Lisiane O. Porciúncula

Neuroprotection by caffeine and adenosine A2A receptor blockade of β‐amyloid neurotoxicity

Adenosine is a neuromodulator in the nervous system and it has recently been observed that pharmacological blockade or gene disruption of adenosine A2A receptors confers neuroprotection under different neurotoxic situations in the brain. We now observed that coapplication of either caffeine (1–25 μM) or the selective A2A receptor antagonist, 4‐(2‐[7‐amino‐2(2‐furyl)(1,2,4)triazolo (2,3‐a)(1,3,5)triazin‐5‐ylamino]ethyl)phenol (ZM 241385, 50 nM), but not the A receptor antagonist, 8‐cyclopentyltheophylline (200 nM), prevented the neuronal cell death caused by exposure of rat cultured cerebellar granule neurons to fragment 25–35 of β‐amyloid protein (25 μM for 48 h), that by itself caused a near three‐fold increase of propidium iodide‐labeled cells. This constitutes the first in vitro evidence to suggest that adenosine A2A receptors may be the molecular target responsible for the observed beneficial effects of caffeine consumption in the development of Alzheimers disease.

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.

Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes

Quinolinic acid (QA) is an endogenous neurotoxin involved in various neurological diseases, whose action seems to be exerted via glutamatergic receptors. However, the exact mechanism responsible for the neurotoxicity of QA is far from being understood. We have previously reported that QA inhibits vesicular glutamate uptake. In this work, investigating the effects of QA on the glutamatergic system from rat brain, we have demonstrated that QA (from 0.1 to 10mM) had no effect on synaptosomal L-[3H]glutamate uptake. The effect of QA on glutamate release in basal (physiological K+ concentration) or depolarized (40 mM KCl) conditions was evaluated. QA did not alter K+-stimulated glutamate release, but 5 and 10mM QA significantly increased basal glutamate release. The effect of dizolcipine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor on glutamate release was investigated. MK-801 (5 microM) did not alter glutamate release per se, but completely abolished the QA-induced glutamate release. NMDA (50 microM) also stimulated glutamate release, without altering QA-induced glutamate release, suggesting that QA effects were exerted via NMDA receptors. QA (5 and 10mM) decreased glutamate uptake into astrocyte cell cultures. Enhanced synaptosomal glutamate release, associated with inhibition of glutamate uptake into astrocytes induced by QA could contribute to increase extracellular glutamate concentrations which ultimately lead to overstimulation of the glutamatergic system. These data provide additional evidence that neurotoxicity of QA may be also related to disturbances on the glutamatergic transport system, which could result in the neurological manifestations observed when this organic acid accumulates in the brain.

Ebselen prevents excitotoxicity provoked by glutamate in rat cerebellar granule neurons

Ebselen is a selenium compound that have glutathione peroxidase-like activity which is neuroprotective in acute stroke ischemia. The efficacy of ebselen to prevent excitotoxicity provoked by glutamate in cerebellar granule neurons was investigated at various time points and concentrations. Simultaneous addition of ebselen with glutamate decreased neuronal death and was completely reversed by 3 microM of ebselen (3 (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide assays). However, when 1 microM of ebselen was added with glutamate and remained in the culture medium until 24 or 48 h, the neuronal survival increased to the control. The mechanism proposed for neuroprotection was the ability of ebselen to prevent lipoperoxidation provoked by glutamate. The present findings propose to amplify the use of ebselen in others neurodegenerative disorders involving glutamatergic system.

Diphenyl diselenide protects rat hippocampal slices submitted to oxygen–glucose deprivation and diminishes inducible nitric oxide synthase immunocontent

Diphenyl diselenide (PhSe)2 is an organic selenium compound that has been little studied. In this study we investigated the effects of (PhSe)2 (0.1-3 microM) in a classical model of in vitro brain ischemia, which consists of exposing rat hippocampal slices to oxygen-glucose deprivation (OGD). Hippocampal slices were exposed for 60 min to OGD and the cellular viability (performed by MTT assay) as well as the immunocontent of nitric oxide synthase inducible (iNOS) were evaluated after 180 min of a recovery period. OGD decreased cellular viability by 50% and increased more than twice the immunocontent of iNOS of hippocampal slices. (PhSe)2 (1 and 3 microM) added during OGD and the recovery period abolished both effects. These results demonstrate for the first time the neuroprotective effects of (PhSe)2. Although the selenium analog--ebselen--has been widely used in ischemia models, our results suggest that other selenoorganic compounds could be investigated as pharmacological tools against brain disorders.

CAFFEINE PREVENTS AGE-ASSOCIATED RECOGNITION MEMORY DECLINE AND CHANGES BRAIN-DERIVED NEUROTROPHIC FACTOR AND TIROSINE KINASE RECEPTOR (TrkB) CONTENT IN MICE

The beneficial effects of caffeine on cognition are controversial in humans, whereas its benefit in rodents had been well characterized. However, most studies were performed with acute administration of caffeine and the tasks used to evaluate cognition had aversive components. Here, we evaluated adulthood administration of caffeine up to old age on recognition memory in mice using the object recognition task (ORT) and on brain-derived neurotrophic factor (BNDF) and tyrosine kinase receptor (TrkB) immunocontent in the hippocampus. Adult mice (6 months old) received either drinking water or caffeine (1 mg/mL) during 12 months. At 18 months of age both groups were tested for ORT. Our results showed that aged mice exhibited lower performance in the recognition memory compared with adults (6 months old). Furthermore, caffeine-treated mice showed similar performance to adult mice in the ORT and an improvement compared with their age-matched control mice. Caffeine also counteracted the age-related increase in BDNF and TrkB immunocontent. Our results corroborate with other studies and reinforce that caffeine consumed in adulthood may prevent recognition memory decline with aging. This preventive effect may involve a decrease in the hippocampal BDNF and TrkB immunocontent.

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.

Caffeine improves adult mice performance in the object recognition task and increases BDNF and TrkB independent on phospho-CREB immunocontent in the hippocampus.

Caffeine is one of the most psychostimulants consumed all over the world that usually presents positive effects on cognition. In this study, effects of caffeine on mice performance in the object recognition task were tested in different intertrial intervals. In addition, it was analyzed the effects of caffeine on brain derived neurotrophic factor (BDNF) and its receptor, TrkB, immunocontent to try to establish a connection between the behavioral finding and BDNF, one of the neurotrophins strictly involved in memory and learning process. CF1 mice were treated during 4 consecutive days with saline (0.9g%, i.p.) or caffeine (10mg/kg, i.p., equivalent dose corresponding to 2-3 cups of coffee). Caffeine treatment was interrupted 24h before the object recognition task analysis. In the test session performed 15min after training session, caffeine-treated mice recognized more efficiently both the familiar and the novel object. In the test session performed 90min and 24h after training session, caffeine did not change the time spent in the familiar object but increased the object recognition index, when compared to control group. Western blotting analysis of hippocampus from caffeine-treated mice revealed an increase in BDNF and TrkB immunocontent, compared to their saline-matched controls. Phospho-CREB immunocontent did not change with caffeine treatment. Our results suggest that acute treatment with caffeine improves recognition memory, and this effect may be related to an increase of the BDNF and TrkB immunocontent in the hippocampus.

Caffeine Consumption Prevents Memory Impairment, Neuronal Damage, and Adenosine A2A Receptors Upregulation in the Hippocampus of a Rat Model of Sporadic Dementia

Intracerebroventricular (icv) streptozotocin (STZ) administration induces pathological and behavioral alterations similar to those observed in Alzheimers disease (AD) and is thus considered an experimental model of sporadic AD. Since caffeine (an adenosine receptor antagonist) and selective antagonists of adenosine A2A receptors modify the course of memory impairment in different amyloid-β-based experimental models of AD, we now tested the impact of caffeine on STZ-induced dementia and associated neurodegeneration in the hippocampus as well as on the expression and density of adenosine receptors. Adult male rats received a bilateral infusion of saline or STZ (3 mg/kg, icv), which triggered memory deficits after four weeks, as gauged by impaired object recognition memory. This was accompanied by a reduced NeuN immunoreactivity in the hippocampal CA1 region and an increased expression and density of adenosine A2A receptors (A2AR), but not A1R, in the hippocampus. Caffeine consumption (1 g/L in the drinking water starting 2 weeks before the STZ challenge) prevented the STZ-induced memory impairment and neurodegeneration as well as the upregulation of A2AR. These findings provide the first demonstration that caffeine prevents sporadic dementia and implicate the control of central A2AR as its likely mechanism of action.

Exercise affects glutamate receptors in postsynaptic densities from cortical mice brain

Physical activity has been proposed as a behavior intervention that promotes mental health and some of the benefits induced by exercise have been related to the glutamatergic system. Indeed, glutamate is the most abundant excitatory neurotransmitter in brain. Thus, we evaluated if voluntary exercise in mice could modulate glutamatergic synapses at level of postsynaptic density (PSD). Through Western blot, we found that exercise during 1 month increased glutamatergic-related protein content in PSD from cortex of mice. Exercise increased the immunocontent of GluR1 (129%), SAP-97 (179%), GRIP-1 (129%), and in less extent, GluR2/3 (118%) and PSD-95 (112%) proteins. The overall content of NMDA subunits R1, R2A and R2B were not altered in mice that had exercised, however, the phosphorylated NMDA subunits, phospho-NMDAR1 (150%), and phospho-NMDAR2B (183%) showed a strong increase. Because exercise increased the content of phosphorylated forms of NMDA receptors, we evaluated the binding of MK-801, a specific ligand that binds to open NMDA channel. Exercise increased the binding of MK-801 in cortical cellular membranes in 51%. Altogether, our results point to a modulation of glutamatergic synapses by exercise with likely implications in the exercise-induced mental health.