International Advisory Board: James Archibald (Translation Studies) - Hugo de Burgh (Chinese Media Studies) - Kristen Brustad (Arabic Linguistics) - Daniel Coste (French Language) - Luciano Curreri (Italian Literature) - Claudio Di Meola (German Linguistics) - Donatella Dolcini (Hindi Studies) - Johann Drumbl (German Linguistics) - Denis Ferraris (Italian Literature) - Lawrence Grossberg (Cultural Studies) - Stephen Gundle (Film and Television Studies) - Tsuchiya Junji (Sociology) - John McLeod (Post-colonial Studies) - Estrella Montolío Durán (Spanish Language) - Silvia Morgana (Italian Linguistics) - Samir Marzouki (Translation, Cultural Relations) - Mbare Ngom (Post-Colonial Literatures) - Christiane Nord (Translation Studies) - Roberto Perin (History) - Giovanni Rovere (Italian Linguistics) - Lara Ryazanova-Clarke (Russian Studies) - Shi-Xu (Discourse and Cultural Studies) - Srikant Sarangi (Discourse analysis) - Françoise Sabban, Centre d'études sur la Chine moderne et contemporaine (Chinese Studies) - Itala Vivan (Cultural Studies, Museum Studies)
Postsynaptic density extraction was done following a protocol published previously 63. Briefly, mouse spinal cord tissue or human cortical tissue was homogenized in cold Sucrose Buffer (320 mM Sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, 30 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, and protease inhibitor cocktail (Roche)) using a tissue grinder and then spun down at 500 g for 6 min at 4℃. The supernatant was re-centrifuged at 10,000 g for 10 min at 4℃. The supernatant was collected as the “Total” fraction, and the pellet was resuspended in cold Triton buffer (50 mM HEPES pH 7.4, 2 mM EDTA, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 1% Triton X-100 and protease inhibitor cocktail (Roche)) and then spun down at 30,000 RPM using a Beckman rotor MLA-130 for 40 min at 4℃. The supernantant was collected as the “Triton” fraction and the pellet was resuspended in DOC buffer (50 mM HEPES pH 9.0, 50 mM NaF, 40 mM β-Glycerophosphate, 10 mM Na3VO4, 20 uM ZnCl2, 1% Sodium Deoxycholate and protease inhibitor cocktail (Roche)) and collected as the “DOC”, PSD-enriched fraction. Collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot. Purity of the PSD preps was analyzed by immunoblotting for PSD-95 (PSD), p53 (non-PSD), and synaptophysin (non-PSD).
To measure the effect of dipeptide repeat protein expression on iMN survival, PR50 and GR50 were cloned into the pHAGE lentiviral vector as fusions with GFP to allow tracking of protein expression. iMN cultures were transduced with PR50 and GR50 lentiviruses at day 17 of reprogramming and longitudinal survival analysis was started the same day. 10 ng/ml of GDNF, BDNF, and CNTF was maintained throughout the experiment, and glutamate treatment was not performed. To measure PR50 turnover, PR50 was cloned into the pHAGE lentiviral vector as a fusion with Dendra2 (Addgene). iPSC-derived fibroblasts were generated according to Daley and colleagues64. Briefly, when C9ORF72−/− iPSC cultures reached 80% confluence, the medium was switched from mTeSR1 (Stem Cell Technologies) to human fibroblast medium containing DMEM (Life Technologies), 10% fetal bovine serum (FBS)(Thermo Fisher Scientific), and 1% penicillin/streptomycin (Life Technologies). Cells were passaged 2 to 3 times using Accutase (Life Technologies) before use in experiments. iPSC-derived fibroblasts were transduced with either pMXs-eGFP or pMXs-C9ORF72 isoform B-T2A-eGFP retrovirus and treated with 10 μg/ml mitomycin C for 3 hrs to inhibit cell proliferation. The cells were then transduced with the PR50–Dendra2 lentivirus and exposed to blue light for 1.5 sec using a lumencor LED light source to initiate photoconversion. The amount of decay (as a fraction of the starting level) of the red fluorescent punctae was monitored by longitudinal time lapse imaging in a Molecular Devices ImageExpress and analyzed using SVCell 2.0 (DRVision Technologies). Fluorescence was quantified at t = 0 and 12 hours after photoconversion. Distinct photoconverted punctae were treated as discrete objects for analysis (n = 20 each for +eGFP and +C9ORF72-T2A-eGFP). For each object, background fluorescence was subtracted and fluorescence was normalized according to object size. The fractional decay was statistically analyzed by two-tailed Student’s t-test. ** - p<.01.

“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.
“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 determine if the survival difference between C9ORF72 patient iMNs and controls was specific to our transcription factor-based reprogramming approach, we also measured the survival of Hb9::RFP+ control and C9ORF72 patient motor neurons derived from iPSCs by small molecule activation of the Sonic Hedgehog and retinoic acid signaling pathways 28 (Supplementary Fig. 3g, h). Similarly to iMNs, morphogen-generated motor neurons showed a significant survival difference between C9ORF72 patients and controls (Supplementary Fig. 3i-l).

To determine if glutamate receptor accumulation occurs on C9ORF72 patient motor neurons in vivo, we measured glutamate receptor expression in ventral horn neurons in lumbar spinal cord samples from 3 C9ORF72 ALS patients and 3 unaffected controls. We identified motor neurons by size and confirmed that most neurons selected in this manner were CHAT+ and SMI-32+ (Supplementary Fig. 12d). Spinal motor neurons from the C9ORF72 ALS patients displayed higher NR1 levels than control neurons (Supplementary Fig. 12e). In addition, post-synaptic densities isolated from the motor corticesof C9ORF72 patients had higher levels of NR1 and GLUR1 than controls (Fig. 4k, l and Supplementary Fig. 5k).
Therapeutic strategies in development for C9ORF72 ALS/FTD target gain-of-function mechanisms. These include ASOs 6–8 and small molecules 13 that disrupt RNA foci formation. However, these approaches have not fully rescued neurodegeneration in human patient-derived neurons 6–8,13, indicating that replacing C9ORF72 function or new therapeutic targets may be required.
(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.
Hb9::RFP+ C9ORF72 ALS/FTD iMNs were generated in 96-well plates. On Day 15 post transduction, neurotrophic factors and RepSox were withdrawn and the small molecule library was added (EMD Millipore kinase collection and Stemselect library, 3.3 µM final concentration) and added fresh every other day until the screen was terminated on Day 25 post-transduction. Identification of neuroprotective compounds was identified using SVcell 3.0 (DRVision Technologies) and further verification by manual iMN tracking.
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
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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).
“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.
Mice were anesthetized with i.p. ketamine (100 mg ⁄ kg) and xylazine (10 mg ⁄ kg), and body temperature kept at 36.9 ± 0.1°C with a thermostatic heating pad. Mice were placed in a stereotactic apparatus (ASI Instruments, USA) and the head is fixed accordingly. A burr hole was drilled, and an injection needle (33 gauge) was lowered into the hippocampus between CA1 and the dentate gyrus (AP −2.0, ML +1.5, DV −1.8). NMDA (20 nmol in 0.3 μl of phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). Simultaneously, or independently, Apilimod (0.3 μl of 20 μM in phosphate-buffered saline, pH 7.4) was infused over 2 min using a micro-injection system (World Precision Instruments, Sarasota, FL, USA). The needle was left in place for an additional 8 min after the injection. Animals were euthanized 48 h later. Brains were quickly removed, frozen on dry ice, and stored at −80°C until processing. Thirty-micrometer-thick coronal sections were prepared using a cryostat. Every fifth section 1 mm anterior and posterior to the site of injection was stained with cresyl violet. The lesion area was identified by the loss of staining, measured by NIH ImageJ software and integrated to obtain the volume of injury.
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