Abstract
ADAM10 is one of the key players in ectodomain-shedding of the amyloid-β protein precursor (AβPP). Previous research with postmortem tissue has shown reduced expression and activity of ADAM10 within the central nervous system (CNS) of Alzheimer’s disease (AD) patients. Determination of cerebral ADAM10 in living humans is hampered by its transmembrane property; only the physiological AβPP cleavage product generated by ADAM10, sAβPPα, can be assessed in cerebrospinal fluid. Establishment of surrogate markers in easily accessible material therefore is crucial. It has been demonstrated that ADAM10 is expressed in platelets and that platelet amount is decreased in AD patients. Just recently it has been shown that platelet ADAM10 and cognitive performance of AD patients positively correlate. In contrast to AD patients, to our knowledge almost no information has been published regarding ADAM10 expression during normal aging. We investigated ADAM10 amount and activity in platelets of cognitively healthy individuals from three different age groups ranging from 22–85 years. Interestingly, we observed an age-dependent increase in ADAM10 levels and activity in platelets.
INTRODUCTION
ADAM10 (a disintegrin and metalloproteinase 10) is well associated with a potential protective function in regard to Alzheimer’s disease (AD) (reviewed in [1]). Data from in vitro analyses [2] as well as from animal experiments [3–5] depicted this zinc metalloproteinase as the major α-secretase in neuronal cells, capable of preventing the formation of toxic amyloid-β (Aβ) peptides. By generating neurotrophic soluble amyloid-beta protein precursor α (sAβPPα) through cleavage within the Aβ stretch, the α-secretase initiates the non-amyloidogenic pathway in contrast to β-secretase-dependent amyloidogenic processing of AβPP. Recent studies have also proposed a potential direct involvement of ADAM10 allelic variations in AD progression: mutations close by the proprotein convertase cleavage site of the enzyme found in individuals with late-onset AD (LOAD) were reported to attenuate ADAM10 function in cell culture [6]. This was confirmed in transgenic mouse models, showing that these mutations shift AβPP processing to the amyloidogenic pathway [7]. These findings are in accordance with the majority of postmortem data that reflect an overall decrease of ADAM10 mRNA, protein, and/or activity in central nervous tissue in AD patients compared to age-matched controls (reviewed in [8]). In addition, a reduction of ADAM10 in peripheral tissue such as platelets has been demonstrated [9] and the enzyme therefore has been discussed as a potential biomarker [10].
Although markers in CSF such as Aβ and tau species can be used for an accurate diagnosis today [11, 12], the necessary invasive lumbar puncture limits their use in routine checks. A more peripheral substitute is needed in order to not only advance the diagnostic toolset for AD but also to be able to diagnose more individuals effectively at earlier stages of the disease and hopefully before clinical onset. As research claims that curative strategies will be more effective the earlier they can be started [13–15], diagnosing AD in its early stages could turn the tides in combatting its fatal neurodegeneration. Platelets prepared from human blood samples seem to represent a reliable source for a diversity of potential biomarkers related to AD (as reviewed in [16]) and just recently it has been shown that the amount of ADAM10 in platelets also correlates with cognitive performance of patients as measured by, e.g., clock drawing test [17] or Mini-Mental State Examination scores [18]. However, to our knowledge, ADAM10 amount in platelets has not yet been investigated during the process of normal, cognitively healthy aging. In the present study, we analyzed protein levels of ADAM10 as well as its catalytic capacity in a cohort of cognitively normal volunteers belonging to three groups of age (mean age of 25, 65, and 80 years) to unravel the fate of the α-secretase in platelets throughout normal aging.
MATERIAL AND METHODS
Subjects
Samples of 36 cognitively healthy subjects were analyzed (for cohort characteristics, see Table 1). Subjects were recruited through advertisement in a local newspaper, as well as notices in medical practices and public institutions. The study was conducted at the Department of Psychiatry and Psychotherapy, University Medical Center of Mainz, Germany. It had been approved by the local Ethics Committee of the Landesärztekammer Rheinland-Pfalz (state medical association of Rhineland-Palatinate) and all subjects gave written informed consent. Participants underwent a preceding psychiatric screening interview (DIA-SSQ) in combination with International Diagnosis Checklists (IDCL) as described before [19, 20]. Exclusion criteria were any psychiatric, neurologic, or cognitive disease observed prior to the study or intake of medication known to influence cognitive performance. APOE genotyping was performed as described before [21]. Consenting to genotyping was no criterion for inclusion into the study, therefore not all participants were genotyped.
Blood sampling and preparation of blood components
For platelet preparation, following a protocol by [9], blood was collected in the morning by puncture of a peripheral arm vein into a 4.3 ml citrate monovette (Sarstedt, Nümbrecht, Germany). The tube was gently inverted to mix blood with anticoagulant and subsequently further processed (at the latest within 25 min of blood collection, handling occurred at room temperature (RT) at all times). To obtain platelet-rich plasma, tubes were centrifuged at 200× g at RT for 10 min and the upper layer was aspirated using a plastic pipette with large opening, carefully avoiding the buffy coat, and transferred into a new tube. Platelets were prepared by centrifugation for 20 min at 1200× g, RT. The pellet was washed twice in buffer A (sterile 10 mM TRIS-HCl, pH 7.4) followed by centrifugation at 1200× g, RT for 10 min. Following the last washing step, platelets were pelleted at 3000× g, 4°C for 15 min and then thoroughly suspended in buffer B (buffer A supplemented with a complete set of protease inhibitors (Complete Mini, Roche, Mannheim, Germany), 1 mM EGTA and 0.1 mM phenylmethanesulfonyl fluoride). Suspensions were stored at –80°C. When all samples were collected, platelet suspensions were thawed on ice and, to achieve homogenization, subjected to three rounds of freezing in liquid nitrogen and subsequent thawing at room temperature before sonication at 0°C for 15 s. An aliquot was used for protein determination by Bradford assay (Rotiquant, Carl Roth, Karlsruhe, Germany).
For preparation of peripheral blood mononuclear cells (PBMCs), blood was collected by venipuncture into evacuated collection tubes (BD Vacutainer CPT, BD Biosciences, Heidelberg, Germany) containing sodium citrate as anticoagulant. A total volume of 8 ml blood per vial was collected, mixed gently and thoroughly immediately and PBMCs were prepared according to the manufacturer’s instructions. An additional washing step was included using ice-cold red blood cell lysis solution (1.5M NH4Cl, 100 mM NaHCO3, 1 mM EDTA tetrasodium salt, pH 7.4). Finally, the PBMC pellet was resuspended in culture medium (RPMI-1640 (Sigma Aldrich, Steinheim, Germany), 5% heat inactivated FCS (Life Technologies, Carlsbad, CA, USA), 1% Penicillin Streptomycin, 1% Glutamine (Sigma Aldrich)). Protein determination of an aliquot of PBMCs was performed by Bradford assay as stated above.
Human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) was cultivated and harvested as described before (e.g., [22]).
Western blotting
For western blot experiments, 10 μg of protein dissolved in NuPAGE buffer (Life Technologies) containing 0.1 M DTT (Carl Roth) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 8% polyacrylamide gels and further transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA) by electro-blotting using a tank blot system (Bio-Rad, Philadelphia, PA, USA). After blocking the membranes in blocking solution, following primary antibodies were applied overnight at 4°C: ADAM10 rabbit polyclonal antibody (used in a dilution of 1:1000, Merck, Darmstadt, Germany), integrin beta 3 (CD61) rabbit monoclonal antibody (1:1000, Epitomics, Burlingame, CA, USA), AβPP N-terminal mouse monoclonal antibody (6E10, 1:1000; Covance, Madison, WI, USA), ADAM17 rabbit polyclonal antibody (1:500, Chemicon, Merck), BACE rabbit monoclonal antibody (1:1000, D10E5, Cell Signaling Technology, Danvers, MA, USA). Blots were developed after incubation with goat anti-rabbit or anti-mouse peroxidase-conjugated antibody (Thermo Scientific, Waltham, MA, USA) with SuperSignal West Femto ECL-reagent (Thermo Scientific). An appropriate picture was taken with a CCD-camera imaging system (Stella camera; Raytest, Straubenhardt, Germany) and quantitative analysis was carried out by AIDA image analyzer 4.26 software (Raytest). To achieve normalization and for comparing age groups, the mean values (ADAM10/CD61) of the youngest age-group on each blot were set to 1.0 and all samples were expressed in relation to that mean.
Fluorescence assay
40 μg of platelet protein were mixed with 10 μM fluorogenic peptide (sequence derived from TNFα: Mca-P-L-A-Q-A-V-Dpa-R-S-S-S-R-NH2, Cat. No. ES003, R&D Systesms, Wiesbaden, Germany) in reaction buffer (final concentrations: 25 mM TRIS-HCl, pH 8.0, 2.5 μM ZnCl2) to yield a total volume of 100 μl per well in a black 96-well plate (Greiner Bio-One, Frickenhausen, Germany). Increase in fluorescence intensity was determined with a microplate reader (Fluostar Omega, BMG Labtech, Ortenberg, Germany) at 320 nm excitation and 405 nm emission wavelength every 2 min for 160 min keeping the plate at 37°C during the measurement. A shaking step before each cycle of measurement was included to provide for a homogenous mixture of components. For comparative analysis, values obtained 30 min after initiation of the reaction were used, subtracted by a blank (buffer without protein). Analyses with recombinant ADAM10 protein (Merck, Schwalbach, Germany) were performed accordingly. To check for specificity of the obtained signal, platelet pool samples or recombinant ADAM10 were analyzed with and without the addition of 10 μM GM6001 (Merck) to the reaction and a pre-incubation of 5 min at 37°C in the dark before adding the fluorogenic peptide.
Statistical analysis
Results of group analyses were tested for statistical significance with Prism 6 (GraphPad Software, La Jolla, CA, USA) by using one-way analysis of variance (ANOVA) followed by Bonferroni’s post-test for multiple comparisons or by unpaired Student’s t test for comparing two groups, as appropriate. For correlation analyses SPSS 22 (IBM, Ehningen, Germany) was used employing Pearson’s test and partial correlation between ADAM10 and age was calculated using intelligence measures (HAWIE-R and LPS4) as covariates. Values of p < 0.05 were considered statistically significant.
RESULTS
Platelets as a tool for investigating AβPP processing
To demonstrate the value of platelets extracted from human blood for their application in AD research, we performed a western blot analysis to compare the commonly used human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266), PBMCs, and platelets. For this analysis, PBMCs and platelets were prepared from blood of the same healthy volunteer to minimize individual differences in protein expression patterns. We were able to show that proteins associated with AβPP processing are favorably expressed in platelets (Fig. 1A). When analyzing ADAM10, we observed a prominent band of about 70 kDa in platelets (188% mean intensity in healthy donor samples as compared to SH-SY5Y cells, Fig. 1B), representing the mature form of the protein, whereas a second band below 98 kDa, representing the enzyme’s pro-form, was considerably fainter. Both bands are visible in SH-SY5Y cells and PBMCs in comparable intensities. When looking at expression of ADAM17 (TACE), the amount of this alternative α-secretase [23] is considerably lower in platelets (43% as compared to SH-SY5Y cells, Fig. 1B), showing only a very faint band around 100 kDa, representing the mature protein, whereas in SH-SY5Y cells and in PBMCs both the 100 kDa band and a band around 120 kDa (pro-form of ADAM17), were detected. When visualizing AβPP with antibody 6E10, only one band was detected in platelets at around 120 kDa. This band was only faintly detected in PBMCs, whereas it was clearly visible in SH-SY5Y cells. The latter also showed a band at around 110 kDa which was not detectable in our platelet and PBMC samples. Together, these bands represent the different isoforms of the protein (AβPP695, 770, and 751) and respective maturation products. In SH-SY5Y cells, also an additional band at around 100 kDa was visible, which has been described before as an immature form of the protein [24]. Finally, the antibody used for detecting β-secretase BACE-1 produced bands both in the human cell line and in platelets, while only marginal amounts were detectable in PBMCs. In SH-SY5Y cells, bands at about 60 kDa and 75 kDa are visible, corresponding to the immature and mature form of the enzyme, respectively [25]. In platelets, we detected bands at around 55 kDa and 70 kDa. Both forms have been described in literature before [26, 27], pointing out that detection of BACE-1 is not trivial and dependent on the antibody used. In PBMCs, only the 70 kDa band was barely visible.
Taken together, this analysis demonstrates that for studying ADAM10 and its processing of AβPP in peripheral human-derived material, platelets represent a valid model. Furthermore, due to the overrepresentation of ADAM10 in platelets in contrast to ADAM17, also shown by quantification of a representative set of platelet samples (protein levels normalized to SH-SY5Y cells, Fig. 1B), analysis of ADAM10 as the α-secretase might even give a more precise result in regard to its functional readouts than in secondary neuronal cell culture.
Amount and enzymatic activity of ADAM10 in cognitively normal subjects during aging
When analyzing the amount of ADAM10 in platelets of cognitively healthy subjects by western blot we used platelet-specific CD61 (integrin beta-3) as a normalization factor (Fig. 2A). Stability of CD61 expression along aging was confirmed by western blot and statistical analysis (p = 0.656 between youngest and middle and p = 0.902 between youngest and oldest age group). Moreover, CD61 correlated well with amounts of β-actin in samples (p = 0.017, western blots not shown), corroborating its use as an age-independent house-keeping protein for platelet samples. After normalization to CD61, a significant 1.2-fold elevation of ADAM10 was detected between the youngest (arbitrarily set to 1 for comparison reasons) and the oldest group (p = 0.003), indicating an increase in platelet ADAM10 expression during the course of cognitively healthy aging (Fig. 2B). Direct correlation of ADAM10 protein amount to the age of the respective subject gave a positive correlation coefficient of r = 0.376 (p = 0.024, Pearson, data not shown).
It is noteworthy that the analyzed cohort was highly intelligent, a fact that could confound interpretation of obtained results for a general population. However, when using general intelligence (as determined by HAWIE-R) or fluid intelligence (as determined by LPS 4 test [20]) as control variables, we still observed a significant positive correlation between platelet ADAM10 and age (p = 0.036 and p = 0.031 for HAWIE-R and LPS 4 as covariates, respectively).
To check if larger amounts of ADAM10 are also accompanied by higher levels of enzymatic activity, we employed an α-secretase activity assay using an artificial pro-fluorescent peptide. Linearity of TNFα-derived peptide cleavage by ADAM10 was demonstrated by using increasing amounts of recombinant ADAM10 (fluorescence determined after 30 min, Fig. 3A). In this test, fluorescence development could also be completely quenched by applying the metalloproteinase inhibitor GM6001 showing specificity of ADAM10 cleavage. Subsequently, we used a pooled platelet sample to measure fluorescence after 30 min of incubation with the pro-fluorescent peptide, where we also saw a significant higher value than when we pre-treated the pool sample with GM6001 (Fig. 3B, p < 0.001), proving that ADAM10 activity is detectable and also relevant in platelets. The reduction in fluorescence detected after 5 min pre-treatment with GM6001 to only 37% of the control activity might be explained by unspecific cleavage of the pro-fluorescent peptide by other proteases present on a cellular membrane background, lacking in experiments with recombinant ADAM10. Therefore, measured fluorescence might not completely be a product of ADAM10-driven cleavage. However, major contribution of ADAM17-based cleavage is not predominant due to relatively much lower amounts of this alternative α-secretase observed in platelets (Fig. 1B).
To test ADAM10 activity in the platelet samples of the study cohort, we incubated them with pro-fluorescent peptide and measured fluorescence generation over time (Fig. 3C, exemplary measurements). For comparison between groups, we used the fluorescence measured after 30 min of incubation with pro-fluorescent peptide, where we saw a significant 1.2-fold increase in fluorescence from the youngest to the oldest group (Fig. 3D, p = 0.032), comparable to the elevation in ADAM10 amount observed in western blot experiments. However, a weak positive correlation between amount and activity of ADAM10 was only found when analyzing values obtained for the oldest group (Pearson r = 0.138). Therefore, further studies have to reveal if the observed increase in activity along healthy aging is solely dependent on an increase in ADAM10 protein amount or if other factors might contribute to this finding.
Correlation of platelet ADAM10 with gender and APOE genotype
According to epidemiological studies, females have a higher prevalence of developing AD [28, 29]. Therefore, we were interested in the association of platelet ADAM10 with gender within the tested age groups. A direct comparison within each age-group gave a slight trend to lower platelet ADAM10 in females than in males (Fig. 4A), although sample sizes were arguably small. When analyzing the complete cohort, no statistical difference was detected between genders (Fig. 4B, p = 0.809).
It has previously been shown that low cholesterol can promote the non-amyloidogenic pathway [30, 31] and furthermore that cholesterol binding in neurons is influenced by APOE genotype [32]. As the APOE genotype ɛ4 also resembles a major risk factor for developing sporadic AD [33, 34], we analyzed our data on platelet amount of ADAM10 in this regard. When splitting the cohort into each of the possible allele combinations, no statement on the influence of APOE makeup can be incurred (Fig. 4C). Since not all participants gave their consent for genotyping, sample sizes were smaller than for other analyses. Furthermore, single allele combinations (e.g., ε2/ε2) were greatly underrepresented in comparison to other combinations (e.g., ε3/ε3), which is in accordance with global allele distribution in populations [35, 36]. However, a comparison between subjects carrying at least one ε4 allele and those who have none (carrier and non-carrier) also gave no significant influence of carrier status on platelet ADAM10 amount (Fig. 4D, p = 0.959).
DISCUSSION
The search for biomarkers in neurodegenerative diseases such as AD is of utmost importance in order to clearly define and diagnose onset of disease. Platelets came into focus lately as a valuable source of potential peripheral biomarkers [16] because of a biochemical resemblance with neurons which can be demonstrated for example by their potential to release neurotransmitters such as serotonin [37] or glutamate [38]. As previous studies have also demonstrated [26], we saw in our own study that AβPP, the α-secretase ADAM10 and β-secretase BACE-1 are expressed in platelets, while the second α-secretase candidate, ADAM17, is only marginally expressed. Furthermore, a reduction in platelet ADAM10 and the product of its cleavage of AβPP, sAβPPα, correlating with CSF status, were previously reported in AD patients [9]. As recent studies have elucidated, lower platelet ADAM10 correlates well with cognitive decline in AD patients [17, 18]. One of the striking results of these studies undisputedly is that a peripherally expressed protein can be used to infer pathogenesis of the CNS. However, to our knowledge, there is no data available concerning platelet ADAM10 during the course of aging. Only one publication reports on ADAM10 during normal aging where the authors found that the amount of α-secretase was increasing with age [39]. For that matter, Bernstein and colleagues compared the amount of ADAM10 in neurons of the temporal cortex in brains of stillborn children and those of normal aged adults. In our work presented here, we used blood samples of a cohort of young, middle-aged and older adults and observed an increase in peripheral ADAM10. Deregulation of ADAM10 has been shown to play a key role in disease pathogenesis [40] and a moderate elevation of this secretase has been demonstrated to ameliorate Aβ plaque load at least in mice [5, 41]. We therefore postulate that the α-secretase might contribute to or is a prerequisite for cognitively healthy aging.
A relationship between the age-dependent depletion of sex hormones [42] and AD pathogenesis is generally accepted and the effects seem to be more dramatic in women than in men [43]. As sex steroids were found to act in a neuroprotective way [44], hormone replacement therapy (HRT) was evaluated as a potential preventive strategy in post-menopausal women. Outcomes of these studies were controversial and a meta-study came to the conclusion that HRT was even contra-indicated [45]. In the cohort we analyzed, no statistical difference in platelet ADAM10 content between genders was observed, pointing at no direct correlation between sex hormones and the peripheral expression of the α-secretase. However, there will have been a certain difference in hormone balance between analyzed groups. Younger individuals (as in the first group) have different levels of sex hormones than older, probably post-menopausal ones (as in the middle aged or the oldest group). Also, one has to keep in mind that the data set we analyzed is rather small and further studies might be needed to verify these results.
The APOE ɛ4 genotype is largely associated with an increased risk of developing AD, whereas the ɛ2 allele is considered as being protective (reviewed in [34]). However, we did not find a relationship between platelet ADAM10 and different APOE makeups in the analyzed cohort. But, as was also true for gender, the analyses might be too preliminary in relation to sample size as to make profound assumptions.
The main finding of the present study is that platelet ADAM10, a factor previously well established to be a potential peripheral correlate to brain status, is increasing during cognitively healthy aging. Together with previous findings from other groups that platelet ADAM10 decreases in AD patients, this might indicate that the α-secretase plays a role in enhancing resilience to neurodegenerative processes. Just recently, the reduction of platelet ADAM10 has been shown to not be caused by reduced mRNA levels [46]. In the present study, we did not assess ADAM10 mRNA levels and therefore are not able to contribute data to elucidate potential underlying mechanisms regarding the increase in α-secretase amount and activity. Combined with the knowledge that despite of their anucleate nature, de novo protein synthesis based on preservation of mRNA is a crucial feature of platelet function [47], this provides fertile ground for further investigations in search of pathways responsible for regulation of ADAM10 in platelets and also in tissue of the CNS.
As different factors such as FoxO3 [48] or klotho [49] have been lately associated with healthy aging and resistance to age-related diseases, a whole new field is currently emerging searching for protectors against degenerative diseases. For example, the cleavage product of klotho is thought to act as a hormone exhibiting anti-aging properties [50]. As klotho has also been reported to be a substrate of ADAM10 [51, 52], an elevation of α-secretase expression in the course of healthy aging might also contribute to the promotion of klotho’s positive properties.
Within this study, we aimed for a naturalistic setting in order to provide results of a representative sample of cognitively healthy individuals. Future studies will also have to elucidate potential lifestyle factors and pharmaceuticals influencing ADAM10 in order to further investigate its potential role as a resilience factor. In this regard, investigations concerning a relationship between cognitive performance and platelet ADAM10 in healthy subjects would be highly interesting.
Footnotes
ACKNOWLEDGMENTS
We are very grateful to participants of the study. Furthermore, the authors would like to acknowledge the help of the study center of the Department of Psychiatry and Psychotherapy with blood drawings. The excellent assistance of K. Duckro in preparing PBMCs is also acknowledged. This study was funded by a scholarship from the Focus Program Translational Neuroscience (FTN), University Medical Center Mainz, Germany, issued to F. Schuck.
