Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Modular structure of NOTCH3, SP indicates signal peptide, red shaded EGF-like modules indicate core ligand binding region, LNR indicates Lin12-Notch repeats, NRR is the negative regulatory region, S1, S2 and S3 are proteolytic cleavage sites, HD-N and HD-C are the heterodimer interface region. RAM and Ankyrin repeat region (Ank) are motifs involved in binding CSL transcription factors, PEST domain is involved in ubiquitin-dependent turnover of the ICD. Location of mutants used in this study are indicated by arrows. Coloured bars indicate locations of regions of NOTCH3 in which epitopes used in this study are located. (B-D) AlphaFold2 predictions of WT NOTCH3 EGF modules 2 (B) , 5 (C) and 11 (D) , and mutant substitutions introduced using the SDM programme. Location of the new Cys residue in R90C indicated in yellow, and other substitutions that replace as cysteine residues are shown in magenta. (E-G) Surface views of the structures from (B-D) depicted as a surface mesh. The introduced cysteine in R90C is surface exposed (E) , but the free cysteines in C212Y and C455R are buried within the module structure. ΔΔG values indicate predicted decreases in stability. (H-K) Expressed WT NOTCH3 (H) and CADASIL mutants (I-K) do not show strong accumulation in the ER when expressed in hTert-RPE1 cells. (L, M) scoring of NOTCH3 localisation in KDEL-positive ER, by % area of ER occupied (L) and by % of total NOTCH3 staining intensity (K) in z-sections through the cytoplasmic region. R90C shows a small increase in %NOTCH3 which is localised to the ER. * indicates P<0.05 by student t-test, error bars SEM, n=10. (N) Antibody surface labelling of NOTCH3 localisation on non-permeabilised cells at time 0 and 60 minutes during a live cell anti-ECD uptake assay. Similar time zero surface distributions, and subsequent surface depletion of WT and CADASIL mutants can be seen.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Ligand Binding Assay, Binding Assay, Mutagenesis, Residue, Staining

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-C) Schematic view of pulse chase labelling protocol. (D-G) WT NOTCH3 localisation in permeabilised cells after antibody uptake at 0 (D) , 15 (E) , 30 (F) , and 60 (G) minutes. Little surface-labelled NOTCH3 is transported to the early endosome by 15 minutes chase but is evident in EEA1 positive endosomes by 30 and 60 minutes. Presence of both full-length and ECD-only staining suggests ECD-shedding can occur by the time NOTCH3 is localised to the EEA1-marked endosome. (H-J) CADASIL mutant NOTCH3 localisation after 60-minute chase shows the mutant proteins labelled at the cell surface also become localised to the EEA1-positive endosome. (K, L) After 60-minute chase surface, little labelled NOTCH3 ECD has reached the CD63-positive late endosome while ICD is present. However full-length NOTCH3 and ECD-only localisations are found adjacent to CD63-marked organelles.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Pulse Chase, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-D) Immunolocalisation of NOTCH3 with anti-ECD and anti-ICD compared with EEA1-marked endosomes in fixed and permeabilised cells of WT (A) and CADASIL mutants ( B-D) . (E) Quantitation of NOTCH3 localisation in the early endosome, showing % endosomal-located NOTCH3 puncta which either full-length NOTCH3, ECD-only, or ICD-only staining. R90C is distinguished from WT and other mutants by a greater proportion of ICD-only puncta compared to ECD or full-length. For WT, n=91 NOTCH3 puncta from198 endosomes scored. For R90C, n=139 NOTCH3 puncta from 180 endosomes score. For C212Y, n=140 NOTCH3 puncta from 240 endosomes scored. For C455R, n=164 NOTCH3 puncta from 302 endosomes scored. Minimum of 7 cells scored for each construct. Data obtained from z sections from at least 7 cells. (F) Full-length NOTCH3, or ECD-only puncta located within or adjacent to Rab11-positive endosomes. (G, H) In CD63 positive endosomes (G) and LAMP1-positive lysosomes (H) , NOTCH3 localisation is predominantly as ICD-only puncta, quantified in (I, J) . Error bars in I and J are SEM, n=11 and n=7 respectively. Statistical significance by student t-test.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Quantitation Assay, Staining, Construct

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Schematic diagram illustrating alternative explanations for visualisation of separated ECD and ICD-positive puncta, including: ECD shedding by S2 cleavage; epitope masking; fragmentation to remove terminal epitopes; non-specific staining. (B) Colocalisation of two different ECD epitopes compared to ICD ab23426 . ICD-stained puncta can be observed which lack both ECD epitopes. (C) Colocalisation of two different ICD epitopes compared to anti-ECD 1E . ECD-only spots are present which lack both ICD epitopes. (D, E) Quantification of the colocalisation between the epitopes used in B, C. In D * indicates P<0.05 compared with double ECD epitope colocalisation by student t-test. Error bars represent SEM, n=5 (D) , n=14 (E) . (F-H) Both ICD epitopes are located to the nucleus in NOTCH3 transfected cells but not the ECD epitope.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) EEA1-positive early endosomes of MCF7 cells costained with anti-ECD, and ICD. Arrowheads indicate endosomes shown enlarged in insets which have separate localisation of ECD and ICD puncta. (B) CD63-positive late endosomes costained with anti-ECD, and ICD. Boxed region is enlarged in inset showing late endosomes predominantly contain ICD-only puncta. (C) After treatment of MCF7 cells with metalloprotease inhibitor BB94, more full-length NOTCH3 staining is observed in the late endosomes, colocalised with CD63. (D) Treatment of cells with gamma-secretase inhibitor DAPT does not alter localisation of ECD in the late endosome. (E-G) Quantification of colocalization of ECD vs ICD (E) , ECD vs CD63 (F) , and ICD vs CD63 ( G) . Error bars, SEM, control DMSO-only treated cells n=10, BB94 n=18, DAPT n=12. * indicates p<0.05 by student t-test compared to control.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A) Schematic figure illustrating luciferase reporter assay methodology. (B) WT and CADASIL mutant NOTCH3 signalling after transfection into hTERT-RPE1 cells compared to empty vector (EV) control. * indicates p<0.05 compared to WT NOTCH3 by student t-test. Error bars SEM, minimum of n=4. (C-F) NOTCH3 signalling after treatment of transfected cells with TRPML inhibitor (ML-Sl1), gamma-secretase inhibitor (RO 4929097 ), or metalloprotease inhibitor (BB94) for WT (C) , R90C (D) , C212Y (E) , and C455R (F) * indicates p<0.05 compared with control by student t-test. Error bars are SEM, minimum of n=3.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Luciferase, Reporter Assay, Mutagenesis, Transfection, Plasmid Preparation, Control

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Mechanisms put forward to explain how the Arg169Cys mutation causes arterial pathology. ( B – E ) Four main types of experiments used to discriminate between a loss/gain of NOTCH3 activity and a neomorphic effect. ( F ) Proposed model emerging from this study. NOTCH3 activity is unaffected and arterial maturation and vascular function proceed normally despite a CADASIL mutation, but mutant Notch3 ECD aggregates with WT Notch3 ECD , possibly via a mechanism involving thiol disulfide exchange reactions. This leads initially to reversible vascular dysfunction owing to an excess of TIMP3, which compromises activity of K v 2.1 voltage-dependent K + channels ( – ). Upon reaching a threshold, accumulated Notch3 ECD aggregates cause arterial SMC loss and irreversible vascular dysfunction, possibly through accumulation and inactivation of other proteins (e.g., HTRA1).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Mutagenesis, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Schematic overview of the protocol for labeling dissected brain arteries. ( B ) Representative confocal images of brain arteries from WT, Notch3 R170C/R170C (KinR170C), and Tg Notch3 R169C (TgN3 R169C ) mice aged 4 months, stained for α-SMA, perlecan, and Notch3 ECD . ( C – E ) Quantification of the mean area ( C ), mean intensity ( D ), and number ( E ) of Notch3 ECD aggregates in brain arterial SMCs of WT, KinR170C, and TgN3 R169C mice ( n = 5 mice per genotype with 20 SMCs per mouse). Shown are violin plots (individual data points represent individual SMCs) and scatter dot plots (individual data points represent individual animals). Data from individual animals were analyzed by 1-way ANOVA and Tukey’s post hoc test. ( F and G ) Intensity distribution of individual Notch3 ECD aggregates ( F ) and intensity versus size distribution of Notch3 ECD aggregates ( G ) in brain arteries of KinR170C and TgN3 R169C mice. Data were analyzed by Kolmogorov-Smirnov test. Scale bars: 9 μm ( B ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Labeling, Staining

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Confocal images of 200-μm-thick brain sections stained for α-SMA, elastin, and perlecan. Shown are representative subcortical brain arteries from WT and Tg Notch3 R169C (TgN3 R169C ) mice. Arteries from TgN3 R169C mice aged 6 months and older exhibit SMC defects, characterized by discontinuous or notched α-SMA staining (yellow dotted lines) as well as gaps in α-SMA staining (brackets). ( B ) Quantification of SMC defects in WT and TgN3 R169C mice aged 6 months in pial, cortical, and subcortical arteries ( n = 5 mice per genotype). Data were analyzed by 2-way ANOVA and Šidák’s post hoc test. ( C and D )Quantification of SMC defects ( C ) and SMC gap numbers ( D ) in brain arteries of WT and TgN3 R169C mice at 6 and 12 months ( n = 5–6 mice per genotype). Data were analyzed by 2-way ANOVA and Tukey’s post hoc test. Scale bars: 20 μm ( A ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Representative confocal images of retinal arteries from WT and Tg Notch3 R169C (TgN3 R169C ) mice, aged 4 months, stained for α-SMA, Notch3 ECD , and perlecan. ( B ) Representative pictures of retinal arteries from WT and TgN3 R169C mice stained for α-SMA, perlecan, and MEF2C. Arteries in mutant mice exhibit a discontinuous SMC coating at 6 months of age onward (dashed yellow lines) that worsens with age, ultimately causing focal SMC gaps (brackets). ( C and D ) Quantification of SMC defects ( C ) and SMC numbers (MEF2C nuclei) ( D ) in retinal arteries of WT and TgN3 R169C mice at 6 and 12 months of age ( n = 8–10 mice per group). Data were analyzed by 2-way ANOVA and Tukey’s post hoc test. ( E ) Representative pictures of retinal arteries from WT and TgN3 R169C mice, stained for α-SMA, cleaved caspase-3 (ClC3), and perlecan. ( F ) Quantification of cleaved caspase-3 in retinal arteries of WT and TgN3 R169C mice at 6 and 12 months of age ( n = 8–10 mice per group). Data were analyzed by 2-way ANOVA and Tukey’s post hoc test. ( G ) Number of TgN3 R169C mice without or with arterial SMC gaps at 6 and 12 months. Data were analyzed by Fisher’s exact test. ( H ) Quantification of SMC gaps in retinal arteries of WT and TgN3 R169C mice at 6 and 12 months of age ( n = 8–10 mice per group). Data were analyzed by 2-way ANOVA and Tukey’s post hoc test. Scale bars: 5 μm ( A ) and 10 μm ( B and E ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Staining, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Representative confocal images of retinal arteries from Notch3 R170C/R170C (KinR170C) mice aged 4 months, stained for α-SMA, Notch3 ECD , and perlecan. ( B ) Representative pictures of retinal arteries (distal segment) from Notch3 +/+ (WT) and KinR170C mice, stained for α-SMA and perlecan, showing SMC defects at 12 months. ( C and D ) Quantification of SMC defects ( C ) and SMC numbers ( D ) in the distal segment (900–1,800 μm) of retinal arteries at 6 and 12 months ( n = 11 mice per genotype per age). Data were analyzed by 2-way ANOVA and Tukey’s post hoc test. ( E ) Number of KinR170C mice without or with SMC gaps at 6 and 12 months of age. Data were analyzed by Fisher’s exact test. Scale bars: 5 μm ( A ) and 10 μm ( B ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A and B ) Relative mRNA levels for genes regulated by Notch3 in brain arteries from Notch3 –/– versus Notch3 +/+ mice ( A ) and brain arteries from Tg Notch3 R169C versus WT mice ( B ), measured by RNA sequencing analysis ( n = 8 mice per genotype). Data are reported as a fold change relative to control mice. Data were analyzed by DESeq2 (Wald test and Benjamini and Hochberg method to correct for multiple testing). ( C and D ) Volcano plot representation of RNA sequencing data comparing brain arteries from Notch3 –/– (N3 –/– ) versus Notch3 +/+ (N3 +/+ ) mice aged 3 months ( C ) and brain arteries from Tg Notch3 R169C (TgN3 R169C ) versus WT mice aged 12 months ( D ) ( n = 8 mice per genotype). Red symbols indicate DEGs. Data were analyzed by DESeq2 (adjusted P value < 0.05). ( E ) Venn diagram showing the overlap of upregulated (red) and downregulated (green) DEGs between TgN3 R169C mice (left) and N3 –/– mice (right). ( F ) Correlation analysis of fold change (log 2 ) values of downregulated genes expressed in N3 –/– mice with their fold change (log 2 ) values in TgN3 R169C mice. Data were analyzed with Spearman’s correlation test.

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: RNA Sequencing, Control

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Schematic depiction of anti-Notch3 ECD antibodies and the Tg HumNotch3 R90C mouse model. ( B ) Representative confocal images of brain tissue from a Notch3 R170C/R170C (KinR170C) mouse and a CADASIL patient stained with the 1E4 anti–human Notch3 ECD (yellow), J53 anti–mouse Notch3 ECD (red), and anti–α-SMA (white) antibodies. ( C ) Representative confocal images of brain arteries from WT and Tg HumNotch3 R90C ( n = 6 per genotype) mice, stained with the 1E4 monoclonal anti–human Notch3 ECD , J53 anti–mouse Notch3 ECD , and anti-perlecan (cyan) antibodies. ( D ) Quantification of the colocalization between 1E4- and J53-positive aggregates. Data were analyzed with SODA. ( E – H ) Quantification of the number of aggregates containing human mutant Notch3 ECD ( E ) or murine WT Notch3 ECD ( G ) and the mean intensity of aggregates containing human mutant Notch3 ECD ( F ) or murine WT Notch3 ECD ( H ) ( n = 6 per genotype with 20 SMCs per mouse). Shown are violin plots (individual data points represent individual SMCs) and scatter dot plots (individual data points represent individual animals). Data were analyzed with a linear mixed-effects model. Scale bars: 40 μm ( B ) and 5 μm ( C ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Staining, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Representative confocal images of brain arteries from Tg Notch3 R169C Notch3 +/+ (TgN3 R169C ;N3 +/+ ) and Tg Notch3 R169C Notch3 +/– (TgN3 R169C ;N3 +/– ) mice stained for α-SMA, perlecan, and Notch3 ECD . ( B – D ) Quantification of the area ( B ), mean intensity ( C ), and number ( D ) of Notch3 ECD aggregates in SMCs of TgN3 R169C ;N3 +/+ and TgN3 R169C ;N3 +/– mice ( n = 6–7 mice per genotype). Shown are violin plots (individual data points represent individual SMCs) and scatter dot plots (individual data points represent individual animals). Data were analyzed with a linear mixed-effects model. ( E ) Intensity distribution of individual Notch3 ECD aggregates in TgN3 R169C ;N3 +/+ and TgN3 R169C ;N3 +/– mice. Data were analyzed by Kolmogorov-Smirnov test. Scale bars: 5 μm ( A ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Protein aggregates containing wild-type and mutant NOTCH3 are major drivers of arterial pathology in CADASIL

doi: 10.1172/JCI175789

Figure Lengend Snippet: ( A ) Representative images of retinal arteries from non-Tg Notch3 +/+ (nonTg;N3 +/+ ), non-Tg Notch3 +/– (nonTg;N3 +/– ), Tg Notch3 R169C Notch3 +/+ (TgN3 R169C ;N3 +/+ ), and Tg Notch3 R169C Notch3 +/– (TgN3 R169C ;N3 +/– ) mice aged 6 months, stained for α-SMA and perlecan. ( B and C ) Quantification of SMC defects ( B ) and SMC numbers ( C ) in retinal arteries from mice aged 6 months ( n = 10–15 mice per genotype). Data were analyzed by Kruskal-Wallis test followed by Dunn’s post hoc test ( B ) and by 1-way ANOVA followed by Tukey’s post hoc test ( C ). ( D ) Number of TgN3 R169C ;N3 +/+ and TgN3 R169C ;N3 +/– mice without or with SMC gaps in retinal arteries. Data were analyzed by Fisher’s exact test. ( E ) Relative mRNA levels of murine Notch3 , Grip2 , and Nrip2 in TgN3 R169C ;N3 +/+ and TgN3 R169C ;N3 +/– mice, measured by quantitative reverse transcriptase PCR ( n = 8 per genotype). Data were analyzed by Student’s t test. Scale bar: 20 μm ( A ).

Article Snippet: Primary antibodies against the following proteins (species, dilution, catalog number, supplier) were used: FITC-conjugated α-SMA (mouse, 1:1,000, clone 1A4, F3777, Sigma-Aldrich), cleaved caspase-3 (Asp175) (rabbit, 1:1,000, 9661, Cell Signaling), elastin (rabbit, 1:1,000; provided by R. Mecham, Washington University School of Medicine, St. Louis, Missouri, USA), MEF2C (rabbit, 1:1,000, clone D80C1, 5030, Cell Signaling), Notch3 ECD (J53: rabbit, 1:16,000 or 1:32,000, ref. ; 1E4: mouse, 1:200, ref. ), heparan sulfate proteoglycan (perlecan) (rat, 1:2,000 or 1:3,000, clone A7L6, MAB1948P, Millipore).

Techniques: Staining, Reverse Transcription

Journal: bioRxiv

Article Title: Multiomic profiling reveals pericyte and smooth muscle cell contributions to CADASIL pathology in cell-specific Notch3 mutant mice

doi: 10.1101/2025.09.10.675140

Figure Lengend Snippet: ( a ) Schematic of workflow: brain tissue was collected, vessels were isolated, and subjected to quantitative LC-MS/MS proteomics. ( b ) Representative images of CD31-labeled brain vasculature confirming successful isolation of vascular fragments. ( c ) Western blot of isolated vessels shows accumulation of full-length NOTCH3 (∼90 kDa) in Per-Notch3 R170C and SMC-Notch3 R170C mice; GAPDH used as loading control. ( d ) Heatmap of differentially expressed proteins reveals distinct profiles between genotypes (Differentially expressed proteins were identified separately in each comparison; the proteins represented in each heatmap are not identical across the two models.) ( e ) Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) show clear separation of mutant and control groups. ( f ) Volcano plots of differentially expressed proteins (Per-Notch3 R170C vs WT and SMC-Notch3 R170C vs WT) highlighting top up- and down-regulated proteins, including shared hits LMO3 (up) and DGAT2 (down). ( g ) Venn diagram summarizing the overlap of most differentially expressed proteins (FDR < 0.05; FC > 0.5) between Per- and SMC-Notch3 R170C mice. ( h ) Gene set enrichment analysis (GSEA) of proteomic data reveals distinct pathway enrichment patterns: Per-Notch3 R170C mice show upregulation of mitochondrial respiration and protein folding pathways, while SMC-Notch3 R170C mice display enrichment in RNA processing, chromatin remodeling, and immune pathways. Data represent normalized enrichment scores (NES) for selected top-ranking gene sets.

Article Snippet: Membranes were blocked in 5% non-fat dry milk (Bio-Rad, 1706404) in TBS-T for 1 h at room temperature, incubated overnight at 4 °C with primary antibodies against NOTCH3 ECD (clone 1E4) (MilliporeSigma, MACB594) and GAPDH (Cell Signaling Technology, 2118), and then with HRP-conjugated secondary antibody (Cell Signaling Technology, 7076, 7074) for 2 h at room temperature.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Labeling, Western Blot, Control, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL

doi: 10.1101/2024.11.17.623993

Figure Lengend Snippet: (A-D) Immunolocalisation of NOTCH3 with anti-ECD and anti-ICD compared with EEA1-marked endosomes in fixed and permeabilised cells of WT (A) and CADASIL mutants ( B-D) . (E) Quantitation of NOTCH3 localisation in the early endosome, showing % endosomal-located NOTCH3 puncta which either full-length NOTCH3, ECD-only, or ICD-only staining. R90C is distinguished from WT and other mutants by a greater proportion of ICD-only puncta compared to ECD or full-length. For WT, n=91 NOTCH3 puncta from198 endosomes scored. For R90C, n=139 NOTCH3 puncta from 180 endosomes score. For C212Y, n=140 NOTCH3 puncta from 240 endosomes scored. For C455R, n=164 NOTCH3 puncta from 302 endosomes scored. Minimum of 7 cells scored for each construct. Data obtained from z sections from at least 7 cells. (F) Full-length NOTCH3, or ECD-only puncta located within or adjacent to Rab11-positive endosomes. (G, H) In CD63 positive endosomes (G) and LAMP1-positive lysosomes (H) , NOTCH3 localisation is predominantly as ICD-only puncta, quantified in (I, J) . Error bars in I and J are SEM, n=11 and n=7 respectively. Statistical significance by student t-test.

Article Snippet: Primary antibodies used were polyclonal sheep anti-NOTCH3 ECD (SF1559, R&D systems, Minneapolis, MN, USA, used 1:500), monoclonal mouse anti-NOTCH3 ECD (1E4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal Rabbit anti-ICD (D11B8, Cell Signaling technology Inc, MA, USA, used 1:500), polyclonal Rabbit anti-ICD (ab23456, Abcam, Cambridge, UK, used 1:500), monoclonal rat anti-ICD (8G5, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-EEA1 (E9Q6G, Merck-Millipore, MA, USA, used 1:200), monoclonal mouse anti-CD63 (RFAC4, Merck Life Science, Gillingham, UK, used 1:500), monoclonal rabbit anti-LAMP1 (D2D11, Cell Signaling technology Inc, MA, USA, used 1:500), monoclonal mouse anti-KDEL (10C3, ENZO Life Sciences, NY, USA, used 1:500), polyclonal anti-Rab11a (2413S, Cell Signaling Technology Inc, MA, USA, used 1:50), mouse monoclonal anti α-smooth muscle actin (1A4, Abcam, Cambridge, UK, used 1:500), polyclonal rabbit anti-Calponin, CNN1 (ab46794, Abcam, Cambridge, UK, used 1:500), monoclonal anti-Oct4 (9B7, R&D systems, MN, USA, used 1:500), mouse monoclonal anti-SOX2 (245610, R&D systems, MN, USA, used 1:500).

Techniques: Quantitation Assay, Staining, Construct