iMNs from healthy controls and ALS patients were collected on day 21 post-transduction in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche). Protein quantity was measured by the BCA assay (Pierce) and samples were run on a 10% SDS gel at 4 °C. The gel was transferred onto an Immobilon membrane (Millipore). The membrane was blocked with 5% milk in 0.1% PBS-Tween 20 (PBS-T)(Sigma-Aldrich), incubated with primary antibodies overnight at 4 °C, washed three times with 0.1% PBS-T, then incubated with horseradish peroxidase (HRP)-conjugated (Santa Cruz). After three washes with 0.1% PBS-T, blots were visualized using an Amersham ECL Western Blotting Detection Kit (GE) or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) and developed on X-ray film (Genesee). The following primary antibodies were used: rabbit anti-C9ORF72 (Proteintech, cat. no. 22637–1-AP), mouse anti-GAPDH (Santa Cruz, cat. no. sc-32233), chicken anti-MAP2 (Abcam, cat. no. ab11267), mouse anti-FLAG (Sigma, cat. no. F1804), rabbit anti-GLUR1 (Millipore, cat. no. 1504), mouse anti-NR1 (Novus, cat. no. NB300118), mouse anti-Transferrin receptor (Thermo, cat. no. 136800), mouse anti-LAMP3 (DSHB, cat. no. H5C6), rabbit anti-LAMP3 (Proteintech, cat. no. 12632), mouse anti-LAMP2 (DSHB, cat. no. H4B4), mouse anti-LAMP1 (Abcam, cat. no. Ab25630), goat anti-HRP (Santa Cruz, cat. no. sc-47778 HRP), mouse anti-EEA1 (BD Biosciences, cat. no. BD610457), mouse anti-TUJ1 (Biolegend, cat. no. MMS-435P), rabbit anti-APP (Abcam, cat. no. ab32136), mouse anti-Tau5 (Thermo, cat. no. AHB0042), mouse anti-PSD-95 (Thermo, cat. no. MA1–045) , mouse anti-p53 (Cell Signaling, cat. no. 2524S), anti-mouse HRP (Cell Signaling, cat. no. 7076S), anti-rabbit HRP (Cell Signaling, cat. no. 7074S). For C9ORF72 western blots, to generate enough motor neurons for C9ORF72 protein detection, we used a directed differentiation method described previously 28.
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).
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]
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]
To determine if PIKFYVE inhibition rescued patient iMN survival by reversing phenotypic changes caused by C9ORF72 haploinsufficiency, we measured glutamate receptor levels with and without PIKFYVE inhibitor treatment. PIKFYVE inhibition significantly lowered NR1 (NMDA receptor) and GLUR1 (AMPA receptor) levels in patient (n=4 patients) and C9ORF72+/− iMNs (Supplementary Fig. 15p-s). PIKFYVE inhibition also reduced electrophysiological activity in patient motor neurons (C9-ALS1) during glutamate treatment (Supplementary Fig. 15t). To determine if small molecule inhibition of Pikfyve rescues C9ORF72 disease processes in vivo, we first established an NMDA-induced hippocampal injury model in C9orf72-deficient mice. In control mice, hippocampal injection of NMDA caused neurodegeneration after 48 hrs as we have shown previously 57 (Supplementary Fig. 17a, b). Consistent with C9orf72-deficient mice having elevated NMDA receptor levels (Fig. 4h, i and Supplementary Fig. 11a-d), injection of NMDA caused significantly greater neurodegeneration in C9orf72+/− and C9orf72−/− mice than in controls (Fig. 6g, h). Importantly, co-administration of Apilimod rescued the NMDA-induced neurodegeneration in C9orf72-deficient mice (Fig. 6g, h).
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).

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,†

With the four components of a chemical heat pump (two solid-gas reactors, an evaporator and a condenser), a cycle of the double-effect type can be applied to continuous refrigeration. The performance of this process is analysed, allowing the infinite sink temperature and the couples of reactive salts to be used, which depend on the production temperature envisaged, to be selected. The results are ... [Show full abstract]Read more


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.
To confirm that reduced C9ORF72 protein levels are sufficient to cause neurodegeneration, we used CRISPR/Cas9-mediated genome editing to introduce a frameshift mutation into one or both alleles of C9ORF72 in control iPSCs (Fig. 2e and Supplementary Fig. 4e). qPCR showed that targeting one allele reduced C9ORF72 transcript levels due to nonsense-mediated decay and transcript levels were more severely reduced in homozygous mutant cells (Supplementary Fig. 4f). Frameshift mutations also decreased C9ORF72 protein expression (Supplementary Fig. 4g, 5c). RNA sequencing of flow-purified Hb9::RFP+ iMNs showed that targeting C9ORF72 did not significantly alter the expression of the top 10 genes with predicted off-target sites for the CRISPR guide RNA (Supplementary Fig. 4h and Supplementary Table 7). In addition, expression levels of the 20 genes nearest C9ORF72 on chromosome 9 were largely unperturbed in either the C9ORF72+/− and C9ORF72−/− iMNs, indicating that this approach specifically inactivated C9ORF72 (Supplementary Fig. 4i).
The tomb murals of the Eastern Wei Dynasty (534–550) in the Southern and Northern Dynasties Period (386–581) unearthed from Xiaomachang Village of Wuqiao County in 1958 depict the performances of handstands, plate spinning, deft horsemanship and so on. However, it was after the Yuan Dynasty (1271–1368) that acrobatics of Wuqiao gained much reputation. Before that, acrobatics in Henan Province was much more influential. After the Yuan Dynasty was established, the capital was moved from Kaifeng of Henan to Beijing, and the acrobatics in Wuqiao of Hebei, which neighbors Beijing, began to prosper and was increasingly influential.
Since glutamate receptor activation and neuronal firing both induce calcium influx, we determined their relative contributions to the increased Gcamp6 activation by. using the ion channel inhibitors TTX and TEA to block neuronal firing. C9ORF72+/− iMNs still displayed more frequent Gcamp6 activation than C9ORF72+/+ iMNs (Supplementary Fig. 13a), indicating that part of the hyperexcitability is due to increased glutamate receptor activation. To determine which receptors were responsible for the increased glutamate response, we tested small molecule agonists of specific glutamate receptor subtypes. Activation of NMDA, AMPA, and kainate receptors was higher in C9ORF72+/− iMNs than controls (Supplementary Fig. 13a).

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.
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).
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.
To confirm that reduced C9ORF72 protein levels are sufficient to cause neurodegeneration, we used CRISPR/Cas9-mediated genome editing to introduce a frameshift mutation into one or both alleles of C9ORF72 in control iPSCs (Fig. 2e and Supplementary Fig. 4e). qPCR showed that targeting one allele reduced C9ORF72 transcript levels due to nonsense-mediated decay and transcript levels were more severely reduced in homozygous mutant cells (Supplementary Fig. 4f). Frameshift mutations also decreased C9ORF72 protein expression (Supplementary Fig. 4g, 5c). RNA sequencing of flow-purified Hb9::RFP+ iMNs showed that targeting C9ORF72 did not significantly alter the expression of the top 10 genes with predicted off-target sites for the CRISPR guide RNA (Supplementary Fig. 4h and Supplementary Table 7). In addition, expression levels of the 20 genes nearest C9ORF72 on chromosome 9 were largely unperturbed in either the C9ORF72+/− and C9ORF72−/− iMNs, indicating that this approach specifically inactivated C9ORF72 (Supplementary Fig. 4i).
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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