NOTCH3 Mutations in CADASIL Alter Lipid Metabolism: Insights from Patient Plasma and In Vitro Models

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common monogenic form of cerebral small vessel disease (CSVD), is caused by mutations in NOTCH3. However, the effects of NOTCH3 mutations on lipid metabolism remain poorly understood. In this study, plasma samples from CADASIL patients and matched healthy controls were subjected to lipidomic analysis. Stable 293T cell lines expressing an empty vector (NC), wild-type (WT) NOTCH3, or CADASIL-associated NOTCH3 mutants (R110C, R133C, R1175W, and R544C), as well as hCMEC/D3 cell lines expressing NC, WT, or R544C, were established to investigate lipid metabolic alterations. Lipid droplet (LD) accumulation, cholesterol levels, and the expression of cholesterol transport-related molecules were assessed. Lipidomic profiling revealed significant alterations in plasma lipid profiles in CADASIL patients compared with healthy controls. In 293T cells, LD accumulation was significantly increased in the R110C, R1175W, and R544C groups, but not in the R133C group, whereas cholesterol levels were elevated in all mutant groups. In hCMEC/D3 cells, expression of the R544C mutant markedly promoted cholesterol accumulation and reduced the expression of the cholesterol transporters ABCA1 and ABCG1. These findings indicate that NOTCH3 mutations are associated with disrupted cellular lipid homeostasis and suggest that lipid metabolic dysregulation may represent a potential mechanism contributing to CADASIL pathogenesis.