Abstract
Our attention is focused on the study of a new model based on the red blood cell (RBC) and on its interaction with amyloid beta peptide 1-42 (Aβ). RBC are highly deformable to assist blood flow in the microcirculation. For this reasons RBC abnormalities could contribute to Alzheimer’s disease (AD) by obstructing oxygen delivery to brain, causing hypoxia. In our work, considering that RBC membrane contains, among blood elements, higher acetylcholinesterase (AChE) levels, we can assume that in blood occurs a mechanism similar to the one which occurs at the neuronal level leading to an increase of Aβ toxicity mediated by its binding with AChE, located on the RBC external face. Furthermore, since mechanical properties of RBC membrane are regulated by a number of molecular components of signalling and/or regulatory pathways, of these, particular interest has been addressed toward Nitric Oxide (NO) metabolism, due to its dependence to AChE.
Keywords
Introduction
Oxidative stress has been a key topic of research concerning cancer, aging, heart diseases, arthritis, diabetes, and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases (AD) [5]. Mechanisms proposed for the neurodegeneration in the brain of AD patients generally focus on the amyloid beta peptide 1-42 (Aβ), a proteolytic product of the ubiquitously distributed amyloid precursor protein (APP) [24, 43].
Neurons use 20% of the total oxygen consumed, therefore the potential contribution of impaired oxygen delivery to the brain, causing associated neuronal AD dysfunction, has been considered an important factor in AD. It has been proposed that AD may originate as a vasculature disorder with the resultant impairment of oxygen delivery and oxidative changes initiating the cascade of neuronal changes found in AD [19]. Oxygen delivery to the brain requires that red blood cells (RBC) deform to pass through the narrow capillaries that supply oxygen to brain. Vascular changes associated with amyloidosis are expected to directly impair blood flow.
In addition to Aβ peptide deposition in neurons, Aβ peptide is generated at high levels in platelets [9] and it is present at nanomolar concentration in blood [44]. RBC are also exposed to amyloid peptides on the luminal surfaces of cerebral microvessels [20] and have been reported to sequester monomeric Aβ [28]. Previous in vitro and in vivo studies showed that important parameters indicative of RBC function and integrity might be negatively affected in AD RBC. In fact, changes in the physical state of membrane proteins [25], alteration of Ca++ permeability [16], alteration of the antioxidant enzyme activities [13], morphological perturbations [2, 23] and an altered ATP efflux [34] have been described in AD RBC.
Our earlier studies [10], show that Aβ affects RBC metabolism. Growing evidences suggest that many abnormalities in vascular could be responsible for AD [11]. In particular, the reduced deformability is one of the most suspected events associated to vascular abnormalities in AD [35, 42]. Nitric oxide (NO) was proposed to be a regulatory factor of RBC mechanical properties [4] and a functional endothelial type nitric oxide synthase (eNOS), which is located in plasma membrane has been reported to be expressed in RBC [8, 27].
Acetylcholinesterase (AChE; EC 3.1.1.7), also known as AChE or acetylhydrolase, is a hydrolase that catalyses hydrolysis of the neurotransmitter, acetylcholine (Ach). AChE is found mainly at neuromuscular junctions and cholinergic brain synapses, where its activity serves to terminate synaptic transmission. It belongs to carboxyl esterase family of enzymes. It is reported that dimeric (G) AChE forms are present in the human RBC [38] and they can be considered among blood elements, cells with the highest membrane-bound enzyme AChE expression [49]. RBC AChE has been found to be firmly attached to the membrane components [31]. AChE has been widely exploited as a primary target of action by organophosphorus compounds such as nerve agents [40]. As a reliable indicator, it is used in the diagnosis of poisoning caused by reversible and irreversible inhibitors including heavy metals and pesticides. The proper binding mechanism of the ACh with AChE has been well documented by kinetics as well as molecular modelling studies with different inhibitors.
RBC AChE, could represent a favorite target for Aβ in blood, similarly to neuronal AChE, that plays a pathogenic role in AD by influencing the mechanism leading to Aβ toxicity [1, 22].
For AChE enzyme, previous data indicate a dependence between external AChE conformational status and NO metabolism, involving biochemical events such as protein kinase C (PKC) activity and band 3 phosphorylation levels [6, 47].
Thus, the aim of this study was to clarify the role played by AChE in Aβ-dependent abnormalities in RBC morphology, that could be responsible for AD by obstructing oxygen delivery to brain causing hypoxia.
Materials and methods
Chemicals
Amyloid beta peptide 1-42 (Aβ) were purchased from Peptide Speciality Laboratories GmbH (Heidelberg, Germany). Analysis of the peptides by reverse-phase high-performance chromatography and mass spectrometry, as supplied by manufacturer, revealed a high degree of purity (>98%). Aβ peptides were dissolved in 100% 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, Sigma) (final concentration, 1 mM) to eliminate any aggregate forms that might have been present. The HFIP was then removed by vacuum evaporation, and the remaining film of disaggregated peptide was dissolved in DMSO, and used immediately thereafter. In all control experiments, DMSO was added to cell cultures at the same concentrations present in the peptide solutions. All other reagents were of the highest grade and obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Ethics statement
Blood samples were obtained from Department of Laboratory Medicine, Catholic University. Blood samples were anonymized, according to the local ethics committee guidelines and Helsinki Declaration. All participants (healthy individuals of 30±5 years old, abstained from drug treatments) gave written informed consent to participate in this study.
Red Blood Cell preparation and incubation conditions
Whole blood (3 ml) was collected with citrate as an anticoagulant, and washed three times with an iso-osmotic NaCl solution. Plasma separation was obtained by centrifuging at low speed (800 g for 5 min), in order to avoid any Red Blood Cell (RBC) morphological alteration induced by mechanical stress. A density gradient centrifugation with Ficoll was used to isolate mature RBC. After washing step, 45 μl of packed RBC were re-suspended, in a final volume of 1.5 ml (haematocrit 3%) of the incubation buffer (mM Na2SO4, 90 mM NaCl, 25 mM HEPES [N-(2-hydroxyethyl)-piperazine-N1-2-ethanesulfonic acid], 1.5 mM MgCl2, glucose 5 mM). RBC suspensions (packed RBC/PBS:1 to 10, in volume) were incubated in a thermostatic shaker at 37°C gently agitated for 6 h, 18 h, 24 h in PBS with or without 1 μM Aβ. After 4°C at 2500 rpm centrifugation samples were used for biochemical and microscopical analysis. After incubation, Met-hemoglobin (met-Hb) and haemolysis levels were determined. To determine nitrite and nitrate levels and the role played by AChE on the effect of Aβ RBC suspensions were incubated for 12 h with Aβ in the presence and absence of propidium (70 μM), a peripheral anionic binding site ligand of AChE, and edrophonium (20 μM), an active site inhibitor of AChE. After incubations, RBC were washed two times with the same incubation buffer (2500 g for 3 min at 4°C) to eliminate unbound Aβ. Cell morphology was recorded in an Nikon microscope (40X objective), images were collected exploiting a digital Nikon camera (Coolpix 5400) using the software X-Pro.
Immuno-histochemical procedure
For immune-histochemical purposes 7 μL of treated and untreated RBC were coated on a microscope slide and heat fixed. Thereafter, they were washed in 0.1 M PBS, permeabilized for 30 min in 0.1% trypsin, placed in a solution of 2% hydrogen peroxide and 80% methanol PBS for 20 min, and treated with 3% milk powder in 0.1 M TBS for 30 min at room temperature. Incubation with the respective primary antibody against activated Endothelial nitric oxide synthase (eNOS) (dilution 1 : 1000, Biomol, Hamburg, Germany), for 45 min, respectively, was performed. Bloch and colleagues demonstrated that the antibody against activated eNOS (Biomol, Hamburg, Germany) is specific for the translocated active form of eNOS [3]. After rinsing with TBS, the sections were incubated with the secondary goat-anti-rabbit antibody (Dako, Glostrup, Denmark) at a dilution 1 : 400 for 30 min. For negative controls, RBC without primary antibody incubation were used. A streptavidin-horseradish-peroxidase complex (Amersham, Buckinghamshire, England) was applied as a detection system (dilution 1 : 150) for 30 min. The staining was developed using 3,3-diaminobenzidine-tetrahydrochloride solution (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M PBS. For further analysis only RBC incubated in the same immunostaining under identical conditions were compared. Magnification for all presented images is 400×.
Acetylcholinesterase enzyme
Acetylcholinesterase (AChE) activity was assayed in RBC suspensions after Aβ treatment using the colorimetric method proposed by Ellman [15]. Fluorescence spectroscopy analysis of recombinant AChE (Sigma Chemical Co. (St. Louis, MO, USA), was performed according previous study [45].
Measurement of nitrite and nitrate concentrations in intra and extra RBC compartments
One of the widely used markers for NO production in biological systems are nitrite/nitrate (NO2–/NO3–) levels because they are stable oxidation products of NO [26]. In our study, we measured NO2– in RBCs using the Griess reaction kit (Sigma-Aldrich, St. Louis, MO, USA). In brief, nitrite and nitrate concentrations were measured in the pellets after submitting each RBC’ suspension to haemolysis and haemoglobin precipitation. Haemolysis was induced with distilled water, and haemoglobin precipitation was induced with cold ethanol and chloroform. After vortex agitation, the mixture was centrifuged at 9600g for 10 min. Clear supernatants were incubated with Griess reagent according to the kit instructions. The nitrite concentrations were determined by comparison with a calibration curve (1–100 μM) of sodium nitrite in deionized water. For nitrate measurement, NO3– was first reduced to NO2– in the presence of NADPH and nitrate reductase. The concentrations of the total nitric oxide oxidation products (NO3–/NO2 –) were determined by comparison with a calibration curve (1–100 μM) of sodium nitrate in deionized water. The nitrate concentrations were determined by the difference between the total nitric oxide oxidation products and nitrite concentrations from the same aliquot.
Statistical analysis
The software Excel (Microsoft, CA, USA) was used for statistical calculation and data are expressed as mean±standard deviation (S.D.). A Wilcoxon test, a non-parametric test used to compare related samples, was used to analyze the differences of measured parameters before and after treatment. In different experiments, we used different RBC specimens that could respond differently to treatments and for this reason we have used the each RBC specimen for control and treatment tests.
Results
Red Blood Cell morphology
When human Red Blood Cells (RBC) were incubated with Amyloid beta peptide 1-42 (Aβ), cell morphology was monitored by means of a phase-contrast microscopic observation. Figure 1B, revealed that after 24-h treatment with Aβ, RBC show an altered morphology, with many irregular shapes in most of the cells. In particular, RBC developed a form characterized by blebs or protuberances over the cell membrane, markers of an accelerated ageing. In contrast, as shown in Fig. 1A, in control cells any significant morphological alteration was evident.
Nitrites and nitrates production in RBC
NO has been hypothesised to regulate RBC deformability [4]. On these basis, in order to test whether Aβ-mediated cell morphology alteration may be linked with an alteration of nitric oxide (NO) production, nitrite and nitrate levels were measured in treated and untreated RBC as markers of NO production [26]. The exposure of human RBC to Aβ alters nitrite and nitrate levels in the cell. As shown in Fig. 2A, Aβ treatment reduces nitrate and nitrite production in a time dependent manner. The maximum effect was observed after 24 h of incubation but it was already significant after 12 h. A shown in Fig. 2B, the inhibitory effect triggered by Aβ at 12 h on nitrite and nitrate production was partially recovered by propidium (70 μM), a peripheral anionic binding site ligand of AChE, but not by edrophonium (20 μM), an active site inhibitor. Propidium and edrophonium alone did not show any significant effect on nitrate and nitrite levels (data not shown).
Hystochemical detection of Endothelial nitric oxide synthase
To evidence whether the decrease in RBC nitrites and nitrates levels may be paralleled by alterations of Endothelial nitric oxide synthase (eNOS) content, staining was performed using an antibody that has been shown to specifically detect the activated form of the eNOS enzyme [3]. In cells not treated with Aβ, eNOS activity was significantly higher than in cells after 24 h Aβ exposure. The results showed in Fig. 3A and B-C provide pictures of the stained RBC for activated eNOS without (B) and with Aβ peptide (A). From analysis, it becomes obvious that activated eNOS is down-regulated after Aβ exposure in RBC
Acetylcholinesterase enzyme
Since we verified, by using specific Acetylcholinesterase (AChE) inhibitors, that Aβ-dependent effects were mediated by AChE, we subsequently investigated whether Aβ affected enzyme activity. We found that in treated RBC, Aβ inhibits AChE activity in a time dependent manner. As shown in Fig. 4A, the maximum effect was observed after 24 h of incubation, but it was already significant after 12 h. Next we investigated, by fluorescence spectroscopy, oxidation of Trp residues in recombinant AChE following Aβ exposure. As shown in Fig. 4B, the fluorescence excitation maximum of Trp in AChE at λ280 nm shows a strong signal as expected. After incubation of the enzyme with Aβ over time peak decreases, indicative of Aβ-mediated oxidation. However, even after 24 h incubation the signal is still present, suggesting that not all Trp residues are affected.
Hemolysis degree
An obvious potential source of extra-cellular nitrite and nitrates is the spontaneous RBC lysis. To determine cell lysis after experiments, RBC suspensions were analysed to evaluate haemoglobin concentration in the supernatant. The percentage of hemolysis was always less than 3%.
Discussion
The aim of this study was to investigate a mechanism of pathogenesis related to Alzheimer’s disease (AD), involving non neuronal tissues. Our hypothesis suggests that morphological abnormalities and oxidative stress [7] in circulating Red Blood Cell (RBC), following to Aβ interaction, result in an oxygen delivery impairment, especially in the brain microvasculature [37], where RBC deform to pass through the narrow capillaries that supply oxygen to brain.
Acetylcholinesterase (AChE) is also a common ligand during Aβ fibril formation process and AChE–Aβ complex was reported to be more toxic for neuronal cells than Aβ alone [22]. We observed that Aβ affects AChE activity in RBC, as previously evidenced [1, 21], suggesting that Aβ/AChE interaction could be an event shared by RBC and neurons. Our data suggest the reduction in activity of AChE can be also based on conformational changes of the enzyme via Aβ-mediated oxidation. Subsequently, we asked if Aβ-mediated AChE conformational changes could cross talk with the signaling pathways responsible for mechanical abnormalities of RBC membrane, occurring following Aβ exposure. RBC mechanical properties are known to be modulated by a number of molecular components of signalling and/or regulatory pathways [36]. Among them, nitric oxide (NO) was proposed to be a regulatory factor of RBC mechanical properties [4] by its capability to alter ion transport across membrane and physiochemical properties of the cytoskeleton. Whether this role is mediated directly by NO through interference with cytoskeletal elements or indirectly via some intermediate such as peroxynitrite [14] is unknown at this time. We observed by immune-histochemical experiments, using an antibody specific for the active form of endothelial nitric oxide synthase (eNOS) [3], that eNOS enzyme content decreases in RBC treated with Aβ. This result is in line with a previous report that evidenced the negative impact of Aβ on eNOS function in endothelial cells [18]. Recent studies demonstrated that human RBC possess an active and functional endothelial type NOS [8, 27] which is located within the plasma membrane. Therefore, it seems to be logical that in RBC, morphological changes of the plasma membrane induced by Aβ might be responsible for down-regulation of eNOS content. In support of this finding, we observed that NO derived metabolites levels (i.e. nitrite and nitrate), widely used markers for NO production in biological systems [26], are reduced in RBC treated with Aβ. It is also known that eNOS enzymatic activity is influenced by phosphorylation status [39, 41]. Mechanisms related to PKC-dependent eNOS inhibition have been explored by recent studies. Several reports indicated that PKC mediated phosphorylation of Thr495 [17, 33] or Thr497 [32] in eNOS calmodulin-binding domain, is correlated to inhibition of eNOS function and to a decreased NO production. Furthermore, it has been reported, but still not completely clarified, a dependence between PKC, cytoskeleton proteins phosphorylation degrees, NO metabolism and AChE [12, 47].
On this issue, we demonstrated that in the presence of edrophonium a specific center inhibitor for AChE [48], no alterations are observed regarding Aβ-mediated effects on NO metabolites formation. On the other hand, propidium, an exclusive peripheral anionic site ligand of AChE [46], abolished Aβ effect on NO formation, suggesting an involvement of AChE in the signaling pathway responsible for Aβ-induced eNOS inhibition.
In conclusion, our results show that Aβ impairs eNOS content in RBC and this event is linked to RBC morphological changes and membrane AChE activity inhibition. Although further experiments are required to fully understand Aβ-AChE interaction, our findings assigned to AChE, located on the external face of RBC, an important role in the Aβ-mediated effects in RBC. Therefore, the presence of high levels of AChE in RBC membranes [49], may render these blood cells more susceptible to injury by Aβ, contributing to vascular abnormalities responsible for AD. Thus, AChE located in RBC could be considered a novel target for the development of therapeutic approachesfor AD.
Footnotes
Acknowledgments
This research was supported by University of Cassino and SL, FAR 2015 to F.M.