Paula M. Canas

Adenosine A2A Receptor Blockade Prevents Synaptotoxicity and Memory Dysfunction Caused by β-Amyloid Peptides via p38 Mitogen-Activated Protein Kinase Pathway

Alzheimers disease (AD) is characterized by memory impairment, neurochemically by accumulation of β-amyloid peptide (namely Aβ1-42) and morphologically by an initial loss of nerve terminals. Caffeine consumption prevents memory dysfunction in different models, which is mimicked by antagonists of adenosine A2A receptors (A2ARs), which are located in synapses. Thus, we now tested whether A2AR blockade prevents the early Aβ1-42-induced synaptotoxicity and memory dysfunction and what are the underlying signaling pathways. The intracerebral administration of soluble Aβ1-42 (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 A2ARs in mice. Moreover, these were synaptic events since purified nerve terminals acutely exposed to Aβ1-42 (500 nm) displayed mitochondrial dysfunction, which was prevented by A2AR 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 Aβ1-42 (500 nm). This A2AR-mediated control of neurotoxicity involved the control of Aβ1-42-induced p38 phosphorylation and was independent from cAMP/PKA (protein kinase A) pathway. Together, these results show that A2ARs play a crucial role in the development of Aβ-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.

Different synaptic and subsynaptic localization of adenosine A2A receptors in the hippocampus and striatum of the rat

Adenosine A(2A) receptors are most abundant in the striatum where they control the striatopallidal pathway thus controlling locomotion. Extra-striatal A(2A) receptors are considerably less abundant but their blockade confers robust neuroprotection. We now have investigated if striatal and extra-striatal A(2A) receptors have a different neuronal location to understand their different functions. The binding density of the A(2A) antagonist, [(3)H]-7-(2-phenylethyl)-5-amino-2-(2-furyl)pyrazolo[4,3e][1,2,4]triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), was enriched in nerve terminals membranes (B(max)=103+/-12 fmol/mg protein) compared with total membranes (B(max)=29+/-4 fmol/mg protein) from the hippocampus, the same occurring with A(2A) receptor immunoreactivity. In contrast, there was no enrichment of [(3)H]SCH 58261 binding or A(2A) receptor immunoreactivity in synaptosomal compared with total membranes from the striatum. Further subcellular fractionation of hippocampal nerve terminals revealed that A(2A) receptor immunoreactivity was enriched in the active zone of presynaptic nerve terminals, whereas it was predominantly located in the postsynaptic density in the striatum, although a minority of striatal A(2A) receptors were located in the presynaptic active zone. These results indicate that A(2A) receptors in the striatum are not enriched in synapses in agreement with the preponderant role of A(2A) receptors in signal processing in striatopallidal neurons. In contrast, hippocampal A(2A) receptors are enriched in synapses, mainly in the active zone, in accordance with their role in controlling neurotransmitter release. This regional variation in the neuronal distribution of A(2A) receptors reinforces the care required to extrapolate our knowledge from striatal A(2A) receptors to other brain preparations.

Adenosine A2A receptor antagonists exert motor and neuroprotective effects by distinct cellular mechanisms

To investigate whether the motor and neuroprotective effects of adenosine A2A receptor (A2AR) antagonists are mediated by distinct cell types in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of Parkinsons disease.

A Critical Role of the Adenosine A2A Receptor in Extrastriatal Neurons in Modulating Psychomotor Activity as Revealed by Opposite Phenotypes of Striatum and Forebrain A2A Receptor Knock-Outs

The function of striatal adenosine A2A receptors (A2ARs) is well recognized because of their high expression levels and the documented antagonistic interaction between A2ARs and dopamine D2 receptors in the striatum. However, the role of extrastriatal A2ARs in modulating psychomotor activity is largely unexplored because of the low level of expression and lack of tools to distinguish A2ARs in intrinsic striatal versus nonstriatal neurons. Here, we provided direct evidence for the critical role of A2ARs in extrastriatal neurons in modulating psychomotor behavior using newly developed striatum-specific A2AR knock-out (st-A2AR KO) mice in comparison with forebrain-specific A2AR KO (fb-A2AR KO) mice. In contrast to fb-A2AR KO (deleting A2ARs in the neurons of striatum as well as cerebral cortex and hippocampus), st-A2AR KO mice exhibited Cre-mediated selective deletion of the A2AR gene, mRNA, and proteins in the neurons (but not astrocytes and microglial cells) of the striatum only. Strikingly, cocaine- and phencyclidine-induced psychomotor activities were enhanced in st-A2AR KO but attenuated in fb-A2AR KO mice. Furthermore, selective inactivation of the A2ARs in extrastriatal cells by administering the A2AR antagonist KW6002 into st-A2AR KO mice attenuated cocaine effects, whereas KW6002 administration into wild-type mice enhanced cocaine effects. These results identify a critical role of A2ARs in extrastriatal neurons in providing a prominent excitatory effect on psychomotor activity. These results indicate that A2ARs in striatal and extrastriatal neurons exert an opposing modulation of psychostimulant effects and provide the first direct demonstration of a predominant facilitatory role of extrastriatal A2ARs.

Adenosine A2A receptors control neuroinflammation and consequent hippocampal neuronal dysfunction

J. Neurochem. (2011) 117, 100–111.

Adenosine A2A receptor blockade prevents memory dysfunction caused by β-amyloid peptides but not by scopolamine or MK-801

Adenosine A2A receptor antagonists alleviate memory deficits caused by aging or by administration of beta-amyloid peptides in rodents, which is in accordance with the beneficial effects of caffeine consumption (an adenosine receptor antagonist) on memory performance in aged individuals and in preventing Alzheimers disease. We now tested if A2A receptor blockade affords a general beneficial effect in different experimental paradigms disturbing memory performance in rodents. The beta-amyloid fragment present in patients with Alzheimers disease (Abeta1-42, 2 nmol, icv) decreased spontaneous alternation in the Y-maze after 15 days (29%) to an extent similar to the decrease of memory performance caused by scopolamine (2 mg/kg, ip) or MK-801 (0.25 mg/kg, ip) after 30 min (28% and 39%, respectively). The selective A2A receptor antagonist SCH58261 (0.05 mg/kg, ip every 24 h, starting 30 min before the noxious stimuli) prevented Abeta1-42-induced amnesia, but failed to modify scopolamine- or MK-801-induced amnesia. Similar conclusions were reached when testing another A2A receptor antagonist (KW6002, 3 mg/kg, ip). These results indicate that A2A receptors do not affect general processes of memory impairment but instead play a crucial role restricted to neurodegenerative conditions involving an insidious synaptic deterioration leading to memory dysfunction.

Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

Significance Epidemiological studies show that individuals exposed to repeated stress, a major trigger of depression, increase their caffeine intake, which correlates inversely with the incidence of depression. However, the mechanism underlying this protective effect is unknown. We used an animal model of chronic unpredictable stress (CUS) to show that caffeine prevents the maladaptive changes caused by CUS in a manner mimicked by the selective blockade of adenosine A2A receptors (A2AR). CUS enhanced A2AR in synapses, and the selective elimination of neuronal A2AR abrogated CUS modifications. Moreover, A2AR blockade also afforded a therapeutic benefit, paving the way to consider A2AR blockers as a strategy to manage the negative impact of chronic stress on mood and memory. The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function.

Increased density and synapto-protective effect of adenosine A2A receptors upon sub-chronic restraint stress.

Stress initially causes adaptive changes in the brain and can lead to neurodegeneration if continuously present. Noxious brain conditions trigger the release of adenosine that can control brain function and neurodegeneration through inhibitory A(1) and facilitatory A(2A) receptors. We tested the effect of restraint stress on the density of adenosine receptors and their effect on the outcome of stress, focusing in a known affected region, the hippocampus. Sub-chronic restraint stress (6 h/day for 7 days) caused a parallel decrease of the density of A(1) receptors (15-20%) and an increase (near 250%) of A(2A) receptor density in rat hippocampal nerve terminals. This indicates that sub-chronic stress unbalances adenosine receptors, up-regulating A(2A) and down-regulating A(1) receptors. Sub-chronic stress did not cause hippocampal neurodegeneration but decreased the immunoreactivity (immunohistochemistry and Western blot) of a synaptic marker, synaptophysin. The blockade of A(2A) receptors with 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (0.05 mg/kg, daily i.p. injection) attenuated the loss of synaptophysin immunoreactivity observed in the hippocampus of rats subjected to sub-chronic restraint stress. This ability of A(2A) receptor antagonists to prevent the earliest stress-induced synaptic modifications provides a neurochemical and morphological correlate for the interest of A(2A) receptor antagonists to attenuate the burden of chronic stress.

Key Modulatory Role of Presynaptic Adenosine A2A Receptors in Cortical Neurotransmission to the Striatal Direct Pathway

Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

Enhanced role of adenosine A2A receptors in the modulation of LTP in the rat hippocampus upon ageing

Adenosine neuromodulation depends on a balanced activation of inhibitory A1 (A1R) and facilitatory A2A receptors (A2AR). Both A1R and A2AR modulate hippocampal glutamate release and NMDA‐dependent long‐term potentiation (LTP) but ageing affects the density of both A1R and A2AR. We tested the effects of selective A1R and A2AR antagonists in the modulation of synaptic transmission and plasticity in rat hippocampal slices from three age groups (young adults, 2–3 month; middle‐aged adults, 6–8 months; aged, 18–20 months). The selective A2AR antagonist SCH58261 (50 nm) attenuated LTP in all age groups, with a larger effect in aged (−63 ± 7%) than in middle‐aged adults (−36 ± 9%) or young adult rats (−36 ± 9%). In contrast, the selective A1R antagonist DPCPX (50 nm) increased LTP magnitude in young adult rats (+42 ± 6%), but failed to affect LTP magnitude in the other age groups. Finally, in the continuous presence of DPCPX, SCH58261 caused a significantly larger inhibition of LTP amplitude in aged (−71 ± 45%) than middle‐aged (−28 ± 9%) or young rats (−11 ± 2%). Accordingly, aged rats displayed an increased expression of A2AR mRNA in the hippocampus and a higher number of glutamatergic nerve terminals equipped with A2AR in aged (67 ± 6%) compared with middle‐aged (34 ± 7%) and young rats (25 ± 5%). The results show an enhanced A2AR‐mediated modulation of LTP in aged rats, in accordance with the age‐associated increased expression and density of A2AR in glutamatergic terminals. This age‐associated gain of function of A2AR modulating synaptic plasticity may underlie the ability of A2AR antagonists to prevent memory dysfunction in aged animals.