Lunatic fringe promotes the aggregation of CADASIL NOTCH3 mutant proteins.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease characterized by NOTCH3 mutation and abnormal aggregation of NOTCH3 mutant proteins around vessel walls. NOTCH3 is a transmembrane receptor that is degraded by JAGGED1 (JAG1) through a process called trans-endocytosis. There are two types of CADASIL-associated NOTCH3 mutations: signal-active (SA) and signal-deficient (SD) mutations. However, the conditions that lead to abnormal aggregation of NOTCH3 mutant proteins remain poorly understood. Performing a coculture assay, we found that the SA NOTCH3 mutants (C49Y, R90C, R141C, and C185R) were degraded and trans-endocytosed by JAG1 similar to wild-type (WT) NOTCH3, but the SD NOTCH3 mutant (C428S) was not degraded or endocytosed by JAG1, suggesting that other environmental factors may be necessary for the aggregation of SA NOTCH3 mutants. Lunatic fringe (LFNG) is a glycosyltransferase of NOTCH3, but whether LFNG affects the aggregation of NOTCH3 mutants remains unknown. Performing a sucrose gradient ultracentrifugation assay, we found that LFNG might decrease the aggregation propensity of WT NOTCH3 but increase that of C185R NOTCH3. In conclusion, the SD NOTCH3 mutant may be more likely to accumulate than the SA NOTCH3 mutants upon interaction with JAG1. Moreover, LFNG may play an important role in promoting the aggregation of SA NOTCH3 mutants.