitro gliosis. These findings suggest that purine-induced gliosis involves the activation of a calcium-independent G-protein-coupled P2Y receptor linked to ERK1/2 and cyclooxygenase-2. Based on the involvement of cyclooxygenase-2 and inflammation in neurodegenerative diseases, these findings open up new avenues in the identification of novel biological targets for the pharmacological manipulation of neurodegeneration.     

Chilling followed by incubation at 37 degrees C causes a reduction in NAD+ levels which can be prevented by the poly(ADP-ribose)transferase inhibitor 3-aminobenzamide

The exposure of cells for 60 min to a serum free medium at ice temperature followed by a return to normal culture conditions (30 min at 37 degrees C) caused a dramatic decrease in NAD+ levels. This decrease in NAD+ was prevented by 3-aminobenzamide. Alkaline elution analysis of DNA from cultures that were sisters to the ones utilized for measuring cellular NAD+ content revealed an absence of DNA breakage. These data suggest that poly(ADP-ribose)transferase may be induced in conditions not involving DNA fragmentation. The induction of this enzyme could therefore represent a cellular emergency reaction and not just a response to DNA damage.     

Superoxide anions are required for the inactivation ofEscherichia coli induced by high concentrations of hydrogen peroxide

The wild-type strain and mutants ofEscherichia coli lacking Mn-superoxide dismutase (Sod A) or Fe-superoxide dismutase (Sod B) are compared for their sensitivity to the H₂O₂ insult (exposure for 15 min at 37°C, in M9 salts). Whereas mode one killing is similar in superoxide dismutase mutants and wild-type cells, the latter strain appears to be more resistant than the former ones to mode two lethality. Furthermore, Sod B cells, as well as wild-type cells but unlike Sod A cells, are capable of reversing the toxicity of the oxidant (even in the presence of chloramphenicol), this effect being observed by gradually increasing the H₂O₂ concentration from 2.5 to 10 mM. It is concluded that (a) superoxide ions may not be involved in the production of mode one killing by H₂O₂, although further experiments are needed to validate or modify this hypothesis; (b) superoxide ions mediate mode two killing by H₂O₂, possibly by reducing trivalent iron to the divalent form; and (c) the intervening zone of partial resistance observed in wild-type and Sod B cells exposed to intermediate H₂O₂ concentrations is not a consequence of Mn-superoxide dismutase induction; it would appear, however, that cells lacking this superoxide dismutase isoenzyme are not proficient in this acquired response.     

The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide

The cytotoxicity of hydrogen peroxide in Escherichia coli was investigated after various conditions of drug exposure. Two modes of killing were detected following a 15-min challenge with H2O2 under either aerated or anoxic conditions. Mode one killing occurred at levels below 2.5 mM and mode two killing at concentrations higher than 10 mM. Whereas mode one killing was similar at the two conditions of drug exposure, mode two lethality differed in that aerated cells were more sensitive than anoxic cells. Independently of O2 tension the hydroxyl radical scavenger, thiourea, prevented mode two but not mode one killing by H2O2. Cells treated with the drug at ice temperature did not display mode one killing and mode two lethality occurred only at very high concentrations. We suggest that hydroxyl radicals mediate mode two but not mode one killing by H2O2.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Inhibition of Chinese hamster ovary cell DNA synthesis by hydrogen peroxide

The DNA synthesis inhibitory effect of hydrogen peroxide has been examined under a number of experimental conditions. Results have indicated that the effect of the oxidant is more pronounced when the treatment is performed at 37 degrees C than at 4 degrees C and in low density as compared to high density cultures. In addition, similar levels of inhibition were achieved by measuring the incorporation of radiolabelled thymidine in the presence of, or following treatment with, the oxidant. Although early events seem to be responsible for the decreased rate of DNA synthesis, it would appear that hydrogen peroxide does not alter thymidine extracellularly and/or decrease the transport of the nucleoside across the plasma membrane, which may actually be slightly augmented. Thus, the previously illustrated results may represent an underestimate of the actual capacity of the oxidant to reduce DNA synthesis. This inference is further supported by the fact that the effect of hydrogen peroxide appears markedly enhanced in cells preloaded with the radiolabelled precursor. A temporal relationship seems to exist between the steady state level of DNA single strand breaks and the extent of DNA synthesis inhibition by hydrogen peroxide. The oxidant has no effect on DNA chain elongation. In conclusion, data presented in this paper suggest that early events, involving selective effects on replicon initiation, mediate the DNA synthesis inhibitory effect of hydrogen peroxide.