We thank the NINDS Biorepository at Coriell Institute for providing the following cell lines for this study: ND12133, ND03231, ND01751, ND11976, ND03719, ND00184, ND5280, ND06769, ND10689, ND12099, ND14954, ND08957, ND12100, and ND014587. We thank Helena Chui and Carol Miller at the University of Southern California Alzheimer’s Disease Research Center and Neil Shneider at the Columbia University Medical Center for control and C9ORF72 patient tissue. We thank the Choi Family Therapeutic Screening Facility for chemical screening support and the Translational Imaging Center at USC for imaging support. We thank Max Koppers, Youri Adolfs, Christiaan van der Meer, and Mark Broekhoven for help with mouse breeding and kainate injection experiments. We thank Prof. Satoshi Waguri for providing the M6PR-GFP construct. We thank Christopher Buser for assistance with electron microscopy. We also thank Sam Alworth (DRVision Technologies, LLC), Katja Hebestreit, and Raj Bhatnagar (Verge Genomics), Bob Baloh, Jacqueline O’Rourke, Christopher Donnelly, Chang Tong, Andrew McMahon and Qing Liu-Michael for reagents, technical support, and discussions. E.Y.S. is a Walter V. and Idun Berry Postdoctoral Fellow. K.A.S. was supported in part by a Muscular Dystrophy Association Development Grant. L.M. was supported by NIH grant T32DC009975–04. This work was supported by NIH grants AG039452, AG023084, and NS034467 to B.V.Z. R.J.P. was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12–08, 15–05), and VICI grant Netherlands Organisation for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, U.S. Department of Defense grant W81XWH-15–1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Tau Consortium, the Frick Foundation for ALS Research, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator.
Hb9::RFP+ C9ORF72 ALS/FTD iMNs were generated in 96-well plates. On Day 15 post transduction, neurotrophic factors and RepSox were withdrawn and the small molecule library was added (EMD Millipore kinase collection and Stemselect library, 3.3 µM final concentration) and added fresh every other day until the screen was terminated on Day 25 post-transduction. Identification of neuroprotective compounds was identified using SVcell 3.0 (DRVision Technologies) and further verification by manual iMN tracking.
Libraries were prepared from total RNA using Clontech SMARTer Stranded RNA-Seq kit, with Clonetech RiboGone ribodepletion performed ahead of cDNA generation. Amounts of input RNA were estimated using the Bioanalyzer and libraries produced according to Clontech’s protocol. Library generation and sequencing were performed at the Norris Cancer Center Sequencing Core at USC. All FASTQ files were analyzed using FastQC (version 0.10.1), trimmed using the FASTQ Toolkit (v 1.0), aligned to the GRCh37/hg19 reference genome using Tophat (version 2), and transcripts assembled and tested for differential expression using Cufflinks (version 2.1.1). Raw data is available for public download in the NCBI database under accession code PRJNA296854.
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).