Abstract
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common forms of hereditary cerebral small vessel diseases and is caused by mutations in NOTCH3. Our group has previously reported incorporation of NOTCH3 extracellular domain (N3ECD) in the CADASIL-specific granular osmiophilic materials and increase of PDGFRβ immunoreactivity in CADASIL postmortem brains. Here, we aimed to establish an in vitro model of CADASIL, which can recapitulate those CADASIL phenotypes, using induced pluripotent stem cells (iPSCs). We have refined a differentiation protocol of endothelial cells to obtain mature mural cells (MCs) with their characteristic properties. iPSCs from three CADASIL patients with p.Arg182Cys, p.Arg141Cys and p.Cys106Arg mutations were differentiated into MCs and their functional and molecular profiles were compared. The differentiated CADASIL MCs recapitulated pathogenic changes reported previously: increased PDGFRβ and abnormal structure/distribution of filamentous actin network, as well as N3ECD/LTBP-1/HtrA1-immunopositive deposits. Migration rate of CADASIL MCs was enhanced but suppressed by knockdown of NOTCH3 or PDGFRB. CADASIL MCs showed altered reactivity to PDGF-BB. Patient-derived MCs can recapitulate CADASIL pathology and are therefore useful in understanding the pathogenesis and developing potential treatment strategies.
Highlights
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary cerebral small vessel diseases of the brain
Over 250 reported NOTCH3 mutations are reported to be distributed throughout 34 epidermal growth factor-like repeats in the NOTCH3 extracellular domain (N3ECD), all resulting in similar phenotypes such as Vascular smooth muscle cell (VSMC) degeneration, deposition of granular osmiophilic materials (GOM) in the vasculature, thickening of vessel wall, enlarged perivascular spaces and white matter abnormalities [3, 4]
Mural cell (MC) differentiated on laminin-coated dish in Smooth Muscle Cell Medium (SMCM) changed their morphology and cellular properties depending on the culture conditions: flat and spreadshaped MCs cultured in SMCM on collagen I-coated dish transformed to elongated spindle-shaped cells, forming a network-like structure, when cultured in Smooth Muscle Cell Growth Medium 2 (SMCGM2) on a laminin-coated dish (Fig. 1g and i)
Summary
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary cerebral small vessel diseases of the brain. Many studies have attempted to unravel how NOTCH3 mutations lead to artery defects, the pathogenesis of CADASIL is still largely unknown. CADASIL-like rat NOTCH3 mutations p.Arg171Cys, p.His184Cys, p.Cys544Tyr and p.Arg560Cys, for example, were reported to produce mutant receptors but without any abnormalities in processing, maturation and ligand interaction [8]. Another mutation, p.Arg141Cys, impaired S1 cleavage and reduced resultant mature heterodimeric mutant receptors on the cell surface, though signaling activity itself was intact [9]. There is no clear consensus on the involvement of canonical Notch signaling pathway in the pathogenesis of CADASIL, though recent studies seem to support gain of toxic function rather than loss of function [5, 12, 13]
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