The Development of an in vivo γ-Secretase Assay using Zebrafish Embryos

Abstract

Aberrant proteolytic processing of amyloid-β protein precursor by γ-secretase complexes may result in an imbalance between production and clearance of the Aβ proteolytic product and promote neuronal dysfunction and death. Presenilin proteins form the catalytic core of γ-secretase complexes. The zebrafish, Danio rerio, is a versatile vertebrate model for investigating the molecular basis of Alzheimer's disease pathology. It possesses genes orthologous to human PSEN1 and PSEN2 (psen1 and psen2 respectively), and AβPP (appa and appb that are duplicates of an ancestral AβPP orthologue). Currently there is no in vivo assay appropriate for directly monitoring γ-secretase activity. Here, we describe such an assay in which the level of a γ-secretase substrate (a modified form of Appa protein) is observed in zebrafish embryos by western immunoblotting relative to a co-expressed protein not subject to γ-secretase activity. We have used the assay to analyze the effects on γ-secretase activity of blocking translation of transcripts of zebrafish psen1 and/or psen2.

Developingin vivoassays for investigation of p75NTRand NRH1 transmembrane domain cleavage events using zebrafish embryos

“…As was previously observed in a similar γ-secretase assay, both p75 NTF C201-dGFP and Nrh1C191-dGFP appeared to induce developmental abnormalities in embryos [13]. When injected, embryos displayed increased mortality (~46% vs. ~5-10% in uninjected controls) and a range of developmental abnormalities, which were increased by DAPT treatment (~57% mortality).…”

“…This preliminary shedding of large extracellular domains by α- or β-secretase may be rate-limiting, creating problems for an in vivo assay as the rate of cleavage may be dependent on these events rather than γ-secretase itself. To overcome this in our assay, we proposed to produce artificially truncated forms of both proteins similar to a strategy we had adopted previously for zebrafish APP in another γ-secretase assay [13].…”

“…Therefore, when examining the stability of a protein expressed from an injected transgene, it can be useful to have an internal reference standard against which the quantity of the protein can be compared. In a previous study assaying γ-secretase activity by monitoring cleavage of zebrafish Appa fused to dGFP (also expressed from the Tol2 transgene vector, pT2AL200R150G), we co-injected a similar Tol2 vector expressing free GFP, as an internal reference standard [13]. However, results from strategy may be prone to variability as the free GFP vector might not transpose into the genome at a constant rate relative to the Appa construct.…”

Using the zebrafish model for Alzheimer’s disease research

“…Recently, the first assay to assess directly γ-secretase cleavage activity was developed by Wilson and Lardelli (2013). α- and β-secretase cleavage of Appa provide substrates for subsequent γ-secretase cleavage.…”

Understanding taurine CNS activity using alternative zebrafish models

“…Zebrafish cells express genes corresponding to those mutated in human familial AD, amyloid-β precursor protein (APP), appa and appb (Musa et al, 2001;Joshi et al, 2009;Newman et al, 2011). Orthologs to human of presenilin (PSEN1 and PSEN2), psen1 and psen2, respectively ( Wilson and Lardelli, 2013) and prion protein (PRP), prp1, and prp2 have also been described (Kaiser et al, 2012). The loss of zebrafish appa and appb function by morpholino knockdown resulted in reduced body length and defective convergent extension movements during gastrulation in embryos.…”