A Novel Mutation in Exon 10 of the NOTCH3 Gene in Human Cerebral Microvascular Endothelial Cells Induces CADASIL-Like Pathology and the Therapeutic Effect of Edaravone Dexborneol on Hereditary and Non-hereditary Cerebral Small Vessel Disease.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene. We reported a novel mutation in the exon 10 of the NOTCH3 gene (NOTCH3 p.C533S) in a Chinese family with CADASIL. This study aimed to identify NOTCH3 p.C533S-induced CADASIL-like pathogenic mechanisms and to observe the therapeutic effects of edaravone dexborneol (EDB) on CADASIL, which is clinically used for acute ischemic stroke in China. We generated human cerebral microvascular endothelial cells (hCMEC) carrying NOTCH3 p.C533S mutation (NOTCH3 p.C533S hCMECs) using CRISPR/Cas9-mediated NOTCH3 knock-in with a donor plasmid. The NOTCH3 p.C533S hCMECs exhibited CADASIL-like pathology, characterized by a loss of tight junctions, diminished proliferative and migratory capacities, and an upregulation in pro-inflammatory cytokines. The NOTCH3 p.C533S hCMECs induced neuronal ferroptosis, pro-inflammatory activation of astrocytes and microglia with accumulated lipid droplets and oligodendrocytes damage. Mechanistically, utilizing metabolomics and multiple biochemical approaches, we identified that downregulated vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway contribute to the reduced activities of glutathione reductase (GR) and argininosuccinate synthase 1 (ASS1), resulting in reduced glutathione and arginine levels, as well as NOTCH3 p.C533S hCMECs degeneration. EDB treatment could partially reverse CADASIL- and non-hereditary cerebral small vessel disease-like pathology by restoring the VEGF/VEGFR-regulated ASS1 and GR. The NOTCH3 p.C533S mutation causes hCMEC degeneration associated with VEGF/VEGFR pathway impairment-mediated cellular metabolic reprogramming and inflammation. The progression of CADASIL is intensified by a cascade of interactions between compromised hCMEC and surrounding neurons and glial cells. EDB may offer a promising therapeutic approach for cerebral small vessel disease including CADASIL and non-hereditary one.