Biotinylation of plasma membrane localized glutamate receptors was performed using the Piece™ Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, Dox-NIL iMNs were incubated with 0.25mg/ml Sulfo-NHS-SS-Biotin in cold room for 1~2 hrs with end-to-end shaking. After quenching, cells were harvested by scraping and lysed with lysis buffer from the Piece™ Cell Surface Protein Isolation Kit or the M-PER™ mammilian protein extraction buffer (Thermo Fisher Scientific). Cell lysate was incubated with High Capacity NeutrAvidin™ agorase beads (Thermo Fisher Scientific), and the bound protein was eluted in 2X SDS-PAGE sample buffer supplemented with 50mM DTT for 1 hr at room temperature with end-to-end rotation, and further analyzed by western blot.
The results of an experimental investigation of the effects of container geometry on the recovery of product water from indirectly frozen salt water are presented. Salt water was frozen in containers having circular or rectangular cross-section, then allowed to melt and drain until the residual ice was potable. Thin rectangular cross-sections were found to be more effective than circular ... [Show full abstract]Read more
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.[citation needed]

Because C9ORF72 activity is required to maintain normal lysosomal function, we measured the effect of C9ORF72 activity on PR50 clearance by monitoring the clearance of PR50-Dendra2 fusion proteins in C9ORF72−/− iPSC-derived fibroblasts with or without exogenous C9ORF72. Dendra2 is a green fluorescent protein that irreversibly converts to red fluorescence when exposed to blue light, enabling quantification of its degradation 49. PR50-Dendra2 formed discrete punctae within cells, indicating that Dendra2 did not prevent intracellular aggregation of PR50 (Supplementary Fig. 14c). Expression of C9ORF72-T2A-GFP in C9ORF72−/− iPSC-derived fibroblasts significantly enhanced the decay of PR50-Dendra2 fluorescence over GFP alone (Supplementary Fig. 14d). To determine if C9ORF72 activity modulates DPR aggregate clearance in human motor neurons, we compared the decay of PR50-Dendra2 in C9ORF72+/+ and C9ORF72+/− iMNs (Fig. 5e and Suppementary Fig. 14e). Consistent with the hypothesis that C9ORF72 activity promotes DPR aggregate clearance, PR50-Dendra2 decayed significantly slower in C9ORF72+/− iMNs (Fig. 5e).
RNA sequencing output was aligned to the GRCh38 Reference Genome and quantified using the STAR aligner.65 Genes were annotated against the GENCODE version 23 Comprehensive Gene Annotation. Quality control was performed using Picard Tools AlignmentSummaryMetrics. Samples passing quality control and having RNA Integrity Number (RIN) > 5 were used in downstream analysis. To identify differentially expressed genes, the R package DESeq2 was used as previously described.66 The function DESeq was used to estimate size factors, estimate dispersion, fit the data to a negative binomial generalized linear model, and generate differential expression statistics using the Wald test. KEGG enrichment analysis was performed for internal analysis using the R package clusterProfiler.67

Local field potentials (LFPs) were recorded from iPSC-derived motor neurons on days 17–21 in culture in 6-well multielectrode chips (9 electrodes and 1 ground per well) using a MultiChannel Systems MEA-2100 multielectrode array (MEA) amplifier (ALA Scientific) with built-in heating elements set to 37°C. Cells were allowed to acclimate for 5 minutes after chips were placed into the MEA amplifier, and after glutamate addition (10 μM final concentration). For 1 μM Apilimod treatments, chips were incubated for 35 min in a humidified incubator in the presence of the particular drug, then returned to the MEA amplifier and acclimated for 5 min before beginning recordings. For each condition, recordings (5 min baseline, 10 min glutamate and/or drug, 40 kHz sampling rate) were filtered between 1–500 Hz, and average LFP frequency per well was determined using the accompanying MC Rack software.
Hb9::RFP+ iMNs appeared between days 13–16 after retroviral transduction. RepSox was removed at day 17 and the survival assay was initiated. For the glutamate treatment condition, 10 µM glutamate was added to the culture medium on day 17 and removed after 12 hours. Cells were then maintained in N3 medium with neurotrophic factors without RepSox. For the glutamate treatment condition with glutamate receptor antagonists, cultures were co-treated with 10 μM MK801 and CNQX, and 2 μM Nimodipine during the 12 hour glutamate treatment. The antagonists were maintained for the remainder of the experiment. For the neurotrophic factor withdrawal condition, BDNF, GDNF, and CNTF were removed from the culture medium starting at day 17. Longitudinal tracking was performed by imaging neuronal cultures in a Nikon Biostation CT or Molecular Devices ImageExpress once every 24–72 hours starting at day 17. Tracking of neuronal survival was performed using SVcell 3.0 (DRVision Technologies). Neurons were scored as dead when their soma was no longer detectable by RFP fluorescence. All neuron survival assays were performed at least twice, with equal numbers of neurons from three individual replicates from one of the trials being used for the quantification shown. All trials quantified were representative of other trials of the same experiment. When iMNs from multiple independent donors are combined into one survival trace in the Kaplan-Meier plots for clarity, the number of iMNs tracked from each line can be found in Supplementary Table 5.

The GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for about 10% of each disease worldwide 1–4. In the central nervous system (CNS), neurons and microglia express the highest levels of C9ORF72 5, suggesting that C9ORF72 acts in part cell autonomously and effects in neurons are a key source of disease etiology. Studies showing that the repeat expansion generates neurotoxic species including nuclear RNA foci 6–8, RNA/DNA G-quadruplexes 9, and dipeptide repeat proteins (DPRs) 10–12 have oriented the field towards a therapeutic focus on blocking the toxicity of these products 6–8,13,14. However, these strategies have not fully rescued the degeneration of patient-derived neurons 7,13. Moreover, tandem GGGGCC repeats are transcribed from over 80 other genomic locations within human spinal motor neurons (Supplementary Tables 1 and 2), yet genetic studies have not linked repeat expansions in these regions to ALS/FTD. In addition, hexanucleotide repeat-mediated toxicity in mice requires supraphysiological expression levels or a specific genetic background 14–16. These observations suggest that there are additional pathogenic triggers caused by repeat expansion within C9ORF72.
To confirm that glutamate receptor levels were increased on the surface of C9ORF72+/− and C9ORF72 patient iMNs, we used CRISPR/Cas9 editing to introduce a Dox-inducible polycistronic cassette containing NGN2, ISL1, and LHX3 into the AAVS1 safe-harbor locus of control, C9ORF72+/− and C9ORF72 patient iPSCs. This enabled large-scale production of iMNs that expressed motor neuron markers and had transcriptional profiles similar to 7F iMNs (Supplementary Fig. 11). Using this approach, we quantified the amount of surface-bound NR1 by immunoblotting after using surface protein biotinylation to isolate membrane-bound proteins. This confirmed that surface NR1 levels were higher on C9ORF72+/− and C9ORF72 patient iMNs (n=2 patients) than controls (n=3 controls)(Fig. 4e-h, Supplementary Fig. 5g, h).
The following antibodies were used in this manuscript: mouse anti-HB9 (Developmental Studies Hybridoma Bank); 81.5C10. chicken anti-TUJ1 (EMD Millipore); AB9354. rabbit anti-VACHT (Sigma); SAB4200559. rabbit anti-C9ORF72 (Sigma-Aldrich); HPA023873. rabbit anti-C9ORF72 (Proteintech); 25757–1-AP. mouse anti-EEA1 (BD Biosciences); 610457. mouse antiRAB5 (BD Biosciences); 610281. mouse anti-RAB7 (GeneTex); GTX16196. mouse anti-LAMP1 (Abcam); ab25630. mouse anti-M6PR (Abcam); ab2733. rabbit anti-GluR1 (EMD Millipore); pc246. mouse anti-NR1 (EMD Millipore); MAB363. chicken anti-GFP (GeneTex); GTX13970. rabbit anti-Glur6/7 (EMD Millipore); 04–921. mouse anti-FLAG (Sigma); F1804. mouse anti-GAPDH (Santa Cruz); sc-32233. chicken anti-MAP2 (Abcam); ab11267, 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), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), 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).
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