All animal use and care were in accordance with local institution guidelines of the University Medical Center Utrecht (Utrecht, the Netherlands) and approved by the Dierexperimenten Ethische Commissie Utrecht with the protocol number DEC 2013.I.09.069. Wild-type C57BL6/J (strain: 000664), C9ORF72 KO (C57BL/6J-3110043021Rikem5Lutzy/J, strain: 027068), and C9-BAC (C57BL/6J-Tg(C9orf72_i3)112Lutzy/J, strain: 023099) were purchased from Jackson Laboratories (The Jackson Laboratory, Bar Harbor, USA). Mice were housed in standard conditions with food and water ad libitum in the conventional vivarium at the University of Southern California. All animal use and care were in accordance with local institution guidelines of the University of Southern California and the IACUC board of the University of Southern California (Los Angeles, USA) with the protocol numbers 20546 and 11938.
To determine if reduced C9orf72 levels leads to glutamate receptor accumulation in vivo, we examined spinal motor neurons deleted of C9orf72 in Nestin-Cre-Stop-Flox-C9orf72 mice 22. Immunofluorescence analysis indicated that Nr1 (NMDA) and GluR1 (AMPA) levels were elevated in C9orf72-null motor neurons (Supplementary Fig. 12a, b). To confirm these findings, we isolated post-synaptic densities from the spinal cords of control and C9orf72 knockout mice. Post-synaptic density fractions contained glutamate receptors and PSD-95, but not p53 or synaptophysin, indicating they were enriched for post-synaptic density proteins (Supplementary Fig. 12c, 5i). Immunoblotting showed that post-synaptic densities in C9orf72 knockout mice contained significantly higher levels of Nr1 and Glur1 than in control mice (Fig. 4i, j and Supplementary Fig. 5j).
iMNs from healthy controls and ALS patients were collected on day 21 post-transduction in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche). Protein quantity was measured by the BCA assay (Pierce) and samples were run on a 10% SDS gel at 4 °C. The gel was transferred onto an Immobilon membrane (Millipore). The membrane was blocked with 5% milk in 0.1% PBS-Tween 20 (PBS-T)(Sigma-Aldrich), incubated with primary antibodies overnight at 4 °C, washed three times with 0.1% PBS-T, then incubated with horseradish peroxidase (HRP)-conjugated (Santa Cruz). After three washes with 0.1% PBS-T, blots were visualized using an Amersham ECL Western Blotting Detection Kit (GE) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) and developed on X-ray film (Genesee). The following primary antibodies were used: rabbit anti-C9ORF72 (Proteintech, cat. no. 22637–1-AP), mouse anti-GAPDH (Santa Cruz, cat. no. sc-32233), chicken anti-MAP2 (Abcam, cat. no. ab11267), mouse anti-FLAG (Sigma, cat. no. F1804), rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), mouse anti-LAMP1 (Abcam, cat. no. Ab25630), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-EEA1 (BD Biosciences, cat. no. BD610457), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045) , mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S). For C9ORF72 western blots, to generate enough motor neurons for C9ORF72 protein detection, we used a directed differentiation method described previously 28.
To measure the effect of dipeptide repeat protein expression on iMN survival, PR50 and GR50 were cloned into the pHAGE lentiviral vector as fusions with GFP to allow tracking of protein expression. iMN cultures were transduced with PR50 and GR50 lentiviruses at day 17 of reprogramming and longitudinal survival analysis was started the same day. 10 ng/ml of GDNF, BDNF, and CNTF was maintained throughout the experiment, and glutamate treatment was not performed. To measure PR50 turnover, PR50 was cloned into the pHAGE lentiviral vector as a fusion with Dendra2 (Addgene). iPSC-derived fibroblasts were generated according to Daley and colleagues64. Briefly, when C9ORF72−/− iPSC cultures reached 80% confluence, the medium was switched from mTeSR1 (Stem Cell Technologies) to human fibroblast medium containing DMEM (Life Technologies), 10% fetal bovine serum (FBS)(Thermo Fisher Scientific), and 1% penicillin/streptomycin (Life Technologies). Cells were passaged 2 to 3 times using Accutase (Life Technologies) before use in experiments. iPSC-derived fibroblasts were transduced with either pMXs-eGFP or pMXs-C9ORF72 isoform B-T2A-eGFP retrovirus and treated with 10 μg/ml mitomycin C for 3 hrs to inhibit cell proliferation. The cells were then transduced with the PR50–Dendra2 lentivirus and exposed to blue light for 1.5 sec using a lumencor LED light source to initiate photoconversion. The amount of decay (as a fraction of the starting level) of the red fluorescent punctae was monitored by longitudinal time lapse imaging in a Molecular Devices ImageExpress and analyzed using SVCell 2.0 (DRVision Technologies). Fluorescence was quantified at t = 0 and 12 hours after photoconversion. Distinct photoconverted punctae were treated as discrete objects for analysis (n = 20 each for +eGFP and +C9ORF72-T2A-eGFP). For each object, background fluorescence was subtracted and fluorescence was normalized according to object size. The fractional decay was statistically analyzed by two-tailed Student’s t-test. ** - p<.01.