Human Molecular Genetics Advance Access originally published online on May 5, 2009
Human Molecular Genetics 2009 18(15):2761-2767; doi:10.1093/hmg/ddp211
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CADASIL mutations enhance spontaneous multimerization of NOTCH3
1 Department of Neurology 2 Department of Neuropathology, Ludwig-Maximilians-University, Munich, Germany
* To whom correspondence should be addressed at: Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, D-81377 Munich, Germany. Tel: +49 8970956670; Fax: +49 8970957802; Email: martin.dichgans{at}med.uni-muenchen.de
Received February 11, 2009; Revised March 31, 2009; Accepted April 30, 2009
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cause of stroke and vascular dementia. Disease-causing mutations invariably affect cysteine residues within epidermal growth factor-like repeat domains in the extracellular domain of the NOTCH3 receptor (N3ECD). The biochemical and histopathological hallmark of CADASIL is the accumulation of N3ECD at the cell surface of vascular smooth muscle cells which degenerate over the course of the disease. The molecular mechanisms leading to N3ECD accumulation remain unknown. Here we show that both wild-type and CADASIL-mutated N3ECD spontaneously form oligomers and higher order multimers in vitro and that multimerization is mediated by disulfide bonds. Using single-molecule analysis techniques (‘scanning for intensely fluorescent targets’), we demonstrate that CADASIL-associated mutations significantly enhance multimerization compared with wild-type. Taken together, our results for the first time provide experimental evidence for N3 self-association and strongly argue for a neomorphic effect of CADASIL mutations in disease pathogenesis.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.