Figure 1

Zebrafish App protein organization, gene expression, and mutant generation

(A) There are 2 App orthologs, appa and appb, in zebrafish. Appa contains the Kunitz-type protease inhibitor (KPI) domain and thus has a similar gene organization to the human APP770 isoform. Zebrafish Appb lacks the KPI domain similar to the brain-enriched human APP695 isoform. Both Appa and Appb have the functional App domains, including the heparin-binding domain (HBD), copper-binding domain (CuBD), extracellular E2 domain (E2), the conserved YENTPY motif, and amyloid-beta region (AB).

(B) Alignment of Aβ regions of zebrafish Appa and Appb to human APP695 and APP770 shows high conservation within the Aβ region and the proteolytic cleavage sites (indicated with black arrows): α-secretase cleavage site (α), β-secretase cleavage site (β), γ-secretase cleavage sites (γ), and ε-cleavage sites (ε). Black, dark gray, and light gray boxes indicate strictly, highly, and moderately conserved amino acid residues, respectively.

(C) As detected by multiplexed hybridization chain reaction (HCR), appa (green) and appb (red) are both expressed widely but in non-overlapping regions of the 5dpf larval brain, including the cerebellum and nuclei in the hindbrain (C, cerebellum; H, hindbrain). Shown is a representative image from a single brain taken at one z-plane (z140/420) (dorsal view, above) and through the midline of the same brain (lateral view, below) from an experiment with a total of n = 22 fish. A, anterior; D, dorsal; L, left.

(D) CRISPR/Cas9 targeting of zebrafish appa resulted in a 5-bp deletion. The target guide RNA (gRNA) sequence is shown in bold, and the obligatory PAM sequence (AGG) is in red. The predicted translation of appaΔ5 leads to a premature stop codon within the Aβ region (Appa amino acid 665). TALEN targeting of zebrafish appb resulted in a 14-bp deletion (dashes) and 4-bp insertion (red). The left and right sites targeted by the TALENs are highlighted, and the spacer sequence, where cleavage occurs, is bolded. The predicted translation of appbΔ14+4 leads to a frameshift and a premature stop codon.

(E) Western blot analysis of APP in brain homogenates from wild-type (WT) and appa^Δ5/Δ5; appb^−/− double mutants.

(F) Elisa detection of Aβ42 (left) and Aβ40 (right) levels in adult brain homogenates from WT controls, WT animals treated with the γ-secretase inhibitor DAPT for 24 h, appa^Δ5/Δ5 and appb^−/− mutants quantified as picograms (pg) of Aβ per μg total protein extracted. Each dot is an independent biological replicate, and error bars represent the mean ± SEM. The bottom panel plots the ratio of Aβ40 to Aβ42 in WT zebrafish adult brain homogenates, n = 5. ∗p ≤ 0.05, ∗∗p ≤ 0.01, Dunnett’s test.