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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.

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Related in: MedlinePlus

Vascular NOTCH3 protein accumulation and GOM deposits in transgenic human NOTCH3 p.Arg182Cys mice. a NOTCH3 immunostaining on brain sections of human NOTCH3 transgenic mice. All four tgN3MUT mouse strains developed a characteristic granular NOTCH3 staining pattern in the brain vasculature. TgN3WT mice showed only a weak, diffuse NOTCH3 staining pattern, which did not increase with age (comparable to non-transgenic litter-mates, data not shown). The NOTCH3 accumulation load in the tgN3MUT strains correlates well with the NOTCH3 expression level and increases with age; in tgN3MUT350 mice, first granular staining is already visible at 6 weeks of age; at 20 months of age nearly the whole vessel wall is packed with big granular NOTCH3 deposits. In the strains with a lower NOTCH3 expression level, the NOTCH3 accumulation starts at a later age and the granular deposits remain smaller. b Positive NOTCH3 staining in a brain vessel of a CADASIL patient. c NOTCH3 immunostaining of extra-cerebral arteries of 20-month-old tgN3MUT350 mice showing clear granular NOTCH3 staining in vessels of the heart, liver and skin. The aortic wall shows a diffuse and faint NOTCH3 staining pattern comparable to that seen in non-transgenic littermates, whereas the smaller vessels around the aorta do show characteristic granular NOTCH3 staining. d Electron microscopy on brain vessels from 12-month-old tgN3MUT350 mice shows characteristic electron dense deposits reminiscent of granular osmiophilic material (GOM). GOM deposits were first seen at 5–6 months of age. e Electron microscopy on brain tissue from a deceased CADASIL patient shows pathognomonic GOM deposits, adjacent to the basement membrane surrounding the VSMCs. * = granular osmiophilic material (GOM), BM = basement membrane, VSMC = vascular smooth muscle cell
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Fig2: Vascular NOTCH3 protein accumulation and GOM deposits in transgenic human NOTCH3 p.Arg182Cys mice. a NOTCH3 immunostaining on brain sections of human NOTCH3 transgenic mice. All four tgN3MUT mouse strains developed a characteristic granular NOTCH3 staining pattern in the brain vasculature. TgN3WT mice showed only a weak, diffuse NOTCH3 staining pattern, which did not increase with age (comparable to non-transgenic litter-mates, data not shown). The NOTCH3 accumulation load in the tgN3MUT strains correlates well with the NOTCH3 expression level and increases with age; in tgN3MUT350 mice, first granular staining is already visible at 6 weeks of age; at 20 months of age nearly the whole vessel wall is packed with big granular NOTCH3 deposits. In the strains with a lower NOTCH3 expression level, the NOTCH3 accumulation starts at a later age and the granular deposits remain smaller. b Positive NOTCH3 staining in a brain vessel of a CADASIL patient. c NOTCH3 immunostaining of extra-cerebral arteries of 20-month-old tgN3MUT350 mice showing clear granular NOTCH3 staining in vessels of the heart, liver and skin. The aortic wall shows a diffuse and faint NOTCH3 staining pattern comparable to that seen in non-transgenic littermates, whereas the smaller vessels around the aorta do show characteristic granular NOTCH3 staining. d Electron microscopy on brain vessels from 12-month-old tgN3MUT350 mice shows characteristic electron dense deposits reminiscent of granular osmiophilic material (GOM). GOM deposits were first seen at 5–6 months of age. e Electron microscopy on brain tissue from a deceased CADASIL patient shows pathognomonic GOM deposits, adjacent to the basement membrane surrounding the VSMCs. * = granular osmiophilic material (GOM), BM = basement membrane, VSMC = vascular smooth muscle cell

Mentions: To analyse the presence and onset of a CADASIL vascular phenotype, NOTCH3 immunohistochemistry was performed on brain slices from mice between the ages of 4 weeks and 20 months. This showed that all tgN3MUT strains developed cerebrovascular NOTCH3 accumulation, as seen by a positive, granular NOTCH3 staining of the vessel wall (Fig. 2a), similar to that which is seen in CADASIL patients (Fig. 2b). There was a considerable difference in age at onset of positive NOTCH3 staining per mouse strain, ranging from 6 weeks in tgN3MUT350 mice to 12 months in tgN3MUT100 mice. The age at onset directly correlated with the level of human NOTCH3 RNA expression for all four tgN3MUT strains i.e. the higher the NOTCH3 RNA expression level, the earlier the onset of NOTCH3 accumulation (Table 1). Furthermore, in each mutant strain, the positive NOTCH3 immunostaining became progressively more intense and granular with age (Fig. 2a). The individual granular NOTCH3 deposits increased not only in number, but also in size. This was most prominent in mice with the highest NOTCH3 RNA expression level (tgN3MUT350), in which the NOTCH3 protein accumulation progressively evolved to a vessel wall packed with intense and big granular NOTCH3 deposits at age 20 months. Characteristic granular NOTCH3 staining was also present in arterioles of the heart, liver, kidney, skin and tail, but not in the aorta (Fig. 2c, data not shown). Overall, the NOTCH3 accumulation observed in the extra-cerebral arterioles was less pronounced than in the brain. Electron microscopy of brain arterioles revealed characteristic electron dense deposits within the basement membrane (Fig. 2d) reminiscent of GOM deposits seen in CADASIL patients (Fig. 2e). Neither GOM nor increased cerebrovascular NOTCH3 staining was found in tgN3WT mice at 20 months of age (Fig. 2a). Taken together, these analyses show that transgenic human NOTCH3 p.Arg182Cys mice develop an early and progressive systemic arteriopathy which closely resembles the vascular pathology seen in CADASIL patients, with age-at-onset correlating with the respective levels of mutant human NOTCH3 expression.Fig. 2


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)

Vascular NOTCH3 protein accumulation and GOM deposits in transgenic human NOTCH3 p.Arg182Cys mice. a NOTCH3 immunostaining on brain sections of human NOTCH3 transgenic mice. All four tgN3MUT mouse strains developed a characteristic granular NOTCH3 staining pattern in the brain vasculature. TgN3WT mice showed only a weak, diffuse NOTCH3 staining pattern, which did not increase with age (comparable to non-transgenic litter-mates, data not shown). The NOTCH3 accumulation load in the tgN3MUT strains correlates well with the NOTCH3 expression level and increases with age; in tgN3MUT350 mice, first granular staining is already visible at 6 weeks of age; at 20 months of age nearly the whole vessel wall is packed with big granular NOTCH3 deposits. In the strains with a lower NOTCH3 expression level, the NOTCH3 accumulation starts at a later age and the granular deposits remain smaller. b Positive NOTCH3 staining in a brain vessel of a CADASIL patient. c NOTCH3 immunostaining of extra-cerebral arteries of 20-month-old tgN3MUT350 mice showing clear granular NOTCH3 staining in vessels of the heart, liver and skin. The aortic wall shows a diffuse and faint NOTCH3 staining pattern comparable to that seen in non-transgenic littermates, whereas the smaller vessels around the aorta do show characteristic granular NOTCH3 staining. d Electron microscopy on brain vessels from 12-month-old tgN3MUT350 mice shows characteristic electron dense deposits reminiscent of granular osmiophilic material (GOM). GOM deposits were first seen at 5–6 months of age. e Electron microscopy on brain tissue from a deceased CADASIL patient shows pathognomonic GOM deposits, adjacent to the basement membrane surrounding the VSMCs. * = granular osmiophilic material (GOM), BM = basement membrane, VSMC = vascular smooth muscle cell
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696336&req=5

Fig2: Vascular NOTCH3 protein accumulation and GOM deposits in transgenic human NOTCH3 p.Arg182Cys mice. a NOTCH3 immunostaining on brain sections of human NOTCH3 transgenic mice. All four tgN3MUT mouse strains developed a characteristic granular NOTCH3 staining pattern in the brain vasculature. TgN3WT mice showed only a weak, diffuse NOTCH3 staining pattern, which did not increase with age (comparable to non-transgenic litter-mates, data not shown). The NOTCH3 accumulation load in the tgN3MUT strains correlates well with the NOTCH3 expression level and increases with age; in tgN3MUT350 mice, first granular staining is already visible at 6 weeks of age; at 20 months of age nearly the whole vessel wall is packed with big granular NOTCH3 deposits. In the strains with a lower NOTCH3 expression level, the NOTCH3 accumulation starts at a later age and the granular deposits remain smaller. b Positive NOTCH3 staining in a brain vessel of a CADASIL patient. c NOTCH3 immunostaining of extra-cerebral arteries of 20-month-old tgN3MUT350 mice showing clear granular NOTCH3 staining in vessels of the heart, liver and skin. The aortic wall shows a diffuse and faint NOTCH3 staining pattern comparable to that seen in non-transgenic littermates, whereas the smaller vessels around the aorta do show characteristic granular NOTCH3 staining. d Electron microscopy on brain vessels from 12-month-old tgN3MUT350 mice shows characteristic electron dense deposits reminiscent of granular osmiophilic material (GOM). GOM deposits were first seen at 5–6 months of age. e Electron microscopy on brain tissue from a deceased CADASIL patient shows pathognomonic GOM deposits, adjacent to the basement membrane surrounding the VSMCs. * = granular osmiophilic material (GOM), BM = basement membrane, VSMC = vascular smooth muscle cell
Mentions: To analyse the presence and onset of a CADASIL vascular phenotype, NOTCH3 immunohistochemistry was performed on brain slices from mice between the ages of 4 weeks and 20 months. This showed that all tgN3MUT strains developed cerebrovascular NOTCH3 accumulation, as seen by a positive, granular NOTCH3 staining of the vessel wall (Fig. 2a), similar to that which is seen in CADASIL patients (Fig. 2b). There was a considerable difference in age at onset of positive NOTCH3 staining per mouse strain, ranging from 6 weeks in tgN3MUT350 mice to 12 months in tgN3MUT100 mice. The age at onset directly correlated with the level of human NOTCH3 RNA expression for all four tgN3MUT strains i.e. the higher the NOTCH3 RNA expression level, the earlier the onset of NOTCH3 accumulation (Table 1). Furthermore, in each mutant strain, the positive NOTCH3 immunostaining became progressively more intense and granular with age (Fig. 2a). The individual granular NOTCH3 deposits increased not only in number, but also in size. This was most prominent in mice with the highest NOTCH3 RNA expression level (tgN3MUT350), in which the NOTCH3 protein accumulation progressively evolved to a vessel wall packed with intense and big granular NOTCH3 deposits at age 20 months. Characteristic granular NOTCH3 staining was also present in arterioles of the heart, liver, kidney, skin and tail, but not in the aorta (Fig. 2c, data not shown). Overall, the NOTCH3 accumulation observed in the extra-cerebral arterioles was less pronounced than in the brain. Electron microscopy of brain arterioles revealed characteristic electron dense deposits within the basement membrane (Fig. 2d) reminiscent of GOM deposits seen in CADASIL patients (Fig. 2e). Neither GOM nor increased cerebrovascular NOTCH3 staining was found in tgN3WT mice at 20 months of age (Fig. 2a). Taken together, these analyses show that transgenic human NOTCH3 p.Arg182Cys mice develop an early and progressive systemic arteriopathy which closely resembles the vascular pathology seen in CADASIL patients, with age-at-onset correlating with the respective levels of mutant human NOTCH3 expression.Fig. 2

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