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Consistent with PIKFYVE being the relevant target in the iMN survival assay, Apilimod increased C9ORF72 patient, but not control, iMN survival in either neurotrophic withdrawal conditions (Fig. 6d) or excess glutamate (n=4 patients, Supplementary Fig. 15f (n=3 controls, Supplementary Fig. 15g). Automated neuron tracking software independently verified Apilimod efficacy on C9ORF72 patient iMNs (Supplementary Fig. 15h). As further confirmation that PIKFYVE was the active target, ASO-mediated suppression of PIKFYVE also rescued C9ORF72 patient iMN survival (Fig. 6d and Supplementary Fig. 15i). In addition, we synthesized a structural analog of Apilimod with a reduced ability to inhibit PIKFYVE kinase activity in a biochemical assay using purified PIKFYVE protein (Fig. 6b and Supplementary Fig. 15j, 16). The reduced activity analog was significantly less effective at rescuing C9ORF72 patient iMN survival (Fig. 6e). Thus, small molecule inhibition of PIKFYVE can rescue patient motor neuron survival.
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.
Minerals 2017, 7, 57; doi:10.3390/min7040057 Article Migration Behavior of Lithium during Brine Evaporation and KCl Production Plants in Qarhan Salt Lake Weijun Song 1,2, Hongze Gang 1, Yuanqing Ma 4, Shizhong Yang 1 and Bozhong Mu 1,3,* 1 Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, China; (W.S.); (H.G.); (S.Y.) 2 School of Chemical Engineering, Qinghai University, Xining 810016, China 3 Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai 200237, China 4 Qinghai Salt Lake Industry Group Co. Ltd., Golmud 816000, China; * Correspondence: Academic Editor: Javier Sánchez-España Received: 8 January 2017; Accepted: 2 April 2017; Published: 11 April 2017 Abstract: 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. Keywords: lithium migration; occurrence status; Qarhan Salt Lake 1. Introduction As an energy metal of the twenty-first century, lithium had attracted more and more attention in the past few decades. Lithium has been widely applied in high energy batteries, controlled thermonuclear reactions, the manufacturing of ceramic and glass, and other fields [1–7]. Lithium consumption for batteries had increased most significantly due to the development of the electric vehicle industry and the popularity of portable electronic products. Stimulated by the political affairs, economic requirements, and environmental conservation, lithium resources have become the focus of the international mining market and lithium’s position as a strategic resource is becoming more prominent. Salt lake brine, thermal spring, and oilfield water are important geological sources of lithium. The commercial exploitation of the lithium resource of brine began at the Searles Lake in the US in 1936. Since then, more focus has been placed on recovering lithium from salt lake brine because of its low economical cost and low environmental impact [8–10]. As a country possessing huge amount of
To determine if reduced C9orf72 levels leads to glutamate receptor accumulation in vivo, we examined spinal motor neurons deleted of C9orf72 in Nestin-Cre-Stop-Flox-C9orf72 mice 22. Immunofluorescence analysis indicated that Nr1 (NMDA) and GluR1 (AMPA) levels were elevated in C9orf72-null motor neurons (Supplementary Fig. 12a, b). To confirm these findings, we isolated post-synaptic densities from the spinal cords of control and C9orf72 knockout mice. Post-synaptic density fractions contained glutamate receptors and PSD-95, but not p53 or synaptophysin, indicating they were enriched for post-synaptic density proteins (Supplementary Fig. 12c, 5i). Immunoblotting showed that post-synaptic densities in C9orf72 knockout mice contained significantly higher levels of Nr1 and Glur1 than in control mice (Fig. 4i, j and Supplementary Fig. 5j).
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).
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.
RNA sequencing output was aligned to the GRCh38 Reference Genome and quantified using the STAR aligner.65 Genes were annotated against the GENCODE version 23 Comprehensive Gene Annotation. Quality control was performed using Picard Tools AlignmentSummaryMetrics. Samples passing quality control and having RNA Integrity Number (RIN) > 5 were used in downstream analysis. To identify differentially expressed genes, the R package DESeq2 was used as previously described.66 The function DESeq was used to estimate size factors, estimate dispersion, fit the data to a negative binomial generalized linear model, and generate differential expression statistics using the Wald test. KEGG enrichment analysis was performed for internal analysis using the R package clusterProfiler.67
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.