Yingxiao Shi,#1,2,3 Shaoyu Lin,#1,2,3 Kim A. Staats,1,2,3 Yichen Li,1,2,3 Wen-Hsuan Chang,1,2,3 Shu-Ting Hung,1,2,3 Eric Hendricks,1,2,3 Gabriel R. Linares,1,2,3 Yaoming Wang,3,4 Esther Y. Son,5 Xinmei Wen,6 Kassandra Kisler,3,4 Brent Wilkinson,3 Louise Menendez,1,2,3 Tohru Sugawara,1,2,3 Phillip Woolwine,1,2,3 Mickey Huang,1,2,3 Michael J. Cowan,1,2,3 Brandon Ge,1,2,3 Nicole Koutsodendris,1,2,3 Kaitlin P. Sandor,1,2,3 Jacob Komberg,1,2,3 Vamshidhar R. Vangoor,7 Ketharini Senthilkumar,7 Valerie Hennes,1,2,3 Carina Seah,1,2,3 Amy R. Nelson,3,4 Tze-Yuan Cheng,8 Shih-Jong J. Lee,8 Paul R. August,9 Jason A. Chen,10 Nicholas Wisniewski,10 Hanson-Smith Victor,10 T. Grant Belgard,10 Alice Zhang,10 Marcelo Coba,3,11 Chris Grunseich,12 Michael E. Ward,12 Leonard H. van den Berg,13 R. Jeroen Pasterkamp,7 Davide Trotti,6 Berislav V. Zlokovic,3,4 and Justin K. Ichida1,2,3,†
Samples were first fixed in 4% PFA (1x PBS) overnight at 4°C and were subsequently washed three times with 1x PBS. Next, cells were permeabilized with 0.3% Triton X-100 (1x PBS) for 10 min at room temperature, followed by three washes with 1x PBS for 10 min each. After permeabilization, the samples were equilibrated in 1x SSC buffer for 10 min at room temperature and then transferred into 50% formamide (2x SSC) for 10 min at 60°C. The repeat expansion-targeting probe and the negative control probe were ordered from Exiqon 58. During this step, the probe mixture (1µl salmon sperm (10 µg/µl), 0.5 µl E. coli tRNA (20 µg/µl), 0.4 µl probe (25 µM), 25 µl 80% formamide/per sample) was made and placed at 95°C for at least 10 min. The samples were submerged in 200 µl of hybridization buffer (4ml 100% formamide, 0.5 ml 20x SSC, 1 ml BSA fraction V, 0.5ml RVC (20 mM), 1ml NaPO4 (0.1 M), 3 ml nuclease-free water) and 27 µl of the probe mixture was added to each sample and incubated for 1 hour at 60°C. After probe hybridization, the samples were washed twice with 50% formamide (2x SSC) for 20 min each at 65°C and once more with 40% formamide (1x SSC) for 10 min at 60°C. The remaining formamide was removed by briefly washing with 1x SSC three times. A final crosslinking step was performed by first incubating the samples with 1x Tris-Glycine for 5 minutes followed by a 5 min incubation in 4% PFA. Samples were washed three times with 1x PBS, stained with DAPI, and imaged using a Zeiss LSM 800 confocal microscope.
(a) Super-resolution microscopy images of immunofluorescence shows NR1+ puncta on neurites of iMNs overexpressing eGFP or C9ORF72 isoform B-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b-d) Number of NR1+ puncta per unit area in control (b-d), patient (b), C9ORF72+/− (c), and C9ORF72+/− (d) iMNs. Mean ± s.d. Each grey open circle represents the number of NR1+ puncta per area unit on a single neurite (one neurite quantified per iMN). For (b), n=75 (CTRL + GFP), 84 (C9-ALS + GFP), 95 (C9-ALS + isoA), and 111 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (c), n=37 (CTRL + GFP), 37 (C9ORF72+/− + GFP), 25 (C9ORF72+/− + isoA), and 27 (C9ORF72+/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. For (d), n=37 (CTRL + GFP), 37 (C9ORF72−/− + GFP), 38 (C9ORF72−/− + isoA), and 23 (C9ORF72−/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction for all comparisons. F-value (DFn, DFd): (3, 360) = 56.63 (b), (3, 122) = 13.42 (c), (3, 131) = 17.11 (d). (e-h) Immunoblotting analysis of surface NR1 after surface protein biotinylation in control (e-h), C9ORF72+/− (e-f), and C9-ALS patient (g-h) iMNs generated with 3 factors (NGN2, ISL1, and LHX3). In (f), n=4 biologically independent iMN conversions from CTRL2 and 2 biologically independent iMN conversions from the C9ORF72+/− line. Mean +/− s.d. In (h), two-tailed Mann-Whitney test. n=11 biologically independent motor neuron cultures from 11 independent control lines and 4 biologically independent motor neuron cultures from 4 independent C9-ALS patient lines. Experiments in (e-h) were repeated twice with similar results. Mean +/− s.e.m. (i-j) Immunoblotting analysis of surface Nr1 and Glur1 in post-synaptic densities (PSDs) from C9orf72 control and knockout mice, two-tailed t-test. t-value: 4.424 (Nr1), 4.632 (Glur1), degrees of freedom: 4 (Nr1), 4 (Glur1). n= 3 control PSD preparations isolated from 3 control mice and 3 C9orf72−/− PSD preparations isolated from 3 C9orf72−/− mice. This experiment was repeated twice with similar results. Mean +/− s.e.m. (k-l) Immunoblotting analysis of surface NR1 and GLUR1 in post-synaptic densities (PSDs) from post mortem control and C9-ALS patient motor cortices, n=3 control and 2 C9-ALS patient PSD preparations isolated from 3 control and 2 C9-ALS patients. This experiment was repeated twice with similar results. Mean +/− s.d. (m) Average Ca2+ flux in the presence of glutamate per minute. n=28 (CTRL1), 15 (CTRL2), 15 (CTRL3), 26 C9-ALS1), 20 (C9-ALS2), 24 (C9-ALS3), and 15 (C9ORF72+/−) iMNs analyzed from two biologically independent iMN conversions for each line. Mean ± s.e.m. One-way ANOVA with Tukey correction between all controls and all patients and C9ORF72+/−. F-value (DFn, DFd): (6, 136) = 11.21.
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.
“The Tale of the Curly-Bearded Guest” 231Studies Bian, Xiaoxuan . “Lun ‘Qiu ran ke zhuan’ de zuozhe, zuonian ji zhengzhi beijing” , in Dongnan daxue xuebao. Vol. 3, 2005, pp. 93-98. Cai, Miaozhen . “Chongtu yu jueze — ‘Qiu ran ke zhuan’ de renweu xingge suzao ji qi yihan” in Xingda renwen xuebao . Vol. 34, 2004, pp. 153-180. Zhang, Hong . “Du Guangting ‘Qiu ran ke zhuan’ de liuchuan yu yingxiang” in Zhongguo daojiao, vol. 1, 1997, pp. 28-31. Liu, Zhiwei . “Gujin ‘Qiu ran ke zhuan’ de yanjiu fansi” in Xibei daxue xuebao. Vol. 1, 2000. Sun, Yiping . Du Guangting pingzhuan. Nanjing: Nanjing daxue chubanshe, 2005. ___. “‘Qiu xu ke’ yu ‘Qiu ran ke’” in Zhongguo daojiao. vol. 6, 2005, pp. 14-17. Luo, Zhengming . Du Guangting daojiao xiaoshuo yanjiu . Chengdu: Bashu shushe, 2005. Wang, Meng’ou . “Qiuran ke yu Tang zhi chuangye chuangshuo” in Tangren xiaoshuo yanjiu siji. Taipei: Yiwen chubanshe, 1978, p. 254. Xu, Jiankun . “‘Qiu ran ke zhuan’ jili jiegou xintan” in Donghai zhongwen xuebao . Vol. 11, 1994, pp. 61-72. Ye, Qingbing . “‘Qiu ran ke zhuan’ de xiezuo jiqiao” in Zhongguo gudian wenxue yanjiu congkan — Xiaoshuo zhi bu . Taipei: Juliu, 1977, pp. 167-79.
Reprogramming was performed in 96-well plates (8 × 103 cells/well) or 13mm plastic coverslips (3.2 × 104 cells/coverslip) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors or 5 iDA factors were added in 100–200 µl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 µM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN and iDA neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.
(a-b) Survival of control and CRISPR-mutant iMNs without excess glutamate with overexpression of eGFP or PR(50)-eGFP (a) or GR(50)-eGFP (b). (c-d) Survival of control and C9-ALS iMNs without excess glutamate with overexpression of eGFP or PR(50)-eGFP (c) or GR(50)-eGFP (d). For (a), n=50 (CTRL1 + GFP AND CTRL1 + PR(50)), 49 (C9ORF72+/− + GFP), and 47 (C9ORF72+/− + PR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line. For (b), n=50 (CTRL1 + GFP AND CTRL1 + GR(50)), 49 (C9ORF72+/− + GFP), and 40 (C9ORF72+/− + GR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line. For (c), n=50 (CTRL1 + GFP AND CTRL1 + PR(50)), 50 (from each of two C9-ALS lines + GFP), and 41 (from each of two C9-ALS lines + PR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line per condition. For (d), n=50 (CTRL1 + GFP AND CTRL1 + GR(50)), 50 (from each of two C9-ALS lines + GFP), and 46 and 47 (from two C9-ALS lines + GR(50)) iMNs per line, iMNs quantified from 3 biologically independent iMN conversions per line per condition. All iMN survival experiments in (a-d) were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. Survival curves for the “+GFP” condition were included as a reference, but were not used in statistical analyses. (e) Relative decay in Dendra2 fluorescence over 12 hours in iMNs of specified genotypes. Mean +/− s.e.m. n = 18 (control) and 24 (C9ORF72+/−) iMNs quantified from two biologically independent iMN conversions each, two-tailed t-test with Welch’s correction between data points at each time point, t-value: 2.739, degrees of freedom: 25.62). (f-h) Immunostaining to determine endogenous PR+ puncta in control or C9-ALS iMNs with or without overexpression of C9ORF72 isoform A or B. Scale bar = 2 μm. This experiment was repeated twice with similar results. (g) Mean +/− s.d. n= 4 biologically independent iMN conversions generated from two different iPSC lines per genotype. Quantified values represent the average number of PR+ puncta in 40 iMNs from a single iMN conversion. Two-tailed t-test, t-value: 5.908, degrees of freedom: 6. (h) Mean +/− s.e.m. n= 3 biologically independent iMN conversions per condition. Quantified values represent the average number of PR+ puncta in 40 iMNs from a single iMN conversion. One-way ANOVA with Tukey correction, F-value (DFn, DFd): (2, 6)=10.5. iMN survival experiments in (a-d) were performed in a Molecular Devices ImageExpress.
To measure the effect of C9ORF72 activity on endogenous DPR levels in human motor neurons, we quantified endogenous PR+ puncta in C9-ALS iMNs with or without C9ORF72 overexpression. Using a validated anti-PR antibody 10,50, we found that the majority of PR+ punctae were localized in the nucleus (Fig. 5f), although we also detected cytoplasmic PR+ punctae to a larger extent than we had previously observed with exogenous PR(50) 10. C9-ALS iMNs (n=2 patients) had higher levels of nuclear PR+ puncta than controls (n=2 controls)(Fig. 5f, g) and overexpression of C9ORF72 isoform B significantly reduced the number of PR+ puncta in C9-ALS iMNs (Fig. 5f, h).
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).
We wondered if reduced C9ORF72 activity renders motor neurons vulnerable to other insults that might be exacerbated by impaired endosomal-lysosomal system function, such as DPR toxicity. Without excess glutamate treatment, C9ORF72+/−, C9ORF72−/−, and C9ORF72 patient iMN survival was similar to control iMNs (Fig. 5a-d and Supplementary Fig. 14a, b). Exogenous expression of PR50 or GR50 DPRs using cassettes that do not contain the GGGGCC repeat expansion induced control iMN degeneration (Fig. 5a, b). Interestingly, C9ORF72+/−, C9ORF72−/−, and C9ORF72 patient iMNs (n=2 patients) degenerated significantly faster than controls in response to PR50 or GR50 expression (Fig. 5a-d and Supplementary Fig. 14a, b).
(a) Production of Hb9::RFP+ iMNs and survival tracking by time-lapse microscopy. (b-d) Survival of control (CTRL) and C9ORF72 patient (C9-ALS) iMNs with neurotrophic factors (b) or in excess glutamate (shown with iMNs from all lines in aggregate (b, c) or for each individual line separately (d)). For (b-d), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. (e) iMNs at day 22 in excess glutamate. This experiment was repeated three times with similar results. (f-g) Survival of control and C9-ALS iMNs in excess glutamate with glutamate receptor antagonists (f) or without neurotrophic factors (g). For (f-g), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. (h) Survival of induced dopaminergic (iDA) neurons in excess glutamate. n=50 iMNs per line for 2 control and 2 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. Except in (d), each trace includes neurons from at least 2 donors with the specified genotype; see full detail in Methods. Scale bar: 100 μm (e). 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-g) were performed in a Nikon Biostation and experiments in (h) were performed in a Molecular Devices ImageExpress.
However, C9orf72-deficient mice do not display overt neurodegenerative phenotypes 14,18,19,22. Moreover, no studies have shown that reduced C9ORF72 activity leads to the degeneration of C9ORF72 ALS patient-derived motor neurons, nor have any provided direct evidence identifying a cellular pathway through which C9ORF72 activity modulates neuronal survival. Additionally, a patient homozygous for the C9ORF72 repeat expansion had clinical and pathological phenotypes that were severe but nonetheless did not fall outside the range of heterozygous patients, leaving it uncertain if reductions in C9ORF72 protein levels directly correlate with disease severity 23. Thus, the role of the C9ORF72 protein in C9ORF72 ALS/FTD disease pathogenesis remains unclear.