Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice
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
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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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 7

Notch3 deficiency results in vascular dysfunction due to impaired contractility.

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Notch3 deficiency results in vascular dysfunction due to impaired contr...
(A) Diagram of experimental design. VSMCs from 1-month control and Notch3–/– mice were isolated and mixed with type I collagen to form a cellular hydrogel, detached, and observed 24 hours later. (B) Images of polymer gels from control and Notch3–/– cells after 24 hours. Note the differences in the contraction of the gel with regard to diameter (white dashed line) and total size of gel (red dashed line). (C) Quantification of percent gel diameter reduction 24 hours after plating. Bars indicate mean ± SD; n = 4 biological replicates; Welch’s t test. (D) Immunofluorescence of phosphorylated myosin light chain 2 (p-MLC2; red). White arrows highlight αSMA (green); yellow arrows highlight p-MLC2 (red). (E) VSMC-enriched lysates from mixed-sex cohorts of Notch3–/– and control aortae at the indicated ages were evaluated for expression of p-MLC2, MLC2, calponin, and lamin A (loading control). (F) Quantification of p-MLC2/MLC2 from a 1-month mixed-sex cohort. Data are shown as the mean ± SD; n = 6–7; Welch’s t test. Female data are indicated by inverted triangles, male data by squares. (G) Pulse pressure in 18-month-old male control and Notch3–/– mice. (H) Heart rate measured in 18-month male control and Notch3–/– mice. (I) Simplified diagram of the molecular pathway of acetylcholine-driven relaxation in VSMCs and quantification of systolic blood pressure response to acetylcholine in 18-month male control and Notch3–/– animals. (J) Simplified diagram of the molecular pathway of phenylephrine-driven contraction in VSMCs and quantification of systolic blood pressure response to phenylephrine in 18-month male control and Notch3–/– animals. For G and H, data are shown as the mean ± SD; n = 9–13, unpaired Student’s t test. For I and J, data are shown as the mean ± SD; n = 5–7, unpaired Student’s t test.