Tol2 gene trap integrations in the zebrafish amyloid precursor protein genes appa and aplp2 reveal accumulation of secreted APP at the embryonic veins

Abstract

Background The single spanning transmembrane amyloid precursor protein (APP) and its proteolytic product, amyloid-beta (Aβ) peptide, have been intensely studied due to their role in the pathogenesis of Alzheimer’s disease. However, the biological role of the secreted ectodomain of APP, which is also generated by proteolytic cleavage, is less well understood. Here, we report Tol2 red fluorescent protein (RFP) transposon gene trap integrations in the zebrafish amyloid precursor protein a (appa) and amyloid precursor-like protein 2 (aplp2) genes. The transposon integrations are predicted to disrupt the appa and aplp2 genes to primarily produce secreted ectodomains of the corresponding proteins that are fused to RFP. Results Our results indicate the Appa-RFP and Aplp2 fusion proteins are likely secreted from the central nervous system and accumulate in the embryonic veins independent of blood flow. Conclusions The zebrafish appa and aplp2 transposon insertion alleles will be useful for investigating the biological role of the secreted form of APP.

Amyloid Beta Precursor Protein and Prion Protein Have a Conserved Interaction Affecting Cell Adhesion and CNS Development

“…Investigating the etiology of the MO-induced phenotypes was warranted in light of previous works reporting a lack of overt phenotypes during appa loss-of-function in zebrafish [24], [25]. We sought to more completely assess if decreases in Appa protein abundance, induced by the appa MO (Fig.…”

“…The data are consistent with our MO reagents against appa being efficacious and specific. Past disruptions of this gene with a different MO were not directly tested for knockdown efficacy [24], and mutants with insertions in appa introns produce protein with partial function and continue to produce wild type proteins and thus are not null alleles [25]. Finally, we note that the interaction of appa with prp1 was confirmed using a second MO against appa designed to work in an independent fashion (by blocking translation) and this independent MO showed the same phenotypes and knockdown of APP immunoreactivity (Figs.…”

“…Zebrafish paralogs both show ∼70% predicted identity to human APP, increasing to ∼90–100% identity in the regions that encode the amyloidogenic, transmembrane and intracellular domains [24], [25]. Zebrafish appa and appb are homologs of mammalian APP , and are less similar to mammalian APP-like proteins ( APLP1 & APLP2 , of which zebrafish have further additional homologs not considered herein).…”

Breeding Based Remobilization of Tol2 Transposon in Xenopus tropicalis

“…An effective scheme for generating reporter lines is enhancer or gene trapping, whereby a transgenic cassette is randomly inserted into the genome and co-opts the expression pattern of a nearby gene [7-14]. Transposon systems are commonly used to introduce these cassettes.…”

The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

“…The Amyloid Precursor Protein (APP) belongs to a family of type I, single pass transmembrane proteins that are highly conserved from mammals to C. elegans, and that have prominent expression in the nervous system (Hornsten et al, 2007;Lorent et al, 1995;Luo et al, 1990;Wiese et al, 2010). Some of the studied family members include APP, APLP1, and APLP2 in mammals (Goldgaber et al, 1987;Kang et al, 1987;Slunt et al, 1994;Tanzi et al, 1987;Wasco et al, 1992Wasco et al, , 1993; APPa, APPb, APLP1, and APLP2 in zebrafish (Liao et al, 2012;Musa et al, 2001); APPL in Drosophila (Luo et al, 1990;Rosen et al, 1989); and APL-1 in C. elegans (Daigle and Li, 1993). Although these proteins are best known for the role of human APP in Alzheimer's disease, the Aβ peptide sequence implicated in the pathogenesis of Alzheimer's is only present in APP itself, and is not found in mammalian APLP1and APLP2, or in C. elegans or Drosophila, suggesting it is a more recent addition in evolution.…”