Fig 2: NOTCH3 ECD and perlecan expression in cerebral capillaries Representative images of TgN3R182C150, sham- and NOTCH3 EGF1–5-immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF1–5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan 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 a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3-month-old TgN3R182C150 (n = 3) and 7-month-old TgN3R182C150 (n = 6) mice and 18-month-old TgN3R182C150 (n = 3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 µm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF1–5-immunized (n = 11), sham (n = 8), and non-vaccinated TgN3R182C150 (n = 6) mice. 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): 3 m.o. vs. 18 m.o. **P = 0.0029; 3 m.o. vs. 7 m.o. ***P = 0.0004; 7 m.o. vs. Sham, ns P = 0.7326; 7 m.o. vs. Vaccinated, **P = 0.003; Sham vs. Vaccinated, ****P < 0.0001. NOTCH3 ECD deposits (number/1,000 µm2): 3 m.o. vs. 18 m.o. **P = 0.0038; 3 m.o. vs. 7 m.o. ***P = 0.0002; 7 m.o. vs. Sham, ns P = 0.9913; 7 m.o. vs. Vaccinated, *P = 0.021; Sham vs. Vaccinated, ***P = 0.001. NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. *P = 0.0215; 3 m.o. vs. 7 m.o. **P = 0.0029; 7 m.o. vs. Sham, ns P = 0.3622; 7 m.o. vs. Vaccinated, *P = 0.0428; Sham vs. Vaccinated, **P = 0.0038, ns = non-significant). Dotted lines indicate quartiles and dashed thicker lines are the median. Source data are available online for this figure.

Fig 3: Quantification of human NOTCH3 ECD protein present in whole blood serum of sham, immunized and non-vaccinated TgN3R182C150 mice (at 3 and 7 months old) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C150 mice at 3 months (n = 9) of age and further increased at 7 months (n = 6) of age. Serum from Notch3-/- and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired Student's t-test with Welch's correction (3 m.o. vs. 7 m.o. ***P = 0.0005). Error bars indicate standard error of the mean (SEM).NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated (n = 10) TgN3R182C150 mice vs. sham (n = 9). Statistical analysis was performed using unpaired Student's t-test with Welch's correction (Sham vs. Vaccinated *P = 0.0196). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Fig 4: Microglia activation and NOTCH3 ECD expression in sham, immunized and non-vaccinated TgN3R182C150 mice at 7 months Representative images show microglia stained with anti-CD68 antibody (red) and Iba1 antibody (green). Scale bar = 20 µm.Quantification of CD68-stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1–5-immunized (n = 6), sham (n = 4), and non-vaccinated TgN3R182C150 (n = 5) mice. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test (% microglia with CD68 staining: 7 m.o. vs. Sham ns P = 0.7393; Sham vs. Vaccinated **P = 0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P = 0.9131; Sham vs. Vaccinated *P = 0.0157, ns = non-significant). Error bars indicate standard error of the mean (SEM).Representative images of TgN3R182C150, sham-, and NOTCH3 EGF1–5-immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar = 20 µm.Quantification of NOTCH3 ECD deposits (numbers per 1,000 µm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1–5-immunized (n = 11), sham (n = 8), and non-vaccinated TgN3R182C150 (n = 6) mice at 7 months of age. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P = 0.7997; Sham vs. Vaccinated ns P = 0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P = 0.5362; Sham vs. Vaccinated ns P = 0.8059. NOTCH3 ECD deposits (number/1,000 µm2): 7 m.o. vs. Sham ns P = 0.8392; Sham vs. Vaccinated ns P = 0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P = 0.6664; Sham vs. Vaccinated ns P = 0.8787, ns = non-significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Fig 5: 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 a-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.