For GSAP overexpression, mammalian expression vector pReceiverM07 with the full-length GSAP and a C-terminal-HA tag was purchased from Genecopoeia (same source as He et al.).
Hela-sweAPP and HEK-wtAPP cells are cultured and used as described 20. N2a-695-APP cells are a kind gift of Prof. G. Thinakaran, University of Chicago.
siRNAs were purchased from Invitrogen (stealth siRNA). The sequences and the gene IDs are supplied in Supplementary Table 1.
siRNA transfection for HeLa-swAPP cells
RNAi silencing was performed in HeLa cells expressing the Swedish APP mutation (HeLa-swAPP). siRNAs were transfected with a final concentration of 5 nM using Oligofectamine (Invitrogen) as a transfection reagent at a concentration of 0.3 μl in a total volume of 100 μl following the manufacturer’s instructions. Each siRNA transfection was performed in quadruplicate. After 24 h the transfection mix was replaced with fresh culture medium. 69 h after transfection, medium was again replaced with 100 μl fresh medium containing 10% Alamar Blue (AbD Serotec). Supernatants were collected and assayed for Aβ and sAPPβ, as described below. The cells were lysed with 50 μl lysis buffer (1% NP-40, 0.1% SDS and protease inhibitor cocktail tablet) for 20 min on ice.
siRNA transfection of HEK-wtAPP cells
Lipofectamine RNAiMAX (Invitrogen) was used as a transfection reagent. 24 h prior to transfection, cells were seeded at an initial seeding density of 3500 cells per well in a 96 well plate pre-coated with poly D lysine (Sigma Aldrich). siRNAs were transfected at a final concentration of 5 nM using 0.3 μl Lipofectamine RNAiMAX in a total volume of 100 μl. Each siRNA transfection was performed in quadruplicate. After 48 h the transfection mix was replaced with fresh medium containing 10% Alamar Blue. Supernatants were collected and assayed for Aβ and sAPPβ as described below. The cells were lysed with 35 μl lysis buffer for 20 min on ice.
The plasmid transfection for GSAP/PION-HA was performed in HeLa-swAPP cells. Lipofectamine 2,000 (Invitrogen) was used as a transfection reagent. Cells were seeded in a 96 well plate at an initial seeding density of 6,000 cells per well 24 h prior to the transfection. 0.3 μg of plasmid DNA was transfected using 0.3 μl of Lipofectamine 2,000 in a total volume of 100 μl. Transfection mix was replaced after 3 h by fresh culture medium. 21 h after transfection, medium was again replaced with fresh medium containing 10% Alamar Blue. 24 h after transfection, Alamar Blue measurements were taken. Supernatant was collected and assayed for Aβ and sAPPβ. The cells were lysed with 35 μl lysis buffer, incubated for 20 min on ice, and stored at -20°C.
Alamar Blue assay
For cell viability measurements using Alamar Blue, the medium of transfected cells (siRNA or plasmid) was replaced with normal medium containing 10% Alamar Blue. The final volume in each well was 100 μL. 3 h after medium change, cell viability was monitored using Fluoroscan Ascent Cf (Labsystems), with excitation wavelength 544 nm and emission at 590 nm. Cell viability was measured using the Alamar Blue assay (Serotec Ltd., Kidlington, Oxford, UK), where the absorbance was monitored at the end of the reaction (after 3 h) (Labsystems Multiscan MS UV visible spectrophotometer).
Electrochemiluminescence (ECL) detection of Aβ and sAPPβ
An electrochemiluminescence assay (Meso Scale Discovery, MD) was performed to determine the amount of secreted Aβ40 and sAPPβ in the cell culture medium. For the measurement of Aβ38, 40 and 42, triplex plates were used from conditioned supernatants collected for 12 h. Pre-coated plates were blocked with TBST (Tris Buffered Saline containing Tween), containing 3% Blocker A, for 1 h at room temperature on a shaker at 750 rpm. After washing, 10 μl of the cell culture supernatant was added to each well along with 10 μl of detection antibody followed by incubation for 2 h at room temperature on a shaker at 750 rpm. After washing, detection was performed in 35 μl of 2x MSDT read buffer and read with the Sector Imager 6000.
After 72 h of siRNA transfection, cells were lysed in buffer containing 1% NP-40 and 0.1% SDS and protease inhibitors (Roche). Equal amounts of the lysate (according to the protein content quantified by BCA assay; Pierce) were run on 4–12% BIS-TRIS gels (Invitrogen). The gel was blotted onto a nitrocellulose membrane (BioRad) and probed with the respective antibodies (anti-HA antibody: Roche diagnostics; GAPDH antibody: Meridian Science).
Total RNA from cultured cells was isolated using TRIzol® Reagent (Life Technologies) following manufacturer’s protocol. 1 μg of total RNA was used for reverse transcription with oligo-dT primer using the Superscript III first-strand synthesis system (Invitrogen) according to the manufacturer’s protocol. Real-time PCR was performed using iTaqTM Universal SYBR® Green supermix (Bio-Rad) following manufacturer’s instructions. Relative gene expression levels were calculated with the ΔΔCt method using GAPDH for normalization.
Protease prediction tool
We used the Swiss Institute of Bioinformatics’ Expassy Peptide cutter tool (http://web.expasy.org/cgi-bin/peptide_cutter/peptidecutter.pl). We pasted the human GSAP sequence (UniProtKB/Swiss-Prot: A4D1B5.2) into the field and chose both enzymes and chemicals that could cleave from 1 up to 20 cleavage sites. The following sequence was provided:
>gi|189041192|sp|A4D1B5.2|GSAP_HUMAN RecName: Full=Gamma-secretase-activating protein; Short=GSAP; AltName: Full=Protein pigeon homolog; Contains: RecName: Full=Gamma-secretase-activating protein 16 kDa C-terminal form; Short=GSAP-16K MALRLVADFDLGKDVLPWLRAQRAVSEASGAGSGGADVLENDYESLHVLNVERNGNIIYTYKDDKGNVVFGLYDCQTRQNELLYTFEKDLQVFSCSVNSERTLLAASLVQSTKEGKRNELQPGSKCLTLLVEIHPVNNVKVLKAVDSYIWVQFLYPHIESHPLPENHLLLISEEKYIEQFRIHVAQEDGNRVVIKNSGHLPRDRIAEDFVWAQWDMSEQRLYYIDLKKSRSILKCIQFYADESYNLMFEVPLDISLSNSGFKLVNFGCDYHQYRDKFSKHLTLCVFTNHTGSLCVCYSPKCASWGQITYSVFYIHKGHSKTFTTSLENVGSHMTKGITFLNLDYYVAVYLPGHFFHLLNVQHPDLICHNLFLTGNNEMIDMLPHCPLQSLSGSLVLDCCSGKLYRALLSQSSLLQLLQNTCLDCEKMAALHCALYCGQGAQFLEAQIIQWISENVSACHSFDLIQEFIIASSYWSVYSETSNMDKLLPHSSVLTWNTEIPGITLVTEDIALPLMKVLSFKGYWEKLNSNLEYVKYAKPHFHYNNSVVRREWHNLISEEKTGKRRSAAYVRNILDNAVKVISNLEARNLGPRLTPLLQEEDSHQRLLMGLMVSELKDHFLRHLQGVEKKKIEQMVLDYISKLLDLICHIVETNWRKHNLHSWVLHFNSRGSAAEFAVFHIMTRILEATNSLFLPLPPGFHTLHTILGVQCLPLHNLLHCIDSGVLLLTETAVIRLMKDLDNTEKNEKLKFSIIVRLPPLIGQKICRLWDHPMSSNIISRNHVTRLLQNYKKQPRNSMINKSSFSVEFLPLNYFIEILTDIESSNQALYPFEGHDNVDAEFVEEAALKHTAMLLGL