Heavy metals contaminating the environment of a progressive supranuclear palsy cluster induce tau accumulation and cell death in cultured neurons

Abstract

Statistical analysis. One-way and two-way ANOVA statistical analyses were performed using Prism GraphPad Prism 6. Bonferroni's analysis was performed to analyze the statistical significance between the groups. Plots show means ± standard error (SEM) of all the experiments performed. Values are considered statistical significant if p < 0.05.

TSC1loss-of-function increases risk for tauopathy by inducing tau acetylation and preventing autophagy-mediated tau clearance

“…Two monoclonal TSC1 +/- iPSC lines were generated by the company Synthego in the background of the WT PGP-1 iPSC using the CRISPRs/Cas9 gene editing system. Briefly, a sgRNA (5’-ACCAAGGUGUUUACAAGCAU-3’) targeting the TSC1 gene was designed and validated before the nucleofection of iPSC, as previously described ( 72–74 ). To select TSC1 +/- iPSC clones, genomic DNA was extracted with the QuickExtract kit (Epicentre, #QE09050).…”

“…For the neuronal differentiation of SH-SY5Y cells, 10,000 cells/cm 2 cells were seeded in tissue culture plates 24 hours before starting the differentiation through the addition of 10μM of retinoic acid (RA) in EMEM/F12 media supplemented with 10% FBS and 1% P/S. After 6 days of RA treatment, cells were treated for 4 days with 50ng/mL of BDNF in EMEM/F12 media supplemented only with 1% P/S ( 74, 75 ). Human iPSCs were differentiated into cortical neurons by infection with virus particles containing doxycycline-inducible neurogenin-2 (Ngn2) and puromycin resistance as previously described ( 74, 76 ).…”

“…After 6 days of RA treatment, cells were treated for 4 days with 50ng/mL of BDNF in EMEM/F12 media supplemented only with 1% P/S ( 74, 75 ). Human iPSCs were differentiated into cortical neurons by infection with virus particles containing doxycycline-inducible neurogenin-2 (Ngn2) and puromycin resistance as previously described ( 74, 76 ). After infection, cells were cultured for 21 days before lysate collection.…”

“…For western blotting, equivalent amounts of protein were separated on a 4-12% Bis-Tris gel (ThermoFisher Scientific). Immunoblotting was performed as previously described ( 74 ) using the antibodies listed in supplementary material (Table S2). Immunoreactive bands were visualized using the LI-COR Odyssey CLx imaging system and quantified using the ImageJ software.…”

“…Two monoclonal TSC1+/-iPSC lines were generated by the company Synthego in the background of the WT PGP-1 iPSC using the CRISPRs/Cas9 gene editing system. Briefly, a sgRNA (5'-ACCAAGGUGUUUACAAGCAU-3') targeting the TSC1 gene was designed and validated before the nucleofection of iPSC, as previously described (72)(73)(74). After the generation, both TSC1-family and CRISPR-engineered iPSC lines were verified for the expression of pluripotency markers (SSEA4, Oct4 and SOX2) by immunofluorescence as previously described (20) (Fig.…”