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Pharmacological BACE1 and BACE2 inhibition induces hair depigmentation by inhibiting PMEL17 processing in mice : Scientific Reports Ethics Statement All experiments were carried out in accordance with authorization guidelines of the Swiss Federal and Cantonal veterinary offices for care and use of laboratory animals and the veterinary office regulations of Baden-Württemberg (Germany) and approved by the local Animal Care and Use Committees. Studies described in this report were approved by the Swiss Cantonal Veterinary Authority of Basel City, Switzerland, under the license number 1094. Animals C57BL/6J animals were source from Charles River Laboratories, France. C57BL/6 control littermates from the APP51 line were bred at Novartis Pharma AG, Basel, Switzerland. APP23, APPS1, and APP51 mouse lines used all express human APP751 under the control of the murine Thy1-promoter, resulting in neuron-specific expression. APP23 mice39 express APP with the K670M/N671L “Swedish” mutation. The mice have been backcrossed with C57BL/6J mice for 20 generations (C57BL/6J- Tg(Thy1-APPK670N;M671L)23). APP23 mice are currently bred at the Hertie Institute for Clinical Brain Research (Tübingen, Germany) and overexpress human APP approximately 7 times over endogenous APP and first develop individual Aβ-amyloid plaques in the neocortex at 7 months (females) or 9 months of age (males). For this study male APP23 mice between 15 and 21 months of age were used. pre bonded hairMale and female APPPS1 mice of ages 1.5 to 7.5 months were bred at the Hertie Institute for Clinical Brain Research (Tübingen, Germany). APPPS1 mice have been initially generated and maintained on a C57BL/6 background and co-express K670M/N671L-mutated APP and L166P-mutated presenilin 1 (PS1) (C57BL/6J-TgN(APPswe and PS1 166)21)40. The mice first develop Aβ plaques after 6 weeks of age, and no effect of gender was found. Female 12.5 to 13.5 month-old APP51/16 mice (C57BL/6J-TgN(Thy1-APP)51; APP51) were bred on a C57BL/6J background at the Hertie Institute for Clinical Brain Research (Tübingen, Germany). APP51 mice41 express human wild type APP at about 7–10-fold over endogenous APP and develop the first plaques between 12 and 15 months of age. All mice were kept under specific pathogen-free conditions. BACE1 (B6,129T2-TgH(Bace1)1Goe) and BACE2 (B6,129P2-TgH(Bace2)1Leu) knock-out mice were received from the lab of B. De Strooper29, with unknown genetic background. Animals were black. For the pharmacological experiment with NB-360 this colony was used. For the detailed histo-pathological caharcaterization knock-outs were backcrossed at least 6 generation into C57BL/6 (>98%). Upon arrival in the laboratory, single-housed mice were maintained under standard conditions in temperature and humidity control rooms under a 12/12 light/dark schedule, with lights on at 06:00 hr. Cage bedding was sawdust, and a red Pespex mouse-house (Nalgene®), nesting materials (Nestlet®) and a wooden gnawing-block were supplied in each cage. Either standard laboratory rodent food (C57BL/6) or food pellets containing NB-360 (APPPS1, APP23, APP51) and tap water were available ad libitum. Compound form, formulation and dosing Crystalline NB-360 was formulated as a suspension. Vehicle or compound were given per os in a volume of 10 ml/kg once daily (mornings). Vehicle: 0.1% Tween80 in 0.5% Methylcellulose in water. All suspensions were homogeneous upon visual inspection. Particle size was in the low micrometer range for all suspensions.

NB-360 was dosed in food pellets (0.5 g/kg) to treat APPPS1, APP23, and APP51 mice. Food pellets (KLIBA NAFAG) were produced at Provimi Kliba SA, Kaiseraugst, Switzerland. Fur color scoring Subjective scoring of any hair color changes was performed once weekly. Scores (% of body with grey fur): 0: No change; 1: Spots; 2: >30%; 3: >50%; 4: >75%; 5: 100%. Animals were photo-documented when a fur color change was observed. Trichogram Mouse hair was placed on glass slides with some drops of oil, cover slipped and examined microscopically. remy hair extensionsHistology on eye samples Both eyes were collected and either fixed in Davidson’s fixative or OCT embedded. From two mice/group retina only was collected instead of the entire left eye. Subsequently the Davidson’s fixed eye samples were paraffin embedded and further processed while the OCT embedded samples were stored at −80 °C. Immunohistochemistry and immunofluorescence were performed using antibodies directed against LAMP2 (1:200, rabbit polyclonal IgG, ThermoFisher Scientific, 51-2200) and Rhodopsin (1:1000, rabbit polyclonal IgG, abcam, ab104760). Lipofuscin accumulation was assessed by comparing Rhodopsin fluorescence to autofluorescence. LAMP-2 immunohistochemistry was performed using the fully automated instrument Discovery XT® (Ventana Medical Systems Inc., Switzerland). All chemicals were provided by Ventana Medical Systems Inc. Briefly, sample slides from Davidson-fixed paraffin embedded tissue were deparaffinized and rehydrated under solvent-free conditions using EZprep™ solution for 8 minutes at 75 °C. Depigmentation was performed using a solution of H2O2 3% (Merck, Germany) at 55 °C during 1 hour. Subsequently, heat induced epitope retrieval pretreatment was performed by successive cycles (4×) at 100 °C for 4 minutes in a Tris-EDTA based buffer optimized for the Discovery XT® instrument (CC1 solution). Before applying the primary antibody, the slides were blocked using 1x Casein solution in PBS (BioFX laboratories, Catalog number PBSC-0100-5×) for 32 minutes at room temperature to avoid background. Endogenous avidin/biotin activity was also quenched by using Ventana A/B blocking reagents for 4 minutes each. A short post-fixation using glutaraldehyde at 0.05% was done before applying a biotin conjugated secondary antibody (Jackson Laboratories, Switzerland) diluted at 1/500 in Discovery antibody diluent and incubated for 16 min. Detection of the biotin was performed with the RedMap streptavidin-alkaline phosphatase detection system (Ventana Medical Systems Inc., Switzerland). The detection system was used according to the manufacturer’s recommendations. Counterstaining with Hematoxylin II and Bluing reagent was performed for 2 × 8 min. Sections were dehydrated and covered using Pertex. For the fluorescence detection of Rhodopsin the above described protocol was applied except for antibody dilution (1:1000, goat anti-rabbit Alexa fluor 647 conjugated). Detection was performed manually using goat anti-rabbit Alexa fluor conjugated antibodies (Life Technologies, Switzerland), diluted 1/200 in Amplifying antibody dilution buffer (Cat No. AA4, ProHisto, USA) and incubated 30 minutes at room temperature.

The nuclei were detected using ProLong® Gold Antifade Reagent with DAPI (Cat. No. P36931, Molecular Probes/Life Technologies, Switzerland) and covered with ProLong® Gold Antifade Reagent (Cat. No. P36934 Molecular Probes/Life Technologies, Switzerland). Lipofuscin accumulation was assessed by comparing Rhodopsin fluorescence to autofluorescence. The specific fluorescence signal was detected using the Leica SP5 confocal microscope (Leica, Germany). Assessment of lipofuscin deposition and distribution in the RPE was performed by recording its autofluorescence at 488 nm excitation wave length using Leica SP5 confocal microscope (Leica AG, Germany). Electron microscopy on eye samples The right eyes from 1 vehicle-treated mouse and two compound-treated mice were sampled and immediately fixed in 3% glutaraldehyde and 0.1 M phosphate buffer, pH 7.4, for 24h at 4 °C. A post-fixation was performed in 1% OsO4 in 0.1 M phosphate buffer, pH 7.4, for 1 h at 4 °C. The eyes were subsequently trimmed and dehydrated in graded acetone solutions. A standard Epon embedding procedure was applied, ultrathin sections were cut and assessed using the Tecnai Spirit Transmission Electron Microscope (FEI, Switzerland). For each investigated retina the RPE, Bruch’s membrane were evaluated. For each retina at least 3 different locations were examined. Ex-vivo samples and sample harvest methodology perruques cheveux naturelsBlood samples were used to analyze whole-blood compound levels and were obtained either from tail vein during the in-life part into EDTA tubes (CB300, Sarstedt, Germany) or from trunk blood on the day of necropsy into EDTA Eppendorf tubes (Milian SA, CatNoTOM-14, Fisher Scientific, Wohlen, Switzerland), frozen on dry ice and stored at −80 °C until analysis. The brain was removed immediately after decapitation, rinsed with saline and sectioned sagitally down the midline. The left half of the cerebellum was used to analyze compound level and was placed into a glass tube (Chromacol, 125 × 5-SV T051, Welwyn Garden City, United Kingdom), weighed and frozen in dry-ice. The left half of the forebrain (without olfactory bulb) was used for Aβ analysis, and was frozen on a metal plate on dry ice and placed into protein Lo-bind tube (003 0108.116, Eppendorf, Hamburg, Germany). Ventral and dorsal skin were taken to analyze compound level, weighed and frozen on dry-ice. Analysis of compound levels NB-360 levels in biological samples were quantified in blood, brain and skin by liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Brain samples were mixed with 2 volumes of KH2PO4 buffer and homogenized using the Covaris® device. Skin samples were mixed with approx. 6-fold volumes of methanol/water and homogenized using Precellys tubes. Either 30 μL of blood, brain or skin homogenate were spiked with a structurally related internal standard and subsequently mixed with an at least 6-fold excess volume acetonitrile for protein precipitation. The supernatant was injected directly into the LC/MS/MS system for analysis.

Brain homogenization Frozen mouse forebrains were weighed and homogenized in 9 volumes (w/v) of ice-cold TBS-Complete (20 mM Tris-HCl pH 7.4, 137 mM NaCl, 1× Complete [Protease Inhibitor Cocktail Tablets: 11836145, Roche Diagnostics GmbH, Penzberg, Germany]) by sonication (90% duty cycle, output control 5, 40–55 pulses, [Sonifier 450, Branson]). After homogenization several 50 μl aliquots were prepared for analysis and were stored at −80 °C. Determination of Aβ40 in mouse brain Human Aβ peptide (1–40) trifluoroacetate salt (H 1194.1000, Bachem, Bubendorf, Switzerland) was used as standard. Endogenous Abeta40 in mice was determined with the Meso Scale Discovery (MSD) 96-well MULTI-ARRAY human/rodent (4G8) Aβ40 Ultrasensitive Assay (#K110FTE-3, Meso Scale Discovery, Gaithersburg, USA). The assay was done according to the manufacturer’s instructions except for the calibration curve and the sample preparations. TritonX-100 (TX-100) soluble Aβ40 was extracted from forebrain with 1% TX-100 using a 50 μl aliquot of each 1:10 forebrain homogenate, mixed with 50 μl 2% TX-100 in TBS complete (20 mM Tris-HCl pH 7.4, 137 mM NaCl, 1× Complete [Protease Inhibitor Cocktail Tablets: 11836145, Roche Diagnostics GmbH, Penzberg Germany]) to reach a final concentration of 1% TX-100 and a 1:20 forebrain dilution. The samples were incubated for 15 min on ice and vortexed every 5 min. The samples were ultra-centrifuged (100000×g, 4 °C, 15 min) and 50 μl of the clear supernatants were transferred to fresh tubes. For the Aβ40 assay the supernatants were further diluted 1:5 in 3% Blocker A solution (from kit) to a final forebrain dilution of 1:100 and applied to the plate. perruques cheveuxThe calibration curve was prepared in a corresponding dilution of 1% Blocker A solution spiked with synthetic Aβ1-40 peptide (1.56–100 pg/ml) except for non-transgenic mouse brain samples: In this case, the calibration curve was prepared in a correspondingly diluted APP knock-out mouse forebrain spiked with synthetic Aβ1–40 peptide (1.56–100 pg/ml). For all samples and standards 25 μl were applied per well. For each determination duplicate wells were done. The mean values from the duplicate wells were used for calculations. Since MSD did not provide quantification software, the relative units for samples and standards were imported into SOFTmax PRO 4.0 for calculation of standard curves and quantification of samples. Compounds for in vitro usage NB-360, NB-449, NB-444, NB-480 and NB-109 are products of Novartis Pharma AG. The γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) was purchased from Sigma (D5942). All compounds were dissolved in DMSO. Stocks of 50 mM solution were prepared and stored as aliquots in −20 °C to reduce several freeze thaw cycles. Cell culture systems Human MNT-1 cells [Cuomo M, 1991] were purchased from MediaPharma, Chieti, Italy. Mouse melanoma B16-F0 cells were bought from ATCC (CRL-6322). The human MNT-1 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Invitrogen) supplemented with 16% fetal bovine serum (Hyclone), 10% AIM-V medium (Invitrogen), 0.1 mM non-essential amino acid mix (Invitrogen) and 1 mM sodium pyruvate (Invitrogen), and 100 U/ml of penicillin/streptomycin (Invitrogen). B16F0 cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 100 U/ml of penicillin/streptomycin (Invitrogen). Both cell lines were cultivated at 37 °C, 5% CO2 and 98% relative humidity in a tissue culture incubator (model Cytoperm 2, Heraeus Instrument, Hanau, Germany). Cells were passaged once to twice weekly by using Trypsin-EDTA solution.

Intracellular ATP measurement Intracellular ATP levels were measured as an indicator of energy homeostasis and cellular viability. Melanocytes were seeded in 96 well plates (B16-F0: 1000 cells/well; MNT-1: 10,000 cells/well). Twenty four hours post-seeding, both cell lines were incubated with increasing concentration of inhibitors over another twenty four hours. ATP measurements were performed using CellTiter-Glo Luminiscent Cell Viability kit (Promega) as described by the manufacturer. Briefly, 100 μl of the CellTiter-Glo Reagent were added to the 100 μl of the medium, placed in a shaker for 2 minutes and incubated at room temperature for 10 minutes to induce complete lysis. The resulting luminescence was measured using the Envision 2104 luminescence plate reader (Perkin Elmer). Data were expressed relative to untreated controls. RNA extraction, cDNA synthesis and real-time PCR lace front wigsTotal RNA samples were extracted from cells by using RNeasy Plus Micro Kit (Qiagen #74034). RNA integrity was controlled by running a Bioanalizer profile (Agilent) and by measuring their spectral profile at λ = 230, 260 and 280 nm. 300 ng of total RNA was reverse transcribed into cDNA by using the High Capacity cDNA Archive Kit and according to the manufacturer instructions (Applied Biosystems, cat. No. 4368813). Real-time PCR was performed by using the TaqMan® fast PCR Master Mix (Applied Biosystems, cat no. 4352042) and the ABI prism™ 7900HT (Applied Biosystems) according to the manufacturer instructions. Relative quantification of gene expression changes was performed by using the standard curve method and normalized to control samples to generate fold changes values. The housekeeping gene used for comparison and normalization of gene expression data was the GAPDH RNA. Results were presented in fold change as compared to BACE1 expression. Taqman assays used to assess gene expression: PMEL Mm00498996_m1, BACE1 Mm00478664_m1, BACE2 Mm00517138_m1, PMEL Hs00173854_m1, BACE1 Hs01121195_m1, BACE2 Hs00273238_m1, 18S Hs99999901_s1 Endogenous control, GAPDH Hs02758991_g1 Endogenous control, GAPDH Mm99999915_g1 Endogenous control

Melanin measurement B16-F0 cells were seeded in 6 well plates (500 cells/well) and left to attach overnight. The following day, melanocytes were treated with different concentrations of the compounds for a total duration of 9 days. After every 3 days, both the compounds and the media were replenished. Melanin concentrations were determined by spectrophotometric measurement of the absorbance at 405 nm (Chu HL 2009; Ishida K 2009; Haywood RM 2006). Briefly, upon completion of the treatment, cells were trypsinized and the melanin content was extracted by incubating the melanocytes with 1N NaOH for 2 hours at 80 °C accompanied by mild shaking. The supernatant was then centrifuged and the absorbance was measured at 405 nm in spectrophotometer (SpectraMax 190, Molecular Devices). Melanin concentrations were then extrapolated using a standard curve (0 – 12.5 μg/ml) obtained with synthetic melanin using SoftMax Pro software. For the standard curve, synthetic melanin (Sigma, Cat. No. M0418, Lot no. 011M1326V) was prepared at 20 mg/ml in 1N NH4OH buffer. Cellular treatments Melanocytes were seeded in 6-well plates. Murine B16-F0 cells were plated at 8 × 104 cells/well and human MNT1 cells were plated at 8 × 104 cells/well for protein extraction and western blot analysis. Twenty four hours post seeding, both cell lines were incubated with increasing concentration of inhibitors over twenty four hours. The cells were washed once with PBS (Invitrogen) and then trypsinized with 222 μl of trypsin (Invitrogen). After trypsinization, the plates were placed on ice and 1778 μl of cold medium was used to stop the action of trypsin. The cell suspensions were transferred in pre-cooled Eppendorf tubes and centrifuged at 4 °C for 7 min at 450g. The pellets were washed with 200 μl of cold PBS to remove debris and remaining media. The pellets were centrifuged once more as described previously. The supernatants were removed and the pellets were lysed on ice as described below. cosplay wigsProtein extraction, concentration measurement and immunoblotting After washing the cell pellets, 60 μl of ice cold RIPA buffer (Pierce 89900) including protease and phosphatase inhibitors (Sigma P8340, P2850, P5726-1/100 each) were added to the pellets and the resulting lysates were vortexed shortly. Pellet suspensions were incubated under agitation over 20 min at 4 °C by using a thermomixer (Eppendorf). Pellet suspensions were finally centrifuged (15 min, 16 000×g, 4 °C) and supernatants were collected and stored at –80 °C. An aliquot was subsequently taken to estimate the protein concentration using the MicroBCA Assay Kit (Pierce, #23235) and separately stored at −20 °C. Absorbance was measured at 562 nm (Envision 2104, Perkin Elmer). SDS-PAGE samples were prepared in NuPAGE 4× buffer (Invitrogen NP0007) and 10× reducing agent (Invitrogen NP0004) or in 6× loading buffer (60 mM Tris pH 6.8, 2% SDS, 9.8% glycerol, bromophenol blue, 0.6 mM DTT). Samples were heated (10 min, 70 °C), centrifuged (3 min, 14 000×g) and resolved on Invitrogen NuPAGE Bis-Tris gels (4–12% or 8% gel, MES running buffer, 160 V, 1 hour). Gels were transferred to nitrocellulose membranes (20 V, 7 min) using the I-Blot system (Invitrogen, #IB1001EU). Membranes were blocked for 1 hour in Odyssey Blocking Buffer (LI-COR 927-40000)/PBS (1:1) and subsequently incubated overnight at 4 °C with primary antibodies (anti-PMEL17, abcam Ab52058, goat polyclonal IgG, 1:250; anti-actin, Sigma A5060, rabbit polyclonal IgG, 1:15000) diluted in Odyssey Blocking Buffer/PBS-Tween 0.1% (1:1). After washing 3 times with PBS-Tween 0.1%, the membranes were incubated for 1 h with secondary antibodies labelled with IRdyes diluted in Odyssey Blocking Buffer/PBS-Tween 0.1% (1:1). The membranes were washed (3 times with PBS-Tween 0.1%, 3 times with PBS) and then dried on absorbent paper before being scanned using the Odyssey Infrared Imaging System (LI-COR). Integrated intensities of the protein bands of each target were quantified in their respective fluorescent channels using Odyssey software (background subtraction top/bottom of quantified band). The integrated intensity for PMEL17 Mβ fragment was normalized to the integrated intensity for actin and displayed as % of control.