Total RNA was extracted from sorted iMNs at day 21 post-transduction with Trizol RNA Extraction Kit (Life Technologies) and reverse transcribed with an Oligo dT primer using ProtoScript® II First Strand Synthesis Kit (NEB). RNA integrity was checked using the Experion system (Bio-Rad). Real-time PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using primers shown in Supplementary Data Table 4.
Consistent with previous studies 3,4,6–8, patient iMNs (n=5 patients) had reduced C9ORF72 expression compared to controls (n=3; Fig. 2a and Supplementary Fig. 4a, 5b). While previous studies have linked low C9ORF72 levels to changes in vesicle trafficking or autophagy 18,20,30–33, it remains unknown if loss of C9ORF72 protein directly contributes to degeneration. Thus, we re-expressed C9ORF72 (isoform A or B) in iMNs using a retroviral cassette (Supplementary Fig. 4b) and found that both isoforms rescued C9ORF72 patient iMN survival in response to glutamate treatment (n=3 patients Fig. 2b and Supplementary Fig. 4c). This effect was specific for C9ORF72 iMNs, as forced expression of C9ORF72 did not rescue SOD1A4V iMN survival (Fig. 2c), nor did it improve the survival of control iMNs (n=2 controls Fig. 2d and Supplementary Fig. 4d).
Mice were anesthetized with i.p. ketamine (100 mg ⁄ kg) and xylazine (10 mg ⁄ kg), and body temperature kept at 36.9 ± 0.1°C with a thermostatic heating pad. Mice were placed in a stereotactic apparatus (ASI Instruments, USA) and the head is fixed accordingly. A burr hole was drilled, and an injection needle (33 gauge) was lowered into the hippocampus between CA1 and the dentate gyrus (AP −2.0, ML +1.5, DV −1.8). NMDA (20 nmol in 0.3 μl of phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). Simultaneously, or independently, Apilimod (0.3 μl of 20 μM in phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). The needle was left in place for an additional 8 min after the injection. Animals were euthanized 48 h later. Brains were quickly removed, frozen on dry ice, and stored at −80°C until processing. Thirty-micrometer-thick coronal sections were prepared using a cryostat. Every fifth section 1 mm anterior and posterior to the site of injection was stained with cresyl violet. The lesion area was identiﬁed by the loss of staining, measured by NIH ImageJ software and integrated to obtain the volume of injury.
Whole cell membrane potential and current recordings in voltage- and current-clamp configurations were made using an EPC9 patch clamp amplifier controlled with PatchMaster software (HEKA Electronics). Voltage- and current-clamp data was acquired at 50 kHz and 20 kHz, respectively, with a 2.9 kHz low-pass Bessel filter, while spontaneous action potential recordings were acquired at 1 kHz sampling frequency. For experiments, culture media was exchanged with warm extracellular solution consisting of (in mM): 140 NaCl, 2.8 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, and 10 glucose, with pH adjusted to 7.3 and osmolarity adjusted to 305 mOsm. Glass patch pipettes were pulled on a Narishige PC-10 puller and polished to 5–7 MΩ resistance. Pipettes were also coated with Sylgard 184 (Dow Corning) to reduce pipette capacitance. The pipette solution consisted of (in mM): 130 K-gluconate, 2 KCl, 1CaCl2, 4 MgATP, 0.3 GTP, 8 phosphocreatine, 10 HEPES, 11 EGTA, adjusted to pH 7.25 and 290 mOsm. Pipettes were sealed to cells in GΩ-resistance whole cell configuration, with access resistances typically between 10–20 MΩ, and leakage currents less than 50 pA. Capacitance transients were compensated automatically through software control. For voltage clamp, cells were held at −70 mV. For Current-voltage traces, a P/4 algorithm was used to subtract leakage currents from the traces. Measurements were taken at room temperature (approximately 20–25 °C). Data was analyzed and plotted in Igor Pro 6 (WaveMetrics) using Patcher’s Power Tools plug-in and custom programmed routines. Current density was obtained by dividing the measured ion channel current by the cell capacitance. For control iMNs, 10/10 tested fired action potentials. For C9-ALS iMNs, 9/10 tested fired action potentials.
Near the cities Beijing, Tianjin, Shijiazhuang, and Jinan, Wuqiao County has many transportation connections. There are many rail and bus services operating in the town. Wuqiao was the first Chinese city to open up its doors to the world under the "Open Door" policy and over many years development, Wuqiao has become a flourishing city with a favorable investment environment.
International Advisory Board: James Archibald (Translation Studies) - Hugo de Burgh (Chinese Media Studies) - Kristen Brustad (Arabic Linguistics) - Daniel Coste (French Language) - Luciano Curreri (Italian Literature) - Claudio Di Meola (German Linguistics) - Donatella Dolcini (Hindi Studies) - Johann Drumbl (German Linguistics) - Denis Ferraris (Italian Literature) - Lawrence Grossberg (Cultural Studies) - Stephen Gundle (Film and Television Studies) - Tsuchiya Junji (Sociology) - John McLeod (Post-colonial Studies) - Estrella Montolío Durán (Spanish Language) - Silvia Morgana (Italian Linguistics) - Samir Marzouki (Translation, Cultural Relations) - Mbare Ngom (Post-Colonial Literatures) - Christiane Nord (Translation Studies) - Roberto Perin (History) - Giovanni Rovere (Italian Linguistics) - Lara Ryazanova-Clarke (Russian Studies) - Shi-Xu (Discourse and Cultural Studies) - Srikant Sarangi (Discourse analysis) - Françoise Sabban, Centre d'études sur la Chine moderne et contemporaine (Chinese Studies) - Itala Vivan (Cultural Studies, Museum Studies)