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Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke in humans. Dominant mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a genetic archetype of cerebral ischemic SVD. Progress toward understanding the pathogenesis of this disease and developing effective therapies has been hampered by the lack of a good animal model. Here, we report the development of a mouse model for CADASIL via the introduction of a CADASIL-causing Notch3 point mutation into a large P1-derived artificial chromosome (PAC). In vivo expression of the mutated PAC transgene in the mouse reproduced the endogenous Notch3 expression pattern and main pathological features of CADASIL, including Notch3 extracellular domain aggregates and granular osmiophilic material (GOM) deposits in brain vessels, progressive white matter damage, and reduced cerebral blood flow. Mutant mice displayed attenuated myogenic responses and reduced caliber of brain arteries as well as impaired cerebrovascular autoregulation and functional hyperemia. Further, we identified a substantial reduction of white matter capillary density. These neuropathological changes occurred in the absence of either histologically detectable alterations in cerebral artery structure or blood-brain barrier breakdown. These studies provide in vivo evidence for cerebrovascular dysfunction and microcirculatory failure as key contributors to hypoperfusion and white matter damage in this genetic model of ischemic SVD.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an autosomal dominant small-vessel disease of the brain caused by mutations in the NOTCH3 receptor. The highly stereotyped nature of the mutations, which alter the number of cysteine residues within the epidermal growth factor-like repeats (EGFR), predicts that all mutations share common mechanisms. Prior in vitro assays and genetic studies in the mouse support the hypothesis that common mutations do not compromise canonical Notch3 function but instead convey a non-physiological and deleterious activity to the receptor through the unpaired cysteine residue. Intriguingly, in vitro studies predict that mutations located in the Delta/Serrate/LAG-2 ligand binding domain-(EGFR10-11) may result in a loss of Notch3 receptor function. However, the in vivo relevance and functional significance of this with respect to the pathogenic mechanisms and clinical expression of the disease remain largely unexplored. To ascertain, in vivo, the functional significance of EGFR10-11 mutations, we generated transgenic mice with one representative mutation (C428S) in EGFR10 of Notch3. These mice, like those with a common R90C mutation, developed characteristic arterial accumulation of Notch3 protein and granular osmiophilic material upon aging. By introducing the mutant C428S transgene into a Notch3 null background, we found that, unlike the R90C mutant protein, the C428S mutant protein has lost wild-type Notch3 activity and exhibited mild dominant-negative activity in three different biological settings. From a large prospectively recruited cohort of 176 CADASIL patients, we identified 10 patients, from five distinct pedigrees carrying a mutation in EGFR10 or 11. These mutations were associated with significantly higher Mini-Mental State Examination and Mattis Dementia Rating Scale scores (P < 0.05), when compared with common mutations. Additionally, we found a strong effect of this genotype on the burden of white matter hyperintensities (P < 0.01). Collectively, these results highlight distinctive functional and phenotypic features of EGFR10-11 mutations relative to the common CADASIL mutations. Our findings are compatible with the hypothesis that EGFR10-11 mutations cause the disease through the same gain of novel function as the common mutations, and lead us to propose that reduced Notch3 signalling acts as a modifier of the CADASIL phenotype.

Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy.

Background and Purpose: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease causing stroke and dementia. The disease is caused by highly stereotyped mutations in NOTCH3, which is restrictively expressed in vascular smooth muscle cells (VSMCs). The mechanisms of compromised cerebral hemodynamics in CADASIL remain to be elucidated. We tested the hypothesis that mutant NOTCH3 impairs the vasomotor function of cerebral vessels., Methods: Vasomotor function was examined in vivo in transgenic mice expressing a mutant NOTCH3 in VSMCs (TgNotch3R90C). Mice develop an age-dependent arteriopathy similar to that seen in CADASIL, without brain parenchyma lesions. Using laser-Doppler flowmetry, we assessed in awake TgNotch3R90C mice and wild-type littermates the cerebrovascular reactivity to 2 potent vasodilator stimuli (acetazolamide and hypercapnia) and cerebral blood flow (CBF) autoregulation during stepwise blood pressure elevations and reductions. Mice were studied at 18 months of age, when the CADASIL features are apparent, and at 10 months of age, before their appearance., Results: Eighteen-month-old TgNotch3R90C mice showed reduced responses to hypercapnia and acetazolamide, higher cerebrovascular resistance during hypertension, and their lower limit of CBF autoregulation was shifted to higher blood pressures. Cerebrovascular responses were similarly impaired in 10-month-old TgNotch3R90C mice., Conclusions: Cerebrovascular reactivity is compromised early in TgNotch3R90C mice. The data show an impaired autoregulation and are suggestive of a decreased relaxation or increased resistance of cerebral vessels. Our findings indicate that vascular dysfunction is an early pathogenic event that may promote the subsequent development of brain ischemia in CADASIL.

Background and Purpose: CADASIL is an inherited small-vessel disease responsible for lacunar strokes and cognitive impairment. The disease is caused by highly stereotyped mutations in Notch3, the expression of which is highly restricted to vascular smooth muscle cells (VSMCs). The underlying vasculopathy is characterized by degeneration of VSMCs and the accumulation of granular osmiophilic material (GOM) and Notch3 protein within the cell surface of these cells. In this study, we assessed early functional changes related to the expression of mutant Notch3 in resistance arteries., Methods: Vasomotor function was examined in vitro in arteries from transgenic mice that express a mutant Notch3 in VSMC. Tail artery segments from transgenic and normal wild-type male mice were mounted on small-vessel arteriographs, and reactivity to mechanical (flow and pressure) forces and pharmacological stimuli were determined. Mice were studied at 10 to 11 months of age when VSMC degeneration, GOM deposits, and Notch3 accumulation were not yet present., Results: Passive arterial diameter, contraction to phenylephrine, and endothelium-dependent relaxation to acetylcholine were unaffected in transgenic mice. By contrast, flow-induced dilation was significantly decreased and pressure-induced myogenic tone significantly increased in arteries from transgenic mice compared with wild-type mice., Conclusions: This is the first study to our knowledge providing evidence that mutant Notch3 impairs selectively the response of resistance arteries to flow and pressure. The data suggest an early role of vascular dysfunction in the pathogenic process of the disease.

Formation of a fully functional artery proceeds through a multistep process. Here we show that Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3-/- mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3-/- mice. We further show that Notch3 is required for arterial specification of vSMC but not of endothelial cells. Our data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterized by the degeneration of smooth-muscle cells in small cerebral arteries. CADASIL is caused by mutations in NOTCH3, one of the four mammalian homologs to the Drosophila melanogaster NOTCH gene. Disease-associated mutations are distributed throughout the 34 epidermal growth factor-like repeats (EGFRs) that compose the extracellular domain of the Notch3 receptor and result in a loss or a gain of a cysteine residue in one of these EGFRs. In human adults, Notch3 expression is highly restricted to vascular smooth-muscle cells. In patients with CADASIL, there is an abnormal accumulation of Notch3 in the vessel. Molecular pathways linking NOTCH3 mutations to degeneration of vascular smooth-muscle cells are as yet poorly understood. In this study, we investigated the effect of CADASIL mutations on Notch3 activity. We studied five naturally occurring mutations: R90C and C212S, located in the previously identified mutational hotspot EGFR2-5; C428S, shown in this study to be located in the ligand-binding domain EGFR10-11; and C542Y and R1006C, located in EGFR13 and EGFR26, respectively. All five mutant proteins were correctly processed. The C428S and C542Y mutant receptors exhibited a significant reduction in Jagged1-induced transcriptional activity of a RBP/JK responsive luciferase reporter, relative to wild-type Notch3. Impaired signaling activity of these two mutants arose through different mechanisms; the C428S mutant lost its Jagged1-binding ability, whereas C542Y retained it but exhibited an impaired presentation to the cell surface. In contrast, the R90C, C212S, and R1006C mutants retained the ability to bind Jagged1 and were associated with apparently normal levels of signaling activity. We conclude that mutations in Notch3 differently affect Jagged1 binding and Notch3 signaling via the RBP/JK pathway.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an increasingly recognized adult-onset autosomal dominant vascular dementia, caused by highly stereotyped mutations in the Notch3 receptor. CADASIL is a widespread angiopathy characterized by a degeneration of vascular smooth muscle cells (VSMCs) and the abnormal accumulation of electron-dense granular material called GOM and Notch3 protein, because of an impaired clearance. Evidence that VSMCs are the primary target of the pathogenic process is supported by the restricted expression of Notch3 in these cells but mechanisms of their degeneration remain essentially unknown. We generated transgenic mice in which the SM22alpha promoter drove, in VSMCs, the expression of a full-length human Notch3 carrying the Arg90Cys mutation, a CADASIL archetypal mutation. Transgenic mice showed no evidence of prominent brain parenchyma damage but demonstrated the two hallmarks of the CADASIL angiopathy, GOM deposits and Notch3 accumulation, within both the cerebral and peripheral arteries. Of interest, arteries of the tail were more severely affected with prominent signs of VSMC degeneration. Time-course analysis of vessel changes revealed that disruption of normal VSMC anchorage to adjacent extracellular matrix and cells, VSMC cytoskeleton changes as well as starting signs of VSMC degeneration, which were detected around 10 months of age, preceded Notch3 and GOM accumulation appearance, which were observed only by 14 to 16 months of age. In conclusion, we have generated transgenic mice that recapitulate the characteristic vascular lesions observed in CADASIL. Our results indicate that Notch3 or GOM accumulation are unlikely to be the prerequisites for the induction of VSMC degeneration and suggest that degeneration of VSMCs may rather be triggered by the disruption of their normal anchorage, based on the important role of adhesion for cell survival.