NOTCH3 mutations associated with CADASIL alter lipid metabolism, with lipidomic profiling revealing significant alterations in plasma lipid profiles in CADASIL patients and in vitro models showing increased lipid droplet accumulation, elevated cholesterol levels, and reduced expression of cholesterol transporters ABCA1 and ABCG1.
Key Findings
Results
Plasma lipidomic profiling revealed significant alterations in lipid profiles in CADASIL patients compared with healthy controls.
Plasma samples from CADASIL patients and matched healthy controls were subjected to lipidomic analysis.
The study compared CADASIL patient plasma against matched healthy control plasma, establishing a clinical baseline for lipid dysregulation in this condition.
The lipidomic analysis identified significant differences in plasma lipid profiles between the two groups.
Results
Lipid droplet (LD) accumulation was significantly increased in 293T cells expressing NOTCH3 mutants R110C, R1175W, and R544C, but not in the R133C mutant group.
Stable 293T cell lines were established expressing an empty vector (NC), wild-type (WT) NOTCH3, or CADASIL-associated NOTCH3 mutants (R110C, R133C, R1175W, and R544C).
Three of the four mutants tested (R110C, R1175W, and R544C) showed significantly increased LD accumulation compared to controls.
The R133C mutant group did not show significant LD accumulation, indicating mutant-specific differences in lipid droplet dysregulation.
These findings suggest that lipid droplet accumulation is not a universal consequence of all NOTCH3 mutations but is mutation-dependent.
Results
Cholesterol levels were elevated in all four NOTCH3 mutant groups in 293T cells.
Unlike lipid droplet accumulation, cholesterol elevation was observed across all mutant groups: R110C, R133C, R1175W, and R544C.
Wild-type NOTCH3 and empty vector (NC) controls were used as comparators.
This finding suggests that cholesterol dysregulation may be a more universal consequence of CADASIL-associated NOTCH3 mutations than lipid droplet accumulation.
Results
Expression of the R544C NOTCH3 mutant in hCMEC/D3 cerebral endothelial cells markedly promoted cholesterol accumulation.
hCMEC/D3 cell lines expressing NC, WT, or R544C NOTCH3 were established to model the disease in a cerebrovascular endothelial cell context.
The R544C mutant was selected for hCMEC/D3 studies to examine lipid metabolic alterations in a cell type relevant to CADASIL pathology.
Cholesterol accumulation was markedly increased in R544C-expressing hCMEC/D3 cells compared to NC and WT controls.
Results
Expression of the R544C NOTCH3 mutant in hCMEC/D3 cells reduced the expression of cholesterol transporters ABCA1 and ABCG1.
Both ABCA1 and ABCG1, which are key cholesterol efflux transporters, showed reduced expression in R544C mutant-expressing hCMEC/D3 cells.
Downregulation of ABCA1 and ABCG1 may contribute to the observed cholesterol accumulation by impairing cholesterol efflux from cells.
These findings implicate disrupted cholesterol transport as a mechanistic link between NOTCH3 mutations and lipid dysregulation in CADASIL.
Conclusions
NOTCH3 mutations are associated with disrupted cellular lipid homeostasis, suggesting lipid metabolic dysregulation as a potential mechanism contributing to CADASIL pathogenesis.
The study integrated findings from patient plasma lipidomics and two in vitro cell models (293T and hCMEC/D3).
Four distinct CADASIL-associated mutations (R110C, R133C, R1175W, R544C) were studied, providing breadth across different mutation sites.
The authors conclude that "lipid metabolic dysregulation may represent a potential mechanism contributing to CADASIL pathogenesis."
The convergence of plasma lipidomic alterations and in vitro lipid homeostasis disruptions supports a role for lipid metabolism in CADASIL disease mechanisms.
What This Means
CADASIL is the most common inherited form of cerebral small vessel disease, caused by mutations in a gene called NOTCH3. It leads to strokes and dementia, but exactly how these mutations damage blood vessels is not fully understood. This research suggests that NOTCH3 mutations significantly disrupt how cells handle fats (lipids), potentially contributing to the disease process. By analyzing blood plasma from CADASIL patients and comparing it to healthy controls, researchers found that the overall lipid composition in patient blood was significantly altered. Laboratory experiments using cells engineered to carry different CADASIL mutations further showed that most of these mutations caused fat droplets to accumulate inside cells and led to elevated cholesterol levels.
In experiments using brain endothelial cells — the type of cells lining blood vessels in the brain and most relevant to CADASIL — one of the mutations (R544C) caused notable cholesterol buildup and reduced the activity of two proteins (ABCA1 and ABCG1) that normally help remove cholesterol from cells. This suggests that when NOTCH3 is mutated, cells may lose their ability to properly clear cholesterol, leading to its abnormal accumulation. Interestingly, not all mutations behaved identically: for example, one mutation (R133C) did not cause fat droplet accumulation even though it still raised cholesterol levels, indicating that different mutations may affect lipid metabolism through slightly different pathways.
This research suggests that abnormal fat metabolism in blood vessel cells may be an important but previously underappreciated part of how CADASIL causes damage to the brain's small blood vessels. Understanding this lipid connection could open new avenues for studying CADASIL and potentially for identifying targets for future treatments, though further research is needed to confirm these mechanisms in living organisms and in a broader range of patients.
Sun R, Ji X, Hu W, Li W, Zhao Y, Wang W, et al.. (2026). NOTCH3 Mutations in CADASIL Alter Lipid Metabolism: Insights from Patient Plasma and In Vitro Models.. Neurochemical research. https://doi.org/10.1007/s11064-026-04866-9