In vitro experiments
Cell culture
Human neuroblastoma SH-SY5Y cells and human neuroblastoma SH-SY5Y-APPwt cells stably transfected with DNA constructs harboring human wild-type APP695 (APPwt) were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 °C [28]. Cells were seeded into plates or 100 mm dishes in Dulbecco modified Eagle medium, supplemented with 10% (vol/vol) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (Fisher Scientific, France) and for the transfected cells with 300 μmg/mL hygromycin (Sigma-Aldrich, France), a selective antibiotic. Between 70 and 90% confluency, cells were incubated during various time periods and/or with various concentrations of compounds to be tested.
RNA isolation and quantitative PCR
Total RNA was extracted from SH-SY5Y cells after 0, 30, 60 and 90 min of treatment with 30 μM 5-HIAA, using the Nucleospin RNA L protocol (Macherey-Nagel, Düren, Germany). This protocol included a treatment of isolated RNA by DNase I. Integrity and purity of RNA was checked by spectrophotometry.
Reverse transcription was performed with 1 μg RNA using Biorad iScript® cDNA synthesis kit. q-PCR was performed in an iCycler thermal cycler (Biorad, Hercules, CA, USA) using SYBR Green dye (iQ SYBR green Supermix, Biorad). For each sample, the reaction mix was a make-up of 320 nM forward primer (F), 320 nM reverse primer (R), 200 nM probe, and 4 μL cDNA template in a total reaction volume of 20 μl [3]. Using the iCycler iQ optical system software (version 3.1, Bio-Rad), a standard curve based on successive cDNA dilutions was performed and was used to calculate starting quantities. To ensure a thorough calculation, starting quantities of genes of interest were reported to those of a housekeeping gene (U6 or Actin) in the same plate.
All samples were analyzed in triplicates, and the mean and standard deviation were calculated. After each q-PCR, specificity of the amplification was controlled by a melting curve ranging from 55 to 95 °C whereby a single peak corresponding to the amplicon was present.
The following primer pairs were used to amplify cDNAs after reverse transcription experiments [26]:
Gene
|
Primer
|
---|
Neprilysin
|
F 5’-CCTGGAGATTCATAATGGATCTTGT-3’
|
R 5’-AAAGGGCCTTGCGGAAAG-3’
|
U6
|
F 5’-CTCGCTTCGGCAGCACA-3’
|
R 5’-AACGCTTCACGAATTTGCGT-3’
|
Actin
|
F 5’-CGCAGCAGTCAGGGACATTT-3’
|
R 5’-TTCACATACAGCTTGGGAAGC-3’
|
Measurement of neprilysin protein level
SH-SY5Y cells were washed with PBS and homogenized with lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). The proteins in whole cell lysates were quantified using the BCA protein assay kit with BSA as a standard (Pierce, Rockford, IL, USA).
NEP protein levels were measured by using a DuoSet ELISA kit (R&D Systems Europe, Oxford, UK) according to the manufacturer’s guidelines with minor modifications. Goat anti-human NEP (1.6 mg/mL) diluted in PBS (pH 7.4) was coated overnight on a high-binding 96 well plate (R&D Systems Europe) at room temperature (RT). The plates were washed 3 times in PBS containing 0.5% Tween-20 (Sigma-Aldrich) (PBS-T). Non-specific binding of antibody was blocked by addition of PBS completed with 1% Bovine serum albumin (Sigma-Aldrich) (1% PBS-BSA) for 3 h at RT, then the plates were washed a further 3 times. Serial dilutions of recombinant human NEP or crude homogenates diluted in 1% PBS-BSA or 1% PBS-BSA alone as a control were incubated for 2 h with continuous shaking at RT. After a further 3 washes, biotinylated anti-NEP (1.6 mg/mL) was added for 2 h before another wash and incubation with streptavidin-peroxidase (1200) for 20 min in the dark. Substrate solution (tetramethyl benzidine; R&D Systems, Europe) was added for 30 min, and the optical density for each well was read at 450 nm and 540 nm or 570 nm in a plate reader (Elisa reader model Sigma 960, Metertech, Taipei, Taiwan).
The NEP protein levels were interpolated from the standard curve generated from serial dilutions of recombinant human NEP (R&D Systems Europe). Each measurement was repeated on 3 occasions, and the average value was calculated.
Cell treatment with precursors or inhibitors of the serotonin pathway
Different concentrations (0 to 100 μM) of 5-HIAA, 5-HTP, Serotonin hydrochloride (5-HT, Sigma-Aldrich) and Tryptophan (Trp, Sigma-Aldrich), were used. Pharmacological treatments were performed with specific inhibitors ((S)-(−)-carbidopa monohydrate (Abcam, Cambridge, UK), tranylcypromine hydrochloride (2-PCPA, Abcam), clorgyline hydrochloride (Abcam) were added to the SH-SY5Y cells 90 min or 24 h before harvest.
ERK inhibitor SCH772984 (Carbosynth, Compton Berkshire, UK), MEK ½ inhibitor GSK 1120212 (Targetmol, USA) or GSK-3 inhibitor CHIR99021 (Sigma-Aldrich, France) was added 30 min before the measurement of NEP protein.
SH-SY5Y-APP cell treatment and Western blot analysis
According to experimental design [26], the SH-SY5Y cells were incubated either with 10 mM ammonium chloride (NH4Cl, Sigma-Aldrich) or 30 μM 5-HIAA (Sigma-Aldrich) for 24 h. Non-treated cells were used as control. After 24 h of treatment, the cells were rinsed twice with cold PBS pH 7.4, harvested and pelleted by centrifugation. The cell’s pellets were lysed in RIPA buffer (10 mM Tris/HCl pH 8.0, 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 5 mM EDTA) with complete inhibitor mix from Roche diagnostics (Basel, Switzerland) at 0 °C for 20 min. The lysates were homogenized through 21-G needles 10 times, and then clarified by centrifugation at 2800 x g for 10 min. Protein concentration of lysates was determined using a BCA protein assay kit.
A total of 50 μg proteins were fractionated by 4–20% TGX SDS polyacrylamide gels (BioRad, Hercules, CA, USA). After electrotransfer to polyvinylidene fluoride membranes (BioRad), these membranes were blocked overnight at 4 °C in Tris-buffered saline (TBS: 50 mM Tris, 150 mM NaCl) containing 0.1% (vol/vol) Tween-20 (Sigma-Aldrich) (TBS-T) and 5% (wt/vol) skimmed milk powder. Membranes were incubated 2 h at RT with primary antibodies, Rabbit antibody against anti-APP C-terminal fragment (APP intracellular domain, AICD, Sigma-Aldrich) and Mouse monoclonal anti-β-actin antibody (Sigma-Aldrich) (1: 1000 and 1: 5000, respectively). After washing 3 times with TBS-T, the membranes were probed with corresponding peroxidase conjugated secondary antibodies rabbit anti-mouse or goat anti-rabbit (Abliance former Paris, France) (1: 4000) at RT for 1 h. Detection was carried out by using a chemiluminescence detection kit (Clarity Western ECL substrate, Biorad). After washing steps, signals were detected with ChemiDoc MP (Biorad).
The relative intensity of bands was densitometrically determined by Image J software 1.46r (NIH, USA). For statistical analysis, all data from 3 independent experiments were expressed as the ratio to optical density values of the corresponding β-actin control [53]. The values were expressed as a percentage of the control group arbitrarily set at 100%. The statistical analysis was done with a Student t-test.
Fluorescence-activated cell sorting (FACS) by flow cytometry
After 24 h of treatment with 100 μM 5-HIAA, SH-SY5Y cells were harvested, centrifuged for 10 min at 1000 x g at RT and re-suspended with 0.5% PBS-BSA before being gently fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) in PBS for 1 h. Thereafter, the cells were submitted to two centrifugation steps (10 min at 1000 x g) separated by a washing step using 0.5% PBS-BSA. Cells were then incubated in permeabilization buffer (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. An additional round of washing and centrifugation was performed before incubating the cells with anti-NEP antibody (Merck-Millipore; 1:200) in PBS for 25 min at 4 °C. At the end of the incubation, the cells were washed and centrifuged again for 10 min at 1000 x g. Supernatants were removed and the pellets were suspended and incubated with a secondary antibody for 25 min at 4 °C. The cells were washed and centrifuged a last time, the supernatants were removed, and the pellets were suspended in PBS before being analyzed.
Proteome profiler Phospho-MAPK array
After ½ hour of treatment with 100 μM 5-HIAA or without treatment, SH-SY5Y cells were harvested, centrifuged for 5 min at 300 x g at RT, and then solubilized at 1 × 107 cells/ml in the Lysis Buffer provided with the kit for ½ hour. The level of phosphorylation was determined by using a Proteome Profiler Antibody Kit (Human Phospho-MAPK Array Kit, R&D Systems Europe, Oxford, UK). Briefly, two nitrocellulose membranes, containing 26 different capture antibodies printed in duplicate, were blocked for 1 h under agitation at RT. During the same time, the protein extracts were incubated with a detection antibody cocktail. After one hour, the blocking buffer was replaced by the mix protein extract - antibody cocktail and incubated overnight at 2–8 °C under agitation. Then the membranes were washed 3 times in wash buffer, and were put in a solution of Streptavidin-HRP for ½ hour under agitation at RT. After a further 3 washes, the membranes were covered with a Chemi-reagent mix for 1 min before detection of the signal with ChemiDoc MP (Biorad). The relative intensity of bands was densitometrically determined by Image J software 1.46r (NIH, USA).
Neprilysin activity assay
SH-SY5Y cells were harvested and washed with PBS after 0, 30, 60 and 90 min of treatment with 30 μM 5-HIAA (Sigma-Aldrich). The cells were then sonicated in iced Tris buffer (50 mM Tris-HCl, pH 7.4) and store at − 80 °C.
Measurement of neprilysin activity was performed according to the technical details given by the manufacturer for the fluorescent SensoLyte 520 kit (AnaSpec Inc., Fremont, CA, US). Briefly, 100 μg of total protein were placed in the wells of a non-binding 96-well plate (Corning) before adding the substrate working solution. The reagents were mixed by shaking the plate gently for 30 s and the fluorescence signal was immediately measured at Ex/Em = 490 nm/520 nm continuously and the data were recorded every 5 min for 1 h.
The initial reaction velocity was determined by the slope of the linear portion of the data plot and the results were expressed in percentage by reference to control condition.
Immunocytochemistry
Coverslips were prepared with SH-SY5Y cells treated or not by 5-HIAA at a concentration of 100 μM. The cells were fixed in 4% PFA for 15 min and then permeabilized with 100% ethanol for 5 min and incubated in PBS with 5% fetal bovine serum overnight at 4 °C. A rabbit polyclonal anti-neprilysin antibody (Merck; 1:200) was applied for 2 h at RT then a pre-adsorbed Goat polyclonal anti-Mouse IgG - H&L (Alexa Fluor® 647) antibody (Abcam; 1:500) was used as secondary antibody. Coverslips were mounted in glycerol mounting medium with DAPI and DABCO™ (Abcam) before microscopic analysis using a Zeiss microscope equipped for fluorescence.
In vivo experiments
Animals and treatments
Procedures involving animals and their care were conducted in compliance with a European Communities Council Directive (86/609/EEC) and under the supervision of authorized investigators. In addition, all the protocols were reviewed and approved by the Alsace Head Office of the French Department of Veterinary and Public Health Guide for the Care and Use of Laboratory Animals. Mice were individually housed per cage in a room with 12/12-h light-dark cycle. The room was maintained under constant temperature and humidity conditions. Water and food were available ad libitum.
Mice models
Two mice models were used:
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Female Swiss albino mice, 3-month-old, outbreeded, about 20 g (Janvier Labs, France),
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Male APPSWE hemizygote mice (B6, SJL-Tg (APPSWE) 2576 Kha, tested for heterozygous RD1, Taconic Europe, Denmark) carrying a transgene coding for the 695-amino acid isoform of human Alzheimer APP (Tg 2576) and the corresponding wild type (WT), 13-month-old. All animals were 30-35 g body weight.
Phosphoramidon model of NEP inhibition and Aβ 1–40 or Aβ 1–42 quantification
Four groups of 5 Swiss Albino mice were treated for 5 consecutive days. Group 1 received the NEP inhibitor Phosphoramidon (Peptide Institute, Osaka, Japan) each day via intranasal route. Phosphoramidon was dissolved in PBS (with 1 mM ascorbic acid) at a concentration of 30 mM and was administered intranasally as previously described [28]. Group 2 was treated with 5-HIAA via intranasal route (24 μL of a 30 mM solution). Group 3 received PBS alone. Group 4 received both phosphoramidon and 5-HIAA.
Aβ 1–40 and Aβ 1–42 quantification
After 5 days of treatment, the mice were euthanized for tissue collection 2 h after the last administration. The brains were removed, the two hemispheres separated and immediately frozen in liquid nitrogen and stored at − 80 °C.
The tissues of one hemisphere were homogenized in 8 volumes of ice-cold guanidine buffer (5.0 M guanidine-HCl/50 mM Tris-HCl, pH 8.0). Homogenates were mixed for 3–4 h at RT. The quantification of total proteins was done with a BCA protein assay kit (Pierce, Rockford, IL, USA). The lysates were then aliquoted and stored at − 80 °C until quantification of Aβ 1–40 and 1–42 peptides, according to the technical details given by the manufacturer (Fisher former Invitrogen, Carlsbad, CA, USA).
Samples were briefly diluted with cold Reaction buffer and centrifuged at 16,000 g for 20 min at 4 C. Supernatant was decanted and sample was stored on ice until use. Hundred μL of the diluted homogenates or of the Aβ peptide standards were incubated on the pre-coated 96-well plate for 2 h at RT. The plate was washed 4 times and 100 μL of the primary antibody was added for 1 h at RT. After 4 further washes, 100 μL of the secondary antibody was added for 30 min at RT. After 4 new washes, 100 μl of Stabilized Chromogen was added to each well for 30 min at RT in the dark. Finally, the reaction was stopped by 100 μL of Stop Solution to each well and the optical density was read at 450 nm in a plate reader (Elisa reader model Sigma 960, Metertech, Taipei, Taiwan).
The β peptides levels were interpolated from the standard curve generated from serial dilutions of synthetic peptide. Each measurement was repeated on 3 occasions, and the average value was calculated.
The results were analyzed with an analysis of variance (ANOVA p < 0.0001) to evaluate the effects of genotype and treatment factors. Post hoc analyses were performed with the Bonferroni test.
Tg 2576 and WT mice treatment
Two groups of mice (12 Tg 2576 and 12 WT) were administered daily via intra-nasal route with 4 μL of a 30 mM solution of 5-HIAA (Sigma-Aldrich, France) in PBS [20, 21]. In parallel, 24 other animals (12 Tg 2576 and 12 WT) were treated for 5 consecutive days with 48 mg/kg 5-Hydroxy-L-tryptophan (5-HTP, Sigma-Aldrich) administrated IP. Control groups were obtained by administration of PBS (12 Tg 2576 and 12 WT). These mice were the used for the neprilysin activity assay or for the spatial novelty task.
Neprilysin activity assay
To measure NEP activity in the mouse brain, the animals were treated daily for 5 consecutive days and euthanized for tissue collection 2 h after the last administration for the Tg 2576 mouse (sub-chronical treatment) and at various times (0 to 120 min) for the Swiss mice (acute treatment). The brains were removed, the two hemispheres separated and immediately frozen in liquid nitrogen and stored at − 80 °C. The tissues of one hemisphere were homogenized in 5 volumes of ice-cold PBS, centrifuged at 15000 x g for 5 min at 4 °C and the supernatant was decanted and stored at − 80 °C. The quantification of total proteins was done by a BCA protein assay kit).
Measurement of neprilysin activity was performed according to the technical details given by the manufacturer for the fluorescent SensoLyte 520 kit (AnaSpec Inc., Fremont, CA, US). Briefly, 100 μg of total protein were placed in the wells of a non-binding 96-well plate (Corning) before adding the substrate working solution. The reagents were mixed by shaking the plate gently for 30 s and the fluorescence signal was immediately measured at Ex/Em = 490 nm/520 nm continuously and the data were recorded every 5 min for 1 h.
The initial reaction velocity was determined by the slope of the linear portion of the data plot and the results were expressed in percentage by reference to control condition.
Spatial novelty task
This task is based on the spontaneous tendency of mice to preferentially explore objects which have been displaced within a familiar arrangement of objects. Tg 2576 mice are deeply impaired in this task as early as 7 to 8 months of age, independently of the rd mutation [38, 57].
The spatial novelty task was performed in a square Plexiglas open field (52 × 52 × 40 cm). For each testing period, a specific set of 7 different objects (4 for the habituation phases and 3 for the spatial recognition task), which differed in shape, color, and materials, were used. Each object was available in duplicate (one for each trial when required) and was wiped with 70% ethanol as the whole open field between each trial. On days 1 and 2, the mice received a habituation trial of 10 min with 2 different objects each day.
On day 3 (5th day of treatment), the mice explored the spatial configuration of 3 new objects during a 10-min acquisition trial, returned in their home cage for 3 min, and then received a 10-min retention trial with a new spatial configuration resulting from the shifting of one of the 3 objects to a new location. Recognition performances were calculated as the additional time spent exploring the displaced object during the retention trial when mice are the most involved in exploring the objects. This value was compared with the 0 value (no detection of the spatial change) with a Student t-test to measure whether spatial novelty detection occurred and was also compared among groups with an analysis of variance (ANOVA p < 0.0001) to evaluate the effects of genotype and treatment factors. Post-hoc analyses were performed with the Newman-Keuls test.
Immunocytochemistry on brain tissue slices
Tg 2576/APPSWE mice chronically treated with 5-HIAA were perfused through the heart with cold 4% PFA. The brains were fixed in a 4% PFA solution for 72 h at 4 °C and afterwards were immersed in PBS containing 20% sucrose for 48 h. The brains were cut in a vibratome Leica VT1000M (80 μm thick) and the slices were collected in a Watson medium.
Floating sections were immunostained as follows: Tissue slides were rehydrated with PBS for 1 h at RT then blocked overnight at 4 °C with 5% (v/v) fetal bovine serum in PBS. Sections were stirred overnight at 4 C with the primary antibodies (mouse monoclonal Anti-β Amyloid 1–40 antibody (Abcam) and rabbit polyclonal anti-Neprilysin antibody (Merck Millipore) at a dilution of 1:200. Sections were then washed 10 to 12 times for 1 h in PBS pH 7.4. Then the sections were stirred with species-specific secondary antibodies (pre-adsorbed Goat polyclonal anti-Mouse IgG - H&L (Alexa Fluor® 647) antibody (Abcam) and pre-adsorbed Goat polyclonal anti-Rabbit IgG - H&L (Alexa Fluor® 488) antibody (Abcam), at a dilution of 1:500, overnight at 4 C in the dark. Sections were washed again (10 to 12 times for 1 h) with PBS pH 7.4. After this second period of washing, the sections were mounted in glycerol mounting medium with DAPI and DABCO™ (Abcam) before microscopic analysis using a microscope (Zeiss AxioImager Z2).
Statistical analysis
GraphPad Prism was used for all statistical analysis. For ANOVA multiple statistical comparisons, Newman-Keuls or Bonferroni tests were used and two-sided unpaired Student’s t-test for single statistical comparison. Statistical distribution for the Post-hoc analyses was: * p < 0.05, ** p < 0.005 and *** p < 0.0001. Errors are standard error of mean (SEM).