PrP-1 regulates the turnover of selected AJ components

During gastrulation, the cell-surface expression of E-cadherin is dynamically regulated to promote tissue cohesiveness while allowing for coordinated cell movements [23]. In zebrafish embryos and mammalian cells, the 120 kDa cell-surface, mature E-cadherin protein is derived from the intracellular cleavage of a 140 kDa precursor polypeptide residing largely in intracellular vesicles [24, 25]. We reported that PrP-1 knockdown leads to depletion of mature E-cadherin but not its precursor form, suggesting that biosynthesis of the protein is normal but either its turnover or maturation are impaired [21]. To characterize this phenomenon, we first analyzed the effect of PrP-1 knockdown on E-cadherin internalization using immunofluorescence and Western blot. Treatment of morphant embryos with Dynasore, an inhibitor of clathrin- and dynamin-dependent endocytosis, restored the cell surface localization of E-cadherin by 6 hpf (Fig. 1a). In addition, Dynasore induced a concentration-dependent recovery in the levels of mature E-cadherin (Fig. 1b), indicating that its depletion in PrP-1 morphants is a consequence of increased endocytosis. Consistent with these molecular changes, Dynasore prompted a clear reduction -up to 40.23 %; p < 0.005- in the proportion of arrested/deformed PrP-1 knockdown embryos (Fig. 1c).

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 Full size image

To determine the mechanisms of E-cadherin depletion in PrP-1 morphants, we treated them with MG-132 and chloroquine, inhibitors of proteasomal and lysosomal protein degradation, respectively. By 6 hpf, MG-132 caused a substantial recovery in the levels of mature E-cadherin, whereas chloroquine only enhanced the levels of its 140 kDa precursor form (Fig. 1d). Moreover, MG-132, chloroquine and ammonium chloride (another lysosomal inhibitor) improved the morphology of knockdown embryos (Fig. 1c), suggesting that both the mature and precursor forms of E-cadherin contribute to embryonic cell adhesion under these conditions. Notably, MG-132 -which restores the mature E-cadherin product- had the strongest rescuing effect of all inhibitors tested (71.26 % reduction in the proportion of arrested embryos, Fig. 1c) and restored E-cadherin localization (Additional file 1: Figure S1B). Together, these experiments indicate that in the absence of PrP-1, mature E-cadherin is increasingly endocytosed and degraded via a proteasome-dependent mechanism.

Binding of β-catenin to the cytoplasmic tail of E-cadherin helps anchor AJs to the actin cytoskeleton [26]. When detached from E-cadherin, free β-catenin accumulates in the cytosol and is rapidly degraded unless induced to activate gene transcription in the nucleus [27]. Given the strong cytosolic localization of β-catenin in PrP-1 morphant gastrulae (Fig. 1a), we examined the stability of this protein pool. Western blot and immunofluorescence analyses confirmed that β-catenin is internalized and strongly down-regulated in these embryos, and that this effect can be reverted by MG-132 or chloroquine (Fig. 1d and Additional file 1: Figure S1B). Interestingly, although β-catenin is not a direct target of the endocytic machinery, Dynasore treatment also restored its cell surface localization (Fig. 1a). Because β-catenin associates to the plasma membrane only indirectly via E-cadherin, this result implies that its internalization and degradation in PrP-1 morphants are secondary effects of E-cadherin endocytosis.

p120-catenin stabilizes E-cadherin at the cell surface by binding to its cytoplasmic tail and preventing its endocytosis [26]. Since p120-catenin depletion leads to the internalization and degradation of the entire AJ complexes, [28], we asked if its levels were reduced in PrP-1 morphants. Interestingly, Western blot revealed that while E-cadherin and β-catenin became increasingly degraded at higher PrP-1 morpholino doses, the levels of p120-catenin remained unaltered (Fig. 1e). Taken together, these data show that, in vivo, PrP-1 prevents the internalization and proteasomal/lysosomal degradation of E-cadherin/β-catenin complexes independently of p120-catenin.

Fyn and Yes act downstream of PrP-1 to maintain embryonic AJ stability

PrP activates Fyn under diverse experimental conditions including cross-linking of PrP via antibodies as well as binding of PrP to other PrP molecules, NCAM or Aβ oligomers [7, 14, 21, 29]. In agreement with these data, we previously observed the accumulation of zebrafish embryonic Fyn at PrP-dependent cell-cell contacts [21]. On the other hand, genetic or chemical inactivation of Fyn and the related SFK Yes have been shown to impair zebrafish gastrulation [30–32]. Given the common involvement of SFKs in PrP signaling, zebrafish gastrulation, and the modulation of cadherin-based cell adhesion [33], we examined whether Fyn and Yes jointly mediate the effect of PrP-1 on AJs in vivo. First, we assessed the extent of the similarities between SFK and PrP loss-of-function phenotypes. At 6 hpf, Fyn and Yes single morphants showed epibolic arrest and deformed blastoderms (55–60 % of injected embryos), clearly resembling the PrP-1 morphant phenotype (Fig. 2a and b). The combined Fyn/Yes knockdown produced a stronger effect than the single knockdowns (82 % of injected embryos, Fig. 2a and b), indicating functional synergy between the two kinases. The protein knockdowns were titrated and verified by Western blot using an anti-Src antibody against the conserved C-terminus of SFKs (Fig. 3b): morphant embryos showed a clear reduction in the 65 and 50 kDa bands representing zebrafish Src/Yes and Fyn, respectively [34] (Fig. 3b). In line with the phenotypic quantifications, SFK down-regulation was more pronounced upon combined Fyn/Yes knockdown. We then asked whether the morphological resemblance between SFK and PrP-1 morphants involved common defects in AJ function. In fact, intracellular accumulation of E-cadherin and β-catenin was evident in Fyn and Yes, single and double knockdown embryos (Fig. 3a and Additional file 1: Figure S2A). Furthermore, Fyn/Yes morphant embryos displayed reduced levels of mature E-cadherin and β-catenin at 6 hpf (Fig. 3c). Hence, the morphological and molecular convergence of Fyn/Yes and PrP-1 knockdown phenotypes suggests that these proteins act cooperatively to stabilize embryonic AJ components.

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 Full size image

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 Full size image

Because SFKs transduce extracellular signals from trans-membrane receptors and GPI-anchored molecules like PrP [35, 36], we verified whether zebrafish Fyn and Yes are downstream effectors of PrP-1 and could therefore rescue the PrP-1 knockdown phenotype. We tested for this genetic interaction by co-injecting zebrafish Fyn and Yes EGFP-tagged mRNAs into PrP-1 morphants. To assess if the rescues were directly related to SFK enzymatic activity, we used wild-type (WT) and constitutively active (CA) forms of Fyn and Yes, the latter exhibiting higher kinase activity due to mutation of the conserved negative regulatory site at tyrosine (Tyr) 527. After verifying the proper localization and expression levels of the SFK constructs (Figs. 2c and 3d), their rescuing ability was quantified by scoring embryonic phenotypes between 6 and 7.5 hpf (Fig. 2a and b). Co-injection of Fyn/Yes mRNAs at 75 pg/embryo significantly reduced the proportion of arrested/deformed morphant embryos (41 % reduction, p < 0.01). Notably, CA Fyn/Yes mRNAs produced a more efficient rescue of the PrP-1 phenotype (64 % reduction; p < 0.01) using less mRNA (6 pg/embryo) (Fig. 2b). The higher enzymatic activity of the CA constructs was confirmed by Western blot using phospho-specific antibodies against activated SFKs (phosphorylated at Tyr416) and their target residue on β-catenin (phosphorylated at Tyr142) [37]. These Western blots show that the depletion of phospho-β-catenin in PrP-1 morphants is reversed upon expression of increasingly active WT and CA Fyn/Yes (Fig. 3d). Finally, we verified if SFK expression induced the recovery of AJ components in PrP-1 morphant embryos. Indeed, exogenous Fyn and Yes restored the cell surface localization and levels of E-cadherin and β-catenin by 6 hpf (Fig. 3a and b). Altogether, these data indicate that Fyn and Yes act downstream of PrP-1 to promote AJ function via tyrosine phosphorylation.

PrP-1 modulates embryonic SFK function

Having identified Fyn and Yes as downstream effectors of PrP-1 in vivo, we next asked whether PrP modulates the levels and activity of SFKs. In fact, Western blots of 6 hpf embryos showed reduced levels of Src/Yes (65 kDa band, 29.77 % reduction; p < 0.05) and Fyn (50 kDa band, 43.36 % reduction; p < 0.05) upon PrP-1 knockdown (Fig. 4a and b). This effect, also evident in whole mount immunostainings (Fig. 4e), became more pronounced at higher morpholino doses (Additional file 1: Figure S3A) and could be partially rescued with chloroquine but not with MG-132 (Fig. 4d). Interestingly, PrP-1 knockdown and chloroquine also modified the levels of phospho-Tyr142-β-catenin, suggesting that PrP-1 effectively modulates SFK catalytic activity. To verify this, we measured the ratios of active and non-active SFKs in PrP-1 morphants. Using phospho-specific antibodies, we found a significant decline in the ratios of active (phosphorylated at Tyr416) vs. total Src/Yes and Fyn (44.28 % and 83.29 % reduction, respectively; p < 0.01) upon PrP-1 knockdown (Fig. 4c). Notably, no significant changes were observed in the ratios of inactive (phosphorylated at Tyr527) vs. total Src/Yes and Fyn (Additional file 1: Figure S3B), indicating that PrP-1 preferentially stabilizes the levels of activated SFKs. Accordingly, overall tyrosine phosphorylation was reduced in the deep cells of PrP-1 morphants (Additional file 1: Figure S3C). Therefore, our experiments show that PrP-1 positively modulates the levels and activity of SFKs in the gastrula, partly by preventing their lysosomal degradation.

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 Full size image

Embryonic PrP gain-of-function phenotype

We have reported that PrP overexpression in zebrafish induces a distinct morphological phenotype characterized by asymmetric epiboly and increased cell-cell adhesion [21, 38]. To mechanistically link the zebrafish PrP gain- and loss-of-function phenotypes, we characterized PrP-overexpressing (PrP-OE) embryos at the cellular and molecular levels. Normally, between 5 and 6 hpf, cellular structures known as the germ ring and the shield define the marginal and dorsal regions of the zebrafish gastrula, respectively [39]. PrP-OE embryos lack these morphological features and develop instead a large dorsal thickening (Fig. 5a). This abnormal accumulation of cells results in mechanical stress and bursting of the embryo before completion of epiboly (Additional file 2: Video S1). We reasoned that if PrP-1 knockdown destabilizes AJ components, PrP overexpression would enhance their cell surface expression and lead to the observed cell clumping. In fact, embryos overexpressing mouse PrP showed normal plasma membrane localization of E-cadherin and β-catenin at 6 hpf (Additional file 1: Figure S5A). Surprisingly, while their levels of mature E-cadherin were increased (Fig. 5b), those of β-catenin were strongly reduced and could not be restored with MG-132 or chloroquine (Fig. 5c). Given the asymmetric deformation of these embryos, we asked if the opposite changes in the levels of E-cadherin and β-catenin might stem from local alterations in their distribution along embryonic axes. Whole mount immunostainings of 6 hpf control embryos revealed an increasing, ventral to dorsal gradient of both proteins (fluorescence profiles in Fig. 5d). In contrast, embryos overexpressing mouse or zebrafish PrPs exhibited increased dorsal E-cadherin accumulation and reduced dorsal β-catenin (Fig. 5d and Additional file 1: Figure S5B). Concomitant with these alterations, dorsal cells appeared deformed and enlarged (Fig. 5e). Thus, cell surface E-cadherin becomes locally up-regulated in the dorsal cells of PrP-OE embryos, whereas the associated β-catenin is down-regulated via a mechanism other than protein degradation.

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 Full size image

Crucially, β-catenin signaling is pivotal to dorsal specification. As early as the 128-cell stage (2.25 hpf), cytosolic β-catenin enters the nuclei of a small group of marginal blastomeres to activate the transcription of dorsalizing genes [40]. Given the lack of shield and reduced levels of dorsal β-catenin in 6 hpf PrP-OE embryos, we considered the possibility that PrP might disturb earlier stages of axis formation by locally depleting dorsal nuclear β-catenin. Indeed, quantitative confocal analysis of dorsal marginal blastomeres at 3 hpf showed a clear reduction in the average number of cells stained for nuclear β-catenin, from ~7 in control embryos to ~1.8 and ~2.5 in embryos overexpressing zebrafish PrP-1 or mouse PrP, respectively (Fig. 6a and b; p < 0.001). Therefore, the dorsalizing nuclear function of β-catenin in these early embryos is compromised as a result of increased PrP activity.

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 Full size image

In mouse embryonic stem cells, cell surface E-cadherin can bind and sequester β-catenin at the plasma membrane, thus preventing its internalization and nuclear translocation [27]. Given the dorsally increased levels of cell surface E-cadherin in 6 hpf PrP-OE embryos, we examined if a similar mechanism could induce the observed reduction of dorsal nuclear β-catenin earlier, at 3 hpf. Fittingly, Western blot analysis of 3 hpf PrP-OE embryos revealed a ~60 % increase in the levels of mature, cell surface E-cadherin (Fig. 6c). Moreover, fluorescence measurements indicated that the ratio of plasma membrane vs. cytosolic β-catenin was 1.8 times higher in the dorsal cells of PrP-OE embryos than in their control counterparts (Fig. 6d). This difference was not due to cytosolic degradation of β-catenin, as control and PrP-OE embryos showed comparable levels of this protein at 3 hpf (Fig. 6c). Rather, the increased plasma membrane localization suggests that β-catenin is retained by cell surface E-cadherin, thus reducing the pool of cytosolic molecules available for translocation to the nucleus.

It is well-established that preventing the nuclear localization of β-catenin in dorsal blastomeres produces ventralized embryos by 1 dpf [41]. This effect could only rarely be observed in PrP-OE embryos because they died from excessive cell adhesion before completing gastrulation. We circumvented this problem by microinjecting lower mRNA amounts to overexpress PrP at sub-lethal levels. The resulting PrP hypomorphic embryos completed epiboly and by 1 dpf presented a distribution of ventralized phenotypes with variable penetrance and expressivity (Fig. 6e). The anterior and dorsal defects varied in strength, ranging from mildly reduced eyes, brain and notochord to completely undifferentiated heads and shortened anterior-posterior axes. This result further supports the notion that PrP overexpression impairs the role of β-catenin in axis formation by exacerbating its adhesive function.

Finally we asked if, consistent with the PrP-1 knockdown data, SFKs also mediate the PrP gain-of-function phenotype. In fact, at 3 hpf, the relative levels of activated (p-Tyr416) Src/Yes and Fyn were elevated in PrP-OE embryos (Fig. 6c). This higher SFK activity was further evident from the corresponding increase in the levels of p-Tyr142-β-catenin (Fig. 6c). Moreover, blocking SFK activity with the pharmacological inhibitor PP2 reduced the proportion of 6 hpf PrP-OE embryos with abnormal morphology from 83.26 % to 49.97 % (Fig. 6f; p < 0.01). To ascertain whether this early increase in SFK activity may contribute to the dorsal accumulation of cell surface E-cadherin, we analyzed their distribution along embryonic axes. Confocal analyses of whole untreated embryos at 3 hpf revealed that SFKs and E-cadherin are similarly distributed in a ventral to dorsal gradient (Additional file 1: Figure S5C). Interestingly, this gradient was more pronounced in PrP-OE embryos, suggesting that exogenous PrP enhances E-cadherin adhesion preferentially in dorsal blastomeres by activating their already higher levels of SFKs. Of note, the conserved link between PrP and SFK activation was also observed in human MCF-7 cells, where expression of mouse or fish PrPs triggered the accumulation of SFKs at contacts sites and the corresponding increase in SFK activity (Additional file 1: Figure S6). Altogether, our data point at the positive regulation of SFK activity and AJ function by PrP as the underlying mechanism behind the zebrafish PrP gain- and loss-of-function phenotypes.

Functionality of mouse PrP mutants in zebrafish embryos

Deletions in the central region (ΔCR, residues 105-125) and the N-terminal polybasic stretch (Δ23-31) of mouse PrP (Fig. 7a) modulate its neurotoxic activities. For instance, PrPΔCR triggers a lethal neurodegenerative phenotype in transgenic mice [42] whereas PrPΔ23-31 abrogates this neurotoxic effect as well as the rescuing (neuroprotective) activity of WT PrP [43–45]. Because mouse PrP can partially revert the zebrafish PrP-1 knockdown phenotype [21, 38], we used mRNA rescue experiments to assess whether the ΔCR and Δ23-31 mutations modify the conserved PrP function required for zebrafish epiboly. After verifying and titrating expression of the mouse constructs in zebrafish embryos (Fig. 7b), mRNAs and PrP-1 morpholinos were co-injected into fertilized eggs, and the resulting phenotypes were scored at 6 hpf (Fig. 7c). Notably, PrPΔCR retained full activity and rescued the PrP-1 morphants as efficiently as WT PrP. In contrast, PrPΔ23-31 showed a strongly reduced rescuing activity (~74 % relative to WT PrP), similar to an additional mutant carrying both deletions, PrPΔCR/Δ23-31. The reduced activity of the Δ23-31-contanining constructs was not due to deficient cell surface expression, as demonstrated by whole-mount immunofluorescence (Fig. 7d).

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 A 570 values in untreated cells ± SEM; statistical significance was assessed using unpaired, two-tailed t-tests, * = p ≤ 0.05, ** = p ≤ 0.01 Full size image

To confirm the rescue data without the confounding effect of the PrP-1 knockdown phenotype, we also conducted gain-of-function experiments and measured the ability of the mutants to induce the conserved PrP overexpression phenotype (Fig. 7e). In these assays, the activity of the ΔCR construct was again comparable to that of WT PrP (~95 % of embryos with overexpression phenotype), whereas deletion of residues 23-31 reduced this effect by ~50 % (Fig. 7f). Importantly, these results reflect the ability of the constructs to inhibit nuclear β-catenin signaling (Additional file 1: Figure S7), as expression of WT and ΔCR PrPs significantly reduced the numbers of marginal blastomeres positive for nuclear β-catenin (2.5 and 3.7 cells/embryo, respectively, vs. 7 cells/untreated embryo). Moreover, β-catenin nuclear translocation was barely affected by overexpression of Δ23-31 or Δ23-31/ΔCR PrPs (7.8 and 7.7 cells/embryo, respectively). Therefore, we conclude that the N-terminal polybasic stretch (residues 23-31) is critical for the control of embryonic cell adhesion by PrP, and that the ΔCR mutation does not significantly impair this PrP function.

Importantly, expression of mouse PrPΔCR in the presence or absence of endogenous PrP-1 did not cause enhanced toxicity or lethality in fish embryos. A possible explanation is that ΔCR toxicity requires the expression of a developmentally regulated PrP partner absent from the zebrafish gastrula. Alternatively, ΔCR toxicity might be a specific property of mammalian PrPs. To address this issue, we asked whether zebrafish PrPΔCRs can induce toxicity in an established mammalian cell model. For this, we generated ΔCR versions of zebrafish PrP-1 and -2 (Additional file 1: Figure S4), and tested them in HEK cells using the drug-based cellular assay (DBCA) [46]; changes in cell viability were assessed by the MTT assay. Notably, cells expressing either zebrafish or mouse PrPΔCRs were less viable than those expressing the corresponding WT PrPs (Fig. 7g; 20 %, 40 % and 45 % reduction in MTT metabolic activity for PrP-1-, PrP-2- and mouse PrP-expressing cells; p < 0.05, p = 0.01, and p = 0.01, respectively). This suggests that the role of the central region as a suppressor of cytotoxicity is evolutionarily conserved and that the corresponding toxic cascade in zebrafish may become active only after gastrulation.

PrP-dependent influence of Aβ oligomers on zebrafish embryonic SFKs and AJs

In mammalian neurons, binding of Aβ oligomers to neuronal PrP activates Fyn and blocks the endocytosis of the NR2B subunit of the NMDA receptor (NMDAR), leading to glutamate excitotoxicity and synaptic damage [7]. In our zebrafish embryos, PrP-mediated activation of Fyn and Yes prevents the endocytosis of E-cadherin to support embryonic cell adhesion. The obvious similarity between these two unrelated events prompted us to ask if Aβ oligomers would modulate zebrafish embryonic SFKs and AJ components in a PrP-dependent manner. As a negative control, we used monomeric Aβ, which unlike oligomeric Aβ, does not induce Fyn activation (Um et al, 2012). To facilitate access of Aβ to embryonic cell surfaces, we dissociated 5 hpf zebrafish control and PrP-1 morphant embryonic cells and allowed them to re-aggregate for one hour in the presence or absence of 0.5 μM Aβ oligomers before analyzing them for changes in the levels of SFKs and AJ components. Notably, Aβ oligomers triggered increased levels of p-Tyr416 (activated) Src/Yes and Fyn (4.4-fold and 10.1-fold increase, respectively; p < 0.001) without significantly affecting their total levels (Fig. 8c-e). In addition, the levels of E-cadherin and β-catenin were elevated by 2.6- and 2.2-fold, respectively (p < 0.001, Fig. 8a and b). The increase in p-Tyr416 SFKs and β-catenin was dependent on endogenous PrP-1 expression and Aβ oligomerization, since it was observed neither in PrP-1 morphant cells nor upon exposure to Aβ monomers. Interestingly, the increase in mature E-cadherin was also PrP-1-dependent but it was indistinctively triggered by Aβ monomers or oligomers (see Discussion). Thus, in zebrafish blastocytes, Aβ oligomers induce biochemical changes consistent with a PrP gain-of-function scenario.