NOTCH3 Deficiency Accelerates the Neurovascular Aging Clock via Innate Immune Activation and Vascular Smooth Muscle Cell Senescence

A. FARIA ASSONI1 , CECILIA GALLOTTINI DE MAGALHAES2 , JOELCIMAR MARTINS DA SILVA1 , ORLANDO SOARES LOUZADA NETO1 , IGOR NEVES1 , JOYCE ESPOSITO1 , ALEXANDER TYSHKOVSKIY2,3, ADRIAN MOLIÈRE2,3, VADIM N. GLADYSHEV2,5, MAYANA ZATZ1 1UNIV. DE SÃO PAULO, SÃO PAULO, BRAZIL; 2DIVISION OF GENETICS, DEPARTMENT OF MEDICINE, BRIGHAM AND WOMEN’S HOSPITAL, HARVARD MEDICAL SCHOOL, BOSTON, USA, 3BELOZERSKY INSTITUTE OF PHYSICO-CHEMICAL BIOLOGY, MOSCOW STATE UNIVERSITY, MOSCOW, RUSSIA, 4 INSTITUTE OF MOLECULAR HEALTH SCIENCES, DEPARTMENT OF BIOLOGY, ETH ZÜRICH, SWITZERLAND, 5BROAD INSTITUTE OF HARVARD AND MIT, CAMBRIDGE, MA, USA

Background: Neurovascular unit (NVU) dysfunction is a hallmark of early Alzheimer’s disease (AD)¹. While NOTCH3 mutations cause CADASIL², the role of NOTCH3 in broader neurovascular aging remains unclear. We investigated how NOTCH3 loss impacts the biological aging trajectory and functional survival of brain vascular smooth muscle cells (vSMCs).

Methods: We reanalised publicly available single cell RNA-sequencing of mice brain and focused on comparisons between vessel Smooth muscle cells (vSMCs) from wild-type (WT) and Notch3 knockout (KO) mice³, applying a transcriptomic aging clock⁴ to assess biological age. DEGs were analyzed for pathway enrichment and AD-GWAS overlap. Findings were validated in human iPSC-derived vascular organoids. Flow cytometry was used to quantify Active Caspase-3 and p21 expression. Genomic integrity was assessed via neutral comet assays (with/without H2O2 challenge) and yH2Ax staining.

Results: Notch3 KO vSMCs exhibited a significant acceleration in biological age, primarily driven by the activation of innate immune pathways. Transcriptomic profiling also revealed a collapse of oxidative phosphorylation, overlapping with AD-associated GWAS loci (e.g., CYCS, BCAS3). In human vascular organoids, NOTCH3 deficiency led to a robust increase in p21-positive senescent cells and Active Caspase-3-mediated apoptosis as measured by flow cytometry. These changes were coupled with impaired migration and increased double-strand breaks in neutral comet assays. Notably, KO cells showed a heightened sensitivity to H2O2, suggesting that immune-driven aging compromises the DNA damage response.

Conclusions: Our results demonstrate that NOTCH3 is a critical gatekeeper of the neurovascular aging clock. We propose that NOTCH3 loss drives a "vulnerable" vascular phenotype through innate immune activation and accelerated senescence, providing a mechanistic bridge between vascular-specific genetic risk and the progression of Alzheimer’s disease.

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3.          Masuda, T. et al. Specification of CNS macrophage subsets occurs postnatally in defined niches. Nature 604, 740–748 (2022).

4.          Tyshkovskiy, A. et al. Universal transcriptomic hallmarks of mammalian ageing and mortality. Nature 2026 654:8117 654, 173–188 (2026).