Abstract
BackgroundSeveral 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.ResultsWe 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.ConclusionThe 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.
Highlights
Several g-secretase inhibitors (GSI) are in clinical trials for the treatment of Alzheimer’s disease (AD)
Compounds were dissolved in 1 mL of egg water (final concentration at 50 μM for DAPT {N- [N-(3,5Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester}, Gleevec, AD28, AD94, AD95, and 10 μM for AD115; 0.1% DMSO was used as a negative control)
An amyloid precursor protein (APP) overexpressing CHO cell line 7W was treated with individual compounds and the levels of secreted amyloid b (Ab) in the media were measured by ELISA
Summary
Several g-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 b protein, Ab, the pathogenic protein in AD. Phenotype-based small molecule screening in zebrafish has been described in a number of studies [1]. For non-fluorescent zebrafish, we have developed algorithms to analyze certain morphological changes in the development of zebrafish somites [4] These changes in morphology were linked to the lack of a component of the g-secretase [5], the key protease involved in the pathogenesis of Alzheimer’s disease (AD) [6]
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