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NOTCH3 signal activation by its extracellular domain accumulation in an iPSC line newly established from a CADASIL

Ruihua Sun1, Zhenxiang Zhao1, Ningning Che1

  • 1Department of Neurology, Henan Provincial People's Hospital & Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China.

Human Cell
|May 24, 2026
PubMed

Insights

Researchers developed a patient-derived induced pluripotent stem cell (iPSC) model for Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) carrying the NOTCH3 R544C mutation. This model reveals early NOTCH3 extracellular domain accumulation and pathway activation.

Area of Science:

  • Neuroscience
  • Genetics
  • Stem Cell Biology

Background:

  • Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a genetic small-vessel disease caused by NOTCH3 mutations.
  • The R544C mutation in NOTCH3 is common in East Asian populations, but patient-specific models are scarce.
  • Understanding CADASIL pathogenesis is hindered by a lack of suitable disease models.

Purpose of the Study:

  • To establish and characterize an induced pluripotent stem cell (iPSC) line from a CADASIL patient with the NOTCH3 R544C mutation.
  • To investigate the early molecular mechanisms of CADASIL using this patient-derived iPSC model.
  • To provide a valuable tool for studying CADASIL pathogenesis and developing therapeutic strategies.

Main Methods:

  • Generated iPSCs from peripheral blood mononuclear cells of a CADASIL patient with a heterozygous NOTCH3 R544C mutation using Sendai virus reprogramming.
  • Characterized iPSCs for morphology, karyotype, pluripotency marker expression (OCT4, NANOG, TRA-1-60, SSEA-4), vector clearance, germ layer differentiation potential, and donor identity (STR analysis).
  • Assessed NOTCH3 protein levels (extracellular domain, full-length, intracellular domain) and downstream gene expression (HEY1, NRARP, HES1) in CADASIL iPSCs.

Main Results:

  • Successfully generated and characterized a patient-derived iPSC line with normal karyotype and pluripotency.
  • CADASIL iPSCs exhibited abnormal accumulation of the NOTCH3 extracellular domain (NOTCH3ECD).
  • NOTCH3 signaling pathway activation was indicated by the upregulation of downstream genes HEY1, NRARP, and HES1.

Conclusions:

  • Established a novel, patient-derived iPSC line for CADASIL research, specifically modeling the NOTCH3 R544C mutation.
  • The iPSC model demonstrates early NOTCH3ECD accumulation and NOTCH3 pathway activation, offering insights into CADASIL pathogenesis.
  • This model serves as a crucial resource for investigating disease mechanisms and exploring potential therapeutic interventions for CADASIL.

Keywords:
CADASILDisease modelInduced pluripotent stem cellsNOTCH3R544C mutation

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