Reduced SUMOylation impairs NOTCH3 signaling and cell survival in the pathogenesis of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebral small vessel disease caused by NOTCH3 mutation. The condition leads to recurrent ischemic strokes, vascular dementia, early-onset and high disability, and its prevalence has long been underestimated. Pathologically, CADASIL involves the degeneration and loss of brain vascular smooth muscle cells (VSMCs), but the mechanisms remain unclear. Using a transgenic mouse model of CADASIL (NOTCH3-R545C) and NOTCH3 mutant (R90C and R544C) cell models, the study identifies impaired NOTCH3 signaling, resulting from reduced SUMOylation, as a pivotal pathogenic mechanism that compromises cell survival and proliferation. We found that the NOTCH3-R545C mice exhibited anxiety-like behaviors, spatial working memory deficits, and reduced mural cell coverage. In primary VSMCs and HEK293 cells, the NOTCH3 mutation diminished cell viability, proliferation and NOTCH3 cleavage. Mechanistically, NOTCH3 mutations reduced NOTCH3 SUMOylation. This reduction diminished the interaction between the NOTCH3 intracellular domain (NOTCH3ICD) and the transcription factor RBPjκ, thereby impairing downstream NOTCH3 signaling. Overexpression of the SUMOylation molecule SUMO1 restored NOTCH3 cleavage, stability, transcriptional activity, target gene expression, and cell survival/proliferation. In contrast, the deSUMOylation enzyme SENP1 and SUMOylation-deficient NOTCH3 mutants exacerbated these impairments. These findings demonstrate that reversible SUMOylation of NOTCH3 serves as a critical regulator of VSMC homeostasis, with SUMO1 and SENP1 functioning as key mediators. This study provides novel insights into CADASIL pathogenesis by linking NOTCH3 SUMOylation to vascular dysfunction and further highlights SUMOylation as a potential target for the therapeutic development of CADASIL.