The repeat expansion suppresses the production of C9ORF72 protein by inhibiting transcription 3,4,6,7,9,17, raising the possibility that haploinsufficiency for C9ORF72 activity triggers disease pathogenesis. Consistent with this hypothesis, elimination of C9orf72 activity alters myeloid cell behavior in mice 14,18,19 and in vitro studies suggest that C9ORF72 activity may enhance autophagy 20,21.
CRISPR/Cas9-mediated genome editing was performed in human iPSCs as previously described, using Cas9 nuclease62. To generate loss-of-function alleles of C9ORF72, control iPSCs were transfected with a sgRNA targeting exon 2 of the C9ORF72 gene. Colonies were picked on day 7 after transfection and genotyped by PCR amplification and sequencing of exon 2. Colonies containing a frameshift mutation were clonally purified on MEF feeders and the resulting clones were re-sequenced to verify the loss-of-function mutation in C9ORF72.
Our results indicate that haploinsufficiency for C9ORF72 activity triggers neurodegeneration in C9ORF72 ALS, and this occurs by at least two mechanisms. First, reduced C9ORF72 activity causes the accumulation of glutamate receptors and excitotoxicity in response to glutamate. Although C9orf72 knockout mice do not display overt neurodegeneration14,18,22, these mice may be protected from excitotoxicity because they lack gain-of-function disease processes such as DPRs, which induce aberrant splicing and dysfunction of the EAAT2 glutamate transporter in astrocytes in vitro 12 and in C9ORF72 ALS patients 4,27. EAAT2 dysfunction causes glutamate accumulation in the cerebrospinal fluid of ALS patients 27, and consistent with this notion, we found that poly(PR) expression in human astrocytes reduced their rate of glutamate uptake. By using human iMNs, mice, and human post mortem tissue, we show for the first time that reduced C9ORF72 activity modulates the vulnerability of human motor neurons to degenerative stimuli and establish a mechanistic link between the C9ORF72 repeat expansion and glutamate-induced excitotoxicity
(a) The levels of C9ORF72 variant 2 mRNA transcript (encoding isoform A). Values are mean ± s.e.m., two-tailed t-test with Welch’s correction. t-value: 5.347, degrees of freedom: 11.08. n= 9 biologically independent iMN conversions from 3 control lines and 12 biologically independent iMN conversions from 5 C9-ALS lines. (b–d) iMN survival in excess glutamate following introduction of C9ORF72 (C9 isoform A or B) into C9ORF72 patient iMNs (b), but not control (b, d) or SOD1-ALS iMNs (c). For (b), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. For (c), n=50 iMNs per condition, iMNs scored from 3 biologically independent iMN conversions. For (d), n=50 iMNs per line per condition for 2 control lines, iMNs quantified from 3 biologically independent iMN conversions. Each trace includes iMNs from 2–3 donors with the specified genotype (except SOD1-ALS (c)); see full details in Methods. (e) Strategy for knocking out C9ORF72 from control iPSCs using CRISPR/Cas9. (f) Survival of control (CTRL2) iMNs, the isogenic heterozygous (C9+/−) and homozygous (C9−/−) iMNs and C9ORF72 patient (C9-ALS) iMNs in excess glutamate. n=50 biologically independent iMNs per line per condition for one control and two C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions. (g) Control iMN survival in excess glutamate with scrambled or C9ORF72 antisense oligonucleotides (ASO). Each trace includes control iMNs from 2 donors. n=50 biologically independent iMNs per line per condition for 2 control lines, iMNs quantified from 3 biologically independent iMN conversions. All iMN survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. iMN survival experiments in (b, d, and g) were performed in a Nikon Biostation, and (e and f) were performed in a Molecular Devices ImageExpress.

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.

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)

×