Skin biopsies were obtained from two VSM-20 family members, one with predominantly bvFTD, and the other a presymptomatic carrier. To protect their privacy, they are simply named as carrier 1 and carrier 2 without disclosing their personal information. Frozen tissues, consisting of 1 cm3 blocks of superior frontal gyrus, were obtained from two middle-aged males with bvFTD due to C9ORF72 hexanucleotide repeat expansion. The study was approved by the Institutional Review Board and Ethics Committees at the University of California, San Francisco (UCSF) and written informed consent was obtained from all participants in this study.
Isolation of primary human skin fibroblasts and generation of iPSCs
Skin biopsies were cut into small pieces and placed on culture dishes to allow fibroblasts to expand. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 1X nonessential amino acids, and penicillin/streptomycin (100 U/ml).
FTD patient-specific iPSCs were generated as described [3, 44]. Briefly, fibroblasts (8 × 105 per 100 mm dish) were transduced with equal volumes of supernatants from cultures of retroviruses expressing human OCT3/4, SOX2, KLF4, and c-MYC. The next day, the medium was removed and replaced with fresh viral supernatants. Seven days after the first infection, cells were collected and seeded (5 × 104 cells per 100 mm dish) on SNL feeder cells treated with mitomycin C. One day later, the medium was replaced with iPSC medium containing 4 ng/ml basic fibroblast growth factor; thereafter, the medium was changed every other day. Five weeks after viral transduction, colonies were picked, transferred to 12-well plates coated with Matrigel (BD Biosciences), and cultured in mTeSR1 medium (StemCell Technologies). For expansion, cells were dissociated with 1:2 accutase/PBS solution for 1 min at room temperature, washed twice with PBS, and scraped with a cell lifter into mTeSR1 medium. Larger colonies were further broken up by pipetting and transferred to 6-well plates.
Neuronal differentiation and immunocytochemistry
Neuronal differentiation of human iPSC lines and immunocytochemistry on postmitotic neurons were performed as described .
For neuronal differentiation, iPSC colonies were detached with accutase (Millipore) and grown as embryoid bodies (EBs) in suspension for 5–6 days in iPSC medium in the absence of basic fibroblast growth factor. EBs were allowed to attach and form rosettes. Ten-day-old rosettes were collected and grown in suspension as neurospheres. Neurospheres were dissociated after 3–4 weeks, and the cells were placed on glass coverslips (BD Biosciences) or plates coated with poly-d-lysine (0.1 mg/ml) and laminin (10 μg/ml). Neurons were used after 2–4 weeks in culture.
For in vitro differentiation, EBs were generated as described above, grown for 8 days in suspension, placed on Matrigel-coated glass coverslips, and allowed to further differentiate for 8 days in mTeSR1 medium. Cells migrating out of the attached EBs were stained and analyzed by fluorescence microscopy (Olympus IX-70 microscope) for markers of the three germ layers. Karyotype analysis was done at the Cytogenetic Laboratory, UMASS Memorial (Worcester, MA), using standard protocols for G-banding.
For immunocytochemistry, cells were fixed in 4 % paraformaldehyde (pH 7.4) for 10 min and permeabilized with 0.2 % Triton X-100. After blocking with 3 % bovine serum albumin for 30 min, cells were incubated with primary antibodies for 1 h at room temperature or overnight at 4C. The primary antibodies were goat anti-Nanog (R&D Systems; 1:100), mouse anti-SSEA4 (Abcam; 1:100), rabbit anti-desmin (Thermo Scientific; 1:100), mouse anti-βIII-tubulin (Promega; 1:500), and mouse anti-α-fetoprotein (R&D Systems; 1:200), mouse anti-MAP2 (Sigma; 1:500), rabbit anti-glial fibrillary acidic protein (Dako; 1:1,000), rabbit anti-VGLUT1 (Synaptic Systems; 1:500), rabbit anti-GABA (Sigma; 1:100), rabbit anti-tyrosine hydroxylase (Millipore; 1:500), mouse anti-hnRNP A2/B1 (Santa Cruz Biotechnologies; 1:700), goat anti-hnRNP H1 (Santa Cruz Biotechnologies; 1:3,000), rabbit anti-hnRNP H2 (Sigma; 1:300), mouse anti-hnRNP F (Santa Cruz Biotechnologies; 1:1,000), rabbit anti-TDP43 (Protein Tech Group; 1:100), rabbit anti-FUS (Protein Tech Group; 1:300), mouse anti-nucleophosmin (Abcam; 1:3,000). After three washes with PBS, the cells were incubated with AlexaFluor-conjugated secondary antibodies (Invitrogen; 1:300) for 1 h at room temperature and counterstained with 1 μg/ml Hoechst or DAPI. Immunostained cells were examined by fluorescence microscopy.
Neurons (3–4 weeks in culture) plated on coverslips were mounted onto on an upright microscope (BX51WI, Olympus). Neurons were bathed in pH 7.4 oxygenated extracellular solution containing: 125 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 26 mM NaHCO3, 11 mM d-Glucose. Neurons were visually identified via infrared (IR) differential interference contrast video microscopy with an IR-CCD camera (Olympus). Depolarization-evoked action potentials and spontaneous EPSCs were recorded at room temperature using the whole-cell configuration of a Multiclamp 700B patch-clamp amplifier (Molecular Devices) in current- or voltage-clamp mode, respectively. The junction potential between the patch pipette and extracellular solution was nullified just prior to obtaining a seal on the neuronal membrane. Internal pipette solution contained: 121 mM KCl, 4 mM MgCl2, 11 mM EGTA, 1 mM CaCl2, 10 mM HEPES, 0.2 mM GTP, and 4 mM ATP. Signals were filtered at 2 kHz using the amplifier’s four-pole, low-pass Bessel filter, digitized at 10 kHz with an Axon Digidata 1,440 A interface and stored on a personal computer. Extracellular solution with or without CNQX (10 μM) or TTX (0.5 μM) was applied onto neurons by gravity superfusion.
qRT-PCR and northern blot
Total RNA was isolated using RNeasy Kit (Qiagen) and 500 ng of RNA were reverse transcribed to cDNA using TaqMan Reverse Transcription Reagents kit (Applied Biosystems) following the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green Master Mix (Applied Biosystems) and 10 μM of forward and reverse primers or TaqMan Gene Expression Master Mix and TaqMan primers (C9ORF72 variant 1, Applied Biosystems). Ct values for each sample and gene were normalized to GAPDH gene. The 2exp (−ΔΔCt) method was used to determine the relative expression of each gene. Primers used in this study can be found in Table S1. Analysis of the transgenes silencing was performed as described .
For northern blot analysis, total RNA (2–5 μg) was loaded into a 0.8 % agarose gel containing 1.8 % formaldehyde. RNA was transferred to a positively charged nylon membrane (Roche) by capillary blotting and crosslinked by UV irradiation. The probe recognizing all three C9ORF72 isoforms was synthesized with T7 RNA polymerase (Roche) from cDNAs obtained by PCR with specific primers (Table S1). RNA probes specifically detecting V2 and V3 isoforms were chemically synthesized and 5′ modified to add Dig label (sequence in Table S1). Hybridization was performed overnight at 62 °C.
Southern blot analysis was performed as described  with small modifications. Briefly, genomic DNA (10 μg) was digested overnight with XbaI, separated by electrophoresis on a 0.8 % agarose gel, transferred to a positively charged nylon membrane (Roche Applied Science), crosslinked by UV, and hybridized overnight at 47 °C with a digoxigenin-labeled PCR probe. The 676 bp probe was amplified from genomic DNA with specific primers (Table S1) and the PCR DIG Probe Synthesis Kit (Roche). The probe was denatured at 95 °C for 5 min and added to the hybridization mix (EasyHyb granules, Roche). The digoxigenin-labeled probe was detected with anti-digoxigenin antibody and CDP-Star reagent as recommended by the manufacturer (Roche).
Fluorescence in situ hybridization
FISH was performed using a Cy3-conjugated (GGCCCC)4 or (CAGG)6 oligonucleotide probes. Briefly, cells on glass coverslips were fixed in 4 % paraformaldehyde for 20 min, permeabilized in 70 % ethanol at 4 °C, incubated with 40 % formamide/2X SSC for 10 min at room temperature, and hybridized for 2 h at 37 °C with a Cy3-conjugated (GGCCCC)4 probe (16 ng/ml) in hybridization buffer consisting of 40 % formamide, 2X SSC, 10 % dextran sulfate, yeast tRNA (1 mg/ml), and salmon sperm DNA (1 mg/ml). The cells were washed once with 40 % formamide/1X SSC for 30 min at 37 °C and twice with 1X SSC at room temperature for 30 min. For the RNase A experiments, after fixation, cells were incubated with 24 μg/ml RNase A for 20 min at room temperature.
Stress-induced toxicity and caspase-3 activity assays
Two-week-old neurons were exposed to: tunicamycin, rotenone, staurosporine, chloroquine, 3-methyladenine or DMSO for 24 h. Cell viability and caspase-3-like activity were determined as described . Cell viability values were expressed as the percentage of the untreated cells or cells treated with DMSO (control) and caspase-like activity was calculated as the increase above control (untreated cells).
Fifteen micrograms of protein were separated by SDS-PAGE followed by immunoblotting with mouse anti-p62 (1:500, BD Biosciences), mouse anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, 1:3,000, Millipore) and HRP-conjugated anti-mouse second antibody (1:5,000). Immunoblots were developed by SuperSignal West Pico Chemiluminescent substrates (Thermo Scientific).
Pull-down of GGGGCC-binding proteins from mouse brain lysates
300 μg of nuclear extract from mouse brain was passed over an in vitro transcribed and biotinylated (Biotin 11 CTP, Perkin Elmer) (GGGGCC)30 RNA bound to streptavidin coated magnetic beads (Dynabeads M-280 streptavidin, Invitrogen) in the presence of 20 mM Hepes, 300 mM NaCl, 2 mM MgCl2, 0.01 % NP40, 1 mM DTT and protease inhibitor (PIC, Roche). The magnetic beads with immobilized RNA and its bound proteins were washed three times with the binding buffer and bound proteins were eluted by boiling 3 min the in sample buffer prior to 4–12 % SDS-PAGE (NuPAGE 4–12 % Bis–Tris Gel, Invitrogen) separation and silver staining (SilverQuest, Invitrogen). The protein bands were excised digested and identified using NanoESI_Ion Trap (LTQ XL Thermo Fisher Scientific).
Detection of RAN translation products
Sequential extractions of neuronal cell pellets were performed as described by Winton et al. . Human neuronal cell pellets were lysed in cold RIPA buffer and sonicated on ice. Cell lysates were then cleared by centrifugation at 100,000 g for 30 min at 4 °C. The supernatant was collected and protein concentration was determined by BCA assay. To prevent carry-over, the resulting pellet was resuspended in RIPA buffer, re-sonicated and re-centrifuged. The RIPA-insoluble pellet was then extracted using 7 M urea buffer (volume of buffer adjusted based on protein concentration of RIPA soluble samples), sonicated and centrifuged at 100,000 g for 30 min at 22 °C. The protein concentration of the urea soluble supernatant was determined by Bradford assay. Two micrograms of urea soluble sample was directly dotted onto a nitrocellulose membrane, which was then dried, blocked, and finally probed with anti-GP rabbit polyclonal antibody. The anti-GP antibody was generated by immunizing a rabbit with C-Ahx-GPGPGPGPGPGPGPGP-amide. Specificity of antibody was verified by immunoassay and by Western blot using previously described methods . First, (GP)8 peptide or (GR)8 peptide (negative control) were diluted in Tris-buffered saline (TBS) and added to duplicate wells (30 μl/well) of a 96-well Meso Scale Discovery (MSD) assay plate. Following overnight incubation at 4 °C, wells were washed with TBS containing 0.2 % Tween 20 (TBSTw) and blocked with TBSTw + 3 % non-fat milk. Blocking buffer containing anti-GP and SULFO-TAG™-rabbit secondary antibody was then added at 25 μl/well. Following a 2 h incubation and final washes, anti-GP binding to immobilized peptides was evaluated by adding MSD Read Buffer and measuring light emission at 620 nm upon electrochemical stimulation using the MSD Sector Imager 2,400.
For Western blotting, HEK293T cells were transfected using LipofectamineTM 2000 with pEGFP plasmids into which oligonucleotides of 5 repeats of GP, GR, or GA were inserted. Cell lysates were resolved by 10 % Tris–Glycine SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes for probing with anti-GP. Blots were stripped and reprobed for GFP (Zymed). Pre-immune serum was tested against the peptide antigen and confirmed negative.
Values are expressed as mean ± SEM. The significance of differences among multiple groups was determined with a one-way analysis of variance (ANOVA) followed by a Tukey–Kramer post hoc test (GraphPad Prism version 6.02). Differences were considered significant at p < 0.05.