Journal: bioRxiv

Article Title: Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

doi: 10.1101/2020.01.30.925222

Figure Lengend Snippet: (A-D) IFN-γ treatment induces LRRK2 levels in human iPSC-derived neurons. (A) LRRK2 mRNA levels in control iPSC-derived neurons treated with IFN-γ for 24 hrs at the indicated concentrations. LRRK2 mRNA levels, measured by qRT-PCR, are expressed as fold changes of untreated (mean ± SEM, one-way ANOVA, Bonferroni post hoc, ***P<0.001, **P <0.01; n=3 independent experiments). (B, C) Representative western blot for LRRK2 expression in control (B) and isogenic LRRK2 G2019S (C) iPSC-derived neurons treated with IFN-γ at the indicated concentrations. (D) LRRK2 mRNA levels in control iPSC-derived neurons treated with 200 IU/mL IFN-γ, 100 ng/mL LPS, or 100 IU/mL IL-1β for 24 hrs (mean ± SEM, one-way ANOVA, Bonferroni post hoc, **P<0.01; n=3 independent experiments). (E, F) IFN-γ transcriptional signature genes in control iPSC-derived neurons treated with 200 IU/mL IFN-γ for 24 hrs measured by qRT-PCR and expressed as fold changes over untreated. Down-regulated (E) and upregulated (F) genes are shown (mean ± SEM, t-test treated vs untreated, ***P<0.001, **P<0.01, *P<0.05; n=3 independent experiments). (G) mRNA levels of AKT3, STAT1, SOCS1, ICAM1, IRF1 in LRRK2 G2019S (G2019S-1) neurons treated with 200 IU/mL IFN-γ, expressed as fold changes over corresponding IFN-μ treated isogenic control (CTRL-1) (mean ± SEM, t-test, **P<0.01; n= 3 independent experiments). (H) mRNA levels of AKT3 in LRRK2 G2019S neurons (G2019S-2, G2019S-3) treated with 200 IU/mL IFN-γ, expressed as fold changes over corresponding treated isogenic controls (CTRL-2, CTRL-3) (mean ± SEM, t-test, **P<0.01; n=3 independent experiments). (I) Representative Western blot of AKT3 in LRRK2 G2019S neurons (G2019S-1, G2019S-2) and corresponding isogenic controls (CTRL-1, CTRL-2). Treatment with 200 IU/mL IFN-γ is indicated. (J) Representative Western blot of p-AKT Ser473 in LRRK2 G2019S neurons (G2019S-1, G2019S-2) and corresponding isogenic controls (CTRL-1, CTRL-2). Treatment with 200 IU/mL IFN-γ is indicated. (K) Quantification of pAKT/AKT level in LRRK2 G2019S neurons and isogenic controls. Data are expressed as fold changes of IFN-γ treated over untreated and normalized to CTRL (mean ± SD, t-test, *P<0.05; n= 4 independent experiments).

Article Snippet: The plasmids SF-tagged LRRK2 wt and SF-tagged LRRK2 G2019S were a kind gift from CJ Gloeckner. pEGFP-C1 NFAT3 was purchased from Addgene (Cat. No.10961).

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

doi: 10.1101/2020.01.30.925222

Figure Lengend Snippet: (A) Western blot for LRRK2 in HEK cells transfected with wt or LRRK2 G2019S. (B) NFAT luciferase assay in HEK293 NFAT reporter cells transfected with wt or LRRK2 G2019S and stimulated with 1 μM ionomycin and 40 ng/mL PMA for 24 hrs (mean ± SD, one-way ANOVA, Bonferroni post hoc, ***P<0.001, **P<0.01, *P<0.05; n=3 independent experiments). (C) NFAT luciferase assay in HEK293 NFAT reporter cells transfected with LRRK2 wt or LRRK2 G2019S and stimulated with PMA/Ionomycin for 24 hrs with or without 200 IU/mL IFN-γ. Data are expressed as fold changes of IFN-γ treated over corresponding PMA/Ionomycin stimulated cells (mean ± SD, t-test, ***P<0.001, *P<0.05; n=3 independent experiments). (D) Representative confocal microscopy images of LRRK2 G2019S, KO and corresponding control neurons transfected with vector encoding NFAT3-GFP (green) treated with 200 IU/mL IFN-γ for 24 hrs or left untreated; blue, DAPI staining; red, βTubIII. (E) Quantification of NFAT3-GFP subcellular localization expressed as nuclear NFAT3-GFP fluorescent intensity normalized on total cell body intensity (mean ± SD, t-test, ***P<0.001; n=5 independent experiments). (F) Quantification of NFAT3-GFP subcellular localization expressed as nuclear NFAT3-GFP fluorescent intensity normalized on total cell body intensity. Data are expressed as ratio of IFN-γ treated over corresponding untreated (mean ± SD, t-test, **P<0.01; n=5 independent experiments). (G) Quantification of NFAT3-GFP subcellular localization expressed as nuclear NFAT3-GFP fluorescent intensity normalized on total cell body intensity (mean ± SD, t-test, *P<0.05, **P<0.01; n=5 independent experiments).

Article Snippet: The plasmids SF-tagged LRRK2 wt and SF-tagged LRRK2 G2019S were a kind gift from CJ Gloeckner. pEGFP-C1 NFAT3 was purchased from Addgene (Cat. No.10961).

Techniques: Western Blot, Transfection, Luciferase, Confocal Microscopy, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

doi: 10.1101/2020.01.30.925222

Figure Lengend Snippet: Intracellular Ca dynamics were measured after KCl stimulation in human iPSC-derived neurons (CTRL-1, CTRL-2, G2019S-1, and G2019S-2) using fura-2 AM ratiometric acquisition. (A) Representative Ca 2+ imaging of single neuron traces is shown (normalized data). KCl indicates time of given stimulus. (B) Intracellular Ca 2+ levels at baseline (left panel), Ca 2+ influx upon KCl stimulation (middle panel), and recovery time after stimulation (right panel) are shown (mean ± SD, t-test, ***P<0.001, **P<0.01; n=3 independent experiments, at least 10-15 single cells per each cell line per experiment were analysed). (C) ER Ca 2+ release upon thapsigargin (TPH) treatment (500 nM, 15 min) in LRRK2 G2019S and isogenic control neurons. Quantification of Ca 2+ peak amplitude (Δ-Ca 2+ release) is shown (mean ± SD, t-test, ***P<0.001; n=3 independent experiments, 10-15 cells per experiment were analyzed). (D) Quantification of TPH mediated Δ-Ca 2+ release in isogenic LRRK2 G2019S and control neurons pretreated with 200 IU/mL IFN-γ for 24 hrs or left untreated (mean ± SD, t-test, ***P<0.001; n=3 independent experiments).

Article Snippet: The plasmids SF-tagged LRRK2 wt and SF-tagged LRRK2 G2019S were a kind gift from CJ Gloeckner. pEGFP-C1 NFAT3 was purchased from Addgene (Cat. No.10961).

Techniques: Derivative Assay, Imaging

Journal: bioRxiv

Article Title: Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

doi: 10.1101/2020.01.30.925222

Figure Lengend Snippet: (A) Knockdown efficiency of IQGAP1 in HEK293 NFAT reporter cells determined by qRT-PCR and normalized to scramble (scrb) non-targeting shRNA (mean ± SD, t-test, ***P<0.001, n=3). (B) Representative Western blot for IQGAP1, showing knockdown efficiency. (C) NFAT luciferase assay in scrb and IQGAP1 KD HEK293 NFAT reporter cells at basal level. Data are expressed as fold change over scrb (mean ± SD, t-test, ***P<0.001, n=3 independent experiments). (D) NFAT luciferase assay in scrb and IQGAP1 KD HEK293 NFAT reporter cells with or without PMA/Ionomycin stimulation. Data are expressed as fold change over corresponding vehicle treated control (mean ± SD, one-way ANOVA, Bonferroni post hoc, ***P<0.001, **P<0.01; n=3 independent experiments). (E) NFAT luciferase assay in scrb and IQGAP1 KD HEK293 NFAT reporter cells stimulated with PMA/Ionomycin for 24 hrs, and 200 IU/mL IFN-γ for 24 hrs, where indicated. Data are expressed as fold changes over corresponding PMA/Ionomycin treated control (mean ± SD, one-way ANOVA, Bonferroni post hoc, *P<0.05; n=3). (F) NFAT luciferase assay in scrb and IQGAP1 KD HEK293 NFAT reporter cells transfected with LRRK2 wt plasmid, with or without PMA/Ionomycin, and with 200 IU/mL IFN-γ for 24 hrs, where indicated. Data are expressed as fold over corresponding LRRK2 wt transfected cell line (mean ± SD, one-way ANOVA, Bonferroni post hoc, **P<0.01, *P<0.05; n=4). (G) NFAT luciferase assay in scrb and IQGAP1 KD HEK293 NFAT reporter cells transfected with LRRK2 G2019S plasmid, with or without PMA/Ionomycin, and with 200 IU/mL IFN-γ for 24 hrs, where indicated. Data are expressed as fold over corresponding LRRK2 G2019S transfected cells (mean ± SD, one-way ANOVA, Bonferroni post hoc, ***P<0.001, **P<0.01, *P<0.05; n=4).

Article Snippet: The plasmids SF-tagged LRRK2 wt and SF-tagged LRRK2 G2019S were a kind gift from CJ Gloeckner. pEGFP-C1 NFAT3 was purchased from Addgene (Cat. No.10961).

Techniques: Quantitative RT-PCR, shRNA, Western Blot, Luciferase, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

doi: 10.1101/2020.01.30.925222

Figure Lengend Snippet: (A) Representative images of βTubIII immunostaining (green) showing neurite elongation in LRRK2 G2019S and isogenic control iPSC-derived neurons after treatment with vehicle, 500 nM MCV1 (NFAT inhibitor) for 24 hrs, or 200 IU/mL IFN-γ for 24 hrs. Blue, DAPI staining. Scale bars, 50 μm. (B) Quantification of neurite elongation is shown (mean ± SD, t-test, ***P<0.001, **P<0.01, *P<0.05; n=5 independent experiments). (C) Immunostaining for βTubIII (green) showing neurite elongation in LRRK2 G2019S and isogenic control iPSC-derived neurons after treatment with vehicle, 500 nM MCV1 24 hrs, or 200 IU/mL IFN-γ for 24 hrs with/without 200 ng/mL NRG1 treatment for 48 hrs. Scale bars, 50 μm. (D) Quantification of neurite elongation is shown (mean ± SD, one-way ANOVA, Bonferroni post hoc; for vehicle vs MCV1/IFN-γ treatment **P<0.01, *P<0.05; for NRG1 treatment §P<0.05, §§P<0.01, §§§ P<0.001; n=5 independent experiments).

Article Snippet: The plasmids SF-tagged LRRK2 wt and SF-tagged LRRK2 G2019S were a kind gift from CJ Gloeckner. pEGFP-C1 NFAT3 was purchased from Addgene (Cat. No.10961).

Techniques: Immunostaining, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

doi: 10.1101/2020.01.30.925222

Figure Lengend Snippet: (A) Representative bright-field image of ramified iPSC-derived microglia. Scale bar, 20 μm. (B) Representative confocal image of iPSC-derived microglia immunostained for IBA1 (red) and nuclear staining DAPI (blue). Scale bar, 20 μm. (C) Representative confocal images of iPSC-derived microglia immunostained for PU.1 (red), and nuclear staining DAPI (blue). Scale bar, 20 μm. (D) Quantification of migrated iPSC-derived microglia cells upon 100 μM ATP stimulation, with or without 200 IU/mL IFN-γ treatment (mean ± SD, one-way ANOVA, Bonferroni post hoc; ***P<0.001, *P<0.05; n=3). (E) Isogenic CTRL, LRRK2 KO, and LRRK2 G2019S iPSC-derived microglia were treated with 100 ng/mL LPS for 24 hrs or left untreated, and cytokine levels were measured by Multiplex Elisa. Concentration of differentially secreted cytokines is shown (mean ± SD, t-test, treated vs untreated, ***P<0.001; G2019S vs CTRL or LRRK2 KO vs CTRL §P<0.05, §§P<0.01, §§§ P<0.001; n=3). (F) Seahorse metabolic analysis of extracellular acidification rate (ECAR) in CTRL and LRRK2 KO microglia treated with 100 ng/mL LPS with or without LRRK2 inhibitor 1.5 μ◻ IN-1 (mean ± SD, t-test, treated vs untreated, §§§ P<0.001; CTRL vs LRRK2 KO, CTRL LPS vs LRRK2 KO LPS or CTRL LPS IN-1, ***P<0.001, *P<0.05; n=8). (G) Isogenic wt (CTRL), LRRK2 KO, and LRRK2 G2019S iPSC-derived microglia were treated with 10 μg/mL poly(I:C) for 24 hrs and IL1-β levels were measured by Elisa (mean ± SD, t-test, treated vs untreated, §§§ P<0.001; CTRL treated vs G2019S treated or LRRK2 KO treated, *P<0.05; n=4). (H) Schematic diagram showing treatment of neurons with either untreated, or LPS-treated, microglial-conditioned medium (MCM); microglial cell lines used are indicated below the scheme. Quantification of neurite elongation in CTRL and G2019S neurons is shown on right (mean ± SD, t-test vs CTRL MG, *P<0.05; n=3 independent experiments).

Article Snippet: The plasmids SF-tagged LRRK2 wt and SF-tagged LRRK2 G2019S were a kind gift from CJ Gloeckner. pEGFP-C1 NFAT3 was purchased from Addgene (Cat. No.10961).

Techniques: Derivative Assay, Staining, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay