HEK 293T cells were used to produce retrovirus, lentivirus, and C9ORF72 protein. HEK cells were used for these purposes based on previous published studies using HEK cells in order to produce viral particles and mammalian proteins. HEK cells were obtained from American Type Culture Collection, catalog number CRL-11268. HEK and iPS cells were tested for mycoplasma before, during, and after the study and were negative.
To determine if C9ORF72 iMNs recapitulate neurodegenerative ALS processes, we examined their survival by performing longitudinal tracking of Hb9::RFP+ iMNs (Fig. 1a). This approach enabled us to distinguish differences in neurogenesis from differences in survival, which could not be addressed using previously-reported cross-sectional analyses6,7,10,26. In basal neuronal medium supplemented with neurotrophic factors, control and C9ORF72 patient iMNs survived equally well (Fig. 1b, Supplementary Fig. 3a, Supplementary Tables 5, 6). As human C9ORF72 ALS patients have elevated glutamate levels in their cerebrospinal fluid (possibly triggered by DPR-mediated aberrant splicing of the astrocytic excitatory amino acid transporter 2 EAAT2 4,27) we stimulated iMN cultures with a high glutamate pulse (12-hour treatment, 10 μM glutamate). This initiated a robust degenerative response in patient, but not control, iMNs (Fig. 1c-e and Supplementary Videos 3, 4) that was consistent across lines from multiple patients (n=6 patients) and controls (n=4 controls)(Fig. 1c, d and Supplementary Fig. 3d, e). While iMN survival varied slightly between live imaging systems, or between independent experiments due to the lengthy time course of neurodegeneration, the relative difference between control and C9-ALS patient iMNs was consistent (Fig. 1c - Nikon Biostation CT and Supplementary Fig. 3b - Molecular Devices ImageExpress). Moreover, iMNs from different iPSC lines derived from the same donor behaved similarly, suggesting genotypic differences accounted for these effects (Supplementary Fig. 3c). Treatment with glutamate receptor antagonists during glutamate administration prevented patient iMN degeneration (Fig. 1f). Alternatively, withdrawal of neurotrophic factors without glutamate stimulation also caused rapid degeneration of patient iMNs (n=3 patients, (Fig. 1g and Supplementary Fig. 3f).
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.
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.
The degree of Li force one can employ in kung fu depends on several variables such as resilience of muscles, strength of bones, speed and timing of attack and so on. An effective way to enhance the Li force is to exercise one's muscles and bones by applying increasing pressure on them (weight training, gym exercises, etc.). The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.
For all experiments, sample size was chosen using a power analysis based on pilot experiments that provided an estimate of effect size (http://ww.stat.ubc.ca/~rollin/stats/ssize/n2.html). Mice used for immunohistochemical analysis were selected randomly from a set of genotyped animals (genotypes were known to investigators). Mouse and human tissue sections used for immunohistochemical analysis were selected randomly. For mouse tissues, sections were prepared using an approximately equal representation of all levels of the spinal cord, and of those, all were imaged and quantified. The sections were only not used if NeuN or Chat immunostaining failed. For iMN survival assays, assays were repeated at least twice, with each round containing 3 biologically independent iMN conversions. iMNs from the 3 biologically independent iMN conversions in one representative round was used to generate the Kaplan-Meier plot shown. iMN survival times were confirmed by manual longitudinal tracking by an individual who was blinded to the identity of the genotype and condition of each sample. To select 50 iMNs per condition for analysis, >50 neurons were selected for tracking randomly at day 1 of the assay. Subsequently, the survival values for 50 cells were selected at random using the RAND function in Microsoft Excel. For quantification of immunofluorescence, samples were quantified by an individual who was blinded to the identity of the genotype of each sample.
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).