Notch3 Arg170Cys Knock-In Mice Display Pathologic and Clinical Features of the Neurovascular Disorder Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy

Abstract

Objective-Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset neurovascular disorder caused by stereotyped mutations in the NOTCH3 receptor. Elucidation of its pathobiology is still incomplete and remains a challenge, in part because the available preclinical mouse models to date do not reproduce the full spectrum of CADASIL pathology and clinical disease. Methods and Results-Here, we report a novel knock-in mouse with Arg170Cys substitution in murine Notch3, corresponding to the prevalent Arg169Cys substitution in CADASIL. The Notch3 Arg170Cys mice displayed late-onset, dominant CADASIL arteriopathy with typical granular osmiophilic material deposition and developed brain histopathology including thrombosis, microbleeds, gliosis, and microinfarction. Furthermore, Notch3Arg170Cys mice experienced neurological symptoms with motor defects such as staggering gait and limb paresis. The main symptoms are migraine with aura, psychiatric disturbances, and recurrent subcortical ischemic strokes causing motor disabilities, cognitive decline, dementia, and premature death. 2,3 The accompanying arteriopathy is characterized by the progressive degeneration of arterial smooth muscle cells (SMCs), deposition of electron-dense granular osmiophilic material (GOM) within the basal lamina of the SMCs and pericytes, and accumulation of the Notch3 extracellular cleavage product at the SMC membrane. 2-7 Although the arteriopathy is systemic, 8 the symptoms are mostly neurological. Brain pathology involves microbleeds, loss of myelin or axons, fibrillar gliosis, and lacunar infarcts in the white and deep gray matter. 2,3,9 CADASIL is caused by mutations in the transmembrane receptor Notch3, which is predominantly expressed in adult arterial SMCs. 10 -13 Pathogenic mutations stereotypically affect the number of cysteine residues in the epidermal growth factor-like repeats of the extracellular domain (ECD). [13][14][15] Thus far, more than 180 mutations are known, nearly 60% of which cluster in exon 4. 3,[13][14][15][16] In vitro and in vivo studies have highlighted a role of Notch3 in artery maturation, responses to vascular injury or ischemia, and regulation of SMC proliferation and apoptosis. [17][18][19][20][21][22][23][24][25] How the various Notch3 mutations effectuate arteriopathy and CADASIL disease development remains enigmatic.Previous approaches to generating a model of CADASIL include knock-in mice carrying the counterpart Arg141Cys CADASIL mutation (Arg142Cys in mouse) in the endogenous Notch3 gene, which failed, however, to develop any phenotype at all. 26 On the other hand, transgenic overexpression in mice of human or rat Notch3 carrying archetypal Notch3 mutations (Arg90Cys human Notch3, 27-30 Cys428Ser human Notch3, 31 Arg169Cys rat Notch3, 32 Cys455Arg and Arg1031Cys human Notch3 33 ) met with more success, with all models displaying the CADASIL arteriopathy. However, Here, we report a novel mutant mouse carrying an Arg170Cys knock-in mutation in the...

Animal Models of Vascular Cognitive Impairment and Dementia (VCID)

“…Histological analysis revealed common features of CADASIL: microbleeds, thrombosis, fibrillar gliosis, and microinfarction. Notch3 Arg170Cys mouse mutants also developed motor deficits (ataxia, paresis) at 13 months (Wallays et al 2011).…”

Attenuation of Myeloid-Specific TGFβ Signaling Induces Inflammatory Cerebrovascular Disease and Stroke

“…Spontaneous stroke has been achieved in several mouse models through germline mutation 22–24 , but is not representative of the clinical conditions when specific genetic alterations are not a factor. Tgfbr2 Myeko mice were used to model human stroke risk factors and prevention.…”

Archetypal Arg169Cys Mutation in NOTCH3 Does Not Drive the Pathogenesis in Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leucoencephalopathy via a Loss-of-Function Mechanism

“…Importantly, we present 3 lines of evidence arguing against the general conclusion that all CADASIL mutations reflect hypomorphic NOTCH3 activity and that reduction in NOTCH3 activity is a major driving force of the brain manifestations of this disease. First, using mice harboring the Arg170Cys mutation (corresponding to Arg169Cys mutation in human NOTCH3) at the endogenous Notch3 locus, 14 we show that the archetypal Arg169Cys NOTCH3 mutation does not mitigate NOTCH3 activity in the brain arteries at an age marked by extensive deposition of Notch3 ECD aggregates.…”

“…Previous studies have examined the consequence of CADASIL mutations on NOTCH3 activity either in vitro, in cultured cells, or in vivo using indirect assays based on the ability of mutant NOTCH3 to rescue the arterial defects of Notch3KO mice or their consequences. [7][8][9]11,14 The approach we used here, quantifying in the brain arteries the expression level of NOTCH3 target genes relevant to NOTCH3 function in SMCs, is quite unique in this regard. Significantly, the subset of genes we have tested has proven to be appropriate to detect NOTCH3-RBPJ haploinsufficiency, which could occur with aging (ie, after normal completion of arterial maturation).…”

“…12 In this study, we tested the hypothesis that this archetypal mutation, which retains normal surface expression, ligand binding, and signaling in vitro, 13,14 acquires, with aging, loss-of-function properties that drive the brain manifestations. To achieve this, we used mouse models with reduced NOTCH3-RBPJ activity and mouse models for human NOTCH3 Arg169Cys mutation (corresponding to Arg170Cys and Arg171Cys mutation in mouse and rat NOTCH3, respectively) and conducted molecular analyses of the brain arteries as well as neuropathological studies.…”