Functional analysis of a recurrent missense mutation in Notch3 in CADASIL

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited vascular dementia linked to Notch3 gene mutations.
• CADASIL mutations often alter cysteine residues in the Notch3 receptor's extracellular domain.
• Accumulation of Notch3 extracellular domain occurs in vessels affected by CADASIL.

Purpose of the Study:

• Investigate functional consequences of a specific CADASIL mutation on Notch3 receptor processing.
• Assess the impact on cell surface expression, ligand binding, and downstream signaling (CBF1 activation).

Main Methods:

• Expressed wild-type and mutant Notch3 receptors in cultured cells.
• Utilized flow cytometry to measure binding to Notch ligands (Delta1, Delta4, Jagged1).
• Employed co-culture systems to assess ligand-dependent activation of a CBF1-luciferase reporter.

Main Results:

• Identified subtle abnormalities in furin processing of the mutant Notch3 receptor.
• Confirmed presence of both heterodimeric and full-length mutant receptors on the cell surface.
• Demonstrated retained ability of the mutant receptor to bind ligands and activate CBF1.

Conclusions:

• Individual CADASIL mutations can exhibit distinct effects on Notch3 receptor expression and function.
• The studied mutation results in subtle Notch3 processing alterations but preserved signaling capacity.