AD is a progressive neurodegenerative disorder which is recognized as the most common form of dementia among the elderly characterized by progressive dysfunction of cognition and memory (Scheltens
Several drugs are used for slowing down the progression of AD, but they do not significantly delay symptom development or cure the disease. However naturally derived therapeutics showed advantages in slowing down AD development and delaying the onset of symptoms (Kim
Rabbit anti-APP antibodies to detection the C-terminal of APP were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). FBS was purchased from ATCC Company (Manassas, VA, USA). DMEM, penicillin/streptomycin, G418, and 0.25% trypsin-EDTA were purchased from GIBCO–BRL Company (Carlsbad, CA, USA). Zeocin were purchased from Invitrogen Company (Carlsbad, CA, USA). Rabbit anti-GAPDH, anti-rabbit horseradish peroxidase linked IgG, anti-ADAM9 antibodies and lysis buffer were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-BACE1 antibody, and anti-APP antibody to detection both mAPP and imAPP were obtained from Abcam Company (Cambridge, UK). Anti-ADAM10 antibody was obtained from Calbiochem Company (San Diego, CA, USA). Anti-TACE and anti-ADAM17 antibodies were obtained from Chemicon Company (Billerica, MA, USA). All other chemicals were of analytical grade obtained from Sigma-Aldrich Co.
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Hela cells stably transfected with an APP carrying Swedish mutation (APPsw) using BioT (Bioland Scientific LLC, Paramount, CA, USA) according to the manufacturer’s instructions. APPsw transfected HeLa cells were grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 260 μg/mL Zeocin and 400 μg/mL G418. The cells were maintained 37°C under a humidified atmosphere of 5% CO2 : 95% air. The stock sample solution was dissolved in DMSO and kept at −20°C, and diluted to the final concentration in fresh media before each experiment. The final DMSO concentration of the sample solutions did not exceed 0.5% in all experiments.
For measuring the cell viability, we used the EZ-Cytox kit according to the manufacturer’s instruction obtained from DAEILLAB Co (Cheongwon, Korea). Hela cells were seeded in 96-well plates for 24 h and then various concentrations of comp 27 (0-10 μM) were treated for 12 h. The microplate reader (BIO-TEK® Dower-Wave; BioTek, Winooski, VT, USA) used to measured absorbance at 450 nm. The results were expressed as the percentage of MTT reduction, assigning the 100% value to the absorbance of the control group.
APPsw-transfected HeLa cells were collected after they were treated with the indicated concentrations of samples and chemicals for the indicated times. After harvested the cells using PBS (pH 7.2), the proteins from cell pellets were lysed in a cold lysis buffer containing protease inhibitor cocktail obtained from Sigma-Aldrich Co. The lysates were centrifuged at 13,000 rpm for 20 min at 4°C. The protein content of the supernatant was determined by the Bradford assay obtained from Bio-Rad Laboratories (Hercules, CA, USA) and used in the subsequent experiments. Protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes. After blocked membranes with 5% non-fat milk for 1 h at room temperature, the membranes incubated overnight at 4°C with primary antibodies including anti-APP, ADAM9, ADAM10, ADAM17, and BACE1. The membranes were washed three times using tris-buffered saline buffer with tween 20 (TBST). The membranes were incubated with horseradish peroxidase-conjugate anti-rabbit IgG antibodies for 1 h. Immunoreactive bounds were visualized using enhanced chemiluminescence (ECL) Advance western blotting detection reagents (34095, Bio-Rad Laboratories). Luminescent Image Analyzer (LAS-4000, Fujifilm, Minato City, Tokyo, Japan) performed for the Imaging and quantitative densitometry analyses. All the protein levels were normalized to that of GAPDH.
APPsw-transfected HeLa cells were cultured with the compound or DMSO in DMEM for 8 h and then the medium was harvested for subsequent analyses. For the secreted Aβ detection, the kits for Aβ42 (KHB3442) and Aβ40 (KHB3482) were obtained from Invitrogen Company and used according to the supplier’s instructions. For sAPPα detection, sAPPα (27734) ELISA kit obtained from IBL Company was used in this study according to the supplier’s instructions as well.
Data were analyzed with Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA) using one-way analysis of variance (ANOVA) followed by the Tukey multiple comparison test. Statistical significance was set at
The structure of comp 27 isolated from
Then, we examined the effect of comp 27 on Aβ secretion. Cells were incubated with 2.5, 5, and 7.5 μM comp 27 for 8 h, and we measured the levels of Aβ42 and Aβ40 used specific ELISA kits from the conditioned media. The production of both were decreased in a dose-dependent manner. The Aβ42 level was reduced by 66.7%, 56.1%, and 46.2% at 2.5, 5, and 7.5 μM of comp 27, respectively (Fig. 1C). The Aβ40 level was also reduced by 75.7%, 64.5%, 52.5% at 2.5, 5, and 7.5 μM of comp 27, respectively (Fig. 1D).
β-Secretase and γ-secretase generated Aβ through sequential cleavage of APP. On the other hand, α-secretase and γ-secretase generated precluding Aβ by cleavage within the Aβ domain. Thus, we further tested the effects of comp 27 on the production of APP proteolytic fragments, sAPPβ and sAPPα, as well as the APP expressions to investigate the two pathways. The secreted level of sAPPα was increased by 116.2%, 121.1%, and 131.2% at 2.5, 5, and 7.5 μM of comp 27, respectively (Fig. 2A). In addition, treatment of 7.5 μM comp 27 significantly decreased the level of sAPPβ to 50.6% (Fig. 2B, 2C). On the other hand, comp 27 did not change the levels of both mature and immature APP (mAPP and imAPP) (Fig. 2C, 2D).
Comp 27 increased sAPPα secretion and decreased the secretion of Aβ and sAPPβ. However, it did not affect total APP expression. Therefore, we expected that comp 27 may affect either ADAM family or BACE1 which are respectively acting as α- and β-secretases.
Next, we investigated whether comp 27 affect ADAM family expressions and activities. Cells were incubated with 2.5, 5, and 7.5 μM of comp 27 for 8 h, and the levels of ADAMs in lysates were measured using Western blot analysis. ADAM9 and ADAM10 exist as a pro-enzyme state, which are converted to mature form to cleavage of the APP (Lammich
We tried to determine whether comp 27 influences BACE1 protein expression. As we expected, compound 27 dose-dependently decreased BACE1 expression. The level of BACE1 was decreased by 77.2%, 68.5%, and 55.5% at 2.5, 5, and 7.5 μM comp 27, respectively (Fig. 4).
Aβ oligomers stimulate the kind of biological signaling pathway involving oxidative stress and neuroinflammation (Agostinho
This work was funded by the National Research Foundation of the Ministry of Science, Information and Communications Technology (ICT) & Future Planning of Republic of Korea (NRF-2015M3A9A5030735) and the Bio-Synergy Research Project (NRF-2012M3A9C4048793).
The authors declare no competing financial interest.