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

Rutten JW, Klever RR, Hegeman IM, Poole DS, Dauwerse HG, Broos LA, Breukel C, Aartsma-Rus AM, Verbeek JS, van der Weerd L, van Duinen SG, van den Maagdenberg AM, Lesnik Oberstein SA - Acta Neuropathol Commun (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. j.w.rutten@lumc.nl.

ABSTRACT

Introduction: CADASIL (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.

Results: We 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.

Conclusions: This 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.

Show MeSH

Related in: MedlinePlus

Generation of transgenic human NOTCH3 mice. a Schematic representation of the BAC construct containing the human NOTCH3 gene and flanking regions, used for generation of tgN3WT and tgN3MUT (c.544C > T, p.Arg182Cys) mice. b Sequencing analysis of PCR products of the human NOTCH3 gene in transgenic mice confirmed the presence of the c.544C > T mutation in tgN3MUT mice. c qPCR analysis of human and mouse NOTCH3 expression in brain. In strain tgN3WT, human NOTCH3 expression was comparable to endogenous mouse Notch3 expression. The four mutant strains showed human NOTCH3 expression levels of 350, 200, 150 and 100 %, as compared to endogenous mouse Notch3 expression. Endogenous mouse Notch3 expression was comparable between the transgenic mouse strains
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4696336&req=5

Fig1: Generation of transgenic human NOTCH3 mice. a Schematic representation of the BAC construct containing the human NOTCH3 gene and flanking regions, used for generation of tgN3WT and tgN3MUT (c.544C > T, p.Arg182Cys) mice. b Sequencing analysis of PCR products of the human NOTCH3 gene in transgenic mice confirmed the presence of the c.544C > T mutation in tgN3MUT mice. c qPCR analysis of human and mouse NOTCH3 expression in brain. In strain tgN3WT, human NOTCH3 expression was comparable to endogenous mouse Notch3 expression. The four mutant strains showed human NOTCH3 expression levels of 350, 200, 150 and 100 %, as compared to endogenous mouse Notch3 expression. Endogenous mouse Notch3 expression was comparable between the transgenic mouse strains

Mentions: For transgenesis, a 142,63 kb BAC clone was used (RP11-456 N16 BAC, Bacpac resources, Oakland, USA) (Ensemble release 59). The BAC contains the full-length human genomic NOTCH3 gene and 44 kb of upstream and 67 kb of downstream sequence, including flanking genes SYDE1, ILVBL, EPHX3 and a part of the BRD4 gene (Fig. 1a). The c.544C > T (p.Arg182Cys) mutation was introduced using two-step Red-mediated recombination as previously described [20]. BAC constructs were injected into fertilized C57BL/6 J Ico oocytes. Positive transgenic founder mice were identified by PCR on DNA isolated from mouse ears using human specific primers (for primer sequences see Additional file 1: Table S1). The presence of the mutation was confirmed by direct Sanger sequencing analysis of PCR products (Fig. 1b). Five transgenic mouse strains were generated: one carrying the wild-type NOTCH3 transgene (tgN3WT) and four carrying the mutant NOTCH3 transgene (tgN3MUT). In each strain, integration of the BAC was confirmed by PCR analysis of NOTCH3 and the flanking genes SYDE1, ILVBL and EPHX3 (for primer sequences see Additional file 1: Table S1). All transgenic mouse strains bred normally. All experiments described in this study were approved by the local ethical committee for animal experimentation.Fig. 1


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

Rutten JW, Klever RR, Hegeman IM, Poole DS, Dauwerse HG, Broos LA, Breukel C, Aartsma-Rus AM, Verbeek JS, van der Weerd L, van Duinen SG, van den Maagdenberg AM, Lesnik Oberstein SA - Acta Neuropathol Commun (2015)

Generation of transgenic human NOTCH3 mice. a Schematic representation of the BAC construct containing the human NOTCH3 gene and flanking regions, used for generation of tgN3WT and tgN3MUT (c.544C > T, p.Arg182Cys) mice. b Sequencing analysis of PCR products of the human NOTCH3 gene in transgenic mice confirmed the presence of the c.544C > T mutation in tgN3MUT mice. c qPCR analysis of human and mouse NOTCH3 expression in brain. In strain tgN3WT, human NOTCH3 expression was comparable to endogenous mouse Notch3 expression. The four mutant strains showed human NOTCH3 expression levels of 350, 200, 150 and 100 %, as compared to endogenous mouse Notch3 expression. Endogenous mouse Notch3 expression was comparable between the transgenic mouse strains
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696336&req=5

Fig1: Generation of transgenic human NOTCH3 mice. a Schematic representation of the BAC construct containing the human NOTCH3 gene and flanking regions, used for generation of tgN3WT and tgN3MUT (c.544C > T, p.Arg182Cys) mice. b Sequencing analysis of PCR products of the human NOTCH3 gene in transgenic mice confirmed the presence of the c.544C > T mutation in tgN3MUT mice. c qPCR analysis of human and mouse NOTCH3 expression in brain. In strain tgN3WT, human NOTCH3 expression was comparable to endogenous mouse Notch3 expression. The four mutant strains showed human NOTCH3 expression levels of 350, 200, 150 and 100 %, as compared to endogenous mouse Notch3 expression. Endogenous mouse Notch3 expression was comparable between the transgenic mouse strains
Mentions: For transgenesis, a 142,63 kb BAC clone was used (RP11-456 N16 BAC, Bacpac resources, Oakland, USA) (Ensemble release 59). The BAC contains the full-length human genomic NOTCH3 gene and 44 kb of upstream and 67 kb of downstream sequence, including flanking genes SYDE1, ILVBL, EPHX3 and a part of the BRD4 gene (Fig. 1a). The c.544C > T (p.Arg182Cys) mutation was introduced using two-step Red-mediated recombination as previously described [20]. BAC constructs were injected into fertilized C57BL/6 J Ico oocytes. Positive transgenic founder mice were identified by PCR on DNA isolated from mouse ears using human specific primers (for primer sequences see Additional file 1: Table S1). The presence of the mutation was confirmed by direct Sanger sequencing analysis of PCR products (Fig. 1b). Five transgenic mouse strains were generated: one carrying the wild-type NOTCH3 transgene (tgN3WT) and four carrying the mutant NOTCH3 transgene (tgN3MUT). In each strain, integration of the BAC was confirmed by PCR analysis of NOTCH3 and the flanking genes SYDE1, ILVBL and EPHX3 (for primer sequences see Additional file 1: Table S1). All transgenic mouse strains bred normally. All experiments described in this study were approved by the local ethical committee for animal experimentation.Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. j.w.rutten@lumc.nl.

ABSTRACT

Introduction: CADASIL (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.

Results: We 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.

Conclusions: This 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.

Show MeSH
Related in: MedlinePlus