ABSTRACT: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a small vessel disease caused by NOTCH3 gene mutations, leading to vascular smooth muscle cell degeneration, arteriopathy, and subcortical ischemic infarcts. Many CADASIL patients, however, also develop cognitive impairment, indicating that neuronal functions are disturbed, but less is known about the cellular and molecular basis of these aspects in CADASIL. In this study, we used a humanized CADASIL mouse model harbouring the R182C mutation (R182C-TgN3), post-mortem human CADASIL brain sections with three different NOTCH3 gene mutations and primary human vascular smooth muscle cells harbouring the R133C NOTCH3 mutation as primary cellular models to characterise the neurovascular contribution to cognitive impairment. To specifically evaluate neuronal, mitochondrial and neurovascular function, we performed ex vivo electrophysiology, immunohistochemistry (confocal and iDISCO+ methods), western blotting, Seahorse assay, quantitative polymerase chain reaction (qPCR), and single-cell RNA sequencing. In CADASIL mice, hippocampal gamma oscillation patterns were impaired along with significant decreases in neuronal fiber length and aberrant neuronal morphology. The latter two phenotypes were also observed in post-mortem human brain tissue from CADASIL patients. Consistent with these findings, we noted significantly lower levels of mitochondrial respiratory complexes in the CADASIL mouse hippocampus, isolated mouse brain vessels and primary human vascular smooth muscle cells. Human vascular smooth muscle cells exhibited reduced oxygen consumption rates leading to reduced ATP production as well as decreased glycolytic capacity in conjunction with increased pro-inflammatory gene expression, suggesting a broader impact on cellular energy metabolism and a neuroinflammatory process. In the CADASIL mice, we also observed extensive accumulation of the NOTCH3 extracellular domain on hippocampal vessels. Light sheet imaging with iDISCO+ clearing demonstrated substantial vascular smooth muscle cell loss and reduced vessel density in the hippocampus at 9 months of age. Additionally, 3D imaging showed increased microglial attachment to vessels and enlargement of the size of the vessel-associated microglia in CADASIL mice. Single-cell RNA sequencing revealed a microglial subcluster expressing genes involved in mitochondria respiration and inflammation. Collectively, our results reveal that exacerbated vascular network damage may mediate cognitive decline observed in the later stages of CADASIL and highlight the critical role of the neurovascular unit. Our findings provide valuable insights into the underlying mechanisms of neuronal dysfunction and pave the way for future research and potential therapeutic strategies.
