Transendocytosis is impaired in CADASIL-mutant NOTCH3.

Mutations in the human NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), but the pathogenesis of CADASIL has remained unclear. Recently, endocytosis of the Notch ectodermal domain into ligand-expressing cells, called transendocytosis, has come to be considered critical for Notch activation. We hypothesized that the mutant NOTCH3 protein, particularly the ectodermal domain of NOTCH3 (N3ECD), may be refractory to degradation on the cell surface due to impaired transendocytosis. We established a co-culture system in which HEK293 cells stably expressing one copy of tetracycline-regulated NOTCH3 were cultured with NOTCH3 ligand Jagged1 (Jag1)-expressing HEK293 cells. We obtained three main results: first, the C185R mutant N3ECD on the cell surface was degraded significantly more slowly than the wild N3ECD when NOTCH3 cells were co-cultured with Jag1-expressing cells. Second, both the wild-type and mutant NOTCH3-expressing cells increased HES1 expression on co-culture with ligand-expressing cells. Third, vesicles containing N3ECD were observed in Jag1-expressing cells. Vesicles of mutant N3ECD within the Jag1-expressing cells were significantly less in number than in the case of wild-type N3ECD. These results indicated that the process of degradation of mutant N3ECD on the cell surface is disturbed due to impairment of transendocytosis. Such disturbance on the surface of vascular smooth muscle cells may contribute to the pathogenesis of CADASIL.