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.
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces 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 leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can 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/FTD.
(a) Phenotypic screening for small molecules that enhance the survival of C9-ALS iMNs. (b) Chemical structure of the PIKFYVE inhibitors YM201636 and Apilimod, and a reduced-activity analog of Apilimod. (c) Live cell images of iMNs at day 7 of treatment with DMSO or YM201636 (scale bar: 1 mm). This experiment was performed 3 times with similar results. (d) Survival effect of scrambled or PIFKVYE ASOs on C9-ALS iMNs in excess glutamate. n=50 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions per condition. (e) Survival effect of Apilimod and the reduced-activity analog on C9-ALS patient iMNs with neurotrophic factor withdrawal. n=50 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions per condition. All iMN survival experiments in (d, e) were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. (f) Activities of therapeutic targets in C9ORF72 ALS. (g, h) The effect of 3 μM Apilimod on NMDA-induced hippocampal injury in control, C9orf72+/−, or C9orf72−/− mice. (Mean +/− s.e.m. of n=3 mice per condition, one-way ANOVA with Tukey correction across all comparisons, F-value (DFn, DFd): (3, 8)=43.55, AP = Apilimod, red dashed lines outline the injury sites). (i, j) The effect of 3 μM Apilimod on the level of GR+ puncta in the dentate gyrus of control or C9-BAC mice. Mean +/− s.d. of the number of GR+ puncta per cell, each data point represents a single cell. n=20 (wild-type + DMSO), 20 (wild-type + Apilimod), 87 (C9-BAC + DMSO), and 87 (C9-BAC + Apilimod) cells quantified from 3 mice per condition, one-way ANOVA with Tukey correction for all comparisons, F-value (DFn, DFd): (3, 180) = 16.29. Scale bars = 2 μm, dotted lines outline nuclei, and white arrows denote GR+ punctae (i). (k) Model for the mechanisms that cooperate to cause neurodegeneration in C9ORF72 ALS/FTD. Proteins in red are known to be mutated in ALS or FTD. iMN survival experiments in (d, e) were performed in a Molecular Devices ImageExpress.
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.

Libraries were prepared from total RNA using Clontech SMARTer Stranded RNA-Seq kit, with Clonetech RiboGone ribodepletion performed ahead of cDNA generation. Amounts of input RNA were estimated using the Bioanalyzer and libraries produced according to Clontech’s protocol. Library generation and sequencing were performed at the Norris Cancer Center Sequencing Core at USC. All FASTQ files were analyzed using FastQC (version 0.10.1), trimmed using the FASTQ Toolkit (v 1.0), aligned to the GRCh37/hg19 reference genome using Tophat (version 2), and transcripts assembled and tested for differential expression using Cufflinks (version 2.1.1). Raw data is available for public download in the NCBI database under accession code PRJNA296854.
Human lymphocytes from healthy subjects and ALS patients were obtained from the NINDS Biorepository at the Coriell Institute for Medical Research and reprogrammed into iPSCs as previously described using episomal vectors61. Briefly, mammalian expression vectors containing Oct4, Sox2, Klf4, L-Myc, Lin28, and a p53 shRNA were introduced into the lymphocytes using the Adult Dermal Fibroblast Nucleofector™ Kit and Nucleofector™ 2b Device (Lonza) according to the manufacturer’s protocol. The cells were then cultured on mouse feeders until iPSC colonies appeared. The colonies were then expanded and maintained on Matrigel (BD) in mTeSR1 medium (Stem Cell Technologies).
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