The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation

Abstract

IntroductionCADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model.ResultsWe generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the ‘NOTCH3 score’, a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing.ConclusionsThis novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0268-1) contains supplementary material, which is available to authorized users.

The developmental biology of genetic Notch disorders

“…The NOTCH3ECD cascade hypothesis (Monet-Leprêtre et al, 2013) suggests that NOTCH3-induced GOM scavenges extracellular matrix proteins, contributing to toxic effects. This hypothesis is also supported by animal models in which the strongest CADASIL phenotype is seen in mice expressing the highest levels of mutated Notch3 (Rutten et al, 2015). OFT endocardium, presumptive AVC endocardium and myocardium OFT, valves, endocardium and myocardium, and around valves and chambers vSMCs surrounding valves, chamber myocardium and valve endocardium ; endothelium and SMCs surrounding coronary arteries, and epicardium (Del Monte et al, 2011) Notch2-βgal not detected (Varadkar et al, 2008) Notch2 mRNA in endocardium (D'Amato et al, 2016b) and myocardium (Yang et al,2012); Notch2-βgal not detected (Varadkar et al, 2008); propericardium (Del Monte et al, 2011) Parts of endocardium; myocardium Notch2 mRNA in endocardium and AVC mesenchyme (D'Amato et al, 2016b); (Yang et al, 2012); Notch2-βgal in AVC and OFT mesenchyme (Varadkar et al, 2008) Weak in compact myocardium and strong in trabecular myocardium (Yang et al, 2012); Notch2 mRNA in AVC and pulmonary artery (Loomes et al, 2002); Notch2-βgal in AVC, OFT mesenchyme, valve mesenchyme, aorta and pulmonary (Varadkar et al, 2008) Atrial and ventricular myocardium; Notch2-βgal/Notch2 SMA + tissue around valves, aorta and pulmonary artery (Varadkar et al, 2008) Several key Notch pathway components, including Jag1, Dll4, Notch1 and Notch2, are expressed during heart development, as summarized.…”

Translational models for vascular cognitive impairment: a review including larger species

“…In only one transgenic model, with 4-fold overexpression of mutant Notch3, the mice developed disturbed cerebrovascular reactivity (from 5 months of age), reduced CBF (from 12 months) and white matter damage (from 18 months) [27]. A novel transgenic mouse strain containing genomic human NOTCH3 has recently been developed [136]; these animals show early-onset vascular Notch3 accumulation (from 6 weeks). A knock-in model, made by introducing a mutation in endogenous Notch3 , developed a CADASIL clinical phenotype (at 20 months) [137].…”

Progression and Classification of Granular Osmiophilic Material (GOM) Deposits in Functionally Characterized Human NOTCH3 Transgenic Mice

“…We have previously described that our humanized CADASIL transgenic NOTCH3 Arg182Cys mouse model, which overexpresses human mutant NOTCH3 protein from a genomic construct, shows granular NOTCH3 ECD immunostaining as early as 4 weeks of age, while GOM deposits first appear around 6 months of age [22]. However, little is known about how GOM deposits evolve over time and what their relation is to other CADASIL-associated vascular pathology and vascular dysfunction.…”