Abstract
Background:
Alzheimer’s disease (AD) is a neurodegenerative pathology covering about 70%of all cases of dementia. Adenosine, a ubiquitous nucleoside, plays a key role in neurodegeneration, through interaction with four receptor subtypes. The A2A receptor is upregulated in peripheral blood cells of patients affected by Parkinson’s and Huntington’s diseases, reflecting the same alteration found in brain tissues. However, whether these changes are also present in AD pathology has not been determined.
Objective:
In this study we verified any significant difference between AD cases and controls in both brain and platelets and we evaluated whether peripheral A2A receptors may reflect the status of neuronal A2A receptors.
Methods:
We evaluated the expression of A2A receptors in frontal white matter, frontal gray matter, and hippocampus/entorhinal cortex, in postmortem AD patients and control subjects, through [3H]ZM 241385 binding experiments. The same analysis was performed in peripheral platelets from AD patients versus controls.
Results:
The expression of A2A receptors in frontal white matter, frontal gray matter, and hippocampus/entorhinal cortex, revealed a density (Bmax) of 174±29, 219±33, and 358±84 fmol/mg of proteins, respectively, in postmortem AD patients in comparison to 104±16, 103±19, and 121±20 fmol/mg of proteins in controls (p < 0.01). The same trend was observed in peripheral platelets from AD patients versus controls (Bmax of 214±17 versus 95±4 fmol/mg of proteins, respectively, p < 0.01).
Conclusion:
AD subjects show significantly higher A2A receptor density than controls. Values on platelets seem to correlate with those in the brain supporting a role for A2A receptor as a possible marker of AD pathology and drug target for novel therapies able to modify the progression of dementia.
INTRODUCTION
Alzheimer’s disease (AD) affects about 33 million people around the world and its prevalence is estimated to quadruple by 2050, affecting 1%of the global population. Age is the principal risk factor for AD affecting 5–10%of people older than 65 and 50%of those older than 85 years [1].
AD is characterized by cognitive impairment progressively involving memory, executive functions, language, and visual-perceptual functions. Personality and behavioral changes are also frequent. Motor function is usually spared until the most advanced stages of the disease. The AD clinical course reflects the progressive spread of lesions within the brain. The macroscopic pathological feature characterizing advanced AD is diffuse brain atrophy due to neuronal and synaptic loss, probably induced by amyloid-β (Aβ) oligomers [2]. The extracellular accumulation of Aβ peptides (Aβ or senile plaques), as well as the hyperphosphorylated tau protein aggregates inside the neurons (neurofibrillary tangles), composes the neuritic plaques, which are typical for AD and frequently associated with neuroinflammation. Indeed, the neuropathological diagnosis of AD requires a combination of scores for
Adenosine is an important endogenous neuromodulator, affecting several neurodegenerative diseases, through the interaction with four G-protein coupled receptors named A1, A2A, A2B, and A3 [7]. In particular, the A2A subtype is the most involved in neuropathological effects and is present mainly in striatal cerebral areas, as well as in cortical and hippocampal districts. Its activation consists in the modulation of excitability at pre-synaptic level by controlling, e.g., glutamate release. In addition, A2A adenosine receptors are important regulators of neuroinflammation being expressed in astrocytes and microglia, where they exert a control of pro-inflammatory cytokines release [8]. Antagonism of A2A adenosine receptors is well known to be neuroprotective in neurodegenerative pathologies, including stroke and Parkinson’s disease [9–11].
More recently, a role for this subtype has been identified in the pathogenesis of AD. A2A adenosine receptors regulate synaptic plasticity, participate in memory deficit and are increased in the brain of AD animal models and patients [12–18]. In postmortem human brains they are highly expressed in hippocampal regions important for neurogenesis, fundamental to avoid neurodegeneration [19]. The first morphological hallmark associated with cognitive impairment in early phase of AD is the synaptic loss in hippocampal regions instead of Aβ plaques, tangle formation, or neuronal loss [20]. Interestingly, it has been demonstrated that overexpression and stimulation of A2A adenosine receptors deletes associative long-term synaptic potentiation (LTP) in hippocampal cells of mice with AD amyloidosis [21]. Specifically, different pharmacological and genetic approaches demonstrate that A2A adenosine receptors impairment prevents synaptic toxicity and cognitive deficits in different animal models treated with Aβ [22–24]. Indeed, A2A adenosine receptors are mainly expressed in hippocampal synapses controlling synaptic plasticity [25–27]. In addition, block of A2A adenosine receptors gene expression or function in mice with tau pathology results in protection from tau hyperphosphorylation, neuroinflammation, and improvement of spatial memory and hippocampal long-term depression [28]. As confirmation of the role of A2A adenosine receptors in AD, it has been reported that the consumption of coffee and caffeine, an adenosine receptor antagonist, reduces risk and incidence of dementia [29–35]. Altogether, this evidence suggests that A2A adenosine receptors could be a drug target for AD and also a marker of pathology [36].
Literature data suggest that several potential biomarkers can be detected in peripheral blood samples and report a positive association between receptor proteins present in tissues and in peripheral blood cells, from control and pathologic subjects [37–41]. Specifically, platelets have often been used as a model in neurobiological research and are an ideal model, useful and accessible, to investigate the mechanisms of Aβ production in neurons. For example, they produce the major part of the circulating amyloid-β protein precursor (AβPP) and present the same enzymes involved in AβPP transformation leading to Aβ production [42, 43].
The aim of this work is to evaluate the expression of A2A adenosine receptors in frontal white matter (FWM), frontal gray matter (FGM), and hippocampus/entorhinal cortex (H/EC) in autoptic human brains from patients affected by AD in comparison to the same regions obtained from control subjects. At the same time, we compare the expression of A2A receptors in platelets from AD and from control subjects, to evaluate whether peripheral A2A adenosine receptors may reflect the status of neuronal A2A adenosine receptors.
MATERIALS AND METHODS
Setting
All brain samples analyzed come from the Abbiategrasso Brain Bank (ABB) that collects brains from donors who joined the donation program before their death. They are patients from ASP Golgi-Redaelli, home to several patients affected by different neurodegenerative diseases or adult volunteers belonging to the same geographic area (Abbiategrasso and surroundings). All donors (cases and controls) undergo the same clinical and neuropathological protocol [44].
Blood samples come from AD patients (cases) belonging to Cognitive Impairment Center of the S. Anna University Hospital of Ferrara, and from control subjects of the local AVIS Blood Bank.
Ethics approval
The ABB procedures were in accordance with the principles outlined in the Declaration of Helsinki of 1964 and the following amendments. The ABB performs its activities following the ethical standards of the BrainNet Europe Code of Conduct [45, 46]. The brain harvesting procedure was submitted to and approved by the Ethics Committee of the University of Pavia in the context of the InveCe.Ab study (“Invecchiamento Cerebrale Abbiategrasso”, namely Brain Aging Abbiategrasso) [47]. Joining the donation program is a personal decision, and complete awareness is needed. In case a person is not deemed competent to sign the consent form, authorization from legal guardian or next-of-kin is warranted, taking into account the wishes previously expressed by the subject. ABB activity is under the supervision of the“Federazione Alzheimer Italia”.
AD patients were recruited from the Cognitive Impairment Center of the S. Anna University Hospital of Ferrara, Department of Neurology. They had mild to moderate dementia and were able and willing to provide valid written informed consent. After written informed consent we collected blood samples from 26 patients and controls, according to the IRB protocol 170480.
Participants
Brain bank participants
Autoptic human brain samples were provided from the ABB of the Golgi Cenci Foundation (Milan, Italy). Before their death, all donors underwent longitudinal multi-dimensional assessments to determine social, neuropsychological, biological, and clinical profile as previously described in detail [44]. Ten different cases were selected including 4 controls with no cognitive impairment and 6 cases with a clinical diagnosis of major Neuro-Cognitive Disorder (major-NCD), according to DSM-5 [48]. Each case has been assigned a Clinical Dementia Rating score (CDR) [49], a 5-point scale used to track the level of impairment of patients with dementia, including Alzheimer’s disease (CDR 0: no dementia, CDR 0.5: mild neurocognitive disorder, CDR 1: mild dementia, CDR 2: moderate dementia, CDR 3: severe dementia, CDR 4: very severe dementia, and CDR 5: terminal dementia) [50, 51]. All 4 control subjects had CDR 0 at death and showed only mild to moderate age-related neuropathological modifications with low or no AD pathology. On the other hand, all 6 pathological cases correspond to a clinical diagnosis of very severe major neurocognitive disorder (CDR 4-5) and had a definite neuropathological diagnosis of AD showing predominant, high to intermediate, AD pathology (Table 1).
Summary of the main clinical and neuropathological data of selected cases
CDR, Clinical Dementia Rating scale (severity range 0–5); AFS, Agonal Factor Score (range: 0–2) [66]; PM,postmortem; A, Amyloid score (severity range 0–3); B, Braak score (severity range 0–3); C, CERAD score (severity range 0–3) [5]; BB,brain bank; NOLD,normal elderly; GBL, glioblastoma; Major-NCD,major neurocognitive disorder; AD, Alzheimer’s disease; bvAD, behavioral variant of AD; LBD, Lewy body disease; VaD,vascular dementia; SVD,small vessel disease; BG, gasalganglia; ILBD, incidental Lewy body disease; amy, amygdala; CAA, cerebral amyloid angiopathy; capCAA, capillary CAA; HS,hippocampal sclerosis; LTS, Lewy type synucleinopathy; ARTAG, age-related tauastro-gliopathy.
Cognitive impairment center and avis blood bank participants
All consecutive subjects with AD, diagnosed after 2015, according to the DSM-5 criteria [48] have been declared eligible for the inclusion in the study. Exclusion criteria were: serious physical illness, including, but not limited to, cancer and myocardial infarction, neurodegenerative, cerebrovascular, or other neurological diseases; psychiatric illness, or substance abuse; systemic or neurological autoimmune disease, treatment with corticosteroids, Mini–Mental State Examination (MMSE) score higher than 26/30. Table 3 reports demographic and clinical characteristics of the 26 AD patients, including age, gender, disease duration, and MMSE scores; AD group’s main features: 13 women and 13 men, 62–89 years old, MMSE corresponding to mild-moderate dementia (CDR 1–2). Control subjects, with an age spanning from 20 to 70 years, were enrolled among control donors of AVIS Blood Bank, kindly provided by Dr. Palma.
Demographic analysis of patients included in the study
Tissue processing and histological analysis
The brain tissue was obtained with an average postmortem-time of about 10 h (from 3 to 29 h). After the brain harvesting both hemispheres were freshly cut into 10 mm thick slices that were alternately fixed in formalin or frozen in liquid nitrogen at–120°C and stored at–80°C. The neuropathological characterization was carried out on formalin fixed slices, included in paraffin and cut into 8μm thick serial sections. Neocortex (frontal, parietal, temporal and occipital lobes, cingulate and insula), basal ganglia, thalamus, Meynert nucleus, amygdala, hippocampus, cerebellar cortex and dentate nucleus, brainstem (substantia nigra, pons, medulla oblongata) were immunoreacted and analyzed. The sections were stained with Hematoxylin and Eosin, Cresil Violet (Nissl), Luxol Fast Blue, and Gallyas to evaluate vascular, architectural, structural tissue abnormalities, myelin loss, and neuritic plaques. For immunohistochemistry analysis, NeuN and GFAP were used to evaluate neuronal and glial compartments. Anti-phosphoTAU (AT8), anti-β amyloid (4G8), anti-αSynuclein and anti-TDP43 primary antibodies were used to assess all the main proteinopathies (Table 2). The sections were deparaffinized and pre-treated with 3%H2O2 in PBS for 10 min to neutralize endogenous peroxidase activity; only for some antibodies was a specific additional treatment necessary (Table 2). To mask non-specific adsorption sites, a preincubation with 5%of normal goat serum was performed for 30 min, and then an incubation with the primary antibodies overnight at room temperature was made (Table 2). Twenty-four hours later the sections have been rinsed in PBS and incubated with the secondary antibody (Envision + System-HRP labeled Polymer) at a dilution of 1:2 in PBS for 1 h at room temperature. After several washes in PBS a chromogen system with diaminobenzidine (Liquid DAB + Substrate Chromogen System) was used to reveal the reaction.
List of the primary antibodies, their characteristics and dilutions
Cerebral tissue membrane preparation
Binding studies in human brain membranes were carried out as follows. Briefly, membranes were freshly prepared from frozen selected brain areas. Samples were homogenized with polytron in 20 volumes of Tris HCl 50 mM pH 7.4 with protease inhibitors and centrifuged twice for 15 min at 48,000×g (Beckman JA25). The membrane pellet was resuspended in 50 mmol/L Tris/HCl buffer, pH 7.4 (50 mmol/L Tris/HCl, 10 mmol/L MgCl2, and 1 mmol/L EDTA), and incubated with 3 IU/mL adenosine deaminase for 30 minutes at 37°C, in order to remove endogenous adenosine from membranes and stored at –80°C. This suspension was used for binding experiments. Protein concentration was measured by the method of Biorad using bovine serum albumin as a standard.
Platelets isolation and membrane preparation
Blood from patients and controls (20 ml) was collected from the cubital vein and placed in vacutainers containing sodium citrate, for subsequent use to collect platelets. Anticoagulated blood was centrifuged at 800×g at 4°C for 10 min, and platelet-rich plasma was collected by centrifugation at 3000×g at 4°C for 10 min. Platelet pellets were washed with Tris-buffered saline and resuspended in a lysis buffer containing Tris–HCl, ethylene glycol tetraacetic acid, phenylmethanesul-phonyl fluoride, and protease inhibitors. Platelet aliquots were stored at –20°C until analysis. Then, membranes from platelets have been prepared as previously described and used for binding experiments [38].
[3H]ZM 241385 binding assays
ZM 241385 (Campro Scientific, specific activity 20 Ci/mmol), is a potent and selective A2A receptor ligand [52]. Briefly, in saturation experiments, membrane homogenates from the human brain and platelets of AD patients and control subjects (80–100μg of protein/assay) were incubated in duplicate with 10–12 different concentrations of [3H]ZM 241385. Non-specific binding was determined in the presence of 1μM ZM 241385. Bound and free radioactivity were separated, after an incubation time of 120 min at 4°C, by filtering the assay mixture using a Brandel cell harvester.
Statistical analysis
Using dissociation equilibrium constants for saturation binding, affinity or Kd values, and the maximum densities of specific binding sites, Bmax values were calculated for a system of one- or two-binding-site populations by nonlinear curve fitting. The analysis was carried out using the GraphPad Prism 5.0 statistical software package (GraphPad Software, La Jolla, CA, USA), and differences were considered statistically significant with a p value less than 0.01. A one-way analysis of variance (ANOVA), followed by Dunnett’s test when required, was used to compare measurement data among controls and AD patients.
Bias
Blood samples came from AD outpatients with mild to moderate dementia (CDR 1-2), while brains belonged to patients with very severe dementia (CDR 4-5). This was expected, considering that death occurs normally when the clinical picture is very serious. Nonetheless, the two groups were studied separately with matched controls, and we investigated the receptor expression in parallel in the brain and blood, to verify whether the trend was similar. AD outpatients and controls from AVIS Blood Bank had a quite different mean age. To counteract this possible bias, a comparison of A2A adenosine receptors expression in different age groups was performed, demonstrating that age, per se, did not affect receptor distribution on platelets.
RESULTS
Immunohistochemical characterization of brain samples from AD patients and control subjects
The AD cases showed a typical high AD pattern characterized by abundant amyloid deposition in the cortex (Fig. 1C), basal ganglia, hippocampus (Fig. 1F), brainstem, and cerebellum. Diffuse, dense, and cored plaques were homogeneously distributed throughout the cortex and cornu ammonis of the hippocampus. Leptomeningeal and parenchymal cerebral amyloid angiopathy was also appreciable in some cases (Fig. 1C). Phospho tau immunoreactivity was clearly evident in hippocampus (Fig. 1E), subiculum (Fig. 1I), entorhinal cortex, and frontal cortex (Fig. 1B). The AT8 antibody revealed neurofibrillary tangles, neuritic plaques, and threads both in the cortex and in the hippocampus. Frequent neuritic plaques have been well identified also with Gallyas staining (Fig. 1A, H). Control cases, without any cognitive disorders, showed negative or very weak and rare immunoreactivity for both 4G8 and tau in all analyzed areas (Fig. 1D, G, J), (Table 1).
First row: frontal lobe; second row: hippocampus; third row: subiculum. A, B, C, E, F, H, and I include Alzheimer’s disease cases; D, G, and J show the neuropathological picture of a control case. Frequent neuritic plaques are present in the frontal cortex (A) and subiculum (H) of BB137 and BB138 cases (Gallyas staining). AT8 antibody against phosphoTau deposits show neurofibrillary tangles, neuritic plaques, and threads in the frontal cortex (B), subiculum (I), and hippocampus (E) of BB47 and BB37 cases. Widespread amyloid deposits are detectable with 4G8 antibody throughout the frontal cortex (C) and hippocampus (F) of BB37 and BB293 cases with diffuse, dense, and cored plaques. Amyloid angiopathy is also detectable in case BB37 (C, arrow). Control case (BB 71) shows negative immunoreactivity for 4G8 in frontal cortex and hippocampus (D and G) and for AT8 in subiculum(J). Scale bar: 116μm (A); 312μm (B-J).
Expression of A2A adenosine receptors in brain samples from AD patients and control subjects
To investigate whether the A2A adenosine receptors could be relevant in AD pathology, we evaluated their protein expression in 6 postmortem brains of AD patients in comparison to 4 control subjects. The cerebral regions under analysis were 3 for each individual and included FWM, FGM, and H/EC. The expression of A2A adenosine receptors was studied through binding experiments performed with the high affinity radioligand [3H] ZM 241385. Figure 2 shows saturation curves towards A2A adenosine receptors in membranes from FWM (A), FGM (B), and H/CE (C) revealing a density (Bmax) of 174±29, 219±33, 358±84 fmol/mg of proteins, respectively, in AD patients in comparison to 104±16, 103±19, 121±20 fmol/mg of proteins, respectively, in control subjects. Both affinity (KD, nM) and BMAX values of [3H] ZM 241385 binding to A2A adenosine receptors in membranes from FWM, FGM, and H/EC were significantly higher in AD versus control subjects (Table 4, p < 0.01). Interestingly, only in AD cases we found a statistically significant difference between the expression of A2A adenosine receptors protein levels in H/EC versus both FGM and FWM areas, with a higher level of protein in H/EC membranes (p < 0.01).
Saturation curves of [3H] ZM 241385 binding to human A2A adenosine receptors on FWM (A), FGM (B), and hippocampus/EC (C) membranes from one AD patient and one control subject. Scatchard plots of the same data are shown in the inset. Results represent the mean±SE from one control subject and one AD patient. *p < 0.01 versus control subject.
Expression of A2A adenosine receptors in cerebral tissues of AD patients versus control subjects
*p < 0.01 versus CTR #p < 0.01 versus AD FGM §p < 0.01 versus AD FWM.
Expression of A2A adenosine receptors in human platelets from young and old control subjects
Before to perform binding experiments in platelets derived from AD patients, with a median age of 78.5 years, an important point to be addressed was the choice of control subjects for comparison. They were usually provided by the AVIS blood bank, having donors with an age in the range 20–70 years. Therefore, the influence of age in the expression of A2A adenosine receptors in human platelets was investigated. Membranes from young (26±7 years) and old (61±4 years) control subjects were used as a substrate for saturation binding experiments of [3H] ZM 241385. Our results show that affinity and density values of [3H] ZM 241385 binding to A2A adenosine receptors did not change with age, having KD and BMAX values of 0.69±0.08 nM and 99±11 fmol/mg of protein in young in comparison to 0.72±0.10 nM and 93±6 fmol/mg of protein in old donors, respectively (Fig. 3A).
Saturation curves of [3H] ZM 241385 binding to human A2A adenosine receptors on platelets membranes from one control subject and one AD patient. Scatchard plot of the same data are shown in the inset. Results represent the mean±SE from one control subject and one AD patient. *p < 0.01 versus control subject.
Expression of A2A adenosine receptors in human platelets from AD patients and control subjects
To evaluate the possibility of A2A adenosine receptors alterations in blood of patients with AD, we measured the affinity and density parameters of A2A adenosine receptors binding sites in their platelets. [3H] ZM 241385 saturation binding experiments in platelets from AD patients revealed KD and Bmax values of 1.14±0.19 nM, 214±17 fmol/mg of protein respectively, in comparison to the parameters obtained in platelets from control subjects, having KD and Bmax values of 0.80±0.10 nM and 95±4 fmol/mg of protein, respectively (Fig. 3, p < 0.01). Interestingly, there was a trend toward a lower A2A adenosine receptors density value in platelets from AD males (Bmax of 188±24, N = 13) in comparison to platelets from AD females (Bmax of 239±22, N = 13), even if it did not reach a statistical significance.
DISCUSSION
The first principal finding of this study is that there was an increase in A2A adenosine receptor density in all the cerebral regions investigated, in AD patients versus matched control subjects, not affected by cognitive impairment (Table 4, p < 0.01). Moreover, in AD cases we found a significantly higher density of A2A adenosine receptors in H/EC in comparison with both FGM and FWM areas (p < 0.01); it is noteworthy that such a difference was not detected in control subjects, being probably related to the presence of AD pathology. Indeed, A2A expression appears to reproduce the same pattern of AD pathology that spreads in the brain from the H/EC structures to other cortical areas with the highest density of pathology in the H/EC areas [4]. Accordingly, the lowest A2A expression is in FWM where the AD pathology is less pronounced. Although to a lesser degree, A2A receptors are also present in the FWM portion, where glial cells predominate, confirming that such receptors may have a role in AD glial alterations. Indeed, in the FWM of AD cases there is a more robust expression of A2A adenosine receptors than in control subjects, according to literature data [54]. In addition, a rapidly expanding scientific evidence has demonstrated an overexpression of A2A adenosine receptor in frontal cortex of aged rats and AD patients, as well as in hippocampus of transgenic or aged mice [12, 55]. There is general consensus that A2A adenosine receptor overexpression is crucial to trigger synaptic and cognitive problems. Recently, in an A model of early AD, an increase of ATP, associated to ecto-5’-nucleotidase (CD73)-induced adenosine generation, has been reported in hippocampal synapses, leading to defective reference memory, impaired LTP and decreased synaptic markers. All these modifications were reverted following CD73 or A2A adenosine receptor inhibition [55]. Notably, the abnormal accumulation of Aβ is responsible for important detrimental effects, including oxidative stress injury, which then results in neuronal death [56]. Therefore, inhibition of Aβ-induced neurotoxicity may be a promising therapeutic approach to counteract AD occurrence. In this context, the A2A adenosine receptor may be employed as a target for drug development in AD, a process that may be accelerated by the presence on the market of the A2A adenosine receptor antagonist istradefylline, for which safety has been already established. Indeed, Istradefylline was recently approved for Parkinson’s disease therapy in Japan (Nouriast) and in the US (Nourianz) [57], being able to protect from cerebral insults. The A2A adenosine receptor is essentially located in the striatal area, where it plays a crucial role in motor control but, according with our results it is also distributed in other brain regions, like the frontal cortex and hippocampus, relevant for memory and cognition.
Considering the peripheral district, platelets from AD patients present a significantly higher expression of A2A receptors than platelets from control subjects (p < 0.01). These observations suggest that A2A adenosine receptors of platelets might be a peripheral indicator of brain AD pathology and deserve further investigations as a promising marker of disease. Indeed, A and phospho-tau, the main determinants of AD pathology, start to accumulate more than a decade before the hallmark symptoms of dementia become evident, arousing the need for early diagnostic markers of disease, and possibly for new therapeutic targets able to modify pathology progression. A novel candidate suitable for both these aims seems to be the A2A adenosine receptor, playing a crucial role in neurodegeneration. An ideal AD biomarker should offer the ability to monitor disease progression with easy accessibility, without being biased by age, compensatory mechanisms, or treatments. It is recognized that peripheral-blood-mononuclear-cells (PBMCs) reflect related changes in the CNS and are a useful and accessible tool to obtain more insight into the pathogenesis of AD [58]. Specifically, platelets contain the whole A PP that is released in blood serum and secrete in blood an A peptide, comparable to A present in AD patients’ plaques [59]. Adenosine receptors in peripheral blood are a mirror of the central compartment in different pathologies, e.g., heart failure, respiratory failure, Parkinson’s diseases, and colon cancer [39–41, 60]. For these reasons, we evaluated the density of A2A adenosine receptors in platelets obtained from patients affected by AD in comparison to control subjects. The second finding of the present study is that the increased expression of A2A adenosine receptor subtype observed at central level in different area of human brain from patients affected by AD was reflected in a significant upregulation of this adenosine receptor subtype in platelets obtained from AD patients. Interestingly, AD females showed a tendency toward higher levels of A2A adenosine receptors in comparison to those expressed in platelets from AD males, suggesting the need to increase the size of the sample under study to more in deep investigate this point.
In the past, a significant increase of A2A adenosine receptor subtype has been found in platelets from patients affected by Huntington’s disease, indicating a potential link between adenosine receptor expression and the brain dysregulation present in a neurodegenerative process [61]. Moreover, A2A adenosine receptor messenger and protein were increased in PBMC from patients affected by mild cognitive impairment, indicating a role for adenosine in the first phases of AD development [62, 63]. Few years later, the same group of researchers demonstrated a downregulation of the A2A receptors’ mRNA in PBMC from elderly patients with vascular dementia compared to patients with AD or amnestic mild cognitive impairment but with no conclusive results about the protein expression and membrane density of the receptors [64]. Our results on brain tissue and platelets confirm the role of the A2A receptors in AD pathology, and demonstrate that A2A receptors are probably involved also in the intermediate and advanced phases of AD. All these observations support a different and complex role of the central and peripheral A2A adenosine receptors in different type of dementia, such as vascular dementia and AD, involving translational and posttranslational cellular processes, and possibly reflecting the different neuropathological changes. The present study reports for the first time the characterization of A2A adenosine receptor in the neuronal tissue from AD patients as well as the expression of this adenosine receptor subtype in platelets, derived from blood, at peripheral level of the AD patient. Therefore, we suggest that platelets from AD patients may be used as a mirror of what happens in the central neurological compartment. Interestingly, it has been reported that platelets share common mechanisms of signaling with the brain, such as the presence of a functional serotonin transporter able to bind numerous neurotransmitters and the existence of the enzyme monoamine oxidase B [42]. In addition, a similar mechanism is responsible for the release of neurotransmitters or proteins in the brain or from platelets, respectively [42]. All this evidence leads to the hypothesis that neurological pathologies such as AD, rather than PD or depression, could be studied in platelets as a helpful cellular model [64]. Obviously, platelets could be considered better than central tissue due to the easier way to get them respect to the central compartment. Therefore, even for their analogies with neurons, platelets are adequate candidates to become a biological substrate for the diagnosis of a neurological disease such as AD, and A2A adenosine receptors expressed in this blood element represent the best biomarker candidate.
In conclusion, although showing cross-sectional data, this study supports a role for A2A adenosine receptors as a marker of AD pathology and drug target for novel therapies able to modify the progression of dementia. To better define their role as a biomarker, longitudinal studies would be appropriate to verify the changes of A2A receptors in the platelets of subjects who pass from a normal cognitive state to a picture of mild neurocognitive disorder to a subsequent picture of dementia. At the same time, it will be important to compare brains with different stages of pathology, to verify if there is a progression in the expression of adenosine receptors that can be correlated with the severity of neuropathological picture.
