Cells, Antibodies, and Reagents
Maintenance of mouse neuroblastoma N2a cells, N2a cells stably expressing human APP Swedish mutation (N2aSwe), human HeLa cells stably expressing human APP Swedish mutation (HeLaSwe), and rat PC12 cells has been described (Lin et al., 2007
; Wang et al., 2006
; York et al., 2000
). Phoenix-Ampho helper cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Antibodies used were: anti-Myc (9E10), anti-adenylate cyclases, anti-His, anti-ADAM10 and anti-TACE from Santa Cruz Biotechnology; anti-GSK-3α, anti-GSK-3β, anti-phospho-GSK-3α/β (Ser21/9), anti-CREB, anti-phospho-CREB (Ser133), anti-PSD-95, and anti-synapsin from Cell Signaling Technology; anti-Aβ40, anti-pT205 tau and anti-total tau from Abcam; anti-Aβ (6E10) from Covance; anti-tau-1 from Chemicon; anti-α-tubulin from Sigma; anti-PHF-1 tau from P. Davies at Albert Einstein School of Medicine; and the FCA18 antibody specifically recognizing the N-terminus of APP βCTF from F. Checler at Institut de Pharmacologie Molecularie et Cellulaire du CNRS (Ancolio et al., 1999
). The rabbit polyclonal antibody 369 against the APP C-terminus (Xu et al., 1997
) and the anti-FG01 antibody were developed in our laboratory. PKA inhibitor H89 and GSK-3 inhibitor lithium chloride were from Sigma.
Random Homozygous Gene Perturbation (RHGP) Strategy for Screening Aβ-reducing Genes
We constructed a new RHGP gene search vector from the original pLLGSV vector (Li and Cohen, 1996
). In both the 5’LTR and the 3’LTR regions of the new RHGP gene search vector, there is a sequence containing a puromycin N-acetyl-tranferase gene (pac
), a TRE (tetracycline-regulated element, tet-off
) regulated CMV promoter driving the pac
gene, a plasmid replication origin and a chloramphenicol resistance marker (Ori-CAT), and a Lox
P site. In addition, there is a Cre recombinase gene (Cre
) between the 5’LTR and the 3’LTR (). This new RHGP gene search vector was transfected into Phoenix-Ampho help cells. Generated infectious retrovirus in the cell culture supernatant was harvested and used to infect N2aSwe cells.
The infected N2aSwe cells with RHGP vector integration were selected with puromycin, live-stained with fluorescence-labeled APP βCTF antibody FCA18 (Ancolio et al., 1999
), and subjected to multiple rounds of FACS sorting for cells with accumulated cell surface APP βCTF. The positive sorted cells were then treated with doxycycline (a derivative of tetracycline) and sorted for cells whose surface APP βCTF level was reversed back to background level in the presence of doxycycline. Resultant cells were cloned individually and assayed by ELISA and Western blotting to confirm surface accumulation of APP βCTF and reduction of Aβ generation. Positive candidate cell clones were further characterized and used for gene isolation.
Fg01 Gene Cloning
Genomic DNA was extracted from FG01 cells, digested with restriction enzyme BamHI or HindIII, and self-ligated overnight with T4 ligase. The ligated DNA was precipitated, dissolved in TE buffer and electroporated into DH10B ElectroMax competent cells. The plasmid DNA from individual colonies was prepared for DNA sequencing. The target gene was identified by using UCSC Genome Browser Program.
We blasted GenBank database with Fg01
cDNA sequence to explore its origin. Homologous sequences between Fg01
and mouse Rps23
were aligned manually. Sequence similarity between Fg01
cDNA sequences of humans, mice and rats were compared using their homologous regions. Potential transmembrane region in the FG01 protein was predicted using PredictProtein (Rost et al., 2004
N2a cells were transfected with FG01, APP or SMAD3 expression vectors (all Myc-tagged). After 48 hrs, cells were washed with ice-cold phosphate-buffered saline, collected with homogenization buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 200 mM sucrose, 1 mM phenylmethylsulfonyl fluoride) and homogenized with a ball bearing cell cracker. Samples were centrifuged at 900 × g for 10 min to remove cell debris and nuclei. Supernatants were centrifuged at 100,000 × g for 60 min at 4°C. After transferring the supernatant (cytosol) to a new tube, the pellet was washed and re-suspended with an equal volume (to that of cytosol) of homogenization buffer.
FG01 transfected N2a cells were washed with ice-cold phosphate-buffered saline containing 1 mM each of CaCl2 and MgCl2 and incubated at 4 °C with 0.5 mg/ml Sulfo-NHS-LC-biotin (Pierce) for 20 min and the process repeated once. Cell lysates were prepared in Nonidet P-40 lysis buffer. After affinity precipitation with streptavidin beads (Pierce), biotinylated proteins were eluted with SDS-PAGE sample buffer (Invitrogen) and loaded directly on SDS-PAGE gels for electrophoresis followed by Western blot analysis with the Myc antibody.
For cell surface immunostaining of FG01, N2a cells were first transfected with the Myc-FG01-His6 plasmid. Cells were then directly incubated with Myc or His6 antibody at 4°C for 2 hrs, followed by washing, fixation, and permeabilization. In some experiments, cells were permeabilized before incubating with antibodies. Treated cells were incubated with Alexa Fluor 488-conjugated secondary antibody and DAPI. Specimens were examined and fluorescence images collected using a Zeiss fluorescence microscope with AxioVision software.
Aβ ELISA Assay
HeLaSwe cells were transfected with FG01 or controls. Conditioned media and lysates from these cells were collected. The levels of Aβ40 and Aβ42 were quantified using ELISA kits (Invitrogen), following the manufacturer’s protocols.
N2aSwe cells were transfected with control vector or FG01 and then equally split. Four hours before collection, cells were treated with the GSK-3 inhibitor lithium chloride (5 mM) or sodium chloride (5 mM, as control). Alternatively, cells were treated with a PKA inhibitor H89 (10 µM) or with DMSO for control.
FG01 RNA Interference and Quantitative Real-Time PCR
The mouse FG01 siRNA used was: 5’-UACUGUUUGUCAUGCCACUUCUGAU-3’. The control siRNA was from Invitrogen. siRNA was transfected into N2a cells using Lipofectamine RNAiMAX reagent (Invitrogen), following the manufacturer’s protocol. After FG01 RNA interference, total RNA was extracted from N2a cells by Trizol reagent (Invitrogen). After reverse transcription into first strand cDNA using standard conditions, samples were analyzed independently by real-time PCR using an iCycler iQ with SYBR green supermix (Bio-Rad). The FG01 primer pair used for real time PCR was: FG01–5’ (5’-TGTTGCATACACATACATGC-3’) and FG01–3’ (5’-TCATTAAGAACGGGAAGAAG-3’). A pair of β-actin primers served as controls (Zhang et al., 2007
In Situ Hybridization
Histological sections from 2-month-old C57Bl6 mice were used for in situ hybridization reactions. Digoxygenin-labeled sense and antisense probes were generated for FG01 (corresponding to nucleotides 1–641 of NM_001024728), and the hybridization signal was detected using an alkaline-phosphatase-conjugated anti-digoxygenin antibody and BCIP/NTB (Roche).
Crossing Brain-Specific Fg01 Transgenic Mice with 3XTg AD Mice
We generated brain-specific Fg01
transgenic mice (Figure S7
). Hemizygous Fg01
transgenic mice were crossed with homozygous triple transgenic (3XTg) AD mice harboring mutations in human App
(tau) genes on a presenilin 1 (PS1) mutant background (Oddo et al., 2003
). Procedures involving animals and their care conformed to institutional guidelines (Animal Resources Department at Burnham Institute for Medical Research).
Immunohistochemistry and Data Analyses
Fg01/3XTg mice and littermate controls on a 3XTg background were sacrificed at 11 months of age. Half of the brain was used for immunoblot analysis and the other half was paraffin-embedded for immunohistochemistry. Coronal brain sections (4 µm) were deparaffinized, hydrated, and then immunostained with anti-Aβ antibodies (an anti-Aβ40 specific antibody and 6E10), anti-phosphorylated tau antibodies (PHF-1 and pT205), or antibodies against PSD-95 and synapsin. After additional incubation with biotinylated secondary antibody, samples were incubated in ABC Elite (HRP) reagent (Vector Laboratories). Reactions were visualized by developing in DAB substrates (Vector Laboratories). All samples were visualized under a light microscope.
For immunohistochemistry comparison of Aβ and tau, immunostained neurons (>400) in were counted from five randomly selected cortical regions. Ratios of Aβ-positive and phosphorylated tau-positive neurons to total neurons were determined and normalized to those of controls. For immunohistochemistry comparison of synapse markers, five hippocampal regions were randomly selected and the images captured. After converting the images to grayscale, the optical density (darkness) of molecular layer staining was measured as an average of the gray value between white (0) and black (255) as described (Mathern et al., 1997
) for comparison, by a computer-based image analysis using the Photoshop software.
In Vitro Activity Assays and cAMP Assay
Commercial kits were used to assay in vitro activities of GSK-3β (Sigma) and PKA (Upstate). For GSK-3α activity, a commercial GSK-3β activity assay kit was used but the procedure to immunoprecititate GSK-3β was replaced with immunoprecipitation of GSK-3α using an anti-GSK-3α antibody (Cell Signaling). cAMP levels were assayed using a commercial kit (Biovision).
Cells transfected with FG01 were lysed in either CHAPSO buffer (1% CHAPSO, 25 mM HEPES, pH7.4, 150 mM NaCl, and 2mM EDTA supplemented with protease inhibitors) or in NP40 buffer (1% NP40 in phosphate buffered saline, supplemented with protease inhibitors). Lysates were immunoprecipitated using mouse IgG, rabbit IgG, and antibodies against Myc or adenylate cyclases and Trueblot™ IP beads (eBioscience), followed by Western blot with antibodies against Myc or adenylate cyclases.