Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice

Abstract

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD₄/TSPAN12 pathway activates WNT/β-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD₄ stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD₄-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD₄-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (Ndp^KO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD₄-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

Fig. 1. Characterization of the monoFZD₄-specific WNT/β-catenin signaling surrogate, L6-F4-2.

a Binding affinity of the recombinant F4-2_Fab molecule to FZD₄ CRD measured by BLI assay. Dotted lines indicate the global fits generated by using a 1:1 Langmuir binding model. b Schematic of L6-F4-2 and the binding affinity of the L6-F4-2 molecule to the FZD₄ CRD measured by BLI assay. Dotted lines indicate the global fits generated by using a 1:1 Langmuir binding model. c The binding specificity of L6-F4-2 against all ten FZD CRDs was examined by BLI assay. d Dose-dependent STF activity of L6-F4-2 or WNT3A in 293STF cells (top) or FZD₄-transfected 293STF cells (bottom, OE = overexpression). e Dose-dependent STF activity of L6-F4-2 or Norrin in 293STF cells (top) or cells transfected with both FZD₄ and LRP6 (bottom, OE = overexpression). f Dose-dependent STF activities of L6-F4-2 in HRMEC cells (top) and bEnd.3 cells (bottom). Inset box in the bEnd.3-STF graph is an enlarged plot of the Norrin response. g Quantitative RT-PCR of Axin2, Mki67, Lef1 and Mfsd2a gene expression in bEnd.3 cells. mRNA expression values were normalized by Actb gene expression. Results are from three independent experiments. Graphs are shown as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001, two- sided Mann–Whitney U test. Source data are provided as a Source Data file.

Fig. 2. L6-F4-2 treatment rescues retinopathy in Ndp^KO mice.

a–d WT (control) and Ndp^KO male and female mice were treated with PBS or L6-F4-2 by single intravitreal injection (0.19 μg) at P0 and retinas were harvested at P8. a Whole-mount isolectin GS-IB4 labeling of retinal veins. Representative images of the whole retinal vascular plexus, quadrant vascular plexus, angiogenic front and central plexus are shown. Scale bars represent 0.5 mm (top), 0.3 mm (middle) and 0.15 mm (bottom two rows). b Quantification of vessel diameter, vessel density and distance from the optic nerve from (a). c Whole-mount anti-smooth muscle actin labeling of retinal arteries at P8. d Quantification of artery branches from (c). Scale bar represents 0.5 mm. In all the quantifications, error bars represent mean ± SEM, Ndp WT n = 7 mice, Ndp^KO n = 8 mice, *p < 0.05, **p < 0.01, ***p < 0.001; two-sided Mann–Whitney U test. e WT (control) and Ndp^KO mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P5 and every three days with retina harvest at P21. Optical sections of isolectin-stained P21 retinas showing the three-layered retinal vasculature (superficial, intermediate, and deep layers) to document vascular architecture. Scale bars represent 100 μm. f Vascular density quantification of retina intermediate and deep layers from (e), n = 3 mice, ***p < 0.001, ****p < 0.0001, one-way ANOVA. Source data are provided as a Source Data file. For box plots, whiskers indicate the minimum and maximum values in the dataset. The center is the median number. Boundaries of boxes are the first quartile and third quartile.

Fig. 3. Effects of L6-F4-2 on retinal endothelial WNT/β-catenin signaling and vascular subtypes.

a–c WT (control) and Ndp^KO male and female mice were treated with PBS or L6-F4-2 by intravitreal injection (0.19 μg) at P0 and 5-7 pooled retinas for each group were harvested at P8. Retinal vessel ECs were purified by anti-CD31 FACS and ultra-low input bulk RNA-seq analysis was performed. a Left: WT (PBS-treated) vs Ndp^KO (PBS-treated). Right: Ndp^KO (PBS-treated) vs Ndp^KO (treated with L6-F4-2). Volcano plot depicting genes with a p-value <0.05 and log₂ fold-change > 1 are indicated by red dots which represent upregulated genes. Genes with a p-value <0.05 and log₂ fold-change < −1 are indicated by blue dots, representing downregulated genes. b Left: Venn diagrams showing overlap in bulk RNA-seq differentially expressed genes following treatment with PBS or L6-F4-2 in WT or Ndp^KO mice. Right: Heat maps of the top expressed genes mapping to the indicated GO terms across treatment groups in the RNA-seq experiment. c qRT-PCR validation of differentially expressed genes identified by RNA-seq. Error bars represent mean ± SEM, triplicate from 5-7 pooled retina EC cells, *p < 0.05, **p < 0.01; one-way ANOVA. d–f Single-cell RNA-seq with the same treatment conditions as (a-c). d scRNA-seq UMAP plots of retinal EC sub-clusters from P8 retinas from all conditions merged (top) and from the WT, Ndp^KO, and Ndp^KO + L6-F4-2 conditions (bottom), 5–7 pooled retinas for each group. Alteration of the D-tip population is indicated by arrows. e Dot plot of NaRnEA enrichment for previously described Zarkada et al. gene sets. Dot size is determined by the absolute value of the normalized enrichment score (NES); large dots indicate more significant enrichment. Dot color is determined by the proportional enrichment score (PES), which indicates the sign of the enrichment. BRB, brain-retinal-barrier. f Master regulators of the Ndp^KO condition versus WT as identified by PISCES analysis of the scRNA-seq data with reduced or reverted activity profiles in the Ndp^KO + L6-F4-2 condition. g Master regulators of the Ndp^KO + L6-F4-2 condition versus WT without significant activity in the Ndp^KO condition as identified by PISCES analysis of scRNA seq data. Source data are provided as a Source Data file.

Fig. 4. L6-F4-2 treatment promotes endothelial blood-brain and blood-retina barrier function.

a WT (control) and Ndp^KO male and female mice were treated with PBS or L6-F4-2 by i.p. injection (2.5 mg/kg) at P0, P7 and P14 and Sulfo-NHS-biotin injected 30 min prior to tissue harvest at P21. Sulfo-NHS-biotin staining from a representative mouse reveals BBB/BRB defects in cerebellum and retina, but not cerebral cortex in Ndp^KO mice, while these defects can be rescued by L6-F4-2 treatment. Biotin labeling of liver and kidney parenchyma serve as a positive control. Scale bar, 500 μm. b Quantification of (a) for BBB/BRB leakage in Ndp^KO cerebellum and retina with rescue by L6-F4-2 treatment. c Mice were treated at P21, P24 and P27 (2.5 mg/kg, i.p.) and Sulfo-NHS-biotin injected 30 min prior to tissue harvest at P30. Sulfo-NHS-biotin staining from a representative mouse reveals BBB/BRB defects in cerebellum and retina but not cerebral cortex in Ndp^KO mice with rescue by L6-F4-2. Scale bar, 200 μm. d Quantification of (c) with BBB/BRB leakage in Ndp^KO cerebellum and retina and reversal by L6-F4-2. e L6-F4-2 rescues barrier phenotypes in P30 Ndp^KO mice with increased expression of the tight junction component CLDN5, P30 cerebellum IF, overlay of CD31 IF and DAPI. f Quantification of (e). g L6-F4-2 decreases expression of the EC fenestration component PLVAP in P30 Ndp^KO mice, with overlay of CD31 IF and DAPI. h Quantitation of (g). For (e) and (g), scale bars represent 100 μm. To quantify CLDN5 and PLVAP in (f) and (h) the density was measured with ImageJ and normalized to vessel area (CD31). Error bars in (b), (d), (f), and (h) represent mean ± SEM, n = 5 mice, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA. Source data are provided as a Source Data file. For box plots, whiskers indicate the minimum and maximum values in the dataset. The center is the median number. Boundaries of boxes are the first quartile and third quartile.

Fig. 5. L6-F4-2 treatment rescues stroke and blood-brain barrier phenotypes in wild-type mice.

a Schematic of tMCAO surgery with 45 min occlusion time, L6-F4-2 treatment time course and brain harvest at day 2 post-stroke. b Simultaneous TTC staining (top), and mouse IgG extravasation (mIgG) (bottom) analysis of coronal sections from representative mice at 48 h post-stoke. c Quantification of infarct size for NIST control mAb (n = 13 mice) and L6-F4-2 (n = 18 mice), males. p = 0.0042, two-sided Mann–Whitney U test. d Quantification of mouse IgG staining, NIST control n = 4 mice, L6-F4-2 n = 5 mice. p = 0.0159, two-sided Mann–Whitney U test. e Representative images of BBB leak in brains of the indicated mice in stroke and non-stroke regions after 45 min tMCAO, 2 days of reperfusion and two L6-F4-2 treatments (3 mg/kg, i.v.), as assessed by the Sulfo-NHS-biotin tracer extravasation assay. Scale bar, 50 μm. f Quantification of extravasated exogenous tracer Sulfo-NHS-biotin using ImageJ. n = 5 mice. g Fractional change in brain edema for NIST control mAb (n = 13 mice) and L6-F4-2 (n = 18 mice). p = 0.0307, two-sided Mann–Whitney U test. h Neurological scores at 48 h after tMCAO surgery for NIST control mAb (n = 13 mice) and L6-F4-2 (n = 18 mice). p = 0.0312, two-sided Mann–Whitney U test. i Co-immunofluorescence staining for PDGFRB and CD31 in infarcted brain (stroke and non-stroke) regions. j Quantification of pericyte coverage from (i). The PDGFRB signal was normalized to CD31; n = 5 mice. Scale bar, 100 μm. Error bars represent mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001; two-sided Mann–Whitney U test was used to compare groups in (c), (d), (g) and (h). Comparisons between multiple groups were made using one-way ANOVA test in (f) and (j). Source data are provided as a Source Data file. For box plots, whiskers indicate the minimum and maximum values in the dataset. The center is the median number. Boundaries of boxes are the first quartile and third quartile.