Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited systemic vascular disorder characterized by recurrent subcortical ischemic strokes leading to vascular dementia. The gold standard to confirm the diagnosis is to identify a mutation in the underlying gene NOTCH3, encoding a transmembrane receptor protein. Granular osmiophilic material (GOM) deposition around vascular smooth muscle cells is a specific diagnostic feature of CADASIL and electron microscopic examination of a skin biopsy is another useful method for its diagnosis. Although accumulation of Notch3 ectodomain on the surface of vascular smooth muscle cells has been reported, the composition of GOM has not been elucidated. To elucidate the relationship between Notch3 protein and GOM, we performed postembedding immunogold electron microscopy using cryofixed and freeze substituted skin taken from two CADASIL patients. Our results demonstrate that GOM around vascular smooth muscle cells was specifically labeled with antibodies against the extracellular portion of Notch3 but not with antibodies recognizing the intracellular Notch3 domain. In non-CADASIL skin sections, no antibody binding was detected around the small dermal arteries. From these results, the major component of GOM in CADASIL patients is the ectodomain of the Notch3 gene product. Our results shed light on the relationship between Notch3 gene mutations and morphological deposition of GOM around the vascular smooth muscle cells.
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