Fig 1: NOTCH3 ECD and alpha‐smooth muscle Actin (ASMA) expression in cerebral arteries/arterioles Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF1–5‐immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF1–5‐immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α‐SMA antibody (green). Scale bar = 20 μm.Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1–5‐immunized (n = 10), sham (n = 8), and non‐vaccinated (n = 6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months (n = 3) of age versus NOTCH3 EGF1–5‐immunized (n = 10), sham (n = 8), and non‐vaccinated (n = 6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P = 0.9424; 7 m.o. vs. Vaccinated, ns P = 0.9809; 7 m.o. vs. 18 m.o. **P = 0.0046; Sham vs. Vaccinated, ns P = 0.9999; Sham vs. 18 m.o. **P = 0.0032; Vaccinated vs. 18 m.o. **P = 0.0032. NOTCH3 ECD deposits (number/1,000 μm2): 7 m.o. vs. Sham, ns P = 0.7877; 7 m.o. vs. Vaccinated, ns P = 0.8358; 7 m.o. vs. 18 m.o. **P = 0.0069; Sham vs. Vaccinated, ns P = 0.9995; Sham vs. 18 m.o. **P = 0.0044; Vaccinated vs. 18 m.o. *P = 0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P = 0.1169; 7 m.o. vs. Vaccinated, ns P > 0.9999; 7 m.o. vs. 18 m.o. ***P = 0.0009; Sham vs. Vaccinated, ns P = 0.0577; Sham vs. 18 m.o. **P = 0.007; Vaccinated vs. 18 m.o. ****P < 0.0001, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.
Fig 2: The number of retinal VSMC is not reduced by active immunization Immunostaining for smooth muscle actin (ASMA) revealed that there were no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in WT (C57Bl6/J) versus TgN3R182C150 mice at 7 months of age. Scale bar = 50 μm.Immunostaining for smooth muscle actin (ASMA) shows no significant differences in the composition of the smooth muscle cell coating of vessels in the retinal vasculature in NOTCH3 EGF1–5‐vaccinated versus sham‐vaccinated TgN3R182C150 mice. Scale bar = 50 μm.Immunostaining for smooth muscle actin (ASMA) shows an extensive loss of VSMC in the Notch3−/− mice when compared with a WT (C57Bl6/J) at 3 months of age. Scale bar = 50 μm.
Fig 3: Quantitative real‐time PCR analysis of the Notch downstream target genesThe target genes NOTCH3, Hes1, Hey1 and Nrip2 on TgN3R182C150 mice at 5 and 12 months of age. Three biological replicates were run in three technical replicates. Error bars indicate standard deviation (SD).
Fig 4: Schematic representation of Notch signaling and Notch3 CADASIL mutations Schematic representation of Notch signaling. (1) Furin (S1 cleavage) cleaves the NOTCH3 precursor protein in the Golgi system, resulting in a non‐covalently bound bipartite protein that is transported to the cell surface. (2) A mechanical traction force is applied to the NOTCH3 ECD when a Notch ligand binds to the EGF repeats 10–11, exposing the extracellular NRR near the cell membrane, which consists of LNR and the heterodimerization domain (in green). Subsequently, ADAM17 cleaves the C‐terminal portion of the heterodimerization domain (S2‐cleavage). (3) The NEXT, which is made up of a RAM domain, the ANK domains, a PEST domain, and a transmembrane domain, is cleaved by the γ‐secretase (S3‐cleavage) releasing the N3ICD. (4) The N3ICD binds to the CSL protein and together with the co‐activator Mastermind‐like (MAM) trigger downstream gene transcription in the nucleus. (5) The NOTCH3 ECD and ligand are normally endocytosed by the ligand‐expressing cell and degraded in the lysosome.Schematic representation of NOTCH3 cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutations. NOTCH3 ECD contains 34 EGF repeat domains, each of which has six cysteine residues (WT). Mutations in CADASIL change the number of cysteines to an uneven number of cysteines (Mutant). These unpaired cysteines residues result in incorrect EGF repeat folding, irregular protein folding which leads to an enhanced NOTCH3 ECD multimerization. Distribution of the cysteine‐altering mutations that cause CADASIL are shown. In the CADASIL mutant NOTCH3 ECD, the endocytosis is hampered, and NOTCH ECD remains outside of the VSMC and starts to accumulate and aggregate around the vessels. ADAM17, a disintegrin and metalloproteinase domain‐containing protein 17; ANK, ankyrin repeats; EGF, epidermal growth factor; HD, heterodimerization domain; LNR, Lin‐Notch repeats; PEST, proline (P), glutamic acid (E), serine (S), and threonine (T) degradation domain; RAM, Rbp‐associated molecule domain; TM, transmembrane domain.
Fig 5: NOTCH3 R182C mutation is signaling neutralNIH3T3 cells were transfected with the control, WT NOTCH3, or NOTCH3 R182C plasmids, as well as the β‐gal and 12XCSL‐luc reporter plasmids and cultured on immobilized jagged2 (Jag2) in the presence of DMSO or DAPT (n = 3 and two technical replicates). Statistical analysis was performed using 2‐way ANOVA followed by Tukey's multiple comparisons tests (Control: Fc + DMSO vs. Jag2 + DMSO ns P = 0.7223; Jag2 + DMSO vs. Jag2 + DAPT ns P = 0.817. N3WT: Fc + DMSO vs. Jag2 + DMSO ***P = 0.0004; Jag2 + DMSO vs. Jag2 + DAPT **P = 0.0069. N3R182C: Fc + DMSO vs. Jag2 + DMSO *P = 0.0135; Jag2 + DMSO vs. Jag2 + DAPT *P = 0.0352. Jag2 + DMSO: Control Jag2 + DMSO vs. N3WT Jag2 + DMSO ***P = 0.0008; Control Jag2 + DMSO vs. N3R182C Jag2 + DMSO *P = 0.0314; N3WT Jag2 + DMSO vs. N3R182C Jag2 + DMSO ns P = 0.4187, ns = non‐significant). Error bars indicate standard error of the mean (SEM). RLU, relative luminescence units. Source data are available online for this figure.
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Available conjugates: Sizes Available: 50 ug