Journal: CNS Neuroscience & Therapeutics
Article Title: NOTCH3 Mutation Causes Glymphatic Impairment and Promotes Brain Senescence in CADASIL
doi: 10.1111/cns.70140
Figure Lengend Snippet: Diminished AQP4 expression in NOTCH3‐R170C astrocyte. (A–C) Brain cells of NOTCH3‐R170C and ‐WT mice (16 weeks of age) were subjected to single cell RNA sequencing (scRNAseq). (A) Left: Merged Uniform manifold approximation and projection (UMAP) plot representing 15 color‐coded cell clusters identified in the combined single‐cell transcriptomes. Cluster names were manually assigned. Right: Feature plots of Notch3 . (B) Expression of Notch3 among brain cells. (C) Violin plots of Notch3 and Aqp4 expression in astrocytes. (D) Brain protein of NOTCH3‐R170C or ‐WT mice (24 weeks of age) was subjected to western blot to evaluate AQP4 expression. N = 3 in each group. ** p < 0.01; by Student's t test (mean ± SEM). (E, F) Primary NOTCH3‐R170C and ‐WT astrocyte cultures were prepared. (E) Aqp4 mRNA level was evaluated with RT‐PCR. (F) AQP4 protein expression in whole cell and plasma membrane of astrocytes was assessed with western blot. Experiments were repeated for three times. * p < 0.05; by Student's t test. Difference of means (± SEM) is displayed. (G) Primary NOTCH3‐R170C and ‐WT astrocyte cultures were subjected to BN page and then 2D SDS page to evaluate the orthogonal arrays of particles (OAPs) of AQP4. (H) Coronal brain sections of NOTCH3‐R170C or ‐WT mice (24 weeks of age) were subjected to immunostaining of GFAP (green) and AQP4 (red). The AQP4 polarization was calculated by AQP4 perivascular endfeet area. White arrowheads indicated the depolarized AQP4 distribution. N = 6 in NOTCH3‐WT group and N = 5 in NOTCH3‐R170C group. * p < 0.05; by Student's t test (mean ± SEM).
Article Snippet: The protein content of the supernatant was diluted by 2X Blue Native PAGE Sample Buffer (Sangon Biotech, C506055‐0005).
Techniques: Expressing, RNA Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Clinical Proteomics, Membrane, SDS Page, Immunostaining
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