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.