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

Quantitative analysis of vascular NOTCH3 protein accumulation in transgenic human NOTCH3 p.Arg182Cys mice and in brain tissue of CADASIL patients. a ImageJ processing of NOTCH3-immunostained brain sections of tgN3MUT350 mice. The images were filtered to reduce background signal and a standardised threshold was applied to determine the NOTCH3- positive area composed of individual granular NOTCH3 deposits, resulting in the NOTCH3 score. b Quantitative analysis of NOTCH3 accumulation in tgN3MUT350 mice. The NOTCH3 score shows an age-dependent increase and allows for a sensitive discrimination between age groups (One-Way ANOVA, Fishers least significant difference). c Validation of the NOTCH3 score in tgN3MUT150 mice, also showing an age-dependent increase. At each time point, the score is lower in tgN3MUT150 than in tgN3MUT350 mice (unpaired t-test), reflecting the correlation between NOTCH3 RNA expression and NOTCH3 protein accumulation. Data represent the average +/− SD of the three mice analysed per time point. d ImageJ analysis of human brain sections double stained with NOTCH3 and CD31. The vessel area was selected based on the staining with the endothelial cell marker CD31, and within this area, the NOTCH3 score was determined. e CADASIL patients show a significantly higher NOTCH3 score than age-matched controls. (unpaired t-test) Data represent the average +/− SD of three CADASIL patients and three control individuals
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Quantitative analysis of vascular NOTCH3 protein accumulation in transgenic human NOTCH3 p.Arg182Cys mice and in brain tissue of CADASIL patients. a ImageJ processing of NOTCH3-immunostained brain sections of tgN3MUT350 mice. The images were filtered to reduce background signal and a standardised threshold was applied to determine the NOTCH3- positive area composed of individual granular NOTCH3 deposits, resulting in the NOTCH3 score. b Quantitative analysis of NOTCH3 accumulation in tgN3MUT350 mice. The NOTCH3 score shows an age-dependent increase and allows for a sensitive discrimination between age groups (One-Way ANOVA, Fishers least significant difference). c Validation of the NOTCH3 score in tgN3MUT150 mice, also showing an age-dependent increase. At each time point, the score is lower in tgN3MUT150 than in tgN3MUT350 mice (unpaired t-test), reflecting the correlation between NOTCH3 RNA expression and NOTCH3 protein accumulation. Data represent the average +/− SD of the three mice analysed per time point. d ImageJ analysis of human brain sections double stained with NOTCH3 and CD31. The vessel area was selected based on the staining with the endothelial cell marker CD31, and within this area, the NOTCH3 score was determined. e CADASIL patients show a significantly higher NOTCH3 score than age-matched controls. (unpaired t-test) Data represent the average +/− SD of three CADASIL patients and three control individuals

Mentions: As we observed such an early and clear age- and NOTCH3 expression level- dependent vascular NOTCH3 accumulation load, we set out to objectify this by developing a quantitative measure for NOTCH3 staining. This was accomplished by capturing and measuring the surface area of CADASIL specific granular NOTCH3 deposits within brain sections using ImageJ software (Fig. 3a), which we called the ‘NOTCH3 score’. This quantification was first performed in tgN3MUT350 mice, which clearly showed that the NOTCH3 score increased with age, confirming our qualitative observations (Fig. 3b). Next, we validated the NOTCH3 score in a second mouse strain, tgN3MUT150, in which the same age-dependent increase in the NOTCH3 score was seen. At each time-point, the NOTCH3 score was lower for the tgN3MUT150 mice compared to the tgN3MUT350 mice, reflecting the correlation between NOTCH3 expression level and NOTCH3 accumulation load (score at age 20 months: 659 ± 51 vs. 1150 ± 107, p = 0.002) (Fig. 3c). Furthermore, progression of NOTCH3 accumulation was slower in tgN3MUT150 mice compared to tgN3MUT350 mice, as shown by a significant difference in the slope of the NOTCH3 scores between the two mouse strains (11.1 ± 0.5 vs. 6.2 ± 0.3, p = 0.002). Finally, we tested the approach in brain sections of three unrelated CADASIL patients. Measurement of the NOTCH3 accumulation load using the NOTCH3 score was technically feasible in human tissue (Fig. 3d) and showed a significantly higher NOTCH3 score in patients than in controls (score 3.81 ± 1.85 vs. 0.24 ± 0.17, p = 0.02) (Fig. 3e).Fig. 3


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)

Quantitative analysis of vascular NOTCH3 protein accumulation in transgenic human NOTCH3 p.Arg182Cys mice and in brain tissue of CADASIL patients. a ImageJ processing of NOTCH3-immunostained brain sections of tgN3MUT350 mice. The images were filtered to reduce background signal and a standardised threshold was applied to determine the NOTCH3- positive area composed of individual granular NOTCH3 deposits, resulting in the NOTCH3 score. b Quantitative analysis of NOTCH3 accumulation in tgN3MUT350 mice. The NOTCH3 score shows an age-dependent increase and allows for a sensitive discrimination between age groups (One-Way ANOVA, Fishers least significant difference). c Validation of the NOTCH3 score in tgN3MUT150 mice, also showing an age-dependent increase. At each time point, the score is lower in tgN3MUT150 than in tgN3MUT350 mice (unpaired t-test), reflecting the correlation between NOTCH3 RNA expression and NOTCH3 protein accumulation. Data represent the average +/− SD of the three mice analysed per time point. d ImageJ analysis of human brain sections double stained with NOTCH3 and CD31. The vessel area was selected based on the staining with the endothelial cell marker CD31, and within this area, the NOTCH3 score was determined. e CADASIL patients show a significantly higher NOTCH3 score than age-matched controls. (unpaired t-test) Data represent the average +/− SD of three CADASIL patients and three control individuals
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Quantitative analysis of vascular NOTCH3 protein accumulation in transgenic human NOTCH3 p.Arg182Cys mice and in brain tissue of CADASIL patients. a ImageJ processing of NOTCH3-immunostained brain sections of tgN3MUT350 mice. The images were filtered to reduce background signal and a standardised threshold was applied to determine the NOTCH3- positive area composed of individual granular NOTCH3 deposits, resulting in the NOTCH3 score. b Quantitative analysis of NOTCH3 accumulation in tgN3MUT350 mice. The NOTCH3 score shows an age-dependent increase and allows for a sensitive discrimination between age groups (One-Way ANOVA, Fishers least significant difference). c Validation of the NOTCH3 score in tgN3MUT150 mice, also showing an age-dependent increase. At each time point, the score is lower in tgN3MUT150 than in tgN3MUT350 mice (unpaired t-test), reflecting the correlation between NOTCH3 RNA expression and NOTCH3 protein accumulation. Data represent the average +/− SD of the three mice analysed per time point. d ImageJ analysis of human brain sections double stained with NOTCH3 and CD31. The vessel area was selected based on the staining with the endothelial cell marker CD31, and within this area, the NOTCH3 score was determined. e CADASIL patients show a significantly higher NOTCH3 score than age-matched controls. (unpaired t-test) Data represent the average +/− SD of three CADASIL patients and three control individuals
Mentions: As we observed such an early and clear age- and NOTCH3 expression level- dependent vascular NOTCH3 accumulation load, we set out to objectify this by developing a quantitative measure for NOTCH3 staining. This was accomplished by capturing and measuring the surface area of CADASIL specific granular NOTCH3 deposits within brain sections using ImageJ software (Fig. 3a), which we called the ‘NOTCH3 score’. This quantification was first performed in tgN3MUT350 mice, which clearly showed that the NOTCH3 score increased with age, confirming our qualitative observations (Fig. 3b). Next, we validated the NOTCH3 score in a second mouse strain, tgN3MUT150, in which the same age-dependent increase in the NOTCH3 score was seen. At each time-point, the NOTCH3 score was lower for the tgN3MUT150 mice compared to the tgN3MUT350 mice, reflecting the correlation between NOTCH3 expression level and NOTCH3 accumulation load (score at age 20 months: 659 ± 51 vs. 1150 ± 107, p = 0.002) (Fig. 3c). Furthermore, progression of NOTCH3 accumulation was slower in tgN3MUT150 mice compared to tgN3MUT350 mice, as shown by a significant difference in the slope of the NOTCH3 scores between the two mouse strains (11.1 ± 0.5 vs. 6.2 ± 0.3, p = 0.002). Finally, we tested the approach in brain sections of three unrelated CADASIL patients. Measurement of the NOTCH3 accumulation load using the NOTCH3 score was technically feasible in human tissue (Fig. 3d) and showed a significantly higher NOTCH3 score in patients than in controls (score 3.81 ± 1.85 vs. 0.24 ± 0.17, p = 0.02) (Fig. 3e).Fig. 3

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