Phenotypic analysis of images of zebrafish treated with Alzheimer's γ-secretase inhibitors

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.

ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer’s disease

“…7B). Consistent with previous findings, we also observed severe developmental defects in DAPT-treated zebrafish embryos, as evidenced by a curved trunk due to the concomitant blockade of Notch signaling by pan-inhibition of γ-secretase (43). In stark contrast to the DAPT-treated zebrafish embryos, CL-387,785-treated embryos exhibited normal somite development with wild-type trunks and tails (Fig.…”

The zebrafish embryo as a model for assessing off-target drug effects

“…1). Several studies applying the zebrafish model in a similar manner have recently been reported in the literature (Ishizaki et al, 2010;Behra et al, 2004;Davidson et al, 2008;Molina et al, 2009;Arslanova et al, 2010;Buckley et al, 2010;Hao et al, 2010;Kokel et al, 2010;Rihel et al, 2010). In the sections below, we summarize some of these examples to illustrate the utility of the zebrafish embryo as a promising system for detecting off-target effects of drug candidates.…”

Automatic Localization of Interest Points in Zebrafish Images with Tree-Based Methods

“…Images are then labeled by experts and classified thanks to the Matlab Gait-CAD toolbox in only two classes. [3] describes a way to automatically obtain images of zebrafish embryos thanks to a motorized microscope, and classifies them manually into phenotypes. [19] developed an automatic system of data acquisition and embryos' analysis in multi-well plates, where manual intervention is still needed to produce analysis routines based on several image segmentations.…”