Mouse primary neuronal cultures
Mixed cortical/hippocampal primary neuronal cultures were prepared from E16 ICR (CD-1®) outbred mice (Harlan Laboratories, Horst, Netherlands). Cortices with adjacent hippocampi were dissected in ice-cold HBSS, incubated in 7 ml TrypLe Express for 10 min at 37°C and triturated in Dulbecco’s modified Eagle’s medium (DMEM) (1 g/l glucose) containing 5% FCS (all from Life Technologies) through repeated pipetting with a 5 ml serological pipette and then passed through a 70 µM cell strainer. Neurons were cultured in Neurobasal medium supplemented with B27 (1:50) and 1 mM GlutaMax (all from Life Technologies, Zug, Switzerland) on 96-well plates coated with poly-D-lysine (Sigma Aldrich, St. Gallen, Switzerland) in a humidified incubator at 37°C in a 5% (vol/vol) CO2 atmosphere.
siRNA transfection
4DIV primary neurons were transfected with 50 nM of siRNA (stealth siRNA, Life Technologies, Zug, Switzerland) using Lipofectamine RNAiMax (Life Technologies, Zug, Switzerland) as transfection reagent. For each gene, a pool of 4 different siRNA duplexes was used. A pool of 3 different non-targeting siRNAs served as negative control. For each well of a 96-well plate 0.4 µl of RNAiMax were mixed with siRNA and Neurobasal (Life Technologies, Zug, Switzerland) in a final volume of 20 µl. The mix was incubated for 25 min at RT and then further diluted with Neurobasal medium to 100 µl. Cells were incubated with the transfection mix for 6 h, after which the medium on the cells was again replaced with Neurobasal medium supplemented with B27 (1:50) and 1 mM GlutaMax (all from Life Technologies, Zug, Switzerland).
siRNAs
negative controls
medGC duplex #1 Life Technologies #12935-111
medGC duplex #2 Life Technologies #12935-112
medGC duplex #3 Life Technologies #12935-113
APP
Sense sequence 1 GCGGAUGGAUGUUUGUGAGACCCAU
Sense sequence 2 UCAGGAUUUGAAGUCCGCCAUCAAA
Sense sequence 3 GACCAGGUUCUGGGCUGACAAACAU
Sense sequence 4 CACACACCCACAUCGUGAUUCCUUA
APOE
Sense sequence 1 GGUUCGAGCCAAUAGUGGAAGACAU
Sense sequence 2 GCAGAGCUCCCAAGUCACACAAGAA
Sense sequence 3 GAUGGAGGAACAGACCCAGCAAAUA
Sense sequence 4 GAGAAUCAAUGAGUAUCCUUCUCCU
BIN1
Sense sequence 1 CCUGGCAGGGAUGAAGCAAACAAGA
Sense sequence 2 UCGGACCUAUCUGGCUUCUGUUAAA
Sense sequence 3 GAUGACGCAUUUGUCCCUGAGAUCA
Sense sequence 4 AAGAGAUGAGUAAGCUCAAUCAGAA
CLU
Sense sequence 1 UCUCUGACAAUGAGCUCCAAGAACU
Sense sequence 2 UGUACUUGAGCAGAGCGCUAUAAAU
Sense sequence 3 ACGCCAUGAAGAUUCUCCUGCUGUG
Sense sequence 4 CCACUCAAGGGAGUAGGUAUAUUAA
PICALM
Sense sequence 1 GGGAGAUCCUUUCUCUGCUACUCUA
Sense sequence 2 GCUUGACUUGCAGCAGCCAACCUUU
Sense sequence 3 UGGCUCCGCGGUAUCUAAGACAGUA
Sense sequence 4 CAGCAGUCUUCUUGAUGCUUUAGAA
PRNP
Sense sequence 1 GGGACAACCUCAUGGUGGUAGUUGG
Sense sequence 2 CCAGUGGAUCAGUACAGCAACCAGA
Sense sequence 3 UGGAGCAGAUGUGCGUCACCCAGUA
Sense sequence 4 CACGACUGCGUCAAUAUCACCAUCA
CST3
Sense sequence 1 CCAGACAAAUUUGACUGACUGUCCU
Sense sequence 2 AGGCACUCUGCUCCUUCCAGAUCUA
Sense sequence 3 GACUUCGCUGUGAGCGAGUACAACA
Sense sequence 4 CAGCUCGUGGCUGGAGUGAACUAUU
MAPT
Sense sequence 1 CAGGAGGUGGCCAGGUGGAAGUAAA
Sense sequence 2 CAGGAGGUGGCAAGGUGCAGAUAAU
Sense sequence 3 CAGUCGAAGAUUGGCUCCUUGGAUA
Sense sequence 4 CAAGACAGACCAUGGAGCAGAAAUU
TARDBP
Sense sequence 1 GCAAUCUGGUAUAUGUUGUCAACUA
Sense sequence 2 CGAAAGGGUUUGGCUUUGUUCGAUU
Sense sequence 3 GAAAUACCAUCAGAAGACGAUGGGA
Sense sequence 4 CCUCCCUGUUGAGUGAGGCUAUUUA
RELN
Sense sequence 1 GCUCUCAAACUGGAUUUCAAGAUAA
Sense sequence 2 CCUGGGUGAUCGACCAGAUUCUUAU
Sense sequence 3 GAGAGCUCAUUAUACAGCCAGGAUA
Sense sequence 4 CAGUUCCAUGAAGCCACCAUUUAUA
KAT5
Sense sequence 1 AGCCUGGACGGAAGCGGAAAUCUAA
Sense sequence 2 CGGCACCCUCCAGGCAAUGAAAUUU
Sense sequence 3 CGUAAUGACGGAGUAUGACUGCAAA
Sense sequence 4 CACACUGCAGUAUCUCAACCUCAU
SNCA
Sense sequence 1 GCAAGUGACAAAUGUUGGAGGAGCA
Sense sequence 2 GGGAGUCCUCUAUGUAGGUUCCAAA
Sense sequence 3 CCAAGACUAUGAGCCUGAAGCCUAA
Sense sequence 4 CACAGGAAGGAAUCCUGGAAGACAU
Alamar Blue assay
Alamar Blue metabolisation was used as an indirect measure of cell number and viability of primary neurons. Alamar BlueTM assay reagent (AbD Serotec Ltd, Bio-Rad, Cressier, Switzerland) was added to the cells at a final concentration of 10% for the final 3 h before termination of the medium collection period. Levels of the metabolite resofurin were assessed by 544EX nm/590EM nm fluorescence measurements with the Spectra MAX-GEMINI-XS spectrofluorometer (Molecular-Devices, Sunnyvale, CA, USA).
RNA isolation
For RNA extraction, adherent neurons were briefly washed with PBS (containing CaCl2 and MgCl2) and lysed in 100 µl TRI-Reagent (Sigma-Aldrich, Buchs, Switzerland) per well. Lysates of technical replicates of each experimental condition were pooled and isolation of RNA was performed according to manufacturer's instructions. All RNA samples were subjected to DNase treatment using 0.5 units of DNaseI (Fermentas; Life Technologies, Zug, Switzerland) for 30 min at 37°C. DNase was heat inactivated by a 10 min incubation at 65°C in the presence of 5 mM EDTA. RNA concentrations were measured with a Nanodrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Protein isolation
Protein was recovered from the organic/phenol phase that was obtained during RNA isolation with TRI-Reagent (Sigma-Aldrich, Buchs, Switzerland) following the manufacturer’s protocol for protein precipitation with acetone. The obtained protein pellet was resuspended in 9.5 M Urea (pH 9.0), 2% CHAPS. For analysis by electrochemiluminescence multiplex assay, samples were diluted 1:100 with TBS (pH 7.5) containing 0.05% Tween and 1% BlockerA (Meso Scale Discovery).
cDNA synthesis
For each sample 500 ng of total RNA was reverse-transcribed using the iScript cDNA-Synthesis-kit (Bio-Rad, Cressier, Switzerland) according to manufacturer’s instructions.
Real-time PCR
Real-time PCR for relative quantification of cDNA levels was performed with the 7900HT Real-Time PCR System (Life-Technologies, Zug, Switzerland), using the iTaq-SybrGreen Supermix with ROX (Bio-Rad, Cressier, Switzerland). Relative gene expression levels were calculated with the ΔΔCt-method using GAPDH for normalization.
Real-time PCR primer
GAPDH
Forward primer ATCACTGCCACCCAGAAGAC
Reverse primer GGATGCAGGGATGATGTTCT
APP
Forward primer ACCGTTGCCTAGTTGGTGAGT
Reverse primer CGGTGTGCCAGTGAAGATG
APOE
Forward primer CTGAACCGCTTCTGGGATTAC
Reverse primer CCATCAGTGCCGTCAGTTCT
BIN1
Forward primer GAAGATCGCCAGCAACGTAC
Reverse primer TGCTCAAACTGCTCGTCCTT
CLU
Forward primer CGTCCAGGGAGTGAAGCA
Reverse primer AATCCCTAGTGTCCTCCAGAGC
PICALM
Forward primer AAGGTTGCACCAACAACTGC
Reverse primer CTATCATGCCCGTTGGTGTAGT
PRNP
Forward primer TCCATTTTGGCAACGACTG
Reverse primer TCGTGCACGAAGTTGTTCTG
CST3
Forward primer TACAACAAGGGCAGCAACG
Reverse primer TAGTTCGGCCCATCTCCAC
MAPT
Forward primer TCGCCAGGAGTTTGACACA
Reverse primer GTCTCCGATGCCTGCTTCT
TARDBP
Forward primer GAAGACGATGGGACGGTGT
Reverse primer TCCACCAGTCGGACTCCTC
RELN
Forward primer CAAGGTGACGACTGCTCTGTC
Reverse primer ACTCCACCCTGGATGGTTTC
KAT5
Forward primer CCTGTGTCTTCTGGCCAAGT
Reverse primer CCCACGATGTGGAAACCTT
SNCA
Forward primer ATGTTGGAGGAGCAGTGGTG
Reverse primer GCCCATCTGGTCCTTCT
Electrochemiluminescence assay
Aβx-40 and Aβx-42 levels in 24 h conditioned medium of 8DIV mouse primary neurons were analyzed with the Aβ-Panel1 (4G8) Kit (Meso Scale Discovery, Maryland, USA) following the manufacturer’s instructions. To each well of a 96-well assay plate 25 µl of 1:50 diluted Sulfo-Tag 4G8 mAb detection antibody and 25 µl of conditioned medium were added and incubated overnight at 4°C. Total Tau and phosphoThr231-Tau from 8DIV mouse primary neuronal cultures were assayed in the recovered protein fraction with the Phospho(Thr231)/Total Tau Kit (Meso Scale Discovery, Maryland, USA) following the manufacturer’s instructions. To each well of a 96-well assay plate 25 µl of sample were added and incubated for 2 h at RT on a shaker at 750 rpm. Incubation with the detection antibody was for 1 h at RT on a shaker at 750 rpm using 25 µl of Sulfo-Tag anti-Tau antibody per well at a concentration of 1 µg/ml. Measurements were taken on a Sector-Imager-6000 (Meso-Scale-Discovery, Maryland, USA). Electrochemiluminescence values were normalized to the corresponding Alamar-Blue assay values.
SDS-PAGE and Western blot
Proteins were separated on 4–12% Bis-Tris gels (Life Technologies, Zug, Switzerland) and blotted on 0.2µm Nitrocellulose membranes (Life Technologies, Zug, Switzerland). Unspecific binding was blocked by preincubation of membranes with TBS-Tween(0.05%) containing 5% w/v nonfat milk powder. Incubation with primary antibodies was performed overnight at 4°C, incubation with secondary antibodies for 1 h at room temperature. Infrared signal at 700 nm and 800 nm were acquired with an Odyssey CLx Imaging System (Li-COR Biosciences, Bad Homburg, Germany).
Primary antibodies
goat anti-ApoE (Santa Cruz # sc-6384) 1:100
rabbit anti-Bin1 (Proteintech # 14647-1-AP) 1:1000
goat anti-ApoJ/Clu (Abcam # ab79280) 1:500
rabbit anti-Picalm (Abcam # ab106409) 1:500
mouse anti-PrnP (Abcam # ab61409) 1:500
rabbit anti-Cst3 (Proteintech # 12245-1-AP) 1:1000
mouse anti-β-Actin (Abcam # ab6276) 1:10000
mouse anti-Gapdh (Life Technologies # AM4300) 1:5000
Secondary antibodies
donkey anti-mouse IRDye 800CW (Li-COR Biosciences # 926-32212) 1:5000
donkey anti-rabbit IRDye 800CW (Li-COR Biosciences # 926-32213) 1:5000
donkey anti-goat IRDye 800CW (Li-COR Biosciences # 926-32214) 1:5000
donkey anti-mouse IRDye 680RD (Li-COR Biosciences # 926-68072) 1:5000