Increase of α-Secretase ADAM10 in Platelets Along Cognitively Healthy Aging

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.

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).