The quantification of human and rodent sAPPα was achieved by adapting the HTRF technology developed by Cisbio international company (Bagnol ssur Cèze, France). It is a single step immunoassay that employs two primary antibodies. The first antibody for both tests is directed to an epitope in the N-terminal domain of the APP protein, which is common to humans and rodents (22C11; MAB 348 from Chemicon, Billerica, MA, USA), and coupled to Europium cryptate, a donor fluorophore. The second antibody in each assay is specific for the C-terminal domain of either h-sAPPα (6E10) or r-sAPPα (SIG-39153; both antibodies are from Signet laboratories, and purchased from Covance, Dedham, MA, USA). Both of the secondaries antibodies were coupled to an acceptor fluorophore, such as D2 (Figure 1). When the donor and acceptor fluorophores are brought together by a biomolecular interaction, i.e., when sAPPα is present in the sample, a portion of the energy captured by the donor fluorophore during excitation is released through a fluorescence emission at 620 nm, while the remaining energy is transferred to the acceptor. This energy is then released by the acceptor as a specific fluorescence at 665 nm and is proportional to the concentration of sAPPα in the sample.
Recombinant h-sAPPα, r-sAPPα and h-sAPPβ were produced in bacteria using a p-Gex vector and were purified from bacterial lysates using the glutathione-S-transferase system (GE Healthcare, Uppsala, Sweden), as previously described . Their purity was checked by 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA). The Aβ1-42 peptide was purchased from Bachem (Weil am Rhein, Germany). Fluorophore labeling of antibodies was done by Cisbio.
The assay was performed in a final volume of 20 μl in a 384-well white plate (Proxiplate; Perkin Elmer, Waltham, MA, USA). 5 μl of conditioned medium from SH-SY5Y neuroblastoma cell cultures or from mouse neurons in primary culture, 5 μl of rodent CSF dilutions (1/2.5, 1/5, 1/10), 5 μl of human CSF dilutions (1/2.5, 1/5, 1/10, 1/20), or a calibration curve (human or rodent recombinants APPα, at concentrations of 3–200 ng/ml) in a 50 mM phosphate buffer pH 7.0 containing 0.2% BSA and 10 mM DTT, were added to 5 ng of the cryptate conjugate antibody and 20 ng of the D2 conjugate antibody. Both antibodies were diluted in 50 mM phosphate buffer pH 7.0 containing 0.4 M potassium fluorure. All measurements were performed in duplicate. For the negative control, phosphate buffer replaced the calibration curve. The plate covered with a plate sealer was centrifuged for 1 min at 1000 x g and incubated overnight at room temperature. Then, the plate sealer was removed, and the plate was read on a compatible HTRF reader (Envision; Perkin Elmer, Waltham, MA, USA) at 620 and 665 nm. Results were calculated from the 665 nm/620 nm ratio and expressed in delta F: Delta F = ([test sample or calibration sample 665 nm/620 nm ratio − negative control 665 nm/620 nm ratio]/[negative control 665 nm/620 nm ratio])×100. Delta F is proportional to the sAPPα concentration.
SH-SY5Y human neuroblastoma cells were obtained from the ATCC and maintained at 37°C under 5% carbon dioxide/95% humidified air incubator in a DMEM medium, supplemented with Glutamax, 10% fetal calf serum (FCS), 0.1% penicillin/streptomycin. Cells were plated (3 × 104 cells per well) in 96-well plates coated with 10 μg/ml collagen. The next day, when cells were 60–80% confluent, the conditioned medium was discarded and replaced by the same medium containing 1% N2 (Gibco, Paisley, UK) in place of FCS. Then, 10 μM GF-109203 (Sigma, St. Louis, MO, USA) and 30 μM TAPI 0 (Calbiochem, San Diego, CA, USA) were added 1 hour before adding 0.3 μM PDBu (Sigma, St. Louis, MO, USA) and the cells were incubated for 24 hours. After this period, conditioned media were collected, centrifuged at 800 ×g for 5 min at room temperature to remove any dead cells and the supernatant was immediately supplemented with 1X complete protease inhibitor cocktail (Roche, Bâle, Switzerland) and 5 μl duplicate aliquots were tested in the h-sAPPα assay or were stored at −20°C until the h-sAPPα assay.
Cortical neuron primary cultures were obtained from E16 Swiss mouse embryos. Neurons were grown in a defined medium free of serum and supplemented with hormones, proteins, and salts as previously described . Briefly, dissociated cells were plated (5 × 104 neurons per well) in 96-well plates coated with 1.5 μg/ml polyornithin. After six days in vitro, 60% of the conditioned medium was discarded and replaced by fresh medium. Then, 30 μM EGCG or 1 μM PACAP-27 (Sigma, St. Louis, MO, USA) were added for a 24 hours incubation. At the end of this period, conditioned media were collected, centrifuged at 800 × g for 5 min at room temperature and the supernatant was immediately supplemented with 1X complete protease inhibitor cocktail and 5 μl duplicate aliquots were tested in the r-sAPPα assay or were stored at −20°C until the r-sAPPα assay.
60 patients were recruited from the Clinical and Research Memory Center (CMRR) in Paris Lariboisière Hospital, which is specialized in the care management of patients with cognitive disorders. We included in this study AD and non-AD patients for whom a lumbar puncture (LP) had been performed between November 1st, 2008 and November 1st, 2011 to investigate potential cognitive disorders. All patients had an extensive examination including clinical and neuropsychological evaluations, biological measurements and brain imaging. Based on all available information, patients were classified into two groups (32 AD and 28 non-AD). Complex or unclear cases were discussed, and diagnoses were then set by a multidisciplinary team of neurologists, geriatricians and neuropsychologists. AD patients fit the criteria for probable AD, as defined by the NINCDS-ADRDA , and had biochemical CSF results associated with AD (low Aβ1-42 levels, and high total Tau and pTau181 levels) [3–6].
Non-AD patients were diagnosed with other cognitive disorders, according to validated criteria, including: vascular dementia (n = 7), frontotemporal degeneration (n = 3), depression (n = 2), anxiety (n = 4), Lewy body disease (n = 2), intoxication (n = 1), supranuclear palsy (n = 1), chronic hydrocephalus (n = 2), alcoholism (n = 5), and sleep apnea syndrome (n = 1). Patients with unknown clinical diagnoses or who were not able to undergo testing or a magnetic resonance imaging were excluded. There was no Mild Cognitive Impairment patients in our study. LPs were performed on fasting patients in the month following their clinical diagnosis. CSF was collected in 12 ml polypropylene tubes (TC10PCS; CML, Nemours, France) in standardized conditions and transferred within 4 hours to the laboratory. It was then rapidly centrifuged at 1800 × g for 10 min at 4°C. A small amount of CSF was used for routine analysis, including total cell count, bacteriologic exam, total protein and glucose concentration. CSF was aliquoted in polypropylene tubes of 1 ml and stored at −80°C until use for further analysis.
Measurements of Aβ1-42, Tau and pTau181 levels in CSF were performed by sandwich ELISA according to the manufacturer’s protocols (Innogenetics, Ghent, Belgium). A neurobiological AD profile was established for all the patients in this study.
SDS-PAGE and western blotting analysis
Human and rodent sAPPα recombinant proteins (0.5 μg), were separated in 10% SDS-PAGE (Criterion; Bio-Rad, Hercules, CA, USA), and stained with SimplyBlueSafeStain (Invitrogen Life Technologies, Paisley, UK).
For western blotting analysis, 0.6 ml of conditioned medium at six days in vitro from 4 × 105 cortical neurons primary cultures were centrifuged at 800 x g for 5 min at room temperature. The supernatant was precipitated overnight at −20°C with five volumes of a cold mixture 10% trichloracetic acid/90% acetone. After centrifugation at 15,000 × g for 20 min at 4°C, the pellet was washed with cold acetone and dried. Then, the pellet was resuspended in Laemmli buffer and an equal volume was loaded in two wells in 10% SDS-PAGE. After migration, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore, Billerica, MA, USA) and blocked in 5% low fat milk in Tris buffer saline, 0.02% Tween 20 (TBST). Membranes were incubated overnight with the appropriate primary antibody, 22C11 (1 μg/ml) or the rodent specific SIG-39153 (2 μg/ml) in TBST. Then after 5 washings in TBST, species-specific peroxidase-conjugated secondary antibodies were incubated 45 min at room temperature. After 5 washings the peroxidase signal was visualized using ECL (Western Blotting Detection Reagents; GE Healthcare, Uppsala, Sweden).
Human cerebrospinal fluid (4 μg protein) was diluted in Laemmli buffer and loaded in 10% SDS-PAGE. After migration, and transfer onto PVDF membranes, the membranes were processed as above with the appropriate primary antibody, 22C11 (1 μg/ml) or human specific 6E10 (2 μg/ml).
All animal procedures followed the French and European Union regulations. The protocols of animal anesthesia and biological fluid punctures were performed according to French government ethical laws decree 86/109 and approved by the local Ethics Committee (Direction départementale des services vétérinaires de Paris, service de la protection et santé animales et de la protection de l’environnement), permit number: 75–354.
For the clinical study, the research project was approved by the Ethics Committee of Paris University Hospitals (CEERB Bichat University Hospital, Paris, France). All patients or caregivers gave their written informed consent for CSF assessment.
Mouse CSF sampling
We used seven triple transgenic mice (3xTg AD, 12-months-old) harboring three mutant genes: β-amyloid precursor protein (APPswe), presenilin-1 (M146V) knock-in, and Tau (P301L)  and five non-transgenic controls (14-months-old). CSF from anesthetized mice was taken from the cisterna magna using a glass capillary, according to the method of Fisher et al. . The procedure was performed to keep blood contamination to a minimum. Samples of 2 to 5 μl were transferred directly to a microvial, centrifuged, and the supernatant immediately frozen on dry ice, and then stored at −20°C until rodent and human sAPPα assays were performed.
Statistical differences between groups were analyzed using the unpaired Student’s t-test. Differences were considered significant if p < 0.05 (GraphPad Prism software, La Jolla, CA, USA).