Adenosine A2A receptor blockade prevents synaptotoxicity and memory dysfunction caused by beta-amyloid peptides via p38 mitogen-activated protein kinase pathway

Abstract

Alzheimer's disease (AD) is characterized by memory impairment, neurochemically by accumulation of beta-amyloid peptide (namely Abeta(1-42)) and morphologically by an initial loss of nerve terminals. Caffeine consumption prevents memory dysfunction in different models, which is mimicked by antagonists of adenosine A(2A) receptors (A(2A)Rs), which are located in synapses. Thus, we now tested whether A(2A)R blockade prevents the early Abeta(1-42)-induced synaptotoxicity and memory dysfunction and what are the underlying signaling pathways. The intracerebral administration of soluble Abeta(1-42) (2 nmol) in rats or mice caused, 2 weeks later, memory impairment (decreased performance in the Y-maze and object recognition tests) and a loss of nerve terminal markers (synaptophysin, SNAP-25) without overt neuronal loss, astrogliosis, or microgliosis. These were prevented by pharmacological blockade [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261); 0.05 mg . kg(-1) . d(-1), i.p.; for 15 d] in rats, and genetic inactivation of A(2A)Rs in mice. Moreover, these were synaptic events since purified nerve terminals acutely exposed to Abeta(1-42) (500 nm) displayed mitochondrial dysfunction, which was prevented by A(2A)R blockade. SCH58261 (50 nm) also prevented the initial synaptotoxicity (loss of MAP-2, synaptophysin, and SNAP-25 immunoreactivity) and subsequent loss of viability of cultured hippocampal neurons exposed to Abeta(1-42) (500 nm). This A(2A)R-mediated control of neurotoxicity involved the control of Abeta(1-42)-induced p38 phosphorylation and was independent from cAMP/PKA (protein kinase A) pathway. Together, these results show that A(2A)Rs play a crucial role in the development of Abeta-induced synaptotoxicity leading to memory dysfunction through a p38 MAPK (mitogen-activated protein kinase)-dependent pathway and provide a molecular basis for the benefits of caffeine consumption in AD.

Figure 1.

Intracerebroventricular administration of soluble β-amyloid peptides leads to an accumulation of soluble but not aggregated forms of Aβ in the hippocampus, causing delayed memory impairment without evident acute effects. The Coomassie R-250 staining and 6E10 antibody-based Western blot analysis of the two different batches of Aβ_1-42 used showed that they were mainly constituted by monomers and oligomer containing up to four monomers (A). Rats were treated with Aβ_1-42 (2 nmol, i.c.v.) or water (control), which accumulated in the hippocampus after 2 and 15 d (B), as measured by ELISA (n = 4 rats treated with water and n = 6 treated with Aβ_1-42). Congo Red and Thioflavin S staining (C) failed to reveal the presence of Aβ aggregates in hippocampal sections collected 15 d after Aβ_1-42 administration (images representative of 3 animals). D, Spontaneous alternation in the Y-maze test of control and Aβ_1-42-treated rats after 2 or 15 d (n = 6 animals treated with water and n = 9 treated with Aβ_1-42). E, Object recognition index in the object recognition test of control and Aβ_1-42-treated rats after 2 or 15 d (n = 4 animals treated with water and n = 6–7 animals treated with Aβ_1-42). Data in bar graphs are mean ± SEM; *p < 0.05.

Figure 2.

β-Amyloid administration causes a selective synaptotoxicity and memory dysfunction, which is prevented by blockade of adenosine A_2A receptors. Rats were treated with Aβ_1-42 (2 nmol, i.c.v.) or water (control). The A_2AR antagonist SCH58261 (0.05 mg/kg, i.p.) was administered daily starting 30 min before Aβ, and rats were behaviorally analyzed after 15 d. A, B, Cresyl violet staining of Nissl bodies (A) and Fluoro-Jade C staining of neuronal death (B) in hippocampal sections from control and Aβ_1-42-injected rats. C, D, Immunohistochemical labeling with anti-synaptophysin in hippocampal sections from rats injected with water (control), Aβ_1-42 (Aβ), SCH58261 (SCH), and Aβ plus SCH (images representative of 5 experiments) (C) and quantification by Western blot analysis (D) of synaptophysin immunoreactivity in hippocampal membranes from these different experimental groups (data are mean ± SEM from 7 experiments; *p < 0.05). E, Spontaneous alternation in the Y-maze test of the same groups of rats, as well as rats injected with the nonamyloidogenic scrambled Aβ_1-42 peptide (scAβ) (data are mean ± SEM from 9 rats; *p < 0.001).

Figure 3.

Genetic inactivation of adenosine A_2A receptors prevents β-amyloid-induced synaptotoxicity and memory impairment. Wild-type C57BL/6 or A_2AR KO mice were treated with Aβ_1-42 (2 nmol, i.c.v.) or water [control (CTR)] and analyzed after 15 d. A, B, Spontaneous alternation in the Y-maze test (A) and spontaneous locomotion evaluated in an open-field arena (B) (data are mean ± SEM of n = 7 mice per experimental group; *p < 0.001). C, Western blot comparing synaptophysin and SNAP-25 immunoreactivity in hippocampal membranes obtained from wild-type or A_2AR KO mice injected with water (CTR) or Aβ_1-42 (data are mean ± SEM of n = 4 mice per experimental group; *p < 0.001). D, Fluoro-Jade C staining of neuronal death, CD11-b immunohistochemistry evaluating microgliosis, and GFAP immunohistochemistry evaluating astrogliosis in hippocampal sections from wild-type or A_2AR KO mice injected with water (CTR) or Aβ_1-42 (images representative of n = 4 mice per experimental group).

Figure 4.

Exposure to Aβ_1-42 directly decreases the function of rat hippocampal synaptosomes, which is prevented by blockade of adenosine A_2A receptors. Synaptosomes were incubated for 2 h with 500 nm Aβ_1-42 or Krebs' buffer, in the absence or presence of the A_2AR antagonist, SCH58261 (50 nm), added 15 min before. A, Synaptosomal viability was measured using the MTT assay (data are mean ± SEM of n = 4; *p < 0.05). B, Measurement of mitochondrial membrane potential Δ (difference between the final and initial baseline) using the TMRM⁺ indicator after adding FCCP and oligomycin (data are mean ± SEM of n = 8; *p < 0.05).

Figure 5.

Temporal analysis of neuronal death caused by Aβ_1-42 and neuroprotection by blockade of adenosine A_2A receptors. Hippocampal neurons were preincubated with the A_2AR antagonist SCH58261 (50 nm) 15 min before addition of 500 nm Aβ_1-42. Neurons were double labeled with Syto-13 and PI probes. Viable neurons presented green nuclei stained with Syto-13, whereas apoptotic neurons presented shrinkage nuclei stained with PI and Syto-13. A, B, Aβ-induced neuronal death is time dependent. C, D, Blockade of A_2AR with SCH58261 prevents neuronal death on 48 h of incubation with Aβ. A total of ∼300 cells per coverslip was counted. Results are means ± SEM of duplicate coverslips from five independent hippocampal cultures; *p < 0.05.

Figure 6.

Temporal analysis of synaptotoxicity caused by Aβ_1-42 and neuroprotection by blockade of adenosine A_2A receptors. Hippocampal neurons were preincubated with the A_2AR antagonist SCH58261 (50 nm) 15 min before addition of 500 nm Aβ_1-42. Hippocampal neurons were double-labeled for MAP-2 (red) and synaptophysin (green) after 12 h (A), 24 h (B), and 48 h (C) of incubation and analyzed by confocal microscopy. Magnification, 400×. Aβ_1-42 causes a decrease of MAP-2 and synaptophysin immunoreactivities at all type points, which is prevented by SCH58261 and is not mimicked by the nonamyloidogenic scrambled peptide Aβ_42-1. D, Western blot analysis (15 μg of protein loaded in each lane) quantifying the loss of synaptophysin and SNAP-25 immunoreactivities in cultures treated with Aβ, which is prevented by SCH58261 (data are mean ± SEM of 6 independent cultures; *p < 0.05). E, Time course analysis of the extracellular levels of adenosine (quantified by HPLC) in hippocampal neurons incubated with Aβ (data are mean ± SEM of 5 independent cultures; *p < 0.05).

Figure 7.

The neuroprotection afforded by blockade of adenosine A_2A receptors against Aβ_1-42-induced neurotoxicity involves the p38 MAPK rather than the cAMP/protein kinase A signaling pathway. Hippocampal neurons were preincubated with the A_2AR antagonist SCH58261 (50 nm) or with the cAMP analog 8-Br-cAMP (200 μm) 15 min before addition of 500 nm Aβ_1-42. All inhibitors tested were added 30 min Aβ_1-42. A, Neuroprotection by SCH58261 does not involve the cAMP/PKA signaling pathway since the PKA inhibitor H-89 (1 μm) prevents the neuroprotection afforded by 8-Br-cAMP, but fails to modify the neuroprotection afforded by SCH58261, as evaluated after 24 h of exposure to Aβ_1-42 (*p < 0.05 vs control ^#p < 0.05 vs Aβ_1-42; ^&p < 0.05 vs Aβ_1-42 + 8-Br-cAMP). B, C, Aβ_1-42 triggered the activation of JNK (B) and p38 MAPK (C), evaluated by their degree of phosphorylation after 2 h, and SCH58261 enhanced JNK phosphorylation, whereas it blocked p38 MAPK phosphorylation (data are mean ± SEM from 6 independent cultures; *p < 0.05 vs control; **p < 0.05 vs effect of Aβ). D, The p38 MAPK inhibitor SB202190 prevents neuronal death induced by Aβ_1-42 (data are mean ± SEM from 5 independent cultures; *p < 0.05 vs control).