Cells were fixed in 6-well culture plates in 2.5 % glutaraldehyde in 0.1M cacodylate buffer, post-fixed in 1% osmium tetroxide for 1 hour and block stained in 1% uranyl acetate in 0.1M acetate buffer pH 4.4 overnight at 4 ˚C. Dehydration was performed in increasing concentrations of ethanol (10%/25%/50%/75%/90%/100%/100%/100%) for 15 minutes each and infiltrated with increasing concentrations of Eponate12 (Ted Pella Inc., Redding, CA, USA), 25% Eponate12 (no catalyst) in ethanol for 3 hours, 50% overnight, 100% for 5 hours, 100% overnight, and polymerized in fresh Eponate12 with DMP-30 for 48 hours at 60 ˚C. Previously marked areas were sawed out, the tissue culture plastic was removed and the selected area sectioned parallel to the substrate at a thickness of 70 nm. Sections at a depth of 3–5 µm were collected on formvar-filmed 50 mesh copper grids and imaged at 80 kV in an FEI 208 Morgagni (FEI is in Hillsboro, OR, USA). Per micrograph, cytosol was used to quantify the number of electron dense spheres that were defined as lysosomes 40.

“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.

To provide a quantitative measure of (GGGGCC)n hexanuceotide expansion in C9ORF72, 100 ng of genomic DNA was amplified by touchdown PCR using primers shown in Supplementary Data Table 4, in a 28-µl PCR reaction consisting of 0.2 mM each of 7-deaza-2-deoxyguanine triphosphate (deaza-dGTP) (NEB), dATP, dCTP and dTTP, 7% DMSO, 1X Q-Solution, 1X Taq PCR buffer (Roche), 0.9 mM MgCl2, 0.7 µM reverse primer (four GGGGCC repeats with an anchor tail), 1.4 µM 6FAM-fluorescently labeled forward primer, and 1.4 µM anchor primer corresponding to the anchor tail of reverse primer (Supplementary Data Table 4). During the PCR, the annealing temperature was gradually decreased from 70 ºC and 56 ºC in 2 ºC increments with a 3 min extension time for each cycle. The PCR products were purified using the QiaQuick PCR purification kit (Qiagen) and analyzed using an ABI3730 DNA Analyzer and Peak Scanner™ Software v1.0 (Life Technologies).


Immunostaining revealed that C9ORF72+/− and C9ORF72−/− iMNs contained elevated levels of NMDA (NR1) and AMPA (GLUR1) receptors on neurites and dendritic spines compared to control iMNs under basal conditions (Fig. 4a, c, d and Supplementary Fig. 5b and 10a, c-e, g, h, j, k). In addition, control iMNs treated with C9ORF72-specific ASOs displayed increased numbers of NMDA and AMPA receptors in their neurites (Supplementary Fig. 10l, m). C9ORF72 patient iMNs (n=3 patients) also showed elevated NR1 and GLUR1 levels compared to controls (n=3 controls), and forced expression of C9ORF72 isoform B reduced glutamate receptor levels in patient iMNs (n=3 patients) to that of controls (n=3 controls) (Fig. 4a-c and Supplementary Fig. 10a-h). mRNA levels of NR1 (GRIN1) and GLUR1 (GRIA1) were not elevated in flow-purified C9ORF72+/− iMNs, indicating that increased transcription could not explain the increased glutamate receptor levels (Supplementary Fig. 10n).
IPSC-MNs at differentiation D35 were harvested in cold Hypotonic buffer (20 mM HEPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1mM EGTA, 1 mM DTT and protease inhibitor cocktail (Roche)) and lysed by passing through G25 needles 25 times and then spun down at 700 x g for 10min at 4℃. The Supernatant was loaded onto pre-made 30% Percoll solution and re-centrifuged at 33,000 RPM using Beckman rotor SWI55 for 50min at 4℃. 300 ul aliquots were taken from top to bottom as fractions and all the collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot.
Hb9::RFP+ iMNs appeared between days 13–16 after retroviral transduction. RepSox was removed at day 17 and the survival assay was initiated. For the glutamate treatment condition, 10 µM glutamate was added to the culture medium on day 17 and removed after 12 hours. Cells were then maintained in N3 medium with neurotrophic factors without RepSox. For the glutamate treatment condition with glutamate receptor antagonists, cultures were co-treated with 10 μM MK801 and CNQX, and 2 μM Nimodipine during the 12 hour glutamate treatment. The antagonists were maintained for the remainder of the experiment. For the neurotrophic factor withdrawal condition, BDNF, GDNF, and CNTF were removed from the culture medium starting at day 17. Longitudinal tracking was performed by imaging neuronal cultures in a Nikon Biostation CT or Molecular Devices ImageExpress once every 24–72 hours starting at day 17. Tracking of neuronal survival was performed using SVcell 3.0 (DRVision Technologies). Neurons were scored as dead when their soma was no longer detectable by RFP fluorescence. All neuron survival assays were performed at least twice, with equal numbers of neurons from three individual replicates from one of the trials being used for the quantification shown. All trials quantified were representative of other trials of the same experiment. When iMNs from multiple independent donors are combined into one survival trace in the Kaplan-Meier plots for clarity, the number of iMNs tracked from each line can be found in Supplementary Table 5.
We also found that Reduced C9ORF72 activity also induces iMN hypersensitivity to DPRs by impairing their clearance. This uncovers a more direct form of cooperative pathogenesis between gain- and loss-of-function mechanisms in C9ORF72 ALS/FTD. Through a potentially similar mechanism, reduced C9orf72 levels can also facilitate cytoplasmic TDP-43 accumulation in mouse neurons 20.

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.
The kung fu component of Li force is limited by one's physical condition. When a person passes his/her prime age, one's kung fu ability will pass the optimum level, too. The degree of kung fu will decline when muscles and bones are not as strong as they used to be. On the other hand, the kung fu aspect of Neijing is said to continually grow as long as one lives.[7]
For experiments other than the comparison of Apilimod and the reduced-activity analog, Apilimod was purchased from Axon Medchem (cat. no. 1369). For the reduced-activity analog assays, Apilimod and the reduced activity analog were synthesized at Icagen, Inc. according to the schemes shown in Supplementary Fig. 16. PIKFYVE kinase inhibition was measured using the ADP-Glo kinase assay from SignalChem according to the manufacturer’s instructions, using purified PIKFYVE kinase (SignalChem cat. no. P17–11BG-05).
To determine if transcriptional changes in C9ORF72+/− and C9ORF72−/− iMNs also reflect the contribution of C9ORF72 protein levels to neurodegeneration, we performed RNA sequencing on flow-purified Hb9::RFP+ iMNs from C9ORF72+/−, C9ORF72−/−, and isogenic control iMNs, as well as C9ORF72 patient iMNs (Supplementary Table 7), and compared them to existing RNA-seq data from postmortem tissue 34,35. When examining consensus genes that were differentially expressed compared to controls in all C9ORF72 patient postmortem datasets (from GSE56504 and GSE67196)34,35, both C9ORF72+/− and C9ORF72 patient iMNs shared similar gene expression changes to the postmortem tissue (Supplementary Fig. 6). Thus, a reduction in C9ORF72 levels induces disease-associated transcriptional changes observed in C9ORF72 patient postmortem samples.
For heating and evaporation of concentrated solutions of electrolytes, the solution itself can serve as a resistor to give uniform heating throughout. Solutions of salts having a negative temperature coefficient of solubility can be concentrated by electrical heating, provided electrodes are maintained at a temperature below that of the solution. Experimental details are given for solutions of ... [Show full abstract]Read more

GCaMP6 was cloned into the pMXs-Dest-WRE retroviral vector and transduced into reprogramming cultures concurrently with the motor neuron factors. To assess GCaMP6 activity, 1.5 μm glutamate was added to iMN cultures and cells were imaged continuously for 2 minutes at 24 frames per second. GFP flashes were scored manually using the video recording. At least 3 different fields of view from three independent cultures, totalling 50–100 iMNs, were scored per condition.
Base text for this translation. ___. Wang Meng’ou’s , ed. Tangren xiaoshuo jiaoshi . Taipei: Zhongzheng Shuju, 1983, 2319-38. For other texts and editions see footnote 1. Translations Birch, Cyril. “The Curly-bearded Hero,” in Anthology of Chinese Literature, v. 1, New York, 1965, pp. 314-322. Chai, Ch’u, and Winberg Chai. “The Curly-Bearded Guest,” in A Treasury of Chinese Literature, New York, 1965, pp. 117-124. Hsu Sung-nien. “Biographie d’un preux barbu,” Anthologie de la littérature chinoise.Paris: Delagrave, 1933, pp. 241-6. Levenson, Christopher, tran., The Golden Casket. Harmondsworth, Middlesex: Penguin Books, 1967, pp. 137-47. Lévy, André. “Barbe-bouclée, L’étranger à la barbe et aux favoris bouclés,” in Histoires extraordinaires et récits fantastiques de la Chine ancienne.Paris: Flammarion, 1993, pp. 177-195 (with notes). Lin Yutang. “Curly-Beard,” in Famous Chinese Short Stories. New York: John Day (Cardinal), 1953, pp. 3-22. Schafer, E.H. “Three Divine Women of South China,” CLEAR, 1 (1979), pp. 31-42. Wang, Elizabeth Te-chen, tran. “The Curly-Bearded Guest,” in Wang’s Ladies of the Tang: 22 Classical Chinese Stories. Taipei: Mei Ya Publications, 1973, pp. 133-50.
Amongst four reproducible hit compounds, we identified a PIKFYVE kinase inhibitor (YM201636) that significantly increased C9ORF72 patient iMN survival (n=2 patients) (Fig. 6b, c and Supplementary Fig. 15a). PIKFYVE is a lipid kinase that converts phosphatidylinositol 3-phosphate (PI3P) into phosphtidylinositol (3,5)-bisphosphate (PI(3,5)P2)51(Fig. 6f). PI3P is primarily generated by PI3-kinases recruited to early endosomes by RAB5, and PI3P anchors EEA1 to early endosomes to drive endosomal maturation 52(Fig 6f). Following endosomal maturation into lysosomes, PI3P drives fusion of lysosomes with autophagosomes 53. PIKFYVE regulates PI3P levels by converting PI3P into PI(3,5)P2 52, which disfavors lysosomal fusion with endosomes and autophagosomes 53,54. Therefore, inhibition of PIKFYVEincreases autophagosome-lysosome fusion 53 and may compensate for reduced C9ORF72 activity and other disease processes by increasing PI3P levels to facilitate removal of glutamate receptors or DPRs (Fig. 6f). Interestingly, FIG4 is a phosphatase that opposes PIKFYVE kinase by converting PI(3,5)P2 to PI3P and loss-of-function mutations in FIG4 cause ALS 55. Thus, genetic evidence suggests that PIKFYVE inhibition may be capable of modulating ALS disease processes in humans.

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,† 
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