Functional analysis of a recurrent missense mutation in Notch3 in CADASIL

T. Haritunians, T. Chow, P. J. De Lange, J. T. Nichols, D. Ghavimi, N. Dorrani, David Malcolm St Clair, G. Weinmaster, C. Schanen

Research output: Contribution to journalArticle

33 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)1242-1248
Number of pages6
JournalJournal of Neurology, Neurosurgery & Psychiatry
Volume76
Issue number9
DOIs
Publication statusPublished - Sep 2005

Fingerprint

CADASIL
Missense Mutation
Ligands
Furin
Mutation
Vascular Dementia
Aptitude
Cell Surface Receptors
Coculture Techniques
Luciferases
Epidermal Growth Factor
Transcriptional Activation
Cysteine
Cultured Cells
Flow Cytometry
Membrane Proteins
Stroke
Genes

Keywords

  • autosomal dominant arteriopathy
  • smooth muscle cells
  • subcortical infarcts
  • intracellular domain
  • physical interaction
  • mammalian notch
  • nuclear access
  • receptor
  • ligand
  • leukoencephalopathy

Cite this

Haritunians, T., Chow, T., De Lange, P. J., Nichols, J. T., Ghavimi, D., Dorrani, N., ... Schanen, C. (2005). Functional analysis of a recurrent missense mutation in Notch3 in CADASIL. Journal of Neurology, Neurosurgery & Psychiatry, 76(9), 1242-1248. https://doi.org/10.1136/jnnp.2004.051854

Functional analysis of a recurrent missense mutation in Notch3 in CADASIL. / Haritunians, T.; Chow, T.; De Lange, P. J.; Nichols, J. T.; Ghavimi, D.; Dorrani, N.; St Clair, David Malcolm; Weinmaster, G.; Schanen, C.

In: Journal of Neurology, Neurosurgery & Psychiatry, Vol. 76, No. 9, 09.2005, p. 1242-1248.

Research output: Contribution to journalArticle

Haritunians, T, Chow, T, De Lange, PJ, Nichols, JT, Ghavimi, D, Dorrani, N, St Clair, DM, Weinmaster, G & Schanen, C 2005, 'Functional analysis of a recurrent missense mutation in Notch3 in CADASIL', Journal of Neurology, Neurosurgery & Psychiatry, vol. 76, no. 9, pp. 1242-1248. https://doi.org/10.1136/jnnp.2004.051854
Haritunians, T. ; Chow, T. ; De Lange, P. J. ; Nichols, J. T. ; Ghavimi, D. ; Dorrani, N. ; St Clair, David Malcolm ; Weinmaster, G. ; Schanen, C. / Functional analysis of a recurrent missense mutation in Notch3 in CADASIL. In: Journal of Neurology, Neurosurgery & Psychiatry. 2005 ; Vol. 76, No. 9. pp. 1242-1248.
@article{871a71762a6148e2a952028706514a75,
title = "Functional analysis of a recurrent missense mutation in Notch3 in CADASIL",
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.",
keywords = "autosomal dominant arteriopathy, smooth muscle cells, subcortical infarcts, intracellular domain, physical interaction, mammalian notch, nuclear access, receptor, ligand, leukoencephalopathy",
author = "T. Haritunians and T. Chow and {De Lange}, {P. J.} and Nichols, {J. T.} and D. Ghavimi and N. Dorrani and {St Clair}, {David Malcolm} and G. Weinmaster and C. Schanen",
year = "2005",
month = "9",
doi = "10.1136/jnnp.2004.051854",
language = "English",
volume = "76",
pages = "1242--1248",
journal = "Journal of Neurology, Neurosurgery & Psychiatry",
issn = "0022-3050",
publisher = "BMJ Publishing Group",
number = "9",

}

TY - JOUR

T1 - Functional analysis of a recurrent missense mutation in Notch3 in CADASIL

AU - Haritunians, T.

AU - Chow, T.

AU - De Lange, P. J.

AU - Nichols, J. T.

AU - Ghavimi, D.

AU - Dorrani, N.

AU - St Clair, David Malcolm

AU - Weinmaster, G.

AU - Schanen, C.

PY - 2005/9

Y1 - 2005/9

N2 - 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.

AB - 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.

KW - autosomal dominant arteriopathy

KW - smooth muscle cells

KW - subcortical infarcts

KW - intracellular domain

KW - physical interaction

KW - mammalian notch

KW - nuclear access

KW - receptor

KW - ligand

KW - leukoencephalopathy

U2 - 10.1136/jnnp.2004.051854

DO - 10.1136/jnnp.2004.051854

M3 - Article

VL - 76

SP - 1242

EP - 1248

JO - Journal of Neurology, Neurosurgery & Psychiatry

JF - Journal of Neurology, Neurosurgery & Psychiatry

SN - 0022-3050

IS - 9

ER -