Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Heatmap depicting significant gene expression differences (Log2 fold-change, p<0.05) between uninfected Lrrk2 KO and HET BMDMs. ( B ) IPA software analysis showing cellular pathways enriched for differentially expressed genes in uninfected Lrrk2 KO vs. HET BMDMs. ( C ) Heatmap depicting significant gene expression differences (Log2 fold-change) between Lrrk2 KO and HET BMDMs during infection with Mtb. ( D ) As in ( B ) but for pathways enriched for differentially expressed genes in Mtb-infected Lrrk2 KO and HET BMDMs, 4 hr post-infection. ( E ) RT-qPCR showing expression of Ifnb and IFN stimulated genes in uninfected and Mtb-infected Lrrk2 KO and HET macrophages. Data are shown as ISG / Actb . ( F ) RT-qPCR of Tnfa in Lrrk2 KO and HET BMDMs. ( G ) RT-qPCR of Apoe and Ldhb normalized to Actb in uninfected BMDMs. Throughout the manuscript, data are expressed as a mean of three or more biological replicates with error bars depicting SEM. Statistical tests used can be found at the end of the legend. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). In ( E–F ) a two-way ANOVA Tukey post-test was applied, and in ( G ) a two-tailed Student’s T test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Gene Expression, Software, Infection, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Heatmap depicting gene expression differences (Log2 fold-change) between Lrrk2 KO and HET BMDMs after infection with Mtb. ( B ) RT-qPCR of Ifit1 and Isg15 in Lrrk2 WT, HET, and KO BMDMs. ( C ) RT-qPCR of Tnf a expression in Lrrk2 WT, HET, and KO BMDMs. RT-qPCR showing expression of Ifnb and IFN stimulated genes in uninfected and Mtb-infected Lrrk2 KO and HET macrophages. Data are shown as ISG / Actb . ( D ) RT-qPCR of LRRK2 in uninfected SCR and LRRK2 KD U937 macrophage-like cells and gene expression of IFNB and IL1B in uninfected and Mtb-infected cells (MOI = 10, 4 hr). ( E ) RT-qPCR of Lrrk2 in uninfected SCR and Lrrk2 KD RAW 264.7 cells and gene expression of Ifnb and Tnfa in uninfected and Mtb-infected cells (MOI = 10, 4 hr). Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( B–E ) One-way and two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Gene Expression, Infection, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) RT-qPCR of Isg15 expression after 4 and 8 hr of infection with M. leprae (MOI = 50) in Lrrk2 KO BMDMs and HET controls. ( B ) RT-qPCR of Ifnb in unstimulated Lrrk2 KO and HET BMDMs alongside cells transfected with 1 μg/ml ISD (dsDNA) for 4 hr. ( C ) As in ( B ) but with peritoneal macrophages (PEMs) from Lrrk2 KO and HET mice elicited for 4 days with 1 ml 3% Brewer’s thioglycolate broth. ( D ) As in ( B ) but with RAW 264.7 Lrrk2 KO cells and WT controls. ( E ) As in ( B ) but with MEFs from day 14.5 Lrrk2 KO or HET embryos. ( F ) As in ( B ) but with RAW 264.7 Lrrk2 KD and scramble (SCR) controls cells. ( G ) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET BMDMs and in cells treated with 50 ng/ml DMXAA for 2 hr. ( H ) Western blot analysis and quantification of IRF3 phosphorylation (Ser396) and STAT1 phosphorylation (Tyr701) in BMDMs from HET and Lrrk2 KO mice compared to total IRF3 and STAT1 with tubulin as a loading control following transfection with 1 μg/ml ISD (dsDNA). ( I ) As in ( G ) but following transfection with 1 μg/ml poly(I:C), 100 ng/ml LPS, transfection with 10 μM CpG 2395, or stimulation with 1 μM CL097, all for 4 hr. ( J ) RT-qPCR of Isg15 expression after treatment with 200 IU IFN-β for 4 hr. ( K ) RT-qPCR of Irf7 gene expression in Lrrk2 HET and KO BMDMs with or without overnight treatment with IFN-β neutralizing antibody (blocking Ab, 1:250). ( L ) RT-qPCR of Irf7 gene expression in BMDMs from WT, Lrrk2 KO, Ifnar KO, and double knockout ( Lrrk2 / Ifnar DKO) mice. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A–L ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Quantitative RT-PCR, Expressing, Infection, Transfection, Western Blot, Phospho-proteomics, Control, Gene Expression, Blocking Assay, Double Knockout

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) RT-qPCR of Isg15 expression after 4 and 8 hr of infection with M. leprae (MOI = 50) in WT and Lrrk2 KO RAW 264.7. ( B ) Western blot analysis and quantification of IRF3 phosphorylation (Ser396) and STAT1 phosphorylation (Tyr701) in BMDMs from HET and Lrrk2 KO mice compared to total IRF3 and STAT1 with tubulin as a loading control following transfection with 1 μg/ml ISD (dsDNA). ( C ) RT-qPCR of Ifit1 in Lrrk2 HET and KO BMDMs transfected with 1 μg/ml ISD (dsDNA) or 1 μg/ml poly(I:C) or treated with 100 ng/ml LPS for 4 hr. ( D ) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET PEMs treated with 100 ng/ml LPS for 4 hr. ( E ) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET MEFs treated with 100 ng/ml LPS or transfected with 1 μg/ml poly(I:C) for 4 hr. ( F ) RT-qPCR of Irf7 expression in Lrrk2 HET and KO BMDMs after treatment with 200 IU IFN-β for 4 hr. ( G ) RT-qPCR of Isg15 expression in Lrrk2 HET and KO BMDMs with or without overnight blocking with IFN-β neutralizing antibody (blocking Ab, 1:250). ( H ) RT-qPCR of Irf7 gene expression in BMDMs from WT, Lrrk2 KO, Ifnar KO, and double knockout ( Lrrk2 / Ifnar DKO) mice. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); %%p<0.01, ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A–H ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Quantitative RT-PCR, Expressing, Infection, Western Blot, Phospho-proteomics, Control, Transfection, Blocking Assay, Gene Expression, Double Knockout

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Isg15 gene expression in Lrrk2 WT, Lrrk2 KO, cGas KO, and double KO ( cGas/Lrrk2 DKO) BMDMs treated with 5 μg/ml DMXAA or transfected with 1 μg poly(I:C) for 4 hr. ( B ) Western blot analysis of STAT1 phosphorylation (Tyr701) in BMDMs from WT, Lrrk2 KO, cGas KO, and cGas/Lrrk2 double knockout (DKO) mice compared to total STAT1 with tubulin as a loading control. ( C ) Immunofluorescent images with anti-TOM20 antibody to visualize the mitochondrial network of Lrrk2 HET and KO MEFs. TOM20 (green), nucleus (DAPI, blue); Scale bar = 10 μm ( D ) qPCR of total 16 s and cytB (mitochondrial DNA) relative to Tert (nuclear DNA). ( E ) As in ( D ) but cytosolic mitochondrial DNA. ( F ) Western blot of ACTIN, TFAM, and VDAC protein levels in total, cytosol, and pellet (organelle and membrane) fractions of Lrrk2 KD and SCR RAW 264.7 cells. ( G ) Irf7 gene expression normalized to Actb in untreated BMDMs from Lrrk2 WT, Lrrk2 KO, Tfam HET, and Lrrk2 KO/ Tfam HET mice. ( H ) qPCR of dLoop (mitochondrial DNA) normalized to Tert (nuclear) to confirm mtDNA depletion in WT and Lrrk2 KO RAW 264.7 cells treated with 10 μM ddC for 4 days. ( I ) RT-qPCR of Ifnb gene expression in WT and Lrrk2 KO RAW 264.7 cells with or without ddC treatment, untreated and at 4 hr post-transfection with 1 μg/ml ISD. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); %p<0.05, ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A and I ) 3-way ANOVA, Tukey post-test; ( D and E ) two-tailed Student’s T test; ( G and H ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Gene Expression, Transfection, Western Blot, Phospho-proteomics, Double Knockout, Control, Membrane, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Rsad2 or Irf7 gene expression in Lrrk2 WT, Lrrk2 KO, cGas KO, and double KO ( cGas/Lrrk2 DKO) BMDMs treated with 5 μg/ml DMXAA or transfected with 1 μg poly(I:C) for 4 hr. ( B ) qPCR of total 16 s and cytB (mitochondrial DNA) relative to Tert (nuclear DNA) in Lrrk2 HET and KO MEFs. ( C ) As in ( B ) but measuring cytosolic mitochondrial DNA. ( D ) RT-qPCR of Irf7 gene expression in WT and Lrrk2 KO RAW 264.7 cells with or without ddC and transfected with 1 μg ISD for 4 hr. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A and D ) 3-way ANOVA Tukey post-test; ( B and C ) two-tailed Student’s T test. *p<0.05, **p<0.01, ***p<0.005. ( E ) Western blot analysis of STAT1 phosphorylation (Tyr701) in BMDMs from wild-type and Lrrk2 KO mice either untreated (top) or treated with ddC (bottom) at 0, 2, 4, and 6h post-ISD transfection. Total STAT1 and tubulin are shown as controls. Densitometry quantitation of shown under blots.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Phospho-proteomics, Quantitation Assay

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of mitochondrial network TOM20 (green) and DRP1 (red) in MEFs from Lrrk2 HET and KO embryos. Nuclei are stained with DAPI (blue). Scale bar = 10 μm. ( B ) Western blot analysis and quantification of DRP1 phosphorylation (Ser616) in SCR and Lrrk2 KD RAW 264.7 cells compared to total DRP1 and actin as a loading control. ( C ) Histograms showing counts of phospho-S616-DRP1 in Lrrk2 HET and KO MEFs treated with 100 μM H 2 O 2 or 50 μM Mdivi-1 for 4 hr. ( D ) RT-qPCR of Isg15 and Irf7 gene expression Lrrk2 KO and HET BMDMs treated with 50 μM Mdivi-1 for 12 hr. ( E ) qPCR of cytosolic and total 16 s (mitochondrial DNA) relative to Tert (nuclear DNA) in Lrrk2 HET and KO MEFs treated with 50 μM Mdivi-1 for 12 hr. Statistical analysis: As in ; *p<0.05, **p<0.01, ***p<0.005.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR, Gene Expression

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Histograms showing counts of phospho-S616-DRP1 in SCR and Lrrk2 KD RAW 264.7 cells as measured by flow cytometry. ( B ) Western blot analysis and quantification of DRP1 phosphorylation (Ser616) in SCR and Lrrk2 KD RAW 264.7 cells compared to total DRP1 and actin as a loading control. ( C ) As in ( A ) but for BMDMs. ( D ) As in ( A ) but for MEFs. ( E ) Basal gene expression of Isg15 and Irf7 in SCR and Lrrk2 KD RAW 267.4 cells treated with Mdivi-1 50 μM for 12 hr. ( F ) qPCR of cytosolic and total 16 s (mitochondrial DNA) relative to Tert (nuclear DNA) in SCR and Lrrk2 KD RAW 264.7 cells treated with 50 μM Mdivi-1 for 12 hr. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ( A, C, and D ) Two-tailed Student’s T test; ( E and F ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Flow Cytometry, Western Blot, Phospho-proteomics, Control, Gene Expression, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Mitochondrial membrane potential in Lrrk2 HET and KO BMDMs as measured by JC-1 dye by flow cytometry. Aggregates (610/20) indicate normal mitochondrial membrane potential and monomers (520/50) indicate low membrane potential. ( B ) Histogram of ( A ) displaying cell counts of JC-1 aggregates for Lrrk2 HET and KO BMDMs. ( C ) JC-1 aggregates measured by flow cytometry in BMDMs treated for 3 hr with 2.5 μM rotenone followed by 5 μM ATP for 0, 5, or 30 min. Histogram of cell counts is on the right. ( D ) Flow cytometry of mitochondrial membrane potential measured by TMRE (585/15) in SCR and Lrrk2 KD RAW 264.7 cells treated for 3 hr with 2.5 μM rotenone followed by 5 μM ATP for 15 min or 50 μM FCCP for 15 min. ( E ) JC-1 aggregates measured by flow cytometry in Lrrk2 KO BMDMs treated with 10 or 50 μM Mdivi-1 for 4 hr. ( F ) The same as in ( E ) but with TMRE. ( G ) Basal gene expression of Irf7 and Isg15 in Lrrk2 HET and KO BMDMs treated overnight with 200 μM mitoTEMPO. JC-1 flow cytometry assays are representative of 3 independent experiments. Statistical analysis: **p<0.01, ***p<0.005, ****p<0.001. ( F ) Two-tailed Student’s T test; ( G ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Membrane, Flow Cytometry, Gene Expression, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Flow cytometry of SCR or Lrrk2 KD RAW 264.7 cells measuring mitochondrial membrane potential by JC-1 dye with 2.5 μM rotenone followed by 5 μM ATP for the indicated times. ( B ) As in ( A ) but with Lrrk2 HET and KO MEFs. Histogram of cell counts on the right. ( C ) As in ( A ) with 50 μM FCCP as a positive control in Lrrk2 HET and KO BMDMs (left) and SCR and Lrrk2 KD RAW 264.7 cells (right). Histogram of cell counts below. ( D ) Flow cytometry of mitochondrial membrane potential measured by TMRE (585/15) in BMDMs (left), MEFs (center), and RAW 264.7 cells (right). Histogram plots with quantifications below. ( E ) Flow cytometry of mitochondrial membrane potential measured by TMRE (585/15) in SCR or Lrrk2 KD RAW 264.7 cells treated with 2.5 μM rotenone followed by 5 μM ATP or 50 μM FCCP. JC-1 flow cytometry assays are representative of 3 independent experiments. Statistical analysis: As in ; *p<0.05.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Flow Cytometry, Membrane, Positive Control

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Irf7 gene expression in HET and Lrrk2 KO BMDMs cultured with or without 1 mM sodium pyruvate. ( B ) Ifnb and Irf7 gene expression in SCR and Lrrk2 KD RAW 264.7 cells cultured with or without 1 mM sodium pyruvate. ( C ) BMDMs from Lrrk2 HET and KO mice were treated with 200 μM mitoTEMPO, IFN-β blocking antibody, and the combination of both overnight followed by analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured using the Seahorse Metabolic Analyzer (Agilent). ( D ) Quantification of maximal respiration and spare respiratory capacity from ( C ). Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. (A, B, and D) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Gene Expression, Cell Culture, Blocking Assay

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) RT-qPCR of Ifnb expression in Lrrk2 HET and KO MEFs cultured with 1 mM sodium pyruvate for 24 hr. ( B ) Seahorse metabolic analysis of Lrrk2 HET and KO BMDMs treated with increasing concentrations of sodium pyruvate (0, 1, and 2 mM). ( C ) Quantitation of maximal respiration and spare respiratory capacity shown in ( B ). Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ( A and C ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Quantitation Assay

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Chromatogram depicting targeted metabolomic analysis of Lrrk2 HET (n = 3) and KO BMDMs (n = 3) with pure molecular weight standard to IMP (top) and hypoxanthine (bottom). Replicate experiments are shown as individual lines (n = 2). Coefficient of variance (CV) for IMP = 8.8% (KO) and 21.7% (HET). CV for hypoxanthine = 9.1% (KO) and 14.0% (HET). ( B ) Diagram of key metabolites produced during purine metabolism oriented to the major steps of the pathway. De novo synthesis (green), salvage (red), breakdown (blue). ( C ) Representative immunofluorescence microscopy image of purinosome formation measured by PFAS puncta (green) in Lrrk2 HET and KO MEFs. Nuclei stained with DAPI (blue). ( D ) Quantification of number of PFAS puncta per cell. 100 cells were counted per coverslip from three coverslips. ( E ) RT-qPCR of Irf7 and Isg15 gene expression in Lrrk2 HET and KO BMDMs treated with increasing concentrations of urate (10, 50, 100, and 250 μM for 24 hr). ( F ) Basal gene expression of Ifnb and Ifit1 in SCR and Lrrk2 KD RAW 264.7 cells treated with 250 μM urate overnight. ( G ) JC-1 aggregate vs. monomer formation measured by flow cytometry in Lrrk2 HET and KO BMDMs treated with 100 μM urate or 200 μM mitoTEMPO overnight. Histograms shown below and merged histograms shown to the right. ( H ) Histograms of Lrrk2 HET and KO BMDMs treated with 100 μM urate or 200 μM mitoTEMPO overnight and then treated with 2.5 μM rotenone for 3 hr followed by 5 μM ATP for 15 min. ( I ) Histograms of DRP1 p-S616 flow cytometry analysis for Lrrk2 KO BMDMs following treatment with 100 μM urate or 200 μM mitoTEMPO. Quantification is shown on the right. JC-1 flow cytometry assays are representative of three independent experiments. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); #p<0.005, ##p<0.001 (comparing treated to untreated of same genotype). ( D ) Two-tailed Student’s T test; ( E and F ) two-way ANOVA Tukey post-test; ( I ) one-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Molecular Weight, Produced, Immunofluorescence, Microscopy, Staining, Quantitative RT-PCR, Gene Expression, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) LC-MS/MS analysis of BMDMS from Lrrk2 HET and KO mice showing IMP peak. ( B ) As in ( A ) but for hypoxanthine.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Mtb colony forming units (CFUs) recovered from Lrrk2 HET and KO BMDMs over the course of 5 days (MOI = 1). ( B ) Survival curves for Lrrk2 HET (n = 8) and KO (n = 11) mice over a 250 day Mtb infection. Survival times not statistically different based on log-rank Mantel-Cox test. ( C ) CFUs recovered from lungs and spleens of Mtb-infected Lrrk2 HET and KO mice at Day 7, 21, 63, and 126 post-infection. ( D ) Circulating serum cytokines measured at Day 21 in Lrrk2 HET and KO mice. ( E ) RT-qPCR of inflammatory cytokines from total RNA recovered from lung homogenates from Day 21 Mtb-infected Lrrk2 HET and KO mice. ( F ) As in ( E ) but detecting ISGs. ( G ) Hematoxylin and eosin (H&E) stain of inflammatory nodules in the lungs of Lrrk2 KO and HET mice 21 days after infection with Mtb. Small scale bar, 500 μm; large scale bar 1 mm. ( H ) Semi-quantitative score of pulmonary inflammation with a score of 0, 1, 2, 3 or 4 assigned based on granulomatous nodules in none, up to 25%, 26–50%, 51–75% or 76–100% of fields, respectively. Perivascular and peribronchial inflammation was scored using an analogous scale based on percentage of medium-caliber vessels or bronchioles with adjacent inflammatory nodules. ( I ) H&E stain of neutrophils within an inflammatory nodule in the lung of Lrrk2 HET and KO mice 21 days after infection with Mtb. Left panel bar is 20 μm. Right panel bar is 200 μm. ( J ) Quantification of neutrophils in the lungs of Lrrk2 HET and KO mice infected with Mtb for 21 or 63 days. Total neutrophil scores were determined by the percentage of fields of view at 20X magnification containing neutrophils. Degenerate neutrophil scores were determined by the percentage of PMN positive fields containing degenerate neutrophils. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); #p<0.005, ##p<0.001 (comparing infected to uninfected of same genotype). ( A ) Two-way ANOVA Tukey post-test; ( B ) Mantel-Cox log-rank; ( C–J ) Mann-Whitney test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Infection, Quantitative RT-PCR, Staining, MANN-WHITNEY

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet:

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 KO , ATCC , ATCC SC-6004 , .

Techniques: Sequencing, Recombinant

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) Scatter plot of genes up- and down-regulated in LRRK2 knockout (KO) and control (CT) BMDMs 4 h post-infection with Mtb. Genes whose expression changes are significant (p<0.001; adjusted p-value is p<0.05) are highlighted in red. (B) IPA software analysis of cellular pathways enriched for differentially expressed genes in LRRK2 KO vs. CT BMDMs during Mtb infection . (C) Heatmap of significant gene expression differences (log2fold-change) in LRRK2 KO vs. CT BMDMs during Mtb infection. (D) RT-qPCR of fold-change in transcripts for type I IFN genes ( Ifnb, Mx1, Isg15 ) and type II IFN genes ( Ifit1, Gbp2, Gbp7 ) during Mtb infection . (E) RT-qPCR of NF-κB genes ( Tnfa and Il1b) during Mtb infection . (F) Volcano plot of genes significantly upregulated (blue) or downregulated (orange) in uninfected (resting) LRRK2 KO BMDMs . (G) As in (B) but comparing uninfected LRRK2 KO vs. CT BMDMs . (H) As in (C) but for uninfected LRRK2 KO vs. CT BMDMs. Zoom 1 is top downregulated genes; Zoom 2 is top upregulated genes. (I) RT-qPCR of type I IFN associated genes ( Ifnb, Irf7, Isg15) normalized to Actb in uninfected BMDMs . (J) RT-qPCR of Apoe and Ldhb normalized to Actb in uninfected BMDMs. (K) As in (E) but in uninfected BMDMs. Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005. See also Figure S1 and Table S1.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Knock-Out, Control, Infection, Expressing, Software, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) RT-qPCR of fold-change in Ifnb and Isg15 expression during Mtb infection in differentiated U937 monocytes stably expressing scramble shRNA (SCR) or shRNA targeted to LRRK2 (KD) . (B) RT-qPCR of fold-change in Isg15 expression at 4 and 8 h post-infection with M. leprae in CT or LRRK2 KO RAW 264.7 cells. (C-F) RT-qPCR of fold-change in Ifnb and Irf7 expression 4 h post-transfection with 1μg/ml ISD (dsDNA) in LRRK2 KO vs. CT (C) BMDMs, (D) peritoneal macrophages (PEMs), (E) RAW 264.7, and (F) primary mouse embryonic fibroblasts (MEFs). (G) RT-qPCR of fold-change in Ifnb expression in LRRK2 CT or KO BMDMs following stimulation with 50 ng/ml DMXAA (2 h) or 1 μg/ml cGAMP (4 h). (H) Quantitative western blot of IRF3 phosphorylated at Ser396 (p-IRF3), total IRF3, and tubulin at 0, 2, 4, and 6h post-transfection with 1 μg/ml ISD in LRRK2 KO vs. CT BMDMs. Lower graphs show quantification of p-IRF3 and IRF3 to tubulin. (I) As in (G) with 100 ng/ml LPS (4 h), transfection of 10 μM CpG 2395 (4 h), 1 mg/ml poly(I:C) (dsRNA) (4 h) or 1 μM CL097 (4 h). (J) RT-qPCR of fold-change in Irf7 and Isg15 expression following stimulation with 200IU IFN-β (4 h) in LRRK2 KO vs. CT BMDMs. (K) RT-qPCR of Irf7 and Isg15 expression normalized to Actb in LRRK2 KO BMDMs in the presence of overnight blocking with IFN-β neutralizing antibody (1:250). (L) As in (J) but fold-change in Irf7 and Isg15 expression after transfection of 1 μg/ml ISD for 4h. (M) As in (K) but in BMDMs from CT, LRRK2 KO, IFNAR KO, and LRRK2 / IFNAR double KO double (DKO) mice. (N) As in (M) but fold-change in Irf7 and Isg15 expression after transfection of 1 μg/ml ISD for 4h. Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005. See also Figure S2.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Quantitative RT-PCR, Expressing, Infection, Stable Transfection, shRNA, Transfection, Western Blot, Blocking Assay

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) RT-qPCR of Ifnb and Isg15 expression normalized to Actb in BMDMs from CT, LRRK2 KO, cGAS KO, and LRRK2 KO/ cGAS double KO (DKO) mice. (B) As in (A) but fold-change in Ifnb and Isg15 expression after transfection of 1 μg/ml ISD for 4h. (C) Immunofluorescence microscopy of mitochondrial networks in LRRK2 KO vs. CT MEFs. TOM20 (green); nucleus (blue). (D) qPCR of total 16s and cytB (mitochondrial DNA) relative to tert (nuclear DNA) in LRRK2 KO vs. CT MEFs. (E) As in (D) but cytosolic 16s and cytB . (F) RT-qPCR of fold-change in Ifnb and Isg15 expression after treatment with 10 μM ABT737 (BCL2 inhibitor) and 10 μM QVD-OPh (caspase inhibitor) in LRRK2 KO vs. CT BMDMs. RT-qPCR of Irf7 expression normalized to Actb and fold-change in Irf7 expression following transfection of 1 μg/ml ISD (4 h) or stimulation with 100 ng/ml LPS in BMDMs from CT, LRRK2 KO, Tfam Het, and LRRK2 KO/ TFAM HET mice. (H) qPCR of ratio of dLOOP (mitochondrial DNA) to Tert (nuclear) in CT and LRRK2 KO RAW 264.7 macrophages (normalized to CT = 1) after 4 days of 10 μM ddC treatment. (I) RT-qPCR of Isg15 expression normalized to Actb and fold-change in Isg15 expression following transfection of 1 μg/ml ISD (4 h) or 50 ng/ml DMXAA (2 h) in LRRK2 KO vs. CT ddC-treated RAW 264.7 macrophages. (J) Histogram of counts of phospho-S616 Drp1 in LRRK2 KD vs. SCR RAW 264.7 cells as measured by flow cytometry. (K-L) As in (J) but in LRRK2 KO vs. CT (K) BMDMs and (L) MEFs. (M) RT-qPCR of Isg15 and Irf7 expression normalized to Actb in LRRK2 KO vs. CT BMDMs with or without 50 μM Mdivi-1 (12 h). Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005. See also Figure S3.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Immunofluorescence, Microscopy, Flow Cytometry

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) Flow cytometry of mitochondrial membrane potential as measured by JC-1 dye aggregates (610/20) (normal membrane potential) vs. monomers (520/50) (low membrane potential) in LRRK2 KO vs. CT BMDMs. (B) Histogram of cell counts and JC-1 aggregates measured by flow cytometry in LRRK2 KO vs. CT BMDMs . (C) Flow cytometry of JC-1 aggregates vs. monomers and histogram of cell counts and JC-1 aggregates in LRRK2 KO vs. CT BMDMs after treatment with 2.5 μM rotenone (3 h) followed by 5 μM ATP for indicated times. (D) Flow cytometry of JC-1 aggregates vs. monomers LRRK2 KO BMDMs treated for 12 h with indicated concentration of Mdivi-1. (E) RT-qPCR of Ifnb and Ifit1 expression normalized to Actb and fold-change in Ifnb and Ifit1 expression following transfection of 1 μg/ml ISD (4 h) in LRRK2 KO vs. CT BMDMs with or without 200μM mitoTEMPO (mitoT) for 16 h. (F) Seahorse metabolic analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in LRRK2 KO vs. CT BMDMs untreated or treated with 200 μM mitoT, IFN-β blocking antibody, or both overnight. Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005. See also Figure S4.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Flow Cytometry, Membrane, Concentration Assay, Quantitative RT-PCR, Expressing, Transfection, Blocking Assay

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) Chromatogram of targeted metabolomic analysis of LRRK2 KO vs. CT BMDMs with pure molecular weight standard for IMP. (B) As in (A) but for hypoxanthine. (C) Diagram of key metabolites produced during the major steps of purine metabolism. De novo synthesis (green), salvage (red), breakdown (blue). (D) Representative immunofluorescence image of purinosome formation in LRRK2 KO vs. CT MEFs as visualized by PFAS puncta (green). Nuclei (blue). (E) Quantification of PFAS puncta per cell as imaged in (D). (F) RT-qPCR of Isg15 and Irf7 expression normalized to Actb in LRRK2 KO vs. CT BMDMs treated with increasing concentrations of urate, (0, 10, 50, 100, and 250 μM) for 24 h. (G) RT-qPCR of fold-change in Isg15 and Irf7 expression following transfection of 1 μg/ml ISD (4 h) in LRRK2 KO vs. CT BMDMs treated with 100 μM urate for 24 h. (H) Flow cytometry of JC-1 aggregates vs. monomers and histograms of cell counts and JC-1 aggregates in LRRK2 KO vs. CT BMDMs after treatment with 100μM urate or 200μM mitoTEMPO overnight. (I) Histograms as in (H) but in the presence of 2.5 μM rotenone (3 h) and 2.5 μM ATP (15 min). (J) Histogram of counts of phospho-S616 Drp1 as measured by flow cytometry in LRRK2 KO vs. CT BMDMs after treatment with 100μM urate or 200μM mitoTEMPO overnight. Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005. See also Figure S5 and Table S2.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Molecular Weight, Produced, Immunofluorescence, Quantitative RT-PCR, Expressing, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) Representative histology images of inflammatory nodules in the lungs of LRRK2 KO and CT mice 21 days post-Mtb. Hematoxylin and eosin (H&E) stain. (B) Semi-quantification of neutrophils in the lungs of LRRK2 KO and CT mice infected with Mtb for 21 or 63 days. Total neutrophil scores were determined by the percentage of 20x magnification fields of containing neutrophils. Degenerate neutrophil scores determined by the percentage of PMN+ fields containing degenerate neutrophils. (C) Schematic representation of brain areas of interest (DLS, SNc, and VTA) in the nigrostriatal dopaminergic pathway in the mouse brain. Created using BioRender. (D-E) Fluorescence images of reactive microglia in the DLS in LRRK2 KO vs. CT mice uninfected or infected with Mtb for 21 or 126 days. Iba1 (green); NeuN (red). (F) Fluorescence images of reactive microglia in the SNc in LRRK2 CT mice uninfected or infected with Mtb for 126 days. Iba1 (green); TH (red). Arrows highlight instances of colocalization of Iba1 and TH. (G) Quantification of microglial reactivity in the DLS and SNc as measured by Iba1 fluorescence relative to NeuN or TH fluorescence in LRRK2 KO and CT mice infected with Mtb for 21 or 126 days compared to uninfected age-matched controls. (H) Fluorescence images of reactive astrocytes in the DLS in LRRK2 KO vs. CT mice uninfected or infected with Mtb for 21 or 126 days. GFAP (green); NeuN (red). (I) As in (H) but in the SNc. GFAP (green); TH (red). (J) Quantification of astrocyte reactivity in the DLS and SNc as measured by GFAP fluorescence relative to NeuN or TH fluorescence in LRRK2 KO and CT mice infected with Mtb for 21 or 126 days compared to uninfected age-matched controls. Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005. See also Figure S6.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Staining, Infection, Fluorescence

Journal: bioRxiv

Article Title: LRRK2 regulates innate immune responses and neuroinflammation during Mycobacterium tuberculosis infection

doi: 10.1101/699066

Figure Lengend Snippet: (A) RT-qPCR of Gfap expression and western blot of Iba1 expression in LRRK2 KO vs. CT astrocyte- and microglia-enriched primary cell cultures. (B) RT-qPCR of Gfap and Ccl5 expression normalized to Actb and fold-change in Gfap and Ccl5 expression following stimulation with 800 IU IFN-β for indicated times in LRRK2 KO vs. CT astrocytes. (C) As in (B) but for Isg15 expression. (D) Flow cytometry of JC-1 aggregates vs. monomers in LRRK2 KO vs. CT astrocytes with 2.5 μM rotenone (3 h) and 2.5 μM ATP for indicated times. (E) Western blot of Iba1 protein levels in LRRK2 KO vs. CT microglia treated with 800IU IFN-β. Bar graph shows quantification of protein levels relative to Actin. (F) RT-qPCR of Irf7 expression normalized to Actb and fold-change in Irf7 following stimulation with 800 IU IFN-β for indicated times in LRRK2 KO vs. CT microglia. (G) As in (D) but for microglia. (H) As in (F) but for Ccl5, KC , and Tnfa expression. Data represented as means +/- S.E.M. *p<0.05, **p<0.01, ***p<0.005.

Article Snippet: RAW 264.7 LRRK2 KO cells (ATCC® SC-6004TM) generated by the MJFF, were obtained from the ATCC and used with wild type control LRRK2 parental RAW 264.7 (ATCC® SC-6003TM).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry