Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.

We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.

Presenilin-1 regulates the neuronal threshold to excitotoxicity both physiologically and pathologically.

A direct pathophysiological role of Familial Alzheimer's Disease (FAD)-associated Presenilin 1 (PS1) mutations in neuronal vulnerability remains a controversial matter. We evaluated the relationship between PS1 and excitotoxicity in four different experimental models of neurotoxicity by using primary neurons from (i) transgenic (tg) mice overexpressing a human FAD-linked PS1 variant (L286V mutation), (ii) tg mice overexpressing human wild-type (wt) PS1, (iii) PS1 knockout mice, and (iv) wt mice in which PS1 gene expression was knocked down by antisense treatment. We found that primary neurons overexpressing mutated PS1 showed an increased vulnerability to both excitotoxic and hypoxic-hypoglycemic damage when compared with neurons obtained from either mice overexpressing human wt PS1 or in wt mice. In addition, reduced excitotoxic damage was obtained in neurons in which PS1 expression was absent or diminished. Data obtained in in vivo experimental models of excitotoxicity partially supported the in vitro observations. Accelerated neuronal death was demonstrated in the hippocampus of mice overexpressing mutated PS1 after peripheral administration of kainic acid in comparison with wt animals. However, measurement of the infarct volume after middle cerebral artery occlusion did not show significant difference between the two animal groups. The results altogether suggest that expression of FAD-linked PS1 variants increases the vulnerability of neurons to a specific type of damage in which excitotoxicity plays a relevant role. In addition, they support the view that reduction of endogenous PS1 expression results in neuroprotection.

Lack of tolerance to motor stimulant effects of a selective adenosine A(2A) receptor antagonist.

It is well known that tolerance develops to the actions of caffeine, which acts as an antagonist on adenosine A(1) and A(2A) receptors. Since selective adenosine A(2A) antagonists have been proposed as adjuncts to 3,4-dihydroxyphenylalanine (L-DOPA) therapy in Parkinson's disease we wanted to examine if tolerance also develops to the selective A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo [1,5-c]pyrimidine (SCH 58261). SCH 58261 (0.1 and 7.5 mg/kg) increased basal locomotion and the motor stimulation afforded by apomorphine. Neither effect was subject to tolerance following long-term treatment with the same doses given intraperitoneally twice daily. There were no adaptive changes in A(1) and A(2A) adenosine receptors or their corresponding messenger RNA or in dopamine D(1) or D(2) receptors. These results demonstrate that the tolerance that develops to caffeine is not secondary to its inhibition of adenosine A(2A) receptors. The results also offer hope that long-term treatment with an adenosine A(2A) receptor antagonist may be possible in man.

Interleukin-10 modulates neuronal threshold of vulnerability to ischaemic damage.

Interleukin-10 (IL-10) is a powerful suppressor of cellular immune responses, with a postulated role in brain inflammation. First, we have evaluated the role of this cytokine in ischaemic brain damage using IL-10 knockout (IL-10-/-) mice. The middle cerebral artery (MCA) was occluded in either IL-10-/- or wild-type animals of corresponding strain (C57Bl/6) and age. Infarct volume was assessed 24 h later in serial brain sections. Brain infarct produced by MCA occlusion was 30% larger in the IL-10-/- than in wild-type mice (21. 8 +/- 1.2 vs. 16.9 +/- 1.0 mm3, respectively; P < 0.01; Student's t-test). To further characterize these findings, studies were extended to in vitro models. Primary neuronal cortical cultures derived from IL-10-/- animals were more susceptible to both excitotoxicity and combined oxygen-glucose deprivation compared with cell cultures from wild-type mice. Moreover, when added to the culture medium, recombinant murine IL-10 (0.1-100 ng/mL) exerted a concentration-dependent prevention of neuronal damage induced by excitotoxicity in both cortical and cerebellar granule cell cultures taken from either strain. The accordance of in vivo and in vitro data allows us to suggest a potential neuroprotective role of IL-10 against cerebral ischaemia when administered exogenously or made available from endogenous sources.

Rapamycin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12 cells by inhibiting cell cycle progression.

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

Nonsteroidal anti-inflammatory drugs increase tumor necrosis factor production in the periphery but not in the central nervous system in mice and rats.

Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three- to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4-1.8-fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE2 did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level-elevating agents or dexamethasone. Thus, a PG-driven negative feedback controls TNF production in the periphery but not in the CNS.

Cardiovascular pharmacology of the A2A adenosine receptor antagonist, SCH 58261, in the rat.

We characterized the in vivo cardiovascular profile of SCH 58261, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c] pyrimidine, a selective A2A adenosine receptor antagonist, in conscious, freely moving rats by use of the telemetry system. In normotensive rats, SCH 58261, at 10 mg/kg i.p., significantly (P < .05) inhibited hypotension and tachycardia induced by the A2A receptor agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (0.01 mg/kg i.p.), but not the bradycardic effect caused by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (0.03 mg/kg i.p.). SCH 58261, when administered alone, at 0.1 and 1 mg/kg i.p., did not induce significant hemodynamic changes, but at 10 mg/kg i.p., it slightly increased both systolic blood pressure (SBP) and diastolic blood pressure (DBP) (+19 +/- 3 and +16 +/- 2 mm Hg, respectively; P < . 01) and heart rate (HR) (+85 +/- 5 beats/min; P < .01). These effects were inhibited by adrenergic blockade with propranolol (30 mg/kg i.p.) and phentolamine (10 mg/kg i.p.): -5 +/- 3 mm Hg on DBP and -12 +/- 11 beats/min on HR (P < .01). In spontaneously hypertensive rats, SCH 58261, at 3 and 10 mg/kg i.p., increased weakly both SBP (+19 +/- 5 mm Hg and +25 +/- 4 mm Hg) and DBP (+14 +/- 4 mm Hg and +23 +/- 4 mm Hg) vs. vehicle (P < .01) and HR (+45 +/- 17 and +64 +/- 18 beats/min vs. vehicle, respectively; P < .01). The data indicate that SCH 58261 retains A2A selective receptor antagonist properties in vivo. Its effect on cardiovascular sympathetic outflow further suggests that endogenous adenosine exerts a tonic vascular regulation through A2A receptors. Therefore, SCH 58261 can be a useful pharmacological tool for clarifying A2A-mediated cardiovascular actions of adenosine.

The A2A adenosine receptor mediates coronary vasodilation.

The coronary vasodilation caused by adenosine is due to activation of A2 adenosine receptors (A2AdoRs), but the subtype or subtypes of A2AdoR (A2A and/or A2B) that mediate this action are uncertain. The purpose of this study was to test the hypothesis that A2AAdoRs mediate coronary vasodilation caused by exogenous or endogenous adenosine in the guinea pig isolated perfused heart. The newly described A2AAdoR antagonist SCH58261 was used to selectively block A2AAdoRs. Attenuations by SCH58261 of increases in coronary conductance (A2 response) and of atrioventricular nodal conduction time (A1 response) caused by exogenous and endogenous adenosine and by agonists with relative selectivity for A2A and A1AdoRs were measured. The CGS21680-induced increase of coronary conductance was antagonized by SCH58261 in a concentration-dependent and competitive manner with a KB value of 5.01 nm. Also reversed by SCH58261 (60 nmol/L) were the increases in coronary conductance caused by the relatively selective A1AdoR agonists CCPA (70 nM), and (R)-(-)N(b)-(2-phenyl-isopropyl)adenosine (60 nM) but not those caused by sodium nitroprusside (1.2 microM) and diltiazem (0.4 microM). SCH58261 (< or = 100 nM) did not attenuate the A1AdoR-mediated prolongations of S-H interval caused by either adenosine or CCPA. SCH58261 attenuated the coronary vasodilation caused by 50 nM adenosine with an IC50 value of 6.8 +/- 0.6 nM. The coronary vasodilations caused by the nucleoside uptake inhibitor draflazine and the adenosine kinase inhibitor iodotubercidin were completely reversed by 60 nM SCH58261 or adenosine deaminase (7 U/ml). Thus, the A2AAdoR plays a major role as mediator of coronary vasodilation caused by exogenous and endogenous adenosine and by AdoR agonists.

Blockade of adenosine A2A receptors by SCH 58261 results in neuroprotective effects in cerebral ischaemia in rats.

Blockade of adenosine receptors can reduce cerebral infarct size in the model of global ischaemia. Using the potent and selective A2A adenosine receptor antagonist, SCH 58261, we assessed whether A2A receptors are involved in the neuronal damage following focal cerebral ischaemia as induced by occluding the left middle cerebral artery. SCH 58261 (0.01 mg/kg either i.p. or i.v.) administered to normotensive rats 10 min after ischaemia markedly reduced cortical infarct volume as measured 24 h later (30% vs controls, p < 0.05). Similar effects were observed when SCH 58261 (0.01 mg/kg, i.p.) was administered to hypertensive rats (28% infarct volume reduction vs controls, p < 0.05). Neuroprotective properties of SCH 58261 administered after ischaemia indicate that blockade of A2A adenosine receptors is a potentially useful biological target for the reduction of brain injury.

Prolonged exposure to 5'-N-ethylcarboxamidoadenosine (NECA) does not affect the adenosine A2A-mediated vasodilation in porcine coronary arteries.

At present, four distinct adenosine receptors (A1, A2A, A2B, and A3) have been cloned and characterized in several species. It is known that prolonged exposure of tissues to receptor agonists induces A1 receptor desensitization. However, controversial data are reported on whether or not prolonged stimulation of A2A adenosine receptors induces tolerance. Using the porcine coronary artery, a sensitive vascular model, studies were designed, with the aim to clarify how prolonged exposure to the adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) affects A2A receptor responsiveness. The arteries were precontracted with PGF2alpha (3 microM) and cumulative dose-response curves to either NECA itself, or the selective A2A agonists, 2-[4-2(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) and 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) were obtained. In separate experiments, coronary rings were incubated with NECA (10 microM) for 30 min or 2 h. After 2 h washout period, functional response was assessed. The arteries showed high sensitivity to adenosine agonist-induced vasorelaxation. EC50 (nM) values were 71.8 (35.5-145), 20.0 (11.2-32.7) and 40.2 (20.4-79.1) for NECA, 2HE-NECA and CGS 21680, respectively. Vasorelaxant response of A2A selective agonists 2HE-NECA and CGS 21680 was not influenced by preincubation with NECA for 30 min or 2 h. Conversely, dose-response curves to NECA were shifted toward the right by preincubation with NECA itself: ED50 (nM) values were 114 (79.2-165), 211 (161-276) and 412 (132-1290) for 30 min, 2 h and 15 h preincubation, respectively. These effects did not occur after 4 h washout. The present results indicate that prolonged stimulation of A2A receptors does not lead to loss of functional response, suggesting that this receptor subtype does not desensitize after prolonged stimulation by agonists.

Hemodynamic changes do not mediate the cardioprotection induced by the A1, adenosine receptor agonist CCPA in the rabbit.

Stimulation of adenosine A1 receptors is known to reduce infarct size in the rabbit heart. The aim of the present study was to verify whether a protective activity similar to that of the selective A1 receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), could also be obtained by inducing comparable hemodynamic effects with drugs having different mechanisms of action. The effects of the beta-adrenoceptor blocker atenolol, the calcium channel blocker felodipine, the A2A-selective adenosine receptor agonist 2-hexynyl-5'-N-ethyl-carboxamidoadenosine (2HE-NECA), and the non-selective adenosine receptor agonist 5'-N-ethyl-carboxamidoadenosine (NECA) were tested. Groups of 12-15 anesthetized open-chest rabbits received a 5-min infusion of CCPA (50 micrograms kg-1 min-1), atenolol (1 mg kg-1 min-1), felodipine (50 micrograms kg-1 min-1), 2HE-NECA (1 microgram kg-1 min-1), and NECA ( 1 microgram kg-1 min-1). Myocardial infarction was induced by a 30-min occlusion of a branch of the left coronary artery, followed by 3-h reperfusion. Infarct size was measured by tetrazolium staining. In controls, infarct size was about 40% of the zone at risk. Pretreatment with CCPA induced a marked decrease in heart rate (-40%) and blood pressure (-48%), and showed antiischemic activity (28% of the zone at risk). The other drugs tested produced similar effects on either heart rate (atenolol, -25%), or blood pressure (felodipine, 2HE-NECA and NECA, about -45%), but did not affect infarct size. IN this model, the reduction in infarct size by CCPA is most likely mediated by A1 receptors, since comparable hemodynamic effects, induced by other means, are not effective. A2A receptor stimulation does not appear to exert a protective effect.

Cardioprotective effects of adenosine A1 and A2A receptor agonists in the isolated rat heart.

It has been postulated that the adenosine A1 receptor subtype, but also A2a receptors, are involved in mediating the beneficial properties of adenosine during ischemia and reperfusion. We investigated the effects of the selective A1 adenosine receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), the selective A2A adenosine receptor agonists, 2-[p-(2-carboxyethyl)phenetylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA), and the non selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA), on ischemia-reperfusion injury in Langendorff-perfused rat hearts. Global ischemia was induced for 15 min in paced hearts followed by 60 min reperfusion. Control hearts developed left ventricular dysfunction, as indicated by the increase in end diastolic pressure to 40.8 +/- 5.1 vs 5.9 +/- 1.0 mm Hg baseline, and in coronary perfusion pressure to 57.6 +/- 8.4 vs 28.8 +/- 2.2 mm Hg before ischemia. After 15 min of reperfusion, ventricular function (LVDP) recovered by 83%, but creatine kinase levels were still significantly increased (294 +/- 55 IUl(-1) vs basal), indicating the occurrence of myocardial injury. All adenosine agonists added to the perfusion medium 15 min prior to ischemia exerted protective effects against myocardial dysfunction and reperfusion injury. Thus, 2HE-NECA (100 nM), CGS 21680 (10 nM), CCPA (3 nM) and NECA (100 nM) significantly (P < 0.05) decreased end diastolic pressure by 50-75% as compared with the control group. Similarly, all compounds significantly (P < 0.05) reduced coronary perfusion pressure by 30-45% vs control. For all drugs, recovery of LVDP occurred immediately after restoration of coronary flow. At 15-min reperfusion the adenosine agonists decreased myocardial creatine kinase release by 80-95% (P < 0.05 vs control). These findings indicate that both A1 and A2A adenosine receptors are involved in protecting the myocardium against ischemia and reperfusion in isolated rat heart, even if through different mechanisms.