TY - JOUR
T1 - Phenotypic analysis of images of zebrafish treated with Alzheimer's γ-secretase inhibitors
AU - Arslanova, Dilyara
AU - Yang, Ting
AU - Xu, Xiaoyin
AU - Wong, Stephen T.
AU - Augelli-Szafran, Corinne E.
AU - Xia, Weiming
N1 - Funding Information:
We would like to thank Dr. Pamela Raymond for the her6 cDNA, Dr. Peter Seubert and Dr. Dale Schenk for antibodies 2G3, 21F12 and 266, Dr. Ananda Kuppana for the synthesis of all fragments of Gleevec, Dr. Elizabeth A Heilig for helpful discussion. This study was supported by NIH grant AG015379 (WX).
PY - 2010/3/22
Y1 - 2010/3/22
N2 - Background: Several γ-secretase inhibitors (GSI) are in clinical trials for the treatment of Alzheimer's disease (AD). This enzyme mediates the proteolytic cleavage of amyloid precursor protein (APP) to generate amyloid β protein, Aβ, the pathogenic protein in AD. The γ-secretase also cleaves Notch to generate Notch Intracellular domain (NICD), the signaling molecule that is implicated in tumorigenesis.Results: We have developed a method to examine live zebrafish that were each treated with γ-secretase inhibitors (GSI), DAPT {N- [N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester}, Gleevec, or fragments of Gleevec. These compounds were first tested in a cell-based assay and the effective concentrations of these compounds that blocked Aβ generation were quantitated. The mortality of zebrafish, as a result of exposure to different doses of compound, was assessed, and any apoptotic processes were examined by TUNEL staining. We then used conventional and automatic microscopes to acquire images of zebrafish and applied algorithms to automate image composition and processing. Zebrafish were treated in 96- or 384-well plates, and the phenotypes were analyzed at 2, 3 and 5 days post fertilization (dpf). We identified that AD95, a fragment of Gleevec, effectively blocks Aβ production and causes specific phenotypes that were different from those treated with DAPT. Finally, we validated the specificity of two Notch phenotypes (pigmentation and the curvature of tail/trunk) induced by DAPT in a dose-dependent manner. These phenotypes were examined in embryos treated with GSIs or AD95 at increasing concentrations. The expression levels of Notch target gene her6 were also measured by in situ hybridization and the co-relationship between the levels of Notch inhibition by DAPT and AD95 and the severity of phenotypes were determined.Conclusion: The results reported here of the effects on zebrafish suggest that this newly developed method may be used to screen novel GSIs and other leads for a variety of therapeutic indications.
AB - Background: Several γ-secretase inhibitors (GSI) are in clinical trials for the treatment of Alzheimer's disease (AD). This enzyme mediates the proteolytic cleavage of amyloid precursor protein (APP) to generate amyloid β protein, Aβ, the pathogenic protein in AD. The γ-secretase also cleaves Notch to generate Notch Intracellular domain (NICD), the signaling molecule that is implicated in tumorigenesis.Results: We have developed a method to examine live zebrafish that were each treated with γ-secretase inhibitors (GSI), DAPT {N- [N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester}, Gleevec, or fragments of Gleevec. These compounds were first tested in a cell-based assay and the effective concentrations of these compounds that blocked Aβ generation were quantitated. The mortality of zebrafish, as a result of exposure to different doses of compound, was assessed, and any apoptotic processes were examined by TUNEL staining. We then used conventional and automatic microscopes to acquire images of zebrafish and applied algorithms to automate image composition and processing. Zebrafish were treated in 96- or 384-well plates, and the phenotypes were analyzed at 2, 3 and 5 days post fertilization (dpf). We identified that AD95, a fragment of Gleevec, effectively blocks Aβ production and causes specific phenotypes that were different from those treated with DAPT. Finally, we validated the specificity of two Notch phenotypes (pigmentation and the curvature of tail/trunk) induced by DAPT in a dose-dependent manner. These phenotypes were examined in embryos treated with GSIs or AD95 at increasing concentrations. The expression levels of Notch target gene her6 were also measured by in situ hybridization and the co-relationship between the levels of Notch inhibition by DAPT and AD95 and the severity of phenotypes were determined.Conclusion: The results reported here of the effects on zebrafish suggest that this newly developed method may be used to screen novel GSIs and other leads for a variety of therapeutic indications.
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U2 - 10.1186/1472-6750-10-24
DO - 10.1186/1472-6750-10-24
M3 - Article
C2 - 20307292
AN - SCOPUS:77950901539
SN - 1472-6750
VL - 10
JO - BMC Biotechnology
JF - BMC Biotechnology
M1 - 24
ER -