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LSR/angulin-1 is a tricellular tight junction protein involved in blood-brain barrier formation.

Sohet F, Lin C, Munji RN, Lee SY, Ruderisch N, Soung A, Arnold TD, Derugin N, Vexler ZS, Yen FT, Daneman R - J. Cell Biol. (2015)

Bottom Line: One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs).Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues.We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Department of Neuroscience, University of California, San Diego, La Jolla, CA 92093 Department of Pharmacology and Department of Neuroscience, University of California, San Diego, La Jolla, CA 92093 fsohet@ucsd.edu rdaneman@ucsd.edu.

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BBB phenotype of LSR embryos. (A) Quantification of Sulfo-NHS biotin leakage through the BBB of wild-type embryos at different ages. At E12.5 and E13.5, biotin leaks through the BBB. From E14.5 and onwards, leakage of Sulfo-NHS biotin is greatly restricted. (B) Quantification of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr +/−, and Lsr −/− embryos at E12.5, E13.5, and E14.5. At E12.5 and E13.5, Sulfo-NHS biotin leakage is comparable between the genotypes. However, at E14.5 Sulfo-NHS biotin leakage is greatly diminished in Lsr+/+ and Lsr+/−, but not Lsr−/−, mice. Data represent mean ± SEM (error bars). ***, P < 0.0001. (C) Visualization of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr+/−, and Lsr−/− E14.5 embryos. PFA fixed tissue sections of E14.5 Sulfo-NHS biotin-injected embryos were stained with fluorophore (Alexa Fluor 488)-conjugated streptavidin. Lsr+/+ (left) and Lsr+/− (middle) embryos present minimal leakage of Sulfo-NHS biotin from the blood vessels into the SC, whereas the dye is observed throughout the neural tissue of Lsr−/− (right) embryos. Bar, 20 µm. (D) Fresh-frozen tissue sections of E13.5 mouse SC from Lsr+/+ (top), Lsr+/− (middle), and Lsr−/− (bottom) embryos were labeled with an anti–claudin 5 antibody (red) and CD31 to stain blood vessels (green). There was no discernable difference in the claudin 5 expression level or localization between genotypes. Bar, 20 µm. (E) Quantification of TJ protein (LSR, Zo1, Occludin, Cldn5) expression in E14.5 CNS Lsr+/+, Lsr+/−, and Lsr−/− embryos. This graph shows no significant differences for levels of Zo1, occludin, and claudin 5 between genotypes. Data represent mean ± SD (error bars). n is between 3 and 6. (F) Quantification of albumin leakage through the BBB of Lsr+/+, Lsr+/−, and Lsr−/− embryos from E12.5 to E14.5. There was no significant difference in leakage of albumin into the brain between genotypes at any age. Data represent mean ± SD (error bars). n is between 3 and 8. (G) Electron micrograph of SC blood vessels from E14.5 Lsr+/+ and Lsr−/− embryos. Arrowheads indicate kissing points of TJs. There were no discernable differences in the ultrastructure of TJs between genotypes. Bar, 50 nm.
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fig3: BBB phenotype of LSR embryos. (A) Quantification of Sulfo-NHS biotin leakage through the BBB of wild-type embryos at different ages. At E12.5 and E13.5, biotin leaks through the BBB. From E14.5 and onwards, leakage of Sulfo-NHS biotin is greatly restricted. (B) Quantification of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr +/−, and Lsr −/− embryos at E12.5, E13.5, and E14.5. At E12.5 and E13.5, Sulfo-NHS biotin leakage is comparable between the genotypes. However, at E14.5 Sulfo-NHS biotin leakage is greatly diminished in Lsr+/+ and Lsr+/−, but not Lsr−/−, mice. Data represent mean ± SEM (error bars). ***, P < 0.0001. (C) Visualization of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr+/−, and Lsr−/− E14.5 embryos. PFA fixed tissue sections of E14.5 Sulfo-NHS biotin-injected embryos were stained with fluorophore (Alexa Fluor 488)-conjugated streptavidin. Lsr+/+ (left) and Lsr+/− (middle) embryos present minimal leakage of Sulfo-NHS biotin from the blood vessels into the SC, whereas the dye is observed throughout the neural tissue of Lsr−/− (right) embryos. Bar, 20 µm. (D) Fresh-frozen tissue sections of E13.5 mouse SC from Lsr+/+ (top), Lsr+/− (middle), and Lsr−/− (bottom) embryos were labeled with an anti–claudin 5 antibody (red) and CD31 to stain blood vessels (green). There was no discernable difference in the claudin 5 expression level or localization between genotypes. Bar, 20 µm. (E) Quantification of TJ protein (LSR, Zo1, Occludin, Cldn5) expression in E14.5 CNS Lsr+/+, Lsr+/−, and Lsr−/− embryos. This graph shows no significant differences for levels of Zo1, occludin, and claudin 5 between genotypes. Data represent mean ± SD (error bars). n is between 3 and 6. (F) Quantification of albumin leakage through the BBB of Lsr+/+, Lsr+/−, and Lsr−/− embryos from E12.5 to E14.5. There was no significant difference in leakage of albumin into the brain between genotypes at any age. Data represent mean ± SD (error bars). n is between 3 and 8. (G) Electron micrograph of SC blood vessels from E14.5 Lsr+/+ and Lsr−/− embryos. Arrowheads indicate kissing points of TJs. There were no discernable differences in the ultrastructure of TJs between genotypes. Bar, 50 nm.

Mentions: We first performed a time course of biotin leakage in wild-type embryos to examine BBB permeability of embryos from E12.5 to E16.5 (Fig. 3 A). This analysis reveals that the dye leaks through the BBB until E13.5 but is greatly restricted from E14.5 to E16.5. Thus, the BBB is permeable for the first few days after the initiation of CNS angiogenesis, then it seals to this small molecule starting at E14.5. Interestingly, this is similar to the time course of LSR expression in the vessels. To confirm that our method doesn’t disrupt the vasculature, we injected 10 kD rhodamine-dextran in E12.5 LSR embryos. Fig. S2 reveals minimal leakage in any of the genotypes. Thus, at E12.5, CNS ECs are permeable to small molecules but have low permeability to large molecules. These results confirm that our method can be used to assess BBB permeability in embryos without disruption of vascular integrity.


LSR/angulin-1 is a tricellular tight junction protein involved in blood-brain barrier formation.

Sohet F, Lin C, Munji RN, Lee SY, Ruderisch N, Soung A, Arnold TD, Derugin N, Vexler ZS, Yen FT, Daneman R - J. Cell Biol. (2015)

BBB phenotype of LSR embryos. (A) Quantification of Sulfo-NHS biotin leakage through the BBB of wild-type embryos at different ages. At E12.5 and E13.5, biotin leaks through the BBB. From E14.5 and onwards, leakage of Sulfo-NHS biotin is greatly restricted. (B) Quantification of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr +/−, and Lsr −/− embryos at E12.5, E13.5, and E14.5. At E12.5 and E13.5, Sulfo-NHS biotin leakage is comparable between the genotypes. However, at E14.5 Sulfo-NHS biotin leakage is greatly diminished in Lsr+/+ and Lsr+/−, but not Lsr−/−, mice. Data represent mean ± SEM (error bars). ***, P < 0.0001. (C) Visualization of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr+/−, and Lsr−/− E14.5 embryos. PFA fixed tissue sections of E14.5 Sulfo-NHS biotin-injected embryos were stained with fluorophore (Alexa Fluor 488)-conjugated streptavidin. Lsr+/+ (left) and Lsr+/− (middle) embryos present minimal leakage of Sulfo-NHS biotin from the blood vessels into the SC, whereas the dye is observed throughout the neural tissue of Lsr−/− (right) embryos. Bar, 20 µm. (D) Fresh-frozen tissue sections of E13.5 mouse SC from Lsr+/+ (top), Lsr+/− (middle), and Lsr−/− (bottom) embryos were labeled with an anti–claudin 5 antibody (red) and CD31 to stain blood vessels (green). There was no discernable difference in the claudin 5 expression level or localization between genotypes. Bar, 20 µm. (E) Quantification of TJ protein (LSR, Zo1, Occludin, Cldn5) expression in E14.5 CNS Lsr+/+, Lsr+/−, and Lsr−/− embryos. This graph shows no significant differences for levels of Zo1, occludin, and claudin 5 between genotypes. Data represent mean ± SD (error bars). n is between 3 and 6. (F) Quantification of albumin leakage through the BBB of Lsr+/+, Lsr+/−, and Lsr−/− embryos from E12.5 to E14.5. There was no significant difference in leakage of albumin into the brain between genotypes at any age. Data represent mean ± SD (error bars). n is between 3 and 8. (G) Electron micrograph of SC blood vessels from E14.5 Lsr+/+ and Lsr−/− embryos. Arrowheads indicate kissing points of TJs. There were no discernable differences in the ultrastructure of TJs between genotypes. Bar, 50 nm.
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fig3: BBB phenotype of LSR embryos. (A) Quantification of Sulfo-NHS biotin leakage through the BBB of wild-type embryos at different ages. At E12.5 and E13.5, biotin leaks through the BBB. From E14.5 and onwards, leakage of Sulfo-NHS biotin is greatly restricted. (B) Quantification of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr +/−, and Lsr −/− embryos at E12.5, E13.5, and E14.5. At E12.5 and E13.5, Sulfo-NHS biotin leakage is comparable between the genotypes. However, at E14.5 Sulfo-NHS biotin leakage is greatly diminished in Lsr+/+ and Lsr+/−, but not Lsr−/−, mice. Data represent mean ± SEM (error bars). ***, P < 0.0001. (C) Visualization of BBB leakage of Sulfo-NHS biotin in Lsr+/+, Lsr+/−, and Lsr−/− E14.5 embryos. PFA fixed tissue sections of E14.5 Sulfo-NHS biotin-injected embryos were stained with fluorophore (Alexa Fluor 488)-conjugated streptavidin. Lsr+/+ (left) and Lsr+/− (middle) embryos present minimal leakage of Sulfo-NHS biotin from the blood vessels into the SC, whereas the dye is observed throughout the neural tissue of Lsr−/− (right) embryos. Bar, 20 µm. (D) Fresh-frozen tissue sections of E13.5 mouse SC from Lsr+/+ (top), Lsr+/− (middle), and Lsr−/− (bottom) embryos were labeled with an anti–claudin 5 antibody (red) and CD31 to stain blood vessels (green). There was no discernable difference in the claudin 5 expression level or localization between genotypes. Bar, 20 µm. (E) Quantification of TJ protein (LSR, Zo1, Occludin, Cldn5) expression in E14.5 CNS Lsr+/+, Lsr+/−, and Lsr−/− embryos. This graph shows no significant differences for levels of Zo1, occludin, and claudin 5 between genotypes. Data represent mean ± SD (error bars). n is between 3 and 6. (F) Quantification of albumin leakage through the BBB of Lsr+/+, Lsr+/−, and Lsr−/− embryos from E12.5 to E14.5. There was no significant difference in leakage of albumin into the brain between genotypes at any age. Data represent mean ± SD (error bars). n is between 3 and 8. (G) Electron micrograph of SC blood vessels from E14.5 Lsr+/+ and Lsr−/− embryos. Arrowheads indicate kissing points of TJs. There were no discernable differences in the ultrastructure of TJs between genotypes. Bar, 50 nm.
Mentions: We first performed a time course of biotin leakage in wild-type embryos to examine BBB permeability of embryos from E12.5 to E16.5 (Fig. 3 A). This analysis reveals that the dye leaks through the BBB until E13.5 but is greatly restricted from E14.5 to E16.5. Thus, the BBB is permeable for the first few days after the initiation of CNS angiogenesis, then it seals to this small molecule starting at E14.5. Interestingly, this is similar to the time course of LSR expression in the vessels. To confirm that our method doesn’t disrupt the vasculature, we injected 10 kD rhodamine-dextran in E12.5 LSR embryos. Fig. S2 reveals minimal leakage in any of the genotypes. Thus, at E12.5, CNS ECs are permeable to small molecules but have low permeability to large molecules. These results confirm that our method can be used to assess BBB permeability in embryos without disruption of vascular integrity.

Bottom Line: One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs).Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues.We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Department of Neuroscience, University of California, San Diego, La Jolla, CA 92093 Department of Pharmacology and Department of Neuroscience, University of California, San Diego, La Jolla, CA 92093 fsohet@ucsd.edu rdaneman@ucsd.edu.

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Related in: MedlinePlus