To determine if C9ORF72 iMNs recapitulate neurodegenerative ALS processes, we examined their survival by performing longitudinal tracking of Hb9::RFP+ iMNs (Fig. 1a). This approach enabled us to distinguish differences in neurogenesis from differences in survival, which could not be addressed using previously-reported cross-sectional analyses6,7,10,26. In basal neuronal medium supplemented with neurotrophic factors, control and C9ORF72 patient iMNs survived equally well (Fig. 1b, Supplementary Fig. 3a, Supplementary Tables 5, 6). As human C9ORF72 ALS patients have elevated glutamate levels in their cerebrospinal fluid (possibly triggered by DPR-mediated aberrant splicing of the astrocytic excitatory amino acid transporter 2 EAAT2 4,27) we stimulated iMN cultures with a high glutamate pulse (12-hour treatment, 10 μM glutamate). This initiated a robust degenerative response in patient, but not control, iMNs (Fig. 1c-e and Supplementary Videos 3, 4) that was consistent across lines from multiple patients (n=6 patients) and controls (n=4 controls)(Fig. 1c, d and Supplementary Fig. 3d, e). While iMN survival varied slightly between live imaging systems, or between independent experiments due to the lengthy time course of neurodegeneration, the relative difference between control and C9-ALS patient iMNs was consistent (Fig. 1c - Nikon Biostation CT and Supplementary Fig. 3b - Molecular Devices ImageExpress). Moreover, iMNs from different iPSC lines derived from the same donor behaved similarly, suggesting genotypic differences accounted for these effects (Supplementary Fig. 3c). Treatment with glutamate receptor antagonists during glutamate administration prevented patient iMN degeneration (Fig. 1f). Alternatively, withdrawal of neurotrophic factors without glutamate stimulation also caused rapid degeneration of patient iMNs (n=3 patients, (Fig. 1g and Supplementary Fig. 3f).

Shi Y1,2,3, Lin S1,2,3, Staats KA1,2,3, Li Y1,2,3, Chang WH1,2,3, Hung ST1,2,3, Hendricks E1,2,3, Linares GR1,2,3, Wang Y3,4, Son EY5, Wen X6, Kisler K3,4, Wilkinson B3, Menendez L1,2,3, Sugawara T1,2,3, Woolwine P1,2,3, Huang M1,2,3, Cowan MJ1,2,3, Ge B1,2,3, Koutsodendris N1,2,3, Sandor KP1,2,3, Komberg J1,2,3, Vangoor VR7, Senthilkumar K7, Hennes V1,2,3, Seah C1,2,3, Nelson AR3,4, Cheng TY8, Lee SJ8, August PR9, Chen JA10, Wisniewski N10, Hanson-Smith V10, Belgard TG10, Zhang A10, Coba M3,11, Grunseich C12, Ward ME12, van den Berg LH13, Pasterkamp RJ7, Trotti D6, Zlokovic BV3,4, Ichida JK1,2,3.


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.


J.K.I. and P.A. are co-founders of Acurastem, Inc. P.A. is an employee of Icagen Corporation. J.K.I. and P.A. declare that they are bound by confidentiality agreements that prevent them from disclosing details of their financial interests in this work. S-J.L. is a founder of DRVision Technologies and T-Y.C. is an employee of DRVision Technologies. A.Z. and J.A.C. are co-founders of Verge Genomics and V.H-S., N.W., and T.G.B. are employees of Verge Genomics.
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
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).

iMNs were fixed in 4% paraformaldehyde (PFA) for 1h at 4 ºC, permeabilized with 0.5% PBS-T overnight at 4 ºC, blocked with 10% FBS in 0.1% PBS-T at room temperature for 2 h, and incubated with primary antibodies at 4 ºC overnight. Cells were then washed with 0.1% PBS-T and incubated with Alexa Fluor® secondary antibodies (Life Technologies) in blocking buffer for 2 h at room temperature. To visualize nuclei, cells were stained with DAPI (Life Technologies) then mounted on slides with Vectashield® (Vector Labs). Images were acquired on an LSM 780 confocal microcope (Zeiss). The following primary antibodies were used: mouse anti-HB9 (Developmental Studies Hybridoma Bank); mouse anti-TUJ1 (EMD Millipore); rabbit anti-VACHT (Sigma); rabbit anti-C9ORF72 (Sigma-Aldrich); mouse anti-EEA1 (BD Biosciences); mouse anti-RAB5 (BD Biosciences); mouse anti-RAB7 (GeneTex); mouse anti-LAMP1 (Abcam); mouse anti-LAMP3 (DSHB, cat. no. H5C6); rabbit anti-LAMP3 (Proteintech, cat. no. 12632); mouse anti-LAMP2 (DSHB, cat. no. H4B4); mouse anti-M6PR (Abcam, cat. no. Ab2733); rabbit anti-GluR1 (EMD Millipore, cat. no. pc246); mouse anti-GluR1 (Santa Cruz); rabbit anti-NR1 (EMD Millipore); mouse anti-NR1 (EMD Millipore, cat. no. MAB363); chicken anti-GFP (GeneTex).

(a) Super-resolution microscopy images of immunofluorescence shows NR1+ puncta on neurites of iMNs overexpressing eGFP or C9ORF72 isoform B-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b-d) Number of NR1+ puncta per unit area in control (b-d), patient (b), C9ORF72+/− (c), and C9ORF72+/− (d) iMNs. Mean ± s.d. Each grey open circle represents the number of NR1+ puncta per area unit on a single neurite (one neurite quantified per iMN). For (b), n=75 (CTRL + GFP), 84 (C9-ALS + GFP), 95 (C9-ALS + isoA), and 111 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (c), n=37 (CTRL + GFP), 37 (C9ORF72+/− + GFP), 25 (C9ORF72+/− + isoA), and 27 (C9ORF72+/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. For (d), n=37 (CTRL + GFP), 37 (C9ORF72−/− + GFP), 38 (C9ORF72−/− + isoA), and 23 (C9ORF72−/− + isoB) iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction for all comparisons. F-value (DFn, DFd): (3, 360) = 56.63 (b), (3, 122) = 13.42 (c), (3, 131) = 17.11 (d). (e-h) Immunoblotting analysis of surface NR1 after surface protein biotinylation in control (e-h), C9ORF72+/− (e-f), and C9-ALS patient (g-h) iMNs generated with 3 factors (NGN2, ISL1, and LHX3). In (f), n=4 biologically independent iMN conversions from CTRL2 and 2 biologically independent iMN conversions from the C9ORF72+/− line. Mean +/− s.d. In (h), two-tailed Mann-Whitney test. n=11 biologically independent motor neuron cultures from 11 independent control lines and 4 biologically independent motor neuron cultures from 4 independent C9-ALS patient lines. Experiments in (e-h) were repeated twice with similar results. Mean +/− s.e.m. (i-j) Immunoblotting analysis of surface Nr1 and Glur1 in post-synaptic densities (PSDs) from C9orf72 control and knockout mice, two-tailed t-test. t-value: 4.424 (Nr1), 4.632 (Glur1), degrees of freedom: 4 (Nr1), 4 (Glur1). n= 3 control PSD preparations isolated from 3 control mice and 3 C9orf72−/− PSD preparations isolated from 3 C9orf72−/− mice. This experiment was repeated twice with similar results. Mean +/− s.e.m. (k-l) Immunoblotting analysis of surface NR1 and GLUR1 in post-synaptic densities (PSDs) from post mortem control and C9-ALS patient motor cortices, n=3 control and 2 C9-ALS patient PSD preparations isolated from 3 control and 2 C9-ALS patients. This experiment was repeated twice with similar results. Mean +/− s.d. (m) Average Ca2+ flux in the presence of glutamate per minute. n=28 (CTRL1), 15 (CTRL2), 15 (CTRL3), 26 C9-ALS1), 20 (C9-ALS2), 24 (C9-ALS3), and 15 (C9ORF72+/−) iMNs analyzed from two biologically independent iMN conversions for each line. Mean ± s.e.m. One-way ANOVA with Tukey correction between all controls and all patients and C9ORF72+/−. F-value (DFn, DFd): (6, 136) = 11.21.
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).
To study the pathogenic mechanism of the C9ORF72 repeat expansion in human motor neurons, we used the forced expression of the transcription factors Ngn2, Isl1, Lhx3, NeuroD1, Brn2, Ascl1, and Myt1l, to convert control and C9ORF72 ALS/FTD patient induced pluripotent stem cells (iPSCs)(for iPSC characterization, see Supplementary Fig. 1 and Supplementary Tables 3, 4) into iMNs (Supplementary Fig. 2a, b) 10,24. Control and patient iMNs labeled with an Hb9::RFP+ lentiviral reporter construct (Supplementary Fig. 2b-d)25 co-expressed spinal motor neuron markers including TUJ1, HB9, and VACHT; were produced at similar rates amongst different iPSC lines; and possessed electrophysiological properties of motor neurons (Supplementary Fig. 2c-i). Depolarizing voltage steps induced currents characteristic of sodium and potassium channels and iMNs fired single or repetitive action potentials (patient - 90%, n=10; control – 100%, n=10)(Supplementary Fig. 2g-i). When co-cultured with primary chick muscle, channel rhodopsin-expressing control and patient iMNs repeatedly induced myotube contraction upon depolarization with green light, indicating they formed neuromuscular junctions and actuated muscle contraction (Supplementary Fig. 2j and Supplementary Videos 1, 2).

Lithium-brine is an important potential source of lithium. Much research and investigation has been carried out aimed at lithium recovery from brine. Although the distribution and occurrence status of lithium in brine have important implications for lithium recovery, few reports had correlated to this issue. In this article, a study was carried out to explore the lithium migration behavior during brine evaporation and KCl production process at Qarhan Salt Lake. The occurrence status of lithium both in fresh mined brine and residual brine after evaporation were also speculated by means of lithium concentration evaluation and theoretical calculation based on the Pitzer electrolyte solution theory. Results showed that, for Qarhan brine mined from the Bieletan region, most lithium was enriched in the residual brine during the brine evaporation process. The concentration of lithium in the residual brine could be more than 400 mg/L. More than 99.93% lithium ions in residual brine exist in free ions state and lithium does not precipitate from brine with a density of 1.3649 g/mL. The results also revealed that lithium concentration in wastewater discharged from KCl plants can reach a level of 243.8 mg/L. The investigation results provide a theoretical basis for comprehensive development and utilization of lithium resources in Qarhan Salt Lake.
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.
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).
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced 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 led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to 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 and FTD.
(a) The levels of C9ORF72 variant 2 mRNA transcript (encoding isoform A). Values are mean ± s.e.m., two-tailed t-test with Welch’s correction. t-value: 5.347, degrees of freedom: 11.08. n= 9 biologically independent iMN conversions from 3 control lines and 12 biologically independent iMN conversions from 5 C9-ALS lines. (b–d) iMN survival in excess glutamate following introduction of C9ORF72 (C9 isoform A or B) into C9ORF72 patient iMNs (b), but not control (b, d) or SOD1-ALS iMNs (c). For (b), n=50 iMNs per line for 2 control and 3 C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions per line. For (c), n=50 iMNs per condition, iMNs scored from 3 biologically independent iMN conversions. For (d), n=50 iMNs per line per condition for 2 control lines, iMNs quantified from 3 biologically independent iMN conversions. Each trace includes iMNs from 2–3 donors with the specified genotype (except SOD1-ALS (c)); see full details in Methods. (e) Strategy for knocking out C9ORF72 from control iPSCs using CRISPR/Cas9. (f) Survival of control (CTRL2) iMNs, the isogenic heterozygous (C9+/−) and homozygous (C9−/−) iMNs and C9ORF72 patient (C9-ALS) iMNs in excess glutamate. n=50 biologically independent iMNs per line per condition for one control and two C9-ALS lines, iMNs quantified from 3 biologically independent iMN conversions. (g) Control iMN survival in excess glutamate with scrambled or C9ORF72 antisense oligonucleotides (ASO). Each trace includes control iMNs from 2 donors. n=50 biologically independent iMNs per line per condition for 2 control lines, iMNs quantified from 3 biologically independent iMN conversions. All iMN survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated using the entire survival time course. iMN survival experiments in (b, d, and g) were performed in a Nikon Biostation, and (e and f) were performed in a Molecular Devices ImageExpress.
Lithium-brine is an important potential source of lithium. Much research and investigation has been carried out aimed at lithium recovery from brine. Although the distribution and occurrence status of lithium in brine have important implications for lithium recovery, few reports had correlated to this issue. In this article, a study was carried out to explore the lithium migration behavior during brine evaporation and KCl production process at Qarhan Salt Lake. The occurrence status of lithium both in fresh mined brine and residual brine after evaporation were also speculated by means of lithium concentration evaluation and theoretical calculation based on the Pitzer electrolyte solution theory. Results showed that, for Qarhan brine mined from the Bieletan region, most lithium was enriched in the residual brine during the brine evaporation process. The concentration of lithium in the residual brine could be more than 400 mg/L. More than 99.93% lithium ions in residual brine exist in free ions state and lithium does not precipitate from brine with a density of 1.3649 g/mL. The results also revealed that lithium concentration in wastewater discharged from KCl plants can reach a level of 243.8 mg/L. The investigation results provide a theoretical basis for comprehensive development and utilization of lithium resources in Qarhan Salt Lake.
Total RNA was extracted from sorted iMNs at day 21 post-transduction with Trizol RNA Extraction Kit (Life Technologies) and reverse transcribed with an Oligo dT primer using ProtoScript® II First Strand Synthesis Kit (NEB). RNA integrity was checked using the Experion system (Bio-Rad). Real-time PCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) using primers shown in Supplementary Data Table 4.
To determine if a deletion of C9ORF72 or the C9ORF72 repeat expansion caused changes in endosomal trafficking in motor neurons, we examined the number of early endosomes (RAB5+, EEA1+), late endosomes (RAB7+), and lysosomes (LAMP1+, LAMP2+, LAMP3+) in control, C9ORF72 patient, C9ORF72+/−, and C9ORF72−/− iMNs. We observed the most significant difference in the lysosomal population, with C9ORF72 patient iMNs (n=4 patients) having fewer LAMP1+, LAMP2+, and LAMP3+ vesicles than control iMNs (n=4 controls)(Fig. 3c, d and Supplementary Fig. 8a-d). C9ORF72+/− and C9ORF72−/− also harbored fewer LAMP1+, LAMP2+, and LAMP3+ vesicles than isogenic control iMNs, indicating that reduced C9ORF72 levels alone leads to a loss of lysosomes (Fig. 3c, e, f and Supplementary Fig. 8a-d). ASO-mediated knockdown of C9ORF72 expression also decreased lysosome numbers in iMNs (Supplementary Fig. 8e). Although membrane fractionation showed that control and patient iMNs have similar amounts of LAMP2 in the lysosomal membrane fraction (Supplementary Fig. 8f), analysis of the immunofluorescence intensity of LAMP proteins suggests that this is likely due to the fact that C9ORF72 patient and C9ORF72+/− iMNs have a higher concentration of LAMP proteins in their lysosomal membranes, possibly as a result of fewer lysosomes being present (Supplementary Fig. 8g). Using electron microscopy to identify lysosomes by their high election density 40, we verified that the vesicles reduced in C9ORF72-deficient cells were lysosomes (Fig. 3g-i). Forced expression of either C9ORF72 isoform restored the number of LAMP1+, LAMP2+, and LAMP3+ lysosomes in patient (n=4 patients) and C9ORF72-deficient iMNs (Fig. 3c-f and Supplementary Fig. 8a-h). To determine if loss of C9ORF72 activity reduces lysosome numbers in motor neurons in vivo, we measured the number of lysosomes in spinal motor neurons in Nestin-Cre-Stop-Flox-C9orf72 mice 22. C9orf72−/− motor neurons contained significantly fewer Lamp1+ lysosomes than control motor neurons (Fig. 3j, k).
On the other hand, the level of the Neijing force depends on the extent one can exercise over one's will power to release an inner qi energy. Within the framework of Chinese martial arts, every person is believed to possess the inborn energy of qi. Martial artists can harness the force of qi so that it is strong enough to be applied in combat. When qi is being directed by one's will, it is called Neijing.[4]

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,†
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