γ-Secretase is responsible for the final cleavage of amyloid precursor protein to generate the amyloid-β protein, the major component of plaques in the brains of Alzheimer's disease patients. γ-Secretase inhibitors (GSI) have been explored for therapeutic inhibition of amyloid β protein generation, but mechanistic toxicity has been documented because of its blockage of γ-secretase cleavage of several dozens of substrates including Notch. This becomes the primary obstacle for most inhibitors during the pre-clinical development and the main concern for several compounds in the clinical trials. To predict potential side effects related to Notch signaling, we examined global effect of GSIs in vertebrate animal zebrafish. We have used two potent GSIs (GSI A and GSI 18) with a sub-μM effective concentration for 50% amyloid β protein inhibition (EC50). Zebrafish embryos were treated with GSI A, 18 or a well characterized GSI N-[N-(3,5-difluorophenacetyl- l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), and transparent animals were examined for up to 7 days. GSI A had less abnormal phenotype in zebrafish, compared to GSI 18-treated embryos that displayed curved tails, a loss of pigmentation, and reduced swim bladder and heart rate. To understand mechanistic effect at the molecular level, we examined Notch signaling in these GSI-treated zebrafish. Notch phenotypes were observed in embryos treated with 50 and 10 μM GSI 18, but not with 10 μM GSI A. In accordance, in situ hybridization with a probe against Notch target gene her6 showed a weaker staining in embryos treated with 10 μM GSI 18 than those treated with 10 μM GSI A. In conclusion, phenotypic profile in whole animals offers important information on Notch related pathways and provides prediction of safe compounds during early development stages of therapeutic GSIs.
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience