Journal: Science Advances

Article Title: Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier

doi: 10.1126/sciadv.aba7457

Figure Lengend Snippet: ( A ) mRNA levels of Mfsd2a were measured by RT-qPCR in Lrp5 −/− and Ndp y/− retinas and their respective controls at P5, P8, P12, and P17. ( B and C ) Laser-captured microdissection (LCM) was used to isolate retinal blood vessels (red), outlined by the white dashed lines in (C) illustrating retinal cross-sections (left). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). RGC, retinal ganglion cells; INL, inner nuclear layer; ONL, outer nuclear layer. (B) Enrichment of Mfsd2a mRNA levels in LCM isolated blood vessels compared with the whole retina. (C) Comparisons of Mfsd2a mRNA levels in the retinal vessels of Lrp5 −/− / Ndp y/− mice with their controls. ( D ) Protein levels of MFSD2A in Lrp5 −/− and Ndp y/− retinas were quantified and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. ( E ) Retinas were costained with MFSD2A and CD31, which colocalize in the WT eyes. MFSD2A staining is barely visible in Lrp5 −/− and Ndp y/− retinas. Scale bars, 50 μm (B) and 100 μm (E). Data are expressed as individual values plus means ± SD. n = 3 to 6 per group. Statistical differences between groups were analyzed using two-tailed unpaired t tests. * P < 0.05, ** P < 0.01.

Article Snippet: A plasmid expressing the full-length sequence of mouse Mfsd2a was optimized and synthesized by Synbio Technologies (Monmouth Junction, NJ).

Techniques: Quantitative RT-PCR, Laser Capture Microdissection, Staining, Isolation, Two Tailed Test

Journal: Science Advances

Article Title: Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier

doi: 10.1126/sciadv.aba7457

Figure Lengend Snippet: ( A and B ) Mfsd2a mRNA levels were increased in HRMEC treated with lithium chloride (LiCl), using control sodium chloride (NaCl). ( C and D ) Mfsd2a mRNA and protein levels were induced by Wnt3a-conditioned medium (Wnt3a-CM) and suppressed by the Wnt inhibitor XAV939. n-p-β-catenin, nonphosphorylated β-catenin. ( E ) Three promoter regions upstream of MFSD2A gene were identified containing putative Wnt-responsive TCF-binding motifs (TTCAAAG): P1 (−2841 to −2211 bp), P2 (−1900 to −1099 bp), and P3 (−1145 to −168 bp), followed by cloning and ligation with a luciferase reporter, and transfected with an active β-catenin plasmid in HEK293 cells. Luciferase activity was measured. ( F ) Mutation of TCF-binding site #1 (Mut-P1) (mutated to CCTGGGT) partially abolished the Wnt/β-catenin–responsive luciferase reporter activity compared with native P1. ( G ) Scheme of in vitro HRP transcytosis assay in ECs. ( H and I ) Transferred HRP in the lower chambers was measured to indicate the transcytosis levels through the EC monolayer. Activation of Wnt signaling activation was achieved by treatment with Wnt3a-CM (H) or human recombinant Norrin (I), and inhibition by XAV939 treatment. ( J and K ) siRNA targeting MFSD2A (si- M2A ) suppressed MFSD2A mRNA (J) and protein levels (K) in HRMEC compared with si-control (si-Ctrl). ( L ) HRP-based in vitro transcytosis was used to detect transcytosis levels in HRMEC with Wnt3a-CM in combination with si- M2A or si-control (si-Ctrl) treatment (K). Data are expressed as individual values plus means ± SD. n = 3 to 4 per group. Statistical differences between groups were analyzed using a one-way analysis of variance (ANOVA) statistical test with Dunnett’s multiple comparisons tests or two-tailed unpaired t tests. * P < 0.05, ** P < 0.01.

Article Snippet: A plasmid expressing the full-length sequence of mouse Mfsd2a was optimized and synthesized by Synbio Technologies (Monmouth Junction, NJ).

Techniques: Control, Binding Assay, Cloning, Ligation, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Mutagenesis, In Vitro, Activation Assay, Recombinant, Inhibition, Two Tailed Test

Journal: Science Advances

Article Title: Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier

doi: 10.1126/sciadv.aba7457

Figure Lengend Snippet: ( A to C ) HRMECs were treated with XAV939 to inhibit Wnt signaling and infected with Lenti-M2A to detect the effect of MFSD2A overexpression on Wnt signaling–induced up-regulation of CAV-1. (A) Protein levels were shown with Western blot. (B) Relative protein levels of nonphosphorylated β-catenin (n-p-β-catenin), MFSD2A, and CAV-1 were normalized by GAPDH. (C) Inhibition of Wnt signaling by XAV939 induced an increase of transferred HRP, while overexpression of MFSD2A reversed the changes. ( D to G ) Lenti-M2A and its control were injected intravitreally into Lrp5 −/− mice at P10. Mice were euthanized at P30. (D) Retinas were used to detect protein levels with Western blot. (E) Quantification of relative protein levels of MFSD2A and Cav-1 in lenti-M2A– and lenti-control–treated Lrp5 −/− retinas, normalized by GAPDH. (F) Fixed Lrp5 −/− retinas with lenti-M2A and lenti-control treatment were prepared for electron microscopy analysis. Transcytotic vesicles in the retinal vessel endothelia of Lrp5 −/− mice were categorized into three groups (representative images of three groups were shown in ). Overexpression of MFSD2A by lentivirus decreased the transcytotic vesicles of type I (arrows) and type II (asterisks), but not type III (arrowheads). (G) CAV-1 was labeled with immunogold in Lrp5 −/− retinas treated with lenti-M2A and lenti-control, and immunogold was detected with electron microscopy (black dots). The numbers of CAV-1–associated vesicles were counted and normalized by EC area. Scale bars, 500 nm (F) and 200 nm (G). Data are expressed as individual values plus means ± SD. n = 3 per group. Statistical differences between groups were analyzed using an ANOVA statistical test with Dunnett’s multiple comparisons tests or two-tailed unpaired t tests. * P < 0.05, ** P < 0.01.

Article Snippet: A plasmid expressing the full-length sequence of mouse Mfsd2a was optimized and synthesized by Synbio Technologies (Monmouth Junction, NJ).

Techniques: Infection, Over Expression, Western Blot, Inhibition, Control, Injection, Electron Microscopy, Labeling, Two Tailed Test

Journal: Science Advances

Article Title: Wnt signaling activates MFSD2A to suppress vascular endothelial transcytosis and maintain blood-retinal barrier

doi: 10.1126/sciadv.aba7457

Figure Lengend Snippet: ( A and B ) Long-chain polyunsaturated fatty acids in the retina or brain were measured. Both DHA and EPA were down-regulated in the retinas of Lrp5 −/− and Ndp y/− mice compared with their respective controls (A), but remained unchanged in the brains of the same mice (B). Data are expressed as individual values plus means ± SD. n = 4 per group. Statistical differences between groups were analyzed using two-tailed unpaired t tests. * P < 0.05, ** P < 0.01. n.s., not significant. ( C ) Scheme illustration on the role of Wnt signaling in controlling inner BRB integrity by limiting MFSD2A-mediated EC caveolar transcytosis. Canonical Wnt signaling is activated by binding of the ligand (Norrin or Wnts) to the receptor complex containing Frizzed4 (FZD4) and co-receptors (LRP5 or LRP6), leading to the prevention of β-catenin ubiquitination and degradation. Stabilized β-catenin then translocates to the nucleus and works with TCF to bind the TCF-responsive motif in the promoter region of MFSD2A, directly regulating its gene transcription. MFSD2A protein is located on cellular plasma membrane to suppress CAV-1 protein levels and block the formation of CAV-1–positive caveolae, thereby limiting EC transcytosis and maintaining inner BRB integrity.

Article Snippet: A plasmid expressing the full-length sequence of mouse Mfsd2a was optimized and synthesized by Synbio Technologies (Monmouth Junction, NJ).

Techniques: Two Tailed Test, Binding Assay, Ubiquitin Proteomics, Clinical Proteomics, Membrane, Blocking Assay