NM DA receptors and Ca 2/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 GIuR1 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 CaMKll-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKll in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity. Key Words: Rat-H ippocampus-Synaptic plasticityPhosphorylation-Ca2/calmodulin-dependent protein kinase ll-NMDA.

The A3 adenosine receptor has been implicated in modulation of cell growth. As a first step to the characterization of the underlying mechanisms, we exposed Chinese hamster ovary (CHO) cells transfected with the human A3 receptor (A3R-CHO) to selective A3 receptor ligands. At micromolar concentrations, the A3 agonists N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and its 2-chloro derivative Cl-IB-MECA reduced cell number, with no effects on either parental CHO cells (not expressing any adenosine receptor), or CHO cells transfected with the human A1 receptor. Cl-IB-MECA also reduced cell number in the human HEK293 cell line transfected with the human A3 receptor cDNA as opposed to the respective untransfected wild-type cells. In A3R-CHO, agonist-induced effects were antagonized by nanomolar concentrations of A3 antagonists, including the triazoloquinazoline derivative MRS 1220, the dihydropyridine derivative MRS 1191, and the triazolonaphthyridine derivative L-249,313. A3 agonist-induced effects were not due to modulation of cell adhesion, nor to necrosis or apoptosis. Growth curves revealed highly impeded growth, and flow-cytometric analysis showed markedly reduced bromodeoxyuridine incorporation into nuclei. The effect on cell cycle was completely antagonized by MRS1191. Hence, activation of the human A3 receptor in A3R-CHO results in markedly impaired cell cycle progression, suggesting an important role for this adenosine receptor subtype in cell cycle regulation and cell growth.

A 3 adenosine receptor activation has been previously demonstrated to result in both neuroprotective and neurodegenerative effects, depending upon specific pathophysiological conditions. This dual effect may depend on receptor regulation mechanisms that are able to change receptor availability and/or function. In the present study, we investigated desensitization, internalization, and down-regulation of native A 3 adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro-N6-(3-iodobenzyl)-N-methyl-5Ј-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentrationdependent inhibition of adenylyl cyclase activity with an EC 50 value of 2.9 Ϯ 0.1 nM. The effect was suggested to be mediated by A 3 adenosine receptor subtype by the use of selective adenosine receptor antagonists. Cell treatment with pertussis toxin abolished Cl-IBMECA-mediated inhibition of adenylyl cyclase activity, evidencing an A 3 receptor coupling to inhibitory G protein. Short-term exposure to the agonist Cl-IBMECA (100 nM) caused rapid receptor desensitization, within 15 min. Agonist-induced desensitization was accompanied by receptor internalization: A 3 adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IB-MECA. The localization of A 3 adenosine receptors on the plasma membrane and in intracellular compartments was directly revealed by immunogold electron microscopy. After desensitization, the removal of agonist led to the restoration of A 3 adenosine receptor functioning through receptor recycling to the cell surface within 120 min. Prolonged agonist exposure (1-24 h) resulted in a marked down-regulation of A 3 adenosine receptors that reached 21.9 Ϯ 2.88% of control value after 24 h. After down-regulation, the recovery of receptor functioning was slow (24 h) and associated with the restoration of receptor levels close to control values. In conclusion, our results demonstrated that A 3 receptors, in astrocytoma cells, are regulated after short-and long-term agonist exposure.Actions of adenosine are mediated by four G protein-coupled membrane receptors (GPCRs): A 1 , A 2A , A 2B , and A 3 receptors (Fredholm et al., 1994). Although expressed at very low levels in mammalian brain, the A 3 adenosine receptor (AR) subtype has been implicated in behavioral depression and modulation of ischemic cerebral damage (for review, see von Lubitz, 1999). Through the development of selective A 3 AR agonists (e.g., Cl-IBMECA) and antagonists (e.g., MRS 1191 and MRS 1220) (Kim et al., 1994(Kim et al., , 1996Jacobson et al., 1997), the putative pathophysiological roles of this receptor have became clear. It has been demonstrated that A 3 AR agonists profoundly affect cell survival, by promoting cell protection or cell death, depending upon the cell type and/or agonist concentration (for reviews, see Jacobson, 1998;Jacobson et al., 1999). In human astrocytoma cells (ADF cells), exposure to nanomolar Cl-IBMECA concentrations increased resistance to apoptosis, by a mechanism...

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We describe here an innovative, non-transgenic animal model of Alzheimer's disease. This model mimics early stages of sporadic disease, which represents the vast majority of cases. The model was obtained by interfering with the complex between a disintegrin and metalloproteinase domain containing protein 10 (ADAM10), the main α-secretase candidate, and its partner, synapse-associated protein 97, a protein of the postsynaptic density-membrane associated guanylate kinase family. Association of ADAM10 with synapse-associated protein 97 governs enzyme trafficking and activity at synapses. Interfering with the ADAM10/synapse-associated protein 97 complex for 2 weeks by means of a cell-permeable peptide strategy is sufficient to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimer's disease. After 2 weeks of treatment, we detected progressive Alzheimer's disease-like neuropathology, with an increase of β-amyloid aggregate production and of tau hyperphosphorylation, and a selective alteration of N-methyl-d-aspartic acid receptor subunit composition in the postsynaptic compartment of mouse brain. Behavioural and electrophysiological deficits were also induced by peptide treatment.