Cerebral autosomal dominant arteriopathy with subcortical infarcts and 
leukoencephalopathy, or CADASIL, one of the most common inherited small vessel 
diseases of the brain, is characterized by a progressive loss of vascular smooth 
muscle cells and extracellular matrix accumulation. The disease is caused by 
highly stereotyped mutations within the extracellular domain of the NOTCH3 
receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While 
CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor 
function and activity, they all are associated with early accumulation of 
Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic 
explanation to link NOTCH3 mutations with small vessel pathology. Herein, we 
hypothesized that excess Notch3(ECD) could recruit and sequester functionally 
important proteins within small vessels of the brain. We performed biochemical, 
nano-liquid chromatography-tandem mass spectrometry and immunohistochemical 
analyses, using cerebral and arterial tissue derived from patients with CADASIL 
and mouse models of CADASIL that exhibit vascular lesions in the end- and 
early-stage of the disease, respectively. Biochemical fractionation of brain and 
artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide 
cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. 
Further proteomic and immunohistochemical analyses identified two functionally 
important extracellular matrix proteins, tissue inhibitor of metalloproteinases 
3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing 
aggregates. Using cultured cells, we show that increased levels or aggregation 
of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 
recruitment and accumulation. In turn, TIMP3 promotes complex formation 
including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with 
CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 
species. Moreover, reverse zymography assays show a significant elevation of 
TIMP3 activity in the brain vessels from mice and patients with CADASIL. 
Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in 
CADASIL disease pathology, which posits that aggregation/accumulation of 
Notch3(ECD) in the brain vessels is a central event, promoting the abnormal 
recruitment of functionally important extracellular matrix proteins that may 
ultimately cause multifactorial toxicity. Specifically, our results suggest a 
dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) 
toxicity by impairing extracellular matrix homeostasis in small vessels.