Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice
Milagros C. Romay, … , Beth A. Kozel, M. Luisa Iruela-Arispe
Milagros C. Romay, … , Beth A. Kozel, M. Luisa Iruela-Arispe
Published November 28, 2023
Citation Information: J Clin Invest. 2024;134(2):e166134. https://doi.org/10.1172/JCI166134.
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Research Article Neuroscience Vascular biology

Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice

Abstract

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Authors

Milagros C. Romay, Russell H. Knutsen, Feiyang Ma, Ana Mompeón, Gloria E. Hernandez, Jocelynda Salvador, Snezana Mirkov, Ayush Batra, David P. Sullivan, Daniele Procissi, Samuel Buchanan, Elise Kronquist, Elisa A. Ferrante, William A. Muller, Jordain Walshon, Alicia Steffens, Kathleen McCortney, Craig Horbinski, Elisabeth Tournier‑Lasserve, Adam M. Sonabend, Farzaneh A. Sorond, Michael M. Wang, Manfred Boehm, Beth A. Kozel, M. Luisa Iruela-Arispe

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Figure 1

The aging vasculature experiences progressive loss of Notch3 that leads to ongoing disorganization, dedifferentiation, and detachment of VSMCs.

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The aging vasculature experiences progressive loss of Notch3 that leads ...
(A) Retinal vasculature from C57BL/6J mice at indicated ages. αSMA (green) identifies VSMCs, and PECAM (red) identifies endothelium. White arrows highlight VSMC loss. Scale bars: 200 μm (top row), 50 μm (bottom row). (B) Quantification of VSMC coverage at each time point from mixed-sex cohorts. Data are shown as the mean ± SD; n = 3–6. Welch’s t test. (C) Experimental design: Meningeal tissue and penetrating arteries were dissected from young (1 month) and aged (24 months) mice for scRNA-Seq. (D) Heatmap visualizing the top 50 differentially expressed genes (DEGs) in VSMCs. Green circles indicate genes that regulate muscle cell contraction. (E–L) Violin plots from selected transcripts. (M) Human brain vessel sections stained with αSMA (green) to visualize smooth muscle and NOTCH3 (white). White arrows indicate NOTCH3 in VSMC nuclei. Scale bars: 2 μm. (N) Quantification of NOTCH3+ nuclei in human brain VSMCs (20–50 μm vessel diameter) at indicated age ranges. (O) NOTCH3 mean intensity per nuclei in human brain VSMCs. For N and O, data are shown as the mean ± SD; n = 43–53 vessels, 6–7 patients per age group. Kruskal-Wallis with multiple testing correction.