Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Flrt2 mRNA detection by fluorescence in situ hybridization (FISH) in P8 wild-type mouse retina in the optic nerve head (ONH) area, where the central artery and vein enter the retina, and in the superficial vascular plexus (SVP) (upper panel); and in P8 wild-type cerebral cortex (upper cortical layers and pial vasculature) (lower panel). Blood vessels were detected by immunostaining with podocalyxin (Podxl) (general vessel marker). (b) Coronal section of the cerebral cortex from P6 wild-type mouse stained for FLRT2 and NeuN as neuronal marker (neuronal layers I-VI annotated). Blood vessels were visualized with isolectin-B4 (IB4) staining. Arrows show FLRT2 positive blood vessels (right). (c) Flat-mounted P7-P8 retinas from control and Flrt2 iΔEC littermate mice injected with 4-hydroxytamoxifen (Tmx) from P1 to P3 and stained with IB4. (d - f) Quantification of radial vascular length ratio (d), total retinal vessel length (e), and total number of branch points (f) per retina. (g) Representative images of P7-P8 control and Flrt2 iΔEC flat-mounted retinas stained for IB4. Veins (V) and arteries (A) are indicated. (h) Quantification of capillary network density between veins and arteries. (i) Glut1 staining of the vasculature in control and Flrt2 iΔEC brain cortices from P7-P8 mice after Tmx administration from P1 to P3. ( j - l ) Quantification of vessel density (j), vessel length (k) and number of branch points (l). Scale bars: 20 μm (a), 50 μm (b), 500 μm (c), 200 μm (g), 100 μm (i). n = 7-12 (d), 6-7 (e, f), 8 (h), 7 (j-k), 5-6 (l) animals per genotype. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Fluorescence, In Situ Hybridization, Immunostaining, Marker, Staining, Control, Injection

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Schematic representation of the Flrt2 endothelial specific knockout strategy in Cdh5(PAC)-CreERT2:Flrt2 lox/lox ( Flrt2 iΔEC ) mice. (b) Experimental design of Flrt2 gene deletion by Cre recombination. 4-hydroxytamoxifen (Tmx) was applied from postnatal day 1 (P1) to 3 and tissue collected at P5 and P7 or 8. Cartoon created with Biorender.com. (c, e) Flat-mounted retina (c) and neocortical brains slice (e) from P7 Cdh5-CreERT2:Rosa26tdTomato mice injected from P1 to P3 with Tmx. Cells undergoing Cre-mediated recombination expressed the fluorescent protein tdTomato, which was used to assess specificity and efficiency in blood vessels, co-stained with IB4 (c) or Podxl (e). (d, f) Quantification of the percentage of tdTomato-positive signal per IB4-positive (d) and Podxl-positive signal (f). (g) Immunoblot showing FLRT2 protein levels in primary lung ECs isolated from P8 control and Flrt2 iΔEC mice. FLRT2-specific antibody detects the full-size protein, the cleaved extracellular domain, and glycosylated forms. Pan-cadherin was used as loading control. (h) Flrt2 and Flrt3 mRNA levels from primary mouse brain ECs (pmBECs) isolated from P5 control and Flrt2 iΔEC mice. (i) pmBECs isolated from P7 control and Flrt2 iΔEC mice stained for FLRT2 and cell nuclei (DAPI). (j) FLRT2 fluorescence intensity quantification in control and Flrt2 iΔEC pmBECs. (k) Body weight measured from control and Flrt2 iΔEC male and female mice at P7-8. (l) Example of whole fixed brains from control and Flrt2 iΔEC P7-8 mice. (m, n) Quantifications of brain length (m) and width (n) of control and Flrt2 iΔEC P7-8 mice. Scale bar: 200 μm (c), 100 μm (e), 10 μm (i), 1 mm (l). n = 8 mice (d), 3 mice (f), 4-10 animals per genotype (h), 14-16 pictures per animal from 2 mice per genotype, one litter (j), 6-13 mice per genotype and sex (k), 9-10 mice per genotype (m, n). Horizontal bar shows the median value (d, f). Data are shown as mean ± SEM. **P < 0.01, ***P > 0.001, ns= not significant, unpaired t-test (h ( Flrt3 ), j, k, m, n), Mann-Whitney test (h, Flrt2 ).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Knock-Out, Injection, Staining, Western Blot, Isolation, Control, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Flat mounted retinas stained for Collagen IV (Col IV) and IB4 to visualize blood vessel regression. Col IV+ IB4-empty sleeves are marked with yellow arrows. (b) Quantification of vessel regression as Col IV+ IB4-empty sleeves per imaging field. (c) Representative images of retinas from control and Flrt2 iΔEC P7-P8 littermates stained with IB4. Arteries and veins are highlighted in pink and light blue, respectively. (d) Quantification of the total number of veins and arteries per retina in mutant and control mice at P7-8. (e) Negative Flrt2 (grey) expression in a cortical artery (A) expressing the Gkn3 marker (red) and positive Flrt2 signal in a vein (V) expressing Scl38a5 (blue). Podxl immunostaining is used as a general marker of the vasculature. (f) Representative images of recombinant tip cells in the retinal vascular front of Cdh5-CreERT2:Rosa26tdTomato (control) and Flrt2 iΔEC :Rosa26tdTomato P7-P8 mice. Upon Cre-mediated recombination ECs express tdTomato reporter protein (depicted as red). (g) Quantification of the number of filopodia per tip cell. (h) Retinal vascular fronts from control and Flrt2 iΔEC P7-P8 mice with proliferative cells labelled with EdU, EC nuclei stained with ERG, and blood vessels with IB4. (i) Quantification of % EdU + ERG + cells in total ERG + cells per image. Scale bars: 100 μm (a), 500 μm (c), 25 μm (e, f, h). n = 6-7 animals per genotype (b, d), 19-21 cells per genotype (g), 7-9 animals per genotype (i). Data are shown as mean ± SEM. ***P > 0.001, ns = not significant, two-way ANOVA (d), unpaired t-test (b, g i).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Staining, Imaging, Control, Mutagenesis, Expressing, Marker, Immunostaining, Recombinant

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Representative images of main arteries and veins (identified by their morphology) from control and Flrt2 iΔEC P7-P8 retinas stained with IB4. Red dots indicate branch points from the corresponding mother vessel. (b) Quantification of the number of branch points in arteries (left) and veins (right) per vessel length. (c) Expanded P7 retina stained for FLRT2 and collagen IV (Col IV) showing FLRT2 expression in retinal vessels. Note the expression of FLRT2 at the EC membrane in the vein but its absence in the artery. (d) Representative images of retinal vascular front from control and Flrt2 iΔEC mice stained with IB4. Red dots indicate cellular protrusions identified as angiogenic sprouts. (e) Quantification of number of sprouts per 100 μm of retinal vascular front. (f) Vascular fronts from control and Flrt2 iΔEC P7-P8 retinas showing blood vessels labelled with IB4 and EC nuclei stained for ERG. (g) Quantification of the number of tip cells per stalk cells at the vascular front. (h) Glut1 staining visualizing vessel sprouts in P7-P8 control and Flrt2 iΔEC cerebral cortices. ( i ) Quantification of the number of sprouts per vessel density in the cerebral cortex. Scale bars: 50 μm (a, f), 90 μm (c), 40 μm (d), 100 μm (h). n = 17-20 (b), 18-22 (e), 8-10 (g), 5-6 (i) animals per genotype. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Staining, Expressing, Membrane

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) P5 flat-mounted retinas from control and Flrt2 iΔEC littermates injected with 4-hydroxytamoxifen (Tmx) from P1 to P3, stained with IB4 to visualize blood vessels. (b , c) Quantification of the vascular radial growth ratio (b), and the capillary density (c) as the area covered by IB4 staining in P5 control and mutant retinas. (d) Glut1 staining of control and Flrt2 iΔEC cortices at P4-P5 after Tmx administration from P1 to P3. (e - g) Quantification of vessel density (e), vessel length (f) and number of branch points (g) in mouse cerebral cortices. (h) Representative images of arteries and veins from control and Flrt2 iΔEC retinas stained with IB4 at P5. Red dots indicate branch points from the mother vessel. (i) Quantification of the number of branch points in main arteries (left) and veins (right) per vessel length. (j) P5 retinal vascular fronts stained with IB4. Red dots indicate cellular protrusions identified as angiogenic sprouts. (k) Quantification of the number of sprouts per 100 μm of vascular front. ( l) Control and Flrt2 iΔEC cerebral cortices at P5 stained for Glut1 to visualize vessel sprouts. ( m ) Quantification of the number of sprouts per vessel density at P5 in Flrt2 iΔEC and control littermates. Scale bars: 500 μm (a), 100 μm (d), 50 μm (h, j, l). n = 10-13 (b, i), 7-9 (c), 10-11 (e), 6 -10 (f, g), 10-12 (k), 6-10 (m) animals per genotype. Data are shown as mean ± SEM. **P < 0.01, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Injection, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a, b) Flat mounted retinas (a) and cortical brain slices (c) stained for apoptosis marker cleaved caspase-3 (cleaved casp-3) and blood vessels (IB4 or Podxl). (b, d) Quantification of cleaved caspase-3+ ECs in control and Flrt2 iΔEC P5 retinas (b) and P4-P5 brain neocortices (d). (e, g) Flat mounted retinas (e) and cortical brain slices (g) stained for cell cycle arrest marker p21 and blood vessels (IB4 or Podxl). (f, h) Quantification of p21+ ECs in control and Flrt2 iΔEC P5 retinas (f) and P4-P5 brain neocortices (h). Scale bars: 50 μm. n = 4-5 animals per genotype (b, f), 6-7 animals per genotype (d), 5-6 animals per genotype (h). Data are shown as mean ± SEM. *P < 0.05, ns = not significant, unpaired t-test (b, d, h), Mann-Whitney test (f).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Staining, Marker, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Proximity ligation assay (PLA) in HUVEC cultures. White puncta indicate FLRT2 and VE-cadherin being in close proximity (< 40 nm). Cells were stained with phalloidin and DAPI to visualize actin filaments and nuclei, respectively. (b) PLA signal quantification as puncta per cell number in the field (identified by DAPI nuclei). (c) Immunoprecipitation (IP) of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from total mouse brain lysates. TL, total lysate. (d) Representative immunoblot showing FLRT2 protein reduced expression in HUVECs treated with Flrt2 siRNA compared to control treated cells. β-actin was used as loading control. (e) Quantification of FLRT2 protein levels in control and Flrt2 siRNA treated HUVECs. (f) Antibody feeding assay in HUVEC transfected with control and Flrt2 -specific siRNA treated with chloroquine. Cells were immunostained for internalized VE-cadherin, total VE-cadherin and cell nuclei (DAPI). Intensity of internalized VE-cadherin is shown in arbitrary units (AU, upper panels). (g) Quantification of the fluorescence intensity of internalized VE-cadherin per cell. (h) Quantification of VE-cadherin intensity per cell-junction length. (i) Representative immunoblot showing VE-cadherin, its cleaved form, and α-tubulin as loading control in HUVEC transfected with control and Flrt2 -specific siRNAs. (j) Quantification of VE-cadherin/loading control and cleaved VE-cadherin/loading control ratios. ( k) Primary mouse brain ECs (pmBEC) from control and Flrt2 iΔEC littermates stained for Calpain-2 and DAPI. (l) Quantification of the fluorescence intensity of Calpain-2 staining per cell. (m) Neocortical blood vessels stained for Calpain-2 and IB4 from control and Flrt2 iΔEC littermates. (n) Quantification of Calpain-2 fluorescence intensity in the blood vessels. Scale bars: 40 μm (a, upper panels), 15 μm (a, lower panels), 10 μm (f, k), 5 μm (m). n = 20-23 pictures per condition from three different experiments (b), 6 independent experiments (e), 84-115 cells from 3 independent experiments (g), 18-22 pictures per condition from 3 independent experiments (h), 8-11 independent experiments (j), 71-72 cells per condition, from 1 control and 1 Flrt2 iΔE littermate (l), 4-5 animals per genotype (n). Data are shown as mean ±. *P < 0.05, **P < 0.01, SEM. ***P < 0.001, ns = not significant, unpaired t-test (b, e, h, j, n), Mann-Whitney test (g, l).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Proximity Ligation Assay, Staining, Immunoprecipitation, Immunodetection, Western Blot, Expressing, Control, Feeding Assay, Transfection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Immunoprecipitation of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from HUVEC cultures. (b) PLA in primary mouse brain EC (pmBECs) cultures. White puncta indicate FLRT2 and VE-cadherin being in close proximity (< 40 nm). Cells were stained with Pecam1 and DAPI to visualize EC junctions and nuclei, respectively. Note the major presence of PLA puncta along the Pecam1+ junctions in the cells isolated form control mice, indicating that FLRT2 and VE-cadherin interact at the cell surface. (c) PLA signal quantification as puncta per cell. (d) HUVEC treated with control and Flrt2 -specific siRNAs immunostained for VE-cadherin and DAPI. (e) Quantification of VE-cadherin intensity per cellular junction length. (f) mRNA expression of Flrt2 and Cdh5 in HUVEC transfected with control and Flrt2 -specific siRNAs measured by qPCR. (g) HUVEC cultures transfected with control and Flrt2 -specific siRNAs stained for Calpain-1 and DAPI. (h) Quantification of the fluorescence intensity of Calpain-1 staining per cell. Scale bars: 10 μm (b), 15 μm (d), 20 μm (g). n = 71-72 cells per condition from 2 animals per genotype (c), 27-30 images per condition from 3 independent experiments (e), 7 independent experiments (f), 33-46 cells, 1 representative experiment from 3 independents experiments (h). Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, Mann-Whitney test (c, h), unpaired t-test (e, f).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Immunoprecipitation, Immunodetection, Staining, Isolation, Control, Expressing, Transfection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Proximity ligation assay (PLA) in EC cultures. White puncta indicate Numb and FLRT2 and Numb and VE-cadherin being in close proximity (< 40 nm). Cells were stained with phalloidin and DAPI to visualize actin filaments and nuclei, respectively. (b) Expansion microscopy on HUVEC cultures stained for FLRT2 and Numb showing expression of both proteins in cell-cell contacts and along filopodia extensions. (c) mRNA expression of Flrt2 and Numb in ECs transfected with control and Flrt2 -specific siRNAs measured by RT-qPCR. (d) Representative immunoblot showing FLRT2 and Numb protein levels in ECs transfected with control and Flrt2 -specific siRNAs. (e) FLRT2 and Numb protein levels quantification in ECs transfected with control and Flrt2 -specific siRNAs. (f) mRNA expression quantification of Numb from primary mouse brain ECs (pmBECs) isolated from control and Flrt2 iΔEC littermates. (g) Scheme showing the quantification of Golgi orientation in migrating cells after scratch assay and in retinal vascular fronts. Cell was classified as polarized if the angle formed between the scratch or vascular front and Golgi located within a 120°. (h) Scratch assay on HUVECs stained for VE-cadherin, Golgi apparatus (GM130) and cell nuclei (DAPI). Yellow stars in lower panels indicate cells polarized towards the wound area. The 3 first cell rows were considered for quantification. (i) Quantification of the percentage of cells per image polarized towards the wound. (j) Representative images of retinal vascular front from control and Flrt2 iΔEC littermates stained for blood vessels, EC nuclei and Golgi apparatus with IB4, ERG and GM130, respectively. White arrows indicate cellular orientation identified with GM130 position relative to ERG staining. (k) Quantification of the percentage of cells polarized towards the vascular front. The 3 first cell rows were considered for quantification. Scale bars: 20 μm (a, b, h, j). n = 4 independent experiments (c), 7 independent experiments (e), 5-10 animals per genotype (f), 27-28 images from 3 independent experiments (i), 4 animals per genotype (k). Data are shown as mean ± SEM. *P > 0.05, ***P < 0.001, unpaired t-test (c, e, f), Mann-Whitney test (i, k).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Proximity Ligation Assay, Staining, Microscopy, Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot, Isolation, Wound Healing Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a and c) Vascular front of P5 wild-type retinas (a) and remodeling capillaries in cerebral cortex (c) from control and Flrt2 iΔEC littermates stained for VE-cadherin antibody. Blood vessels visualized with IB4. (b and d) Quantification of VE-cadherin activity in retina (b) and cerebral cortex (d) blood vessels. 15 x 15 μm regions of interest (ROIs) were blindly classified to a VE-cadherin activity category: low (smooth pattern), medium (irregular pattern), high (rough pattern). Example images of the three VE-cadherin activity categories in retina (b) and cortex (d) vessels are shown in correlation with the graph. Scale bars: 20 μm (a), 15 μm (c). n = 53-66 images per genotype from three litters b), 78-80 images per genotype from two litters (d). Data are shown as mean ± SEM. *P > 0.05, **P < 0.01, ns = not significant, two-way ANOVA (b and d).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Staining, Activity Assay

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Expansion microscopy 3D-visualization of a large vessel stained for VE-cadherin, FLRT2 and Numb in the cerebral cortex. Higher magnification (lower panels) showing the colocalization of the three proteins at EC junction. (b and c) Expansion microscopy 3D-visualization of a large vessel stained for VE-cadherin and FLRT2 (b) and VE-cadherin and Numb (c) in the retina. (d) Expansion microscopy 3D-visualization of cerebral cortex capillaries stained for VE-cadherin and Glut1 in control (left) and Flrt2 iΔEC (right) mice. Higher magnifications (1-5) showing x-y planes (first row) and y-z planes (second row) exposing VE-cadherin pattern in a control capillary (1-2), a control tip cells (3), and a Flrt2 iΔEC capillary (4-5). Scale bars: 4 μm (a upper panel), 1 μm (a lower pannels), 15 μm (b, c), 30 μm (d).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Microscopy, Staining, Control

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Representative immunoblot of the cytosolic and nuclear fractions obtained from bEnd.3 cells treated with control and Flrt2 siRNA showing protein levels of β-catenin and FoxO1, and α-tubulin and Lamin A/C as cytosolic and nuclear controls, respectively. (b and c) β-catenin (b) and FoxO1 (c) protein levels relative to the loading controls. (d) Representative immunoblot showing FLRT2 and Claudin-5, and β-actin as loading control in bEnd.3 cells treated with control and Flrt2 siRNA. (e) Quantification of FLRT2 and Claudin-5 protein levels relative to the loading control. ( f) Representative immunoblot showing Claudin 5, and α-tubulin as loading control in total brain lysates from control and Flrt2 iΔEC littermates. (g) Quantification of Claudin-5 protein levels relative to the loading control. (h) Neocortical blood vessels stained for Claudin-5 and VE-cadherin. Note the colocalization of both protein in the control vessel, compared to the split Claudin-5 signal in Flrt2 iΔEC blood vessel. (i) Quantification of the ratio of split Claudin-5 junction length to the total junctional length. (j) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor TM 555-conjugated cadaverine at P7-P8. (k) Quantification of cadaverine whole-brain intensity in P7-P 8control and Flrt2 iΔEC littermates. (l) Representative images of Collagen IV (Col IV) immunostaining and cadaverine signal in control and Flrt2 iΔEC cortices showing cadaverine leakage in Flrt2 iΔEC mice. (m) TEM representative images of brain capillaries showing an EC junction in control and Flrt2 iΔEC mice. Note the abnormal junctions often associated with the presence of vacuoles (red arrows) in the Flrt2 iΔEC vessels. (n) Incidence (in percentage) of abnormal junctions (left) and junctions with vacuoles (right) in TEM images of control and Flrt2 iΔEC brain capillaries. Scale bars: 10 μm (h), 1 mm (j), 100 μm (l), 200 nm (m). n = 3 independent experiments (b, c), 5-6 independent experiments (e), 8 animals per genotype (g), 5-7 animals per genotype (i), 16-18 animals per genotype (k), 3 animals per genotype (n). Data are shown as mean ± SEM. *p < 0.05, **P < 0.01, ***P > 0.001, unpaired t-test (b, c, e, i, k), Mann-Whitney test (g), 2-way ANOVA (n).

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Western Blot, Control, Staining, Injection, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) mRNA fold change levels of Flrt2 and tight junction protein claudin5 ( Cldn5 ) in bEnd.3 cells treated with control and Flrt2 siRNA. (b) mRNA expression quantification of Cldn5 in control and Flrt2 iΔEC total brains at P7-P8. (c, e) Representative immunoblots from total brain lysates of control and Flrt2 iΔEC mice showing tight junctions proteins ZO-1 (c) and JAM-A (e) levels. (d, f) Quantification of protein ZO-1 (d) and JAM-A (f) levels in total brain lysates from control and Flrt2 iΔEC mice. (g, h) Neocortical blood vessels stained for ZO-1 (g) and JAM-A (h) showing no differences in tight junction proteins distribution between control and Flrt2 mutant mice. (i) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor555nm-conjugated ovalbumin (45 kDa) at P7-8. (j) Quantification of ovalbumin whole-brain intensity in control and Flrt2 iΔEC mice at P7-8. (k) Representative fluorescent whole-brain images of control and Flrt2 iΔEC littermates injected with AlexaFluor555nm-conjugated cadaverine at P5. (l) Quantification of cadaverine whole-brain intensity in control and Flrt2 iΔEC mice at P5. Scale bars: 10 μm (g, h), 1 mm (i, k). n = 5 independent experiments (a), 6 animals per genotype (b), 5 animals per genotype (d), 4 animals per genotype (f, j), 7-8 animals per genotype (l). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Expressing, Western Blot, Staining, Mutagenesis, Injection

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a, c, e,) Cerebral cortices of control and vascular Flrt2 mutant mice stained with the pericyte marker PDGFRβ (a), astrocytic end-feet marker Aquaporin-4 (Aqp4) (c) and extracellular matrix marker Collagen IV (Col IV) (e) and blood vessel markers Glut1 (a) or Podxl (c, e). (b, d, f) Quantification of PDGFRβ (b), Aqp4 (d) or Col IV (f) coverage of cortical vasculature. Scale bars: 50 μm (a), 10 μm (c, e). n = 7 animals per genotype (b), 6 animals per genotype (d, f). Data are shown as mean ± SEM. ns = not significant, unpaired t-test.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Control, Mutagenesis, Staining, Marker

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: Representative image of an endothelial-specific Flrt2 mutant cerebral cortex section showing a leakage area where the punch was performed and further processed for transmission electron microscopy (TEM) analysis. After assessing the quality of the tissue, imaging was processed focusing in capillary EC tight junctions. Cartoon created with Biorender.com. Scale bars: 1mm, 250 μm, 2500 nm, 250 nm

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Mutagenesis, Transmission Assay, Electron Microscopy, Imaging

Journal: bioRxiv

Article Title: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis

doi: 10.1101/2024.08.27.609862

Figure Lengend Snippet: (a) Schematic timeline representation of the vascular events derived from FLRT2 deletion in ECs. (b) Schematic representation of the molecular mechanisms regulated by FLRT2 in postnatal CNS vasculature. In control conditions, FLRT2 forms a complex with VE-cadherin and the endocytic adaptor Numb which allows the dynamic turnover of VE-cadherin necessary for angiogenic sprouting. By contrast, in FLRT2-deficient cells VE-cadherin cytoplasmic tail is cleaved by Calpains and subsequently fated to lysosomal degradation, while a compensatory biosynthesis accumulates VE-cadherin at the cell membrane impairing vascular sprouts. In addition, FLRT2 deletion facilitates the nuclear translocation of β-catenin, repressing Claudin-5 expression and triggering increased size-selective BBB permeability.

Article Snippet: Quantitative PCR assays were performed using TaqMan Fast Universal PCR master mix (4304437, ThermoFisher) and TaqMan Gene Expression probes for human FLRT2 (Hs00544171_s1), human Cdh5 (Hs00901470_m1), mouse Flrt2 (Mm03809571_m1), mouse Flrt3 (Mm01328142_m1), mouse Numb (Mm00477927_m1), and mouse Cldn5 (Mm00727012_s1).

Techniques: Derivative Assay, Control, Membrane, Translocation Assay, Expressing, Permeability

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 1. Mn-induced movement impairment and locomotor deficits is further worsened in mice with enhanced LRRK2 kinase activity (LRRK2 G2019S KI). A–D, After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), locomotor activity was measured by open-field traces (A), total distance traveled (B), walking speed (C), and vertical activity count (D). The mouse’s movement is depicted by traces and red dots indicate the location of vertical activity in the open-field arena. E, Motor coordination is measured by fall latency as time spent on the rotating rod. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 5). Data are expressed as mean ± SD.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activity Assay

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 3. Mn-induced dysfunction of nigrostriatal dopaminergic pathway is greater in LRRK2 G2019S than in WT mice. A and B, After acute Mn exposure, extracellular dopamine levels in the mouse striatum were measured by microdialysis and HPLC-ECD, as described in the Experimental procedures section. A, Extracellular dopamine release was stimulated after 100 mM KCl, for dopamine release for 20 min between 80 to 100 min intervals. B, Striatal dopamine levels were compared among different groups. C, After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), coronal sections of the substantia nigra were immunostained for TH proteins by IHC as described in the Experimental procedures section. D, TH fluorescence intensities were compared among different treatment groups. E, After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), protein levels of TH, cleaved caspase-3, Bcl-2, and Bax were measured in the striatum and midbrain. β-actin was used as a loading control for protein. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, @@@@p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n =3–5). Data are expressed as mean ± SD.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Control

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 2. Mn-induced cognitive impairment is exacerbated in LRRK2 G2019S mice. A–C, After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), NO recognition was evaluated as described in the Experimental procedures section. A, NO recognition in mice was assessed by comparing the time spent on the NO (bottom-right) compared to the FO (top-left). Traces in the arena depict the mouse’s movement and interaction with FO and NO over a 10-min period. Red dots on the FO and NO shows a point of exploration with an object. B–C, The time spent in each object in the open- field arena was used to calculate the NO exploration time (B) and discrimination index (C). #p < 0.05, ###p < 0.001, compared with the controls; @p < 0.05, @@p < 0.01, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 5). Data are expressed as mean ± SD.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques:

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 4. Mn increased microglial LRRK2 expression and LRRK2 activity in the mouse nigrostriatal region. A and B, After Mn exposure (MnCl2, 30 mg/ kg, intranasal instillation, daily for 3 weeks), protein levels of LRRK2 and Iba1 were analyzed in the striatum and midbrain regions of mouse brains by Western blotting (A) and IHC of the substantia nigra region (B). A, Protein levels of LRRK2 and Iba1 in striatum and midbrain of Mn-treated WT and G2019S mice. B, Expression and colocalization of LRRK2 and Iba1, a microglial marker, were visualized with red and green fluorescence signals, respectively. White arrows depict the co-localization of LRRK2 and Iba1. C and D, PLA for protein-protein interactions of LRRK2 with RAB10 (C) and LRRK2 with 14-3-3ε (D) in the substantia nigra was visualized with red fluorescence signals. Insets show a higher magnification of the PLA puncta in the substantia nigra region. Yellow arrows depict PLA fluorescence signals showing the interaction of LRRK2 with RAB10 or 14-3-3ε that do not overlap with DAPI. β-actin was used as a loading control for protein. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the controls; @p < 0.05, @@@p < 0.001, @@@@p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3). Data are expressed as mean ± SD.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Expressing, Activity Assay, Western Blot, Marker, Protein-Protein interactions, Control

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 6. LRRK2 kinase activity plays a role in regulating Mn-induced proinflammatory TNF-α in mice and microglia. A, After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), striatum and midbrain regions of mouse brains were analyzed for TNF-α protein levels by western blotting. B–E, Following LRRK2 inhibitors MLi-2 (50 nM, 0.5 h) and LRRK2-IN-1 (10 nM, 0.5 h) pre-treatment and Mn exposure (250 μM, 12 h), LRRK2 WT- and G2019S-expressing BV2 cells were analyzed for TNF-α mRNA (B) and protein levels (C and D) by qPCR and western blotting, respectively. GAPDH and β-actin were used as normalization and loading control for RNA and protein, respectively. E, Protein levels of secreted TNF-α were measured by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, @@@@p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3–5). Data are expressed as mean ± SD. The data shown in BV2 cells are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activity Assay, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 5. Mn further increases LRRK2 kinase activity in LRRK2 G2019S-expressing BV2 microglia cells. A and B, Validation of transfection of vectors for LRRK2 WT and G2019S in BV2 microglia. A, Fluorescence imaging of LRRK2 (red) in non-transfected control, LRRK2 WT-, and G2019S-expressing (green) BV2 cells. B, Levels of LRRK2 phosphorylation (p-LRRK2, S1292) and protein in BV2 cells. Following LRRK2 WT and G2019S overexpression, LRRK2 inhibitors MLi-2 (50 nM, 0.5 h) and LRRK2-IN-1 (10 nM, 0.5 h) pre-treatment and Mn exposure, BV2 cells were analyzed for phosphorylation of LRRK2 at S1292 and RAB10 at T73 using western blotting. C–E, Mn increased LRRK2 and RAB10 phosphorylation in LRRK2 WT BV2 cells, which were exacerbated in LRRK2 G2019S BV2 cells. Quantification of LRRK2 (D) and RAB10 (E) phosphorylation in BV2 cells. MLi-2 and LRRK2-IN-1 were used as LRRK2 inhibitors. β-actin was used as a loading control for protein. *p < 0.05, **p < 0.01, ****p < 0.0001, #p < 0.05, ##p < 0.01, compared with the controls; @p < 0.05, @@p < 0.01, @@@@p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3). Data are expressed as mean ± SD. The data shown are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activity Assay, Expressing, Biomarker Discovery, Transfection, Fluorescence, Imaging, Control, Phospho-proteomics, Over Expression, Western Blot

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 7. LRRK2 plays a role in Mn-induced NLRP3-IL-1β inflammasome pathway by modulating lysosomal function in mice and microglia. A, After Mn exposure (MnCl2, 30 mg/kg, intranasal instillation, daily for 3 weeks), striatum and midbrain tissues were analyzed for the protein levels of NLRP3, cleaved CASP1, active CTSB, and LAMP1 by western blotting. B, BV2 cells were examined for protein-protein interactions of LRRK2 with RAB10, fluorescence intensity, and colocalization of NLRP3 and LRRK2, RAB10 in the microglia. C, Following Mn exposure for 12 and 24 h, LRRK2 WT and G2019S BV2 cells were assessed for autophagic flux with LC3-mcherry-GFP fluorescence assay. Cells with high autophagic flux were determined and quantified by red fluorescence using flow cytometry. D, Following pre-treatment of LRRK2 inhibitor MLi-2 (50 nM, 0.5 h) and Mn exposure (250 μM, 12 h), LRRK2 WT and G2019S-expressing BV2 cells were analyzed for lysosomal activity by lysotracker assays. #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, @@@@p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3–5). Data are expressed as mean ± SD. The data shown are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Western Blot, Protein-Protein interactions, Cytometry, Expressing, Activity Assay

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 8. Mn increased NLRP3 inflammasome activation and proinflammatory IL-1β production via CTSB activity. A–C, After transfection of LRRK2 WT and G2019S vectors following Mn exposure, BV2 cells were analyzed for the fluorescence intensity and colocalization of p-RAB10, NLRP3, and CTSB in microglia by immunofluorescence. B, BV2 cells were analyzed for proteins by western blotting. C, Protein levels of NLRP3, cleaved CASP1, mature IL-1β, and active CTSB were quantified in BV2 cells. β-actin was used as a loading control for protein. D, Protein levels of secreted IL-1β in BV2 cell-free media were measured using ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, @@@@p < 0.0001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3–5). Data are expressed as mean ± SD. The data shown are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activation Assay, Activity Assay, Transfection, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 9. Mn alters lysosomal function in microglia via LRRK2-RAB10 activation. BV2 cells were transfected with RAB10 and DN-RAB10 vectors to modulate RAB10 function, followed by exposure to Mn (250 μM, 12 h). A, Validation of RAB10 and DN-RAB10 transfection in BV2 cells visualized with GFP fluorescence. B and C, After Mn exposure, RAB10- and DN-RAB10-expressing BV2 cells were analyzed for proteins by western blotting. B, Proteins for p- RAB10, RAB10, p-LRRK2, and LRRK2 were determined in RAB10- and DN-RAB10-expressing BV2 cells. C and D, Following MLi-2 (50 nM, 0.5 h) pre-treatment and Mn exposure (250 μM, 12 h), RAB10- and DN-RAB10-expressing BV2 cells were assessed for cell viability (C) and lysosomal activity (D) and by resazurin and lysotracker assays, respectively. E, Proteins for NLRP3, cleaved CASP1, LAMP1, active CTSB, and mature IL-1β were assessed in RAB10- and DN-RAB10- expressing BV2 cells after MLi-2 (50 nM, 0.5 h) pre-treatment and Mn exposure (250 μM, 12 h). ##p < 0.01, ####p < 0.0001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 5). Data are expressed as mean ± SD. The data shown are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activation Assay, Transfection, Biomarker Discovery, Expressing, Western Blot, Activity Assay

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 10. Microglial LRRK2 kinase activity mediates Mn-induced cytotoxicity in catecholaminergic neuron-like cells using a microglia-neuron co-culture. A, BV2 cells were transfected with LRRK2 WT and G2019S vectors to modulate LRRK2 kinase activity, followed by LRRK2 inhibitors MLi-2 (50 nM, 0.5 h) and LRRK2-IN-1 (10 nM, 0.5 h) pre-treatment and Mn exposure (250 μM). After Mn exposure for 6 h, BV2 experimental media were replaced with fresh media and incubated for an additional 6 h prior to media collection as described in the Methods. These CM were applied to differ- entiated CAD cells. After CM exposure (12 h for cell viability; 3 h for ROS), CAD cells were analyzed for cell viability was determined by resazurin assays (B) and ROS levels by CM-H2DCFDA fluorescence (C, imaging; D, quantification). ***p < 0.001, ****p < 0.0001, ###p < 0.001, ####p < 0.0001, compared with the controls; @p < 0.05, @@@p < 0.001, compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 6). Data are expressed as mean ± SD. The data shown are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activity Assay, Co-Culture Assay, Transfection, Incubation, Imaging

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 11. Mn-induced apoptosis is exacerbated by enhanced LRRK2 kinase activity in microglia. BV2 cells were transfected with LRRK2 WT and G2019S vectors to modulate LRRK2 kinase activity, followed by exposure to Mn (250 μM). A, After treatment with LRRK2 inhibitors MLi-2 (50 nM, 0.5 h) and LRRK2-IN-1 (10 nM, 0.5 h) and Mn exposure (250 μM, 12 h) of BV2 cells for 24 h (for cell viability), cell viability was determined by resazurin assays. B and C, After transfection with LRRK2 WT and G2019S, followed by LRRK2 inhibitors MLi-2 (50 nM, 0.5 h) and LRRK2-IN-1 (10 nM, 0.5 h) pre-treatment, and Mn exposure, protein levels for Bcl-2, Bax, active caspase-3 were analyzed by western blotting in BV2 cells. C, Quantification of Bax/Bcl-2 ratio and active CASP3 in LRRK2 WT and G2019S were compared. β-actin was used as a loading control for protein. **p < 0.01, ***p < 0.001, ****p < 0.0001, ###p < 0.001, ####p < 0.0001, compared with the controls; @p < 0.05, @@p < 0.01, @@@p < 0.001, @@@@p < 0.0001 compared with each other (two-way ANOVA followed by Tukey’s post hoc test; n = 3–6). Data are expressed as mean ± SD. The data shown are representative of three independent experiments.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activity Assay, Transfection, Western Blot, Control

Journal: The Journal of biological chemistry

Article Title: The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction.

doi: 10.1016/j.jbc.2023.104879

Figure Lengend Snippet: Figure 12. Proposed mechanism of LRRK2 kinase activity in Mn-induced NLRP3 inflammasome activation via RAB10 dysfunction in autophagy impairment and lysosomal enzyme leakage. Mn increases LRRK2 kinase activity, leading to autophosphorylation and phosphorylation of its target substrate, RAB10. RAB10 dysfunction contributes to Mn-induced autophagy impairment, reducing lysosomal membrane integrity and causing lysosomal cathepsin B (CTSB) leakage. The increased CTSB co-localizes and activates the NLRP3 inflammasome, resulting in the CASP1-mediated maturation of interleukin-1β (IL-1β) and subsequent neurotoxicity. This proposed mechanism provides insight into the potential role of LRRK2 kinase activity in Mn- induced neuroinflammation and highlights potential therapeutic targets for treating Mn-induced neurotoxicity.

Article Snippet: Male C57BL/6 (WT, 8 weeks old) and LRRK2 G2019S knock-in (#13940-M, C57BL/ 6-Lrrk2tm4.1Arte, 8 weeks old) mice were purchased from Taconic Biosciences.

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Membrane, Biomarker Discovery

Journal: Nature Communications

Article Title: Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

doi: 10.1038/s41467-020-19603-1

Figure Lengend Snippet: a Overview of RICS and ccRICS. Confocal image series are acquired on a laser scanning confocal microscope, containing spatiotemporal fluorescence information on the microsecond and millisecond timescales. A spatial autocorrelation function (SACF) is calculated from the fluorescence image and fit to a diffusive model. The cross-correlation of intensity between two channels is used to estimate the co-occurrence of two fluorescent molecules in live cells. The mean cross-correlation of the fluctuations is calculated and shown in the 3D plot color-coded according to the correlation value. b – e Representative plots of the spatial cross-correlation function (SCCF) between the depicted fluorescent molecules in cells from each cell line measured: ( b ) wild-type (U1WT:D3 WT ) and ( c ) K85E/R85E/R87E DPPA3 mutant (U1WT:D3 KRR ), and control ESCs expressing ( d ) free eGFP, free mScarlet (eGFP + mScarlet) and ( e ) an eGFP-mScarlet tandem fusion (eGFP-mScarlet). f , g Mobile fraction of ( f ) mScarlet and ( g ) eGFP species in the cell lines depicted in ( b , c , and e ) and in Uhrf1KO ESCs expressing free eGFP and wild-type DPPA3-mScarlet (U1KO:D3 WT ). The mobile fraction was derived from a two-component model fit of the autocorrelation function. Data are pooled from three (U1WT:D3 WT , U1WT:D3 KRR ) or two (U1KO:D3 WT , eGFP-mScar) independent experiments. h Mean cross-correlation values of mobile eGFP and mScarlet measured in the cell lines depicted in ( b – e ). The spatial lag in the x-dimension (sensitive to fast fluctuations) is indicated by ξ , and the spatial lag in the y-dimension (sensitive to slower fluctuations) is indicated by ψ . Data are pooled from two independent experiments. i Microscale thermophoresis measurements of UHRF1-eGFP binding to GST-DPPA3 WT (D3 WT ) or GST-DPPA3 1–60 (D3 1–60 ). Error bars indicate the mean ± SEM of n = 2 technical replicates from n = 4 independent experiments. In ( f – h ), each data point represents the measured and fit values from a single cell where n = number of cells measured (indicated in the plots). In the boxplots, darker horizontal lines within boxes represent median values. The limits of the boxes indicate the upper and lower quartiles; the whiskers extend to the most extreme value within 1.5 x the interquartile range from each hinge. Source data are provided as a Source Data file.

Article Snippet: Data for Raster Image Correlation Spectroscopy (RICS) was acquired on a home-built laser scanning confocal setup equipped with a 100x NA 1.49 NA objective (Nikon) pulsed interleaved excitation (PIE) as used elsewhere .

Techniques: Microscopy, Fluorescence, Mutagenesis, Control, Expressing, Derivative Assay, Microscale Thermophoresis, Binding Assay