Practitioners of kung fu refer to two separate forms of personal force: Li (Traditional Chinese: 力) refers to the more elementary use of tangible physical (or "external") force, such as that produced by muscles. Neijing (Traditional Chinese:內勁) or Neigong (Traditional Chinese: 內功), in contrast, refer to "internal" forces produced via advanced mental control over psychic energy (the qi).
Previously described Nestin‐Cre+/− C9orf72loxP/loxP mice22 (18 months old) and age-matched controls (n=2 per genotype, 1 male and 1 female for C9orf72−/− and 2 males for C9orf72+/+) were transcardially perfused with phosphate buffered saline (PBS) and subsequently with 4% formaldehyde. Cryoprotection occurred in 30% sucrose. Additionally, 6 month old C9-BAC and wildtype controls were transcardially perfused with phosphate buffered saline (PBS) and subsequently with 4% formaldehyde. Cryoprotection occurred in 20% sucrose. After snap freezing, tissue was sectioned by cryostat at 20 µm thickness and stained with the following primary antibodies: NMDAR1, GluR1, GluR6/7, Lamp1, NeuN, Chat, and GR. Antigen retrieval by sodium citrate occurred prior to GR staining. Images were collected using a Zeiss LSM780 or LSM800 confocal microscope. Glutamate subunit intensity measurements occurred with ImageJ where the mean cytosolic intensity was divided by a background measurement collected near to the measured neuron. Lamp1 and GR quantification occurred with the help of ImageJ. The scientist performing the glutamate receptor subunit intensity, LAMP1 vesicle, and GR quantification was blinded to the genotypes or treatment of the samples.
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).
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.
Local field potentials (LFPs) were recorded from iPSC-derived motor neurons on days 17–21 in culture in 6-well multielectrode chips (9 electrodes and 1 ground per well) using a MultiChannel Systems MEA-2100 multielectrode array (MEA) amplifier (ALA Scientific) with built-in heating elements set to 37°C. Cells were allowed to acclimate for 5 minutes after chips were placed into the MEA amplifier, and after glutamate addition (10 μM final concentration). For 1 μM Apilimod treatments, chips were incubated for 35 min in a humidified incubator in the presence of the particular drug, then returned to the MEA amplifier and acclimated for 5 min before beginning recordings. For each condition, recordings (5 min baseline, 10 min glutamate and/or drug, 40 kHz sampling rate) were filtered between 1–500 Hz, and average LFP frequency per well was determined using the accompanying MC Rack software.
Removal of TTX and TEA during glutamate receptor agonist treatment revealed additional increases in Gcamp6 activation in C9ORF72+/− iMNs compared to controls, suggesting that C9ORF72+/− iMNs also fire action potentials more frequently than controls (Supplementary Fig. 13a), although we did not detect large changes in sodium or potassium current amplitudes in C9ORF72+/− iMNs (Supplementary Fig. 13b, c). To determine if increased neuronal activity due in part to elevated glutamate receptor levels contributes to neurodegeneration in C9ORF72 patient and C9ORF72+/− iMNs, we measured iMN survival in the presence or absence of retigabine. Retigabine is approved by the U.S. Food and Drug Administration for the treatment of epilepsy and reduces neuronal excitability by activating Kv7 potassium channels 48. In the glutamate treatment assay, retigabine increased the survival of C9ORF72 patient (n=2 patients) and C9ORF72-deficient iMNs, but not controls (n=2 controls)(Supplementary Fig. 13d-g).
Consistent with previous studies 3,4,6–8, patient iMNs (n=5 patients) had reduced C9ORF72 expression compared to controls (n=3; Fig. 2a and Supplementary Fig. 4a, 5b). While previous studies have linked low C9ORF72 levels to changes in vesicle trafficking or autophagy 18,20,30–33, it remains unknown if loss of C9ORF72 protein directly contributes to degeneration. Thus, we re-expressed C9ORF72 (isoform A or B) in iMNs using a retroviral cassette (Supplementary Fig. 4b) and found that both isoforms rescued C9ORF72 patient iMN survival in response to glutamate treatment (n=3 patients Fig. 2b and Supplementary Fig. 4c). This effect was specific for C9ORF72 iMNs, as forced expression of C9ORF72 did not rescue SOD1A4V iMN survival (Fig. 2c), nor did it improve the survival of control iMNs (n=2 controls Fig. 2d and Supplementary Fig. 4d).

“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.
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.
Human EEA1 (1–209) with an N-terminal GST tag in pGEX-6P-1 vector or GST only were expressed in E. Coli BL21 (DE3) cells (Thermo Fisher Scientific) for 12 hr at 18℃. Harvested cells were lysed by sonication in cold GST Purification Buffer (50 mM Tris pH 8.0, 200 mM NaCl, 2 mM DTT, 0.5 mg/ml Lysozyme, 0.2% Triton X-100 and protease inhibitor cocktail (Roche)). After centrifugation at 15,000 g for 30 min at 4℃, clarified lysate was incubated with Glutathione Sepharose 4B beads (GE Healthcare Life Science) for 3 hours to purify GST-EEA1 or GST. HEK cells were transfected with C-terminal 3XFLAG tagged C9ORF72 isoform A or B, or eGFP constructs and harvested 36–48 hr post-transfection in cold Lysis Buffer (25 mM HEPES pH 7.4, 100 mM NACl, 5 mM MgCl2, 1 mM DTT, 10% Glycerol, 0.1% Triton X-100 and protease inhibitor cocktail (Roche)).After centrifugation at 8,000 g for 10 min at 4℃, the clarified supernatant was incubated with washed GST-EEA1 or GST beads for 2 hr at 4℃ with end-to-end rotation. Beads were then boiled in 2X SDS-PAGE sample buffer and pulled-down protein was analyzed by western blot.
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
Live imaging of iMNs expressing a M6PR-GFP fusion protein that localizes to M6PR+ vesicles 44 confirmed that C9ORF72 patient and C9ORF72-deficient iMNs possess increased numbers of M6PR+ vesicle clusters, and that overexpression of C9ORF72 isoform A or B rescues this phenotype (Supplementary Fig. 9c-g and Supplementary Videos 5-9). Clusters did not disperse over the time course of the assay, suggesting that they are relatively stable and not in rapid flux (Supplementary Videos 5-9). In addition, M6PR+ puncta moved with a slower average speed in C9ORF72 patient and C9ORF72+/− iMNs than controls (Supplementary Fig. 9h, i). Thus, reduced C9ORF72 levels lead to fewer lysosomes in motor neurons in vitro and in vivo, and this may be due in part to altered trafficking of M6PR+ vesicles.
During lysosomal biogenesis, lysosomal proteins are transported in Mannose-6-Phosphate Receptor (M6PR)+ vesicles from the trans-Golgi Network to early and late endosomes for eventual incorporation into lysosomes 41. Disruption of M6PR+ vesicle trafficking can lead to a reduction in lysosome numbers 42 and altered localization of M6PR+ vesicles 43. In control iMNs (n=3 controls), M6PR+ vesicles were distributed loosely around the perinuclear region and to a lesser extent in the non-perinuclear cytosol (Supplementary Fig. 9a, b). In contrast, C9ORF72 patient (n=4 patients), C9ORF72+/−, and C9ORF72−/− iMNs frequently harbored densely-packed clusters of M6PR+ vesicles (Supplementary Fig. 9a, b). This was not due to a reduced number of M6PR+ vesicles in patient and C9ORF72-deficient iMNs (Supplementary Fig. 9c). Forced expression of C9ORF72 isoform B restored normal M6PR+ vesicle localization in patient (n=4 patients) and C9ORF72-deficient iMNs, confirming that a lack of C9ORF72 activity induced this phenotype (Supplementary Fig. 9a, b).
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.
To generate Dox-NIL iMNs, the Dox-NIL construct was integrated into the AAVS1 safe harbor locus of the control, C9+/−, and C9-ALS patient iPSC lines using CRISPR/Cas9 editing (gRNA sequence shown in Supplementary Table 4). Dox-NIL iMNs were generated by plating at ~25% confluency on matrigel coated plates and adding 1 μg/ml of doxycylin in N3 media +7.5 μM RepSox 1 day after plating. Mouse primary mixed glia were added to the cultures at day 6, and doxycyline was maintained throughout conversion. iMN cultures were harvested at day 17.
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.
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(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.
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).
Eliminating C9ORF72 protein expression from one or both alleles reduced iMN survival to levels comparable to patient iMNs (Fig. 2f). Antisense oligonucleotide (ASO)-mediated suppression of C9ORF72 expression levels also reduced control iMN survival (Fig. 2g and Supplementary Fig. 4j), suggesting that reduced iMN survival was not due to an off-target effect of the CRISPR/Cas9 genome editing. Exogenously restoring C9ORF72 expression in C9ORF72+/− and C9ORF72−/− iMNs rescued survival (Supplementary Fig. 4k, l), verifying that depletion of C9ORF72 caused the observed neurodegeneration.
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.
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