Activation of zebrafish Src family kinases by the prion protein is an amyloid-β-sensitive signal that prevents the endocytosis and degradation of E-cadherin/β-catenin complexes in vivo

Abstract

Background: Prions and amyloid-β (Aβ) oligomers trigger neurodegeneration by hijacking a poorly understood cellular signal mediated by the prion protein (PrP) at the plasma membrane. In early zebrafish embryos, PrP-1-dependent signals control cell-cell adhesion via a tyrosine phosphorylation-dependent mechanism.

Results: Here we report that the Src family kinases (SFKs) Fyn and Yes act downstream of PrP-1 to prevent the endocytosis and degradation of E-cadherin/β-catenin adhesion complexes in vivo. Accordingly, knockdown of PrP-1 or Fyn/Yes cause similar zebrafish gastrulation phenotypes, whereas Fyn/Yes expression rescues the PrP-1 knockdown phenotype. We also show that zebrafish and mouse PrPs positively regulate the activity of Src kinases and that these have an unexpected positive effect on E-cadherin-mediated cell adhesion. Interestingly, while PrP knockdown impairs β-catenin adhesive function, PrP overexpression enhances it, thereby antagonizing its nuclear, wnt-related signaling activity and disturbing embryonic dorsoventral specification. The ability of mouse PrP to influence these events in zebrafish embryos requires its neuroprotective, polybasic N-terminus but not its neurotoxicity-associated central region. Remarkably, human Aβ oligomers up-regulate the PrP-1/SFK/E-cadherin/β-catenin pathway in zebrafish embryonic cells, mimicking a PrP gain-of-function scenario.

Conclusions: Our gain- and loss-of-function experiments in zebrafish suggest that PrP and SFKs enhance the cell surface stability of embryonic adherens junctions via the same complex mechanism through which they over-activate neuroreceptors that trigger synaptic damage. The profound impact of this pathway on early zebrafish development makes these embryos an ideal model to study the cellular and molecular events affected by neurotoxic PrP mutations and ligands in vivo. In particular, our finding that human Aβ oligomers activate the zebrafish PrP/SFK/E-cadherin pathway opens the possibility of using fish embryos to rapidly screen for novel therapeutic targets and compounds against prion- and Alzheimer's-related neurodegeneration. Altogether, our data illustrate PrP-dependent signals relevant to embryonic development, neuronal physiology and neurological disease.

Fig. 1

PrP-1 prevents the endocytosis and degradation of AJ components. a. E-cadherin (green) and β-catenin (magenta) localization in 6 hpf deep cells upon treatment with 10 μM Dynasore (DYNA); MO = lissamine-tagged PrP-1 morpholino (red); scale bar = 10 μm. b and d. Total levels of E-cadherin and β-catenin at 6 hpf upon treatment with Dynasore, MG-132 and Chloroquine (Chlq). c. Phenotypic analysis upon treatment with inhibitors. Top: progression of epiboly by 7.5 hpf is assessed by the downward extension of the embryonic margin (arrowheads) relative to the control (dashed horizontal line); brackets show the thickness of the blastoderm. Bottom: phenotypic quantification. Mean values are shown. e. AJ protein levels in 6 hpf embryos injected with increasing PrP-1 morpholino doses. WB = Western blot; IF = immunofluorescence. Red and black arrowheads in 1B, D and E indicate mature E-cadherin (120 kDa) and its more abundant precursor form (140 kDa), respectively. Densitometric analysis of Western blot bands is expressed in arbitrary units (AU) in B, D and E; average values of three independent experiments ± SEM are shown; statistical significance was assessed using unpaired, two-tailed t-tests; ns = not significant (p > 0.05), * = p ≤ 0.05, ** = p ≤ 0.01. In (e), E-cadherin and β-catenin levels were significantly reduced (p ≤ 0.05) at all three morpholino concentrations; p120ctn levels were not significantly changed at any morpholino dose (p > 0.05). See also Additional file 1: Figure S1

Fig. 2

Fyn and Yes act downstream of PrP-1 during epiboly. a. 7.5 hpf embryos injected with different morpholinos or co-injected with PrP-1 morpholino and WT or CA Fyn/Yes EGFP mRNAs. Transmission and fluorescence images are shown merged (morpholinos in red). Expression of WT or CA Fyn/Yes-EGFP (green) is displayed separately on the lower right. Arrowheads and dashed horizontal line as in Fig. 1c. b. Corresponding quantification of embryonic phenotypes at 7.5 hpf. Mean values are shown. c. Expression pattern of WT or CA Fyn- and Yes-EGFP in deep cells of 7.5 hpf PrP-1 morphants. Scale bar = 10 μM

Fig. 3

PrP-1 regulates AJs via Fyn and Yes. a. Localization of AJ components (immunofluorescence) in deep (top) and EVL cells (bottom) of 6 hpf PrP-1, Fyn/Yes morphants or PrP-1 morphants co-injected with Fyn/Yes mRNAs. Scale bars = 10 μM. See also Additional file 1: Figure S2. b. Reduction in Src/Yes (65 kDa) and Fyn (50 kDa) total levels in 6 hpf Fyn and Yes single or double knockdowns. c, d. Levels of AJ components in 6 hpf embryo lysates. Activated WT or CA Fyn- and Yes-EGFP were detected with an anti-phospho-Y416 Src antibody. E-cadherin arrowheads as in Fig. 1. WB = Western blot. Densitometric analysis of Western blot bands is expressed in arbitrary units (AU) in b, c and d; average values of three independent experiments ± SEM are shown; statistical significance was assessed using unpaired, two-tailed t-tests; ns = not significant (p > 0.05), * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001

Fig. 4

PrP-1 modulates SFK levels and activation. a-c. Levels of total and activated (phosphor-Y416) SFKs in 6 hpf embryos (a), and densitometric analysis of Western blot bands (b and c). Phospho-SFK values were normalized to those of total SFKs. Average values ± SEM of four independent experiments are shown. d. SFK levels in 6 hpf embryos upon PrP-1 knockdown and additional treatment with degradation inhibitors. e. SFK localization in deep (top) and EVL cells (bottom) of 6 hpf embryos. WB = Western blot; IF = immunofluorescence. Scale bar = 10 μM. Densitometric analysis of Western blot bands (b, c and d) is expressed in arbitrary units (AU); average values of four independent experiments ± SEM are shown; statistical significance was assessed using unpaired, two-tailed t-tests; ns = not significant (p > 0.05), * = p ≤ 0.05, ** = p ≤ 0.01. See also Additional file 1: Figure S3

Fig. 5

Morphological and molecular phenotypes upon PrP overexpression. a. Shield formation (red arrowhead) in 6 hpf embryos. See also Additional file 2: Video S1. b. Levels of E-cadherin, β-catenin and SFKs in 6 hpf embryos expressing mouse PrP. E-cadherin arrowheads as in Fig. 1. c. Effect of protein degradation inhibitors on the β-catenin levels of PrP-OE embryos. d. Dorsoventral distribution of AJ molecules upon PrP overexpression. Lateral views (midsections) of whole 6 hpf embryos, along with fluorescence profiles through the dorsoventral axis (indicated by red arrows; V = ventral, D = dorsal). See also Additional file 1: Figures S4 and S6. e. Close-up from (d) showing aberrant morphology of dorsal deep cells expressing mouse PrP. WB = Western blot; IF = immunofluorescence. Densitometric analysis of Western blot bands (b and c) is expressed in arbitrary units (AU); average values of four experiments ± SEM are shown; statistical significance was assessed using unpaired, two-tailed t-tests; ns = not significant (p > 0.05), * = p ≤ 0.05

Fig. 6

PrP overexpression up-regulates E-cadherin, inhibits nuclear β-catenin and produces ventralized embryos. a. Localization of β-catenin in the nuclei of dorsal marginal blastomeres at 3 hpf (immunofluorescence). Arrowheads indicate β-catenin-positive nuclei. Scale bar = 20 μm. b. Quantification of cells with nuclear β-catenin in 3 hpf embryos expressing zebrafish and mouse WT PrP constructs. Average numbers of cells with nuclear β-catenin per embryo ± SEM are shown (n = 10, three independent experiments); statistical significance was assessed using unpaired, two-tailed t-tests; *** = p ≤ 0.001. c. Levels of E-cadherin, β-catenin and SFKs in 3 hpf embryos expressing mouse PrP (Western blot and densitometric analysis). E-cadherin arrowheads as in Fig. 1. Densitometric analysis of Western blot bands expressed in arbitrary units (AU); average values of three independent experiments ± SEM are shown; statistical significance was assessed using unpaired, two-tailed t-tests; ns = not significant (p > 0.05), * = p ≤ 0.05, ** = p ≤ 0.01. d. Ratios of plasma membrane to cytosolic β-catenin immunofluorescence in dorsal blastomeres of 3 hpf embryos. Mean ratios ± SEM are from 10 cells/embryo (n = 7, three independent experiments); statistical significance assessed by unpaired, two-tailed t-test, *** = p < 0.001. e. Ventralized hypomorphic phenotypes (v1-3) of 1 dpf embryos injected with 20 ng/μl mouse PrP mRNA. f. Quantification of 6 hpf embryos with abnormal dorsoventral specification upon injection of mouse PrP alone, together with PP2, or with its inactive analog PP3

Fig. 7

Effect of Δ23-31 and ΔCR deletions on PrP activity. a. Mouse PrP deletion mutants. Protein domains are marked with different colors: SP = signal peptide, R = repetitive domain, H = hydrophobic stretch, G = globular domain; green triangles = GPI-anchor. b. Detection of mouse WT or mutant PrPs using the D18 anti-PrP antibody on 6 hpf embryo protein lysates. c. Phenotypic quantification of 7 hpf embryos co-injected with PrP-1 morpholinos and PrP mRNAs. d. Localization of mouse PrP constructs in the deep cells of 6 hpf embryos (6H4 anti-PrP antibody staining). Scale bar = 10 μm. e. Embryonic phenotypes upon expression of zebrafish (ZF) or mouse (mo) PrP mRNAs (7 hpf, lateral views); arrowheads show abnormal dorsal thickening; V = ventral, D = dorsal. f. Quantification of 7 hpf embryos with dorsoventral phenotypes upon expression of mouse PrPs. Mean values are shown in (c) and (f). g. MTT viability assay of HEK cells expressing mouse or zebrafish PrPΔCRs after Zeocin treatment. WB = Western blot; IF = immunofluorescence. Data are shown as the average percentage of A₅₇₀ values in untreated cells ± SEM; statistical significance was assessed using unpaired, two-tailed t-tests, * = p ≤ 0.05, ** = p ≤ 0.01

Fig. 8

Aβ oligomers induce a PrP gain-of-function effect in zebrafish embryonic cells. Levels of AJ proteins (a and b) and SFKs (c-e) in 6 hpf embryonic cells upon treatment with Aβ monomers (m) or oligomers (o). Actin was used as a protein loading control (f). E-cadherin arrowheads as in Fig. 1; arrows = distinct SFKs bands (black: Src/Yes; grey: Fyn). Densitometric analysis of Western blot bands is shown in arbitrary units (AU). Data were collected from three independent experiments (average values ± SEM); statistical significance was assessed using unpaired, two-tailed t-tests, * = p ≤ 0.05, *** = p ≤ 0.001