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.
The fabrication of composite cathode with boroxine ring for all-solid-polymer lithium cell was described. Composite polymer electrolyte (CPE) was applied between the lithium metal anode and the composite cathode in a coin-shaped cell in order to prepare the solid-polymer electrolyte cell. The CPE films were cast on a flat polytetrafluoroethylene vessel from an acetonitrile slurry containing BaTiO ... [Show full abstract]Read more
Whole cell membrane potential and current recordings in voltage- and current-clamp configurations were made using an EPC9 patch clamp amplifier controlled with PatchMaster software (HEKA Electronics). Voltage- and current-clamp data was acquired at 50 kHz and 20 kHz, respectively, with a 2.9 kHz low-pass Bessel filter, while spontaneous action potential recordings were acquired at 1 kHz sampling frequency. For experiments, culture media was exchanged with warm extracellular solution consisting of (in mM): 140 NaCl, 2.8 KCl, 10 HEPES, 1 MgCl2, 2 CaCl2, and 10 glucose, with pH adjusted to 7.3 and osmolarity adjusted to 305 mOsm. Glass patch pipettes were pulled on a Narishige PC-10 puller and polished to 5–7 MΩ resistance. Pipettes were also coated with Sylgard 184 (Dow Corning) to reduce pipette capacitance. The pipette solution consisted of (in mM): 130 K-gluconate, 2 KCl, 1CaCl2, 4 MgATP, 0.3 GTP, 8 phosphocreatine, 10 HEPES, 11 EGTA, adjusted to pH 7.25 and 290 mOsm. Pipettes were sealed to cells in GΩ-resistance whole cell configuration, with access resistances typically between 10–20 MΩ, and leakage currents less than 50 pA. Capacitance transients were compensated automatically through software control. For voltage clamp, cells were held at −70 mV. For Current-voltage traces, a P/4 algorithm was used to subtract leakage currents from the traces. Measurements were taken at room temperature (approximately 20–25 °C). Data was analyzed and plotted in Igor Pro 6 (WaveMetrics) using Patcher’s Power Tools plug-in and custom programmed routines. Current density was obtained by dividing the measured ion channel current by the cell capacitance. For control iMNs, 10/10 tested fired action potentials. For C9-ALS iMNs, 9/10 tested fired action potentials.
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).
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,†
To verify that PIKFYVE-dependent modulation of vesicle trafficking was responsible for rescuing C9ORF72 patient iMN survival, we tested the ability of a constitutively active RAB5 mutant to block C9ORF72 patient iMN degeneration. Active RAB5 recruits PI3-kinase to synthesize PI3P from PI and therefore, like PIKFYVE inhibition, increases PI3P levels 56. Constitutively active RAB5 did not improve control iMN survival (n=2 controls)(Supplementary Fig. 15k), but successfully rescued C9ORF72 patient iMN survival (n=3 patients)(Supplementary Fig. 15l). In constrast, dominant negative RAB5, wild-type RAB5, or constitutively active RAB7 did not rescue C9ORF72 patient iMN survival (n=1, 3, 3 patients, respectively)(Supplementary Fig. 14m-o).
To examine C9ORF72 function, we determined its localization in iMNs. We first used an HA-tagged C9ORF72 construct to verify that the C9ORF72 antibody specifically recognizes C9ORF72 in cells (Supplementary Fig. 7a). In iMNs, C9ORF72 co-localized to cytoplasmic puncta and ASO-mediated knockdown of C9ORF72 expression reduced the number of antibody-detected cytoplasmic puncta in iMNs, indicating that the antibody specifically recognizes C9ORF72 in these puncta (Supplementary Fig. 7b, c). Super-resolution microscopy and z-stack imaging showed that about 80% of the C9ORF72+ vesicles also expressed the early endosomal proteins RAB5 and EEA1 (Fig. 3a and Supplementary 7d-h). Only rarely did C9ORF72 co-localize with the lysosomal marker LAMP1 (20%)(Supplementary Fig. 7e), and control and patient iMNs showed similar C9ORF72 localization (Supplementary Fig. 7h). We performed density gradient centrifugation on lysates from iPSC-derived motor neurons to separate light (endosomal) and heavy (lysosomal) membrane fractions. C9ORF72 co-segregated with EEA1 and not LAMP1, supporting the notion that C9ORF72 localizes predominantly in early endosomes (Fig. 3b, Supplementary Fig. 5d). In addition, we found that C9ORF72 isoform B bound strongly to an immobilized N-terminal fragment of EEA1 (Supplementary Fig. 7i). C9ORF72 isoform A did not interact as strongly with EEA1 (Supplementary Fig. 7i). The fact that not all EEA1+ vesicles contained high levels of C9ORF72 is consistent with this hypothesis and suggests that C9ORF72 may not localize to all types of EEA1+ vesicles (Fig. 3a).
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
“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 Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
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).
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