Biochemical Characterization and Cellular Effects of CADASIL Mutants of NOTCH3

He Meng,Genggeng Yu,Soo Jung Lee,Michael M Wang,Igor Prudovsky,Y Eugene Chen,Xiaojie Zhang,Huaibin Cai

doi:10.1371/journal.pone.0044964

Abstract

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells.

Highlights

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited arteriopathy marked by migraine headaches, premature stroke, and vascular dementia [1,2,3]
We sought to answer four questions: 1) do wildtype or mutant NOTCH3 ectodomains physically interact with NOTCH1, 3, and 4 ectodomains; 2) do mutations in NOTCH3 affect detergent solubility; 3) do mutations in NOTCH3 impair protein clearance; and 4) does NOTCH3 overexpression affect Notch regulation of smooth muscle genes?
Mixing of NOTCH3 proteins expressed separately did not result in coprecipitated protein, indicating that interactions are favored by co-expression of proteins within the same cell

Summary

Introduction

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited arteriopathy marked by migraine headaches, premature stroke, and vascular dementia [1,2,3]. Tissues from patients with CADASIL display damage to smooth muscle cells of small arteries and arterioles, characterized by cellcell separation and cell loss, accumulation of granular osmiophilic material (GOM) around cells, fibrosis, and marked accumulation of NOTCH3 protein in the media [17,18,19,20]. In spite of this detailed description of pathology, a comprehensive view of the molecular and cellular changes induced by NOTCH3 excess has yet to emerge. Since NOTCH regulates crucial smooth muscle genes, we tested whether the overexpression of NOTCH3 affects the regulation of genes known to regulate smooth muscle homeostasis

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