human notch3 extracellular domain  (R&D Systems)

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: 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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Membrane, Expressing, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic representation of NOTCH3 and NOTCH3 EGF 1–5 . NOTCH3 represents the full‐length protein, and NOTCH3 EGF 1–5 represents the NOTCH3 protein with exon 1–5 fused with a myc‐His‐Tag at the C‐terminus used for purification of the aggregated protein. Western blot of the NOTCH3 EGF 1–5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α‐myc antibody. Western blot of NOTCH3 EGF 1–5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF 1–5 WT and R133C were visualized on a western blot using an α‐myc antibody. The purified proteins and the aggregates were visualized after 1–5 days incubation by western blot using α‐myc antibody under non‐reducing conditions. Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Purification, Western Blot, Incubation

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Schematic pipeline of the subcutaneous active immunization in the TgN3R182C150 mouse model. Antibody titer validation of serum from TgN3R182C150 CADASIL mice immunized with NOTCH3 EGF 1–5 aggregates (vaccinated, n = 11) and PBS (sham, n = 9) at 4, 5, and 7 months old. A direct ELISA with NOTCH3 aggregate‐coated plates and different dilutions of serum was performed. Error bars indicate standard error of the mean (SEM).

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Biomarker Discovery, Direct ELISA

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐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 EGF 1–5 ‐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 μm 2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF 1–5 ‐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 μm 2 ) 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 EGF 1–5 ‐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 μm 2 ): 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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: The 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).

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF 1–5 ‐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 μm 2 ) 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 μm 2 ) 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 EGF 1–5 ‐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 μm 2 ): 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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: 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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Serum from 7‐month‐old non‐vaccinated TgN3R182C 150 mice ( n = 4) was monitored in the absence or presence of serum (1:1 dilution) from sham‐and vaccinated C57Bl6/J WT mice. No significant changes in NOTCH3 ECD concentration was observed after dilution adjustment. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Dunnett's multiple comparisons test (TgN3R182C 150 vs. TgN3R182C 150 + C57BL6/J WT Sham, ns P = 0.8383; TgN3R182C 150 vs. TgN3R182C 150 + C57BL6/J WT Vaccinated, ns P = 0.7000; ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: 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 EGF 1–5 ‐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 EGF 1–5 ‐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 μm 2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF 1–5 ‐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 μm 2 ): 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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: 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 EGF 1–5 ‐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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Immunostaining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: NIH3T3 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.

Article Snippet: Whole blood serum samples were diluted 1:40 in 100 μl of reagent diluent (R&D Systems) and recombinant human NOTCH3 ECD (1559‐NT‐050; R&D Systems) was used as standard protein.

Techniques: Transfection, Control, Cell Culture