# Transformation of NOTCH3 protein in cerebral small vessel disease

> **NIH VA I01** · VETERANS HEALTH ADMINISTRATION · 2020 · —

## Abstract

ABSTRACT
Cerebral small vessel disease (SVD) affects over half of Americans over 65 and is a major risk
factor for stroke and dementia. Although the pathological changes in blood vessels in SVD have
been described for decades, the precise molecular defects are unclear. CADASIL is the most
common inherited form of SVD and is caused by mutations in NOTCH3. We investigate the
pathogenesis of CADASIL as a potential window into uncovering targetable mechanisms of
vascular degeneration of the brain. Molecular mapping of affected families has provided
important clues to the molecular pathways leading to arterial degeneration in CADASIL. The
SVD-causing mutations in NOTCH3 either create or delete one cysteine residue, supporting the
hypothesis that disulfide dependent conformational alterations in NOTCH3 trigger pathology of
brain small vessels. In new preliminary data, we use a set of new NOTCH3 antibodies,
including monoclonal probes, that bind to conformations of NOTCH3 that are strongly
expressed in pathologically affected CADASIL arteries. These antibodies specifically bind to
reduced NOTCH3 protein; other denaturants fail to induce the CADASIL conformational change.
Site-directed mutagenesis of NOTCH3 demonstrates that the CADASIL conformation of
NOTCH3 is generated after mutation of multiple cysteines, leading us to hypothesize that the
unique NOTCH3 protein expressed in CADASIL results from reduction of more than one
disulfide bond. We call this protein Multiple Reduced Cysteine NOTCH3 (mrc-N3). The
experiments of this proposal aim to characterize mrc-N3. We propose to test the following
hypotheses (Figure 1): (1) mrc-N3 is generated from reduction of at least two disulfide bonds
and genesis of mrc-N3 is facilitate by NOTCH3 binding proteins from the vessel wall; (2) mrc-N3
activates transcription of disease related proteins and triggers smooth muscle cell stress
pathways; (3) mrc-N3 functions in vivo to cause vascular dysfunction and pathology. These
Aims will be tested using biochemical techniques incorporating purified NOTCH3 proteins, cell
culture using primary human cerebral smooth muscle, pathological analysis of unique tissue
resources available to our lab, and in vivo testing of mrc-N3 function in genetically modified
mice. Successful execution of these studies will shed new light on key molecular mechanisms
of small vessel disease.

## Key facts

- **NIH application ID:** 9815430
- **Project number:** 5I01BX003855-03
- **Recipient organization:** VETERANS HEALTH ADMINISTRATION
- **Principal Investigator:** Michael M Wang
- **Activity code:** I01 (R01, R21, SBIR, etc.)
- **Funding institute:** VA
- **Fiscal year:** 2020
- **Award amount:** —
- **Award type:** 5
- **Project period:** 2017-10-01 → 2021-09-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9815430

## Citation

> US National Institutes of Health, RePORTER application 9815430, Transformation of NOTCH3 protein in cerebral small vessel disease (5I01BX003855-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9815430. Licensed CC0.

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