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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

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 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Sequencing, Recombinant

Journal: Nature Communications

Article Title: The functional organization of excitatory synaptic input to place cells

doi: 10.1038/s41467-021-23829-y

Figure Lengend Snippet: a Schematic of experiments using 2P microscopy to image CA1 pyramidal neuron somatic firing patterns with jRGECO1a (red) and excitatory synaptic inputs to dendrites with iGluSnFR (green) during spatial behaviors in VR. b Example image of jRGECO1a fluorescence from labeled CA1 pyramidal neurons imaged during behavior. c Somatic jRGECO1a ΔF/F versus track position for each traversal of a single session (top) and mean ΔF/F versus position across all traversals (bottom) for three different neurons from different mice. Place cell at the left (cell highlighted in panel ( b )) with place field track location between dashed lines, silent cell in the middle, and active–nonplace cell at the right. Significant transients highlighted in bold. d Mean somatic jRGECO1a ΔF/F versus track position across all traversals of a single session for all recorded neurons (each row represents single-neuron mean ΔF/F). Plotted via cross-validation within each cell category. e Left, example z-projection image of iGluSnFR fluorescence from labeled CA1 pyramidal neurons imaged during behavior (same neurons and field of view as shown in panel ( b ). Right, top, mean images from time series acquired at two different single-imaging planes (from regions shown at the left). Right, bottom, same as top, but with 106 1-µm ROIs shown in green. Similar results were obtained in 54 sessions from 11 mice. f iGluSnFR ΔF/F vs track position for each traversal of a single session (top) and mean ΔF/F versus position across all traversals (mean ROI map, bottom) for five example ROIs shown in panel( e , right), from place cell shown in panels ( b , c ). Significant transients highlighted in bold. Place-ROIs: 29, 44, 70; Silent-ROI: 10, Active–nonplace-ROI: 56. g Mean iGluSnFR ΔF/F versus track position across all traversals of a single session (mean ROI map) for all ROIs (each row represents a single ROI mean ΔF/F) shown in ( e , right), from place cell shown in panels ( b , c ). Somatic place field track location between dashed lines. The percentage of ROIs in each ROI category also shown. Plotted via cross-validation within each ROI category. h , i . Same as panel ( g ), but for all ROIs from all 62 branches of all 23 place cells ( h ) or all 41 branches of all 23 nonplace cells ( i ). j Percentage of ROIs in each ROI category for place vs nonplace cells. Each circle represents a single branch. Mean ± bci across branches. (* p < 3.2e−3, likelihood ratio test, two-sided). n = 62 dendrites from 23 place cells from 35 independent sessions from 11 mice; n = 41 dendrites from 23 nonplace cells from 24 independent sessions from 11 mice. k Spatial dispersion of iGluSnFR transients in each ROI for all ROIs in place cells vs. nonplace cells (* p = 1.24e−4, likelihood ratio test, two-sided). l Mean amount of excitatory input per ROI per second (integral of all significant iGluSnFR transients in each ROI divided by recording time) for all ROIs in place cells vs. nonplace cells (* p = 9.1e−4, Rank-sum test, two-sided, place

Article Snippet: A low-titer Cre virus (AAV1- CaMKII -Cre, 1.51 × 10 8 GC/mL, Addgene) was injected (1 injection of ~60 nL at a depth of ~1250 µm below the dural surface using a beveled glass micropipette: ~1–2 MΩ after beveling) in combination with a high titer of flexed-iGluSnFR.A184S virus (AAV2/1- hSyn -FLEX.SF-iGluSnFR.A184S, 5.87 × 10 12 GC/mL) and flexed-jRGECO1a virus (AAV1- hSyn -FLEX.NES-jRGECO1a, 4.05 × 10 12 GC/mL), leading to expression of SF-iGluSnFR.A184S and jRGECO1a in a sparse subset of the CA1 pyramidal neuron population.

Techniques: Microscopy, Fluorescence, Labeling, Imaging

Journal: Investigative ophthalmology & visual science

Article Title: VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

doi: 10.1167/iovs.61.6.45

Figure Lengend Snippet: FIGURE 4. Immunofluorescence staining for LRRK2 in SC endothelial cells stimulated with VIP in vivo and the mRNA and protein expression of LRRK2 in HUVECs stimulated with VIP in vitro. (A) Representative immunofluorescence staining (red) for LRRK2 in SC endothelium cells. The immunofluorescence optical density of LRRK2 surrounding the SC endothelium increased significantly 6 hours after the start of local VIP application and returned to baseline values 12 hours after the start of local VIP application (each group n = 5). (B) The results of qPCR for LRRK2 in HUVECs upon VIP stimulation and (C) representative western blots of LRRK2 in HUVECs upon VIP stimulation. VIP increased the mRNA and protein expression levels of LRRK2 in HUVECs. All groups were compared with the control. ***P < 0.001, ****P < 0.0001. Scale bars: 50 μm.

Article Snippet: HUVECs were first treated with the Sp1 inhibitor plicamycin (50 μM) (ab142723; Abcam) for 24 hours or the LRRK2 inhibitor, LRRK2-IN-1 (3 μM) (S7584; Selleck Chemicals, Houston, TX, USA) for 120 minutes.

Techniques: Immunofluorescence, Staining, In Vivo, Expressing, In Vitro, Western Blot, Control

Journal: Investigative ophthalmology & visual science

Article Title: VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

doi: 10.1167/iovs.61.6.45

Figure Lengend Snippet: FIGURE 6. Western blotting for Sp1 and LRRK2 in HUVECs stimulated with VIP + Sp1/LRRK2 inhibitor. (A) Representative western blots of Sp1 and LRRK2 in HUVECs treated with the VIP + Sp1 inhibitor plicamycin. Plicamycin inhibited the expression of Sp1 by targeting DNA and RNA polymerase and DNA binding. The application of plicamycin decreased the protein expression of both Sp1 and LRRK2. (B) Representative western blots of Sp1 and LRRK2 in HUVECs treated with VIP + LRRK2 inhibitor (LRRK2-IN-1). LRRK2-IN-1 inhibited only the biological activity of LRRK2 and thus had no influence on the protein expression of Sp1 and LRRK2 induced by VIP. All groups were compared with the control. **P < 0.01, ****P < 0.0001.

Article Snippet: HUVECs were first treated with the Sp1 inhibitor plicamycin (50 μM) (ab142723; Abcam) for 24 hours or the LRRK2 inhibitor, LRRK2-IN-1 (3 μM) (S7584; Selleck Chemicals, Houston, TX, USA) for 120 minutes.

Techniques: Western Blot, Expressing, Binding Assay, Activity Assay, Control

Journal: Investigative ophthalmology & visual science

Article Title: VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

doi: 10.1167/iovs.61.6.45

Figure Lengend Snippet: FIGURE 8. Immunofluorescence staining for F-actin and G-actin in SC endothelial cells stimulated with VIP and with VIP + LRRK2-IN-1. (A) Representative immunofluorescence staining of F-actin (red), G-actin (green), and DAPI (blue) in the SC endothelium. VIP decreased the F-/G-actin ratio in the SC endothelium, whereas the application of LRRK2-IN-1 eliminated this effect. (B) Representative HE staining of the SC lumen, and (C) immunohistochemical (CD31) staining of the SC lumen. VIP expanded the SC lumen, whereas the application of LRRK2-IN-1 abolished this effect. All groups were compared with the control (each group n = 4). **P < 0.01, ****P < 0.0001. Scale bars: 50 μm.

Article Snippet: HUVECs were first treated with the Sp1 inhibitor plicamycin (50 μM) (ab142723; Abcam) for 24 hours or the LRRK2 inhibitor, LRRK2-IN-1 (3 μM) (S7584; Selleck Chemicals, Houston, TX, USA) for 120 minutes.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Control

Journal: Investigative ophthalmology & visual science

Article Title: VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

doi: 10.1167/iovs.61.6.45

Figure Lengend Snippet: FIGURE 7. Western blotting and immunofluorescence staining of F-actin and G-actin in HUVECs stimulated with VIP and with VIP+LRRK2- IN-1. Representative (A) western blots and (C) immunofluorescence staining (red for F-actin and green for G-actin) for F-actin and G-actin in HUVECs. (B, D) VIP induced a decrease in the F-/G-actin ratio, and this effect was abolished by the application of LRRK2-IN-1. All groups were compared with the control. ****P < 0.0001. Scale bars: 50 μm.

Article Snippet: HUVECs were first treated with the Sp1 inhibitor plicamycin (50 μM) (ab142723; Abcam) for 24 hours or the LRRK2 inhibitor, LRRK2-IN-1 (3 μM) (S7584; Selleck Chemicals, Houston, TX, USA) for 120 minutes.

Techniques: Western Blot, Immunofluorescence, Staining, Control

Journal: Investigative ophthalmology & visual science

Article Title: VIP Induces Changes in the F-/G-Actin Ratio of Schlemm's Canal Endothelium via LRRK2 Transcriptional Regulation.

doi: 10.1167/iovs.61.6.45

Figure Lengend Snippet: FIGURE 9. Schematic diagram of the effect of VIP on the cytoskele- ton. VIP induced an increase in the expression of transcription factor Sp1 and then recruited Sp1 into the nuclei. After that, the migration of Sp1 to nuclei upregulated the expression of LRRK2, which would further change the ratio of F-actin and G-actin.

Article Snippet: HUVECs were first treated with the Sp1 inhibitor plicamycin (50 μM) (ab142723; Abcam) for 24 hours or the LRRK2 inhibitor, LRRK2-IN-1 (3 μM) (S7584; Selleck Chemicals, Houston, TX, USA) for 120 minutes.

Techniques: Expressing, Migration