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Functional analysis of a recurrent missense mutation in Notch3 in CADASIL
  1. T Haritunians1,
  2. T Chow2,
  3. R P J De Lange3,
  4. J T Nichols4,
  5. D Ghavimi5,
  6. N Dorrani1,
  7. D M St Clair3,
  8. G Weinmaster4,
  9. C Schanen5
  1. 1Departments of Human Genetics, University of California, Los Angeles, CA, USA
  2. 2The Rotman Research Institute, University of Toronto, Toronto, ON, Canada
  3. 3Department of Mental Health, University of Aberdeen, Medical School, Foresterhill, Aberdeen, UK
  4. 4Departments of Biological Chemistry, University of California, Los Angeles, CA, USA
  5. 5Nemours Biomedical Research, Alfred I duPont Hospital for Children, Nemours Children’s Clinic, Wilmington, DE, USA
  1. Correspondence to:
 Dr C Schanen
 Nemours Biomedical Research, Alfred I duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE 19803, USA; schanenmedsci.udel.edu

Abstract

Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterised by recurrent ischemic strokes in the deep white matter. Mutations in the gene encoding the cell surface receptor, Notch3, have been identified in CADASIL patients, and accumulation of the extracellular domain of Notch3 has been demonstrated in affected vessels. Almost all CADASIL mutations alter the number of cysteine residues in the epidermal growth factor (EGF)-like repeats in the extracellular domain of the protein.

Objectives: To understand the functional consequences of a recurrent CADASIL mutation on furin processing, cell surface expression, ligand binding, and activation of a downstream effector CBF1 by the Notch3 receptor.

Methods: We expressed wild type and mutant Notch3 receptors in cultured cells and examined cell surface expression of the proteins. We also applied a new flow cytometry based approach to semi-quantitatively measure binding to three Notch ligands. Additionally, we used a well characterised co-culture system to examine ligand dependent activation of transcription from a CBF1-luciferase reporter construct.

Results: These studies revealed subtle abnormalities in furin processing of the mutant receptor, although both heterodimeric and full length receptors are present on the cell surface, are capable of interacting with soluble forms of three ligands, Delta1, Delta4, and Jagged1, and retain the ability to activate CBF1 in a ligand dependent manner.

Conclusions: By comparison with other mutant forms of Notch3, these data indicate that individual CADASIL mutations can have disparate effects on Notch3 expression and function.

  • CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
  • CBF-1, C promoter binding factor-1
  • DMEM, Dulbecco’s modified Eagle’s medium
  • ECD, extracellular domain
  • EGF, epidermal growth factor
  • EM, electron microscopy
  • FBS, fetal bovine serum
  • GOM, granular osmiophilic material
  • HA, haemagglutinin
  • ICD, intracellular domain
  • MI, myocardial infarction
  • MRI, magnetic resonance imaging
  • VSMC, vascular smooth muscle cell
  • cell surface receptor
  • stroke
  • EGF-like repeat
  • hereditary dementia
  • multi-infarct

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Footnotes

  • Competing interests: none