The <i>Drosophila</i> Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling

<div><p>Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. <i>Drosophila</i> possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of <i>Drosophila</i> APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.</p> </div

APPL interacts with PCP signaling during MB development.

<p>(A–D) Structure of αβ neurons labeled with the FasII antibody. All the images are <i>z</i>-projections of confocal image stacks (scale bar, 50 µm). (A) Morphologically normal αβ neurons in a control adult brain. (B) Morphologically aberrant αβ neurons in <i>Appl^dw*/Y;;Fz^KD/+</i> adult brains. Loss of one copy of Fz in the <i>Appl</i>^−/− background increases moderately, but not significantly, the penetrance of β-lobe loss (indicated by the arrow) to 21% (<i>n</i> = 28); <i>p</i> value = 0.2166 calculated with G-test. (C) Morphologically aberrant α/β neurons in <i>Appl^d w*/Y;UAS-Fz-DN/+;P247Gal4/+</i> adult brains. Overexpression of a dominant negative form of Fz in the <i>Appl</i>^−/− background increases moderately, but not significantly, the penetrance of the phenotype to 18% (<i>n</i> = 28); <i>p</i> value = 0.4226 calculated with G-test. (D) Morphologically aberrant αβ neurons in <i>Appld w*/Y;Vang^stbm-6/+</i> adult brains. Loss of one copy of Vang in the <i>Appl</i>^−/− background increases the penetrance of the β-lobe loss up to 33% (<i>n</i> = 21); <i>p</i> value = 0.02304 calculated with G-test. (E) The graph shows the penetrance of β-lobe loss in genetic interaction experiments, normalized against the penetrance in the <i>Appl^dw*</i> background. (F, G) APPL and Vang localization during development in brain of flies <i>Act-Stbm-EYFP</i> expressing an EYPF tagged form of Vang under the control of the Actin promoter. Immuno-fluorescence analysis using anti-APP-Cterm (F, G in blue), anti-GFP (F′, G′ in green), and anti-FasII (F″, G″ in red) antibodies. The images are single confocal stacks (scale bar, 25 µm). The F‴ and G‴ panels show the merge of the FasII and GFP channel for the indicated samples. (F) At 48 h APF, Vang is broadly expressed along the MB β axons, where also APPL is expressed as indicated by the arrow. (G) At adult stage, the level of Vang detectable in the β axons is strongly reduced compared to the previously analyzed time point, while APPL is enriched in the MB neurons (as indicated by the arrow). (H) The graph shows the penetrance of the β-lobe loss in the <i>Appld^w*</i> flies overexpressing Dsh or Wnt5 (<i>p</i> value equal to 0.1287 and 0.0006692, respectively). Activation of Wnt-PCP signaling upon Wnt5 signaling rescues the β-lobe phenotype. ** Indicates a <i>p</i> value<0.001 calculated with G-test.</p

APP is required for the proper response to Wnt5 and forms complexes with PCP core proteins.

<p>(A) Analysis of the responsiveness of wt MEFs and MEFs lacking all the APP isoforms to Wnt5 treatment. MEFs were treated for 2 h with rmWnt5a and subsequently analyzed by Western blot. After the Wnt5 treatment, Dvl2 is phosphorylated and this modification is indicated by a shift of the band detected by Dvl2 Ab. KO MEFs respond less efficiently to Wnt5. Re-introduction of hAPP rescues the responsiveness to Wnt5. The graph shows a quantification of the ratio between phospho-Dvl2 and Dvl2 in the analyzed samples. * Indicates a <i>p</i> value<0.05 calculated with one-way ANOVA plus Tukey's multiple comparison test. (B) Co-immunoprecipitation (Co-IP) of Appl-FLAG and Vang-Myc. The tagged proteins were co-expressed in HEK293T cells and immunoprecipitated with anti-FLAG antibody. Vang-Myc can be precipitated upon IP of Appl-FLAG. (C) Co-IP of Appl-FLAG and human Vangl2-HA. Human Vangl2-HA can be precipitated upon IP of Appl-FLAG. (D) Co-IP of human APP (C99)-FLAG and human Vangl2-HA. Human Vangl2-HA can be precipitated upon IP of APP (C99)-FLAG, indicating that interaction with PCP proteins is a conserved feature of APP proteins. (E) Co-IP of Appl-FLAG and dFz-GFP. <i>Drosophila</i> Fz can be precipitated upon IP of Appl-FLAG. (F) Co-IP of human APP (C99)-FLAG and human V5-Fzd5. V5-Fzd5 can be precipitated upon IP of APP (C99)-FLAG.</p

APPL is a robustness factor required for Mushroom Bodies development.

<p>(A–D) Developmental APPL expression in MB. Immuno-fluorescence analysis using anti-APP-Cterm (A–D in magenta) and anti-FasII (A′–D′ in green) antibodies. The A‴–D‴ panels show the merge of the FasII and GFP channel for the indicated samples. APPL is expressed at high levels in the developing brain and is enriched along MB axons both during development and in adult stages. (A, B) 48 h APF MB lobes. The images are single confocal stacks; zoom shows 63× magnification of the boxed area (scale bar, 50 µm in A, 25 µm in B). (C) Adult MB axons. The images are single confocal stacks (scale bar, 50 µm). (D) Zoom shows 63× magnification of the boxed area in panel C. The images are <i>z</i>-projections of two confocal image stacks (step size, 0.6 µm; scale bar, 25 µm). (E–G) Adult MB lobes labeled with FascilinII antibody (FasII). All images are <i>z</i>-projections of confocal image stacks (scale bar, 50 µm). (E) Morphologically normal α/β neurons in control adult brain. (F–G) Structure of α/β neurons in <i>Appl^dw*</i> null mutant adult brains. In the absence of APPL, MB lobes show an aberrant pattern of growth in 26% of the analyzed sample (<i>n</i> = 101). In particular, in 14% of the cases (F), the α lobe fails to project towards the dorsal side of the brains (as indicated by the arrow), whereas in 12% of the cases (G), the β lobe fails to project towards the midline (as indicated by the arrow). (H) Morphologically normal α/β neurons of a 48APF Canton S brain. (I–J) Morphologically aberrant α/β neurons of an Appld w*48APF brain. The α- (I) and β-lobe (J) loss observed in the adult brain is already present at 48 APF, thus suggesting that is not due to degeneration but rather to failure in axon growth.</p

Dsh phosphorylation is required for MB β-lobe development.

<p>Structure of αβ neurons labeled with FasII antibody. All the images are <i>z</i>-projections of confocal image stacks (scale bar, 50 µm). (A) Morphologically aberrant αβ neurons in <i>dsh¹</i> adult brains. Flies homozygous for a PCP specific allele of dsh (<i>dsh¹</i>) show a phenotype comparable to <i>Appl</i>^−/− mutants but with increased penetrance of β-lobe loss as indicated by the arrow (30%; <i>n</i> = 36). (B) Morphologically normal αβ neurons in <i>dsh¹/Y</i>;<i>dsh-DshGFP-wt/+</i> adult brains. Reintroduction of wild-type Dsh in the <i>dsh¹</i> mutant background completely rescues β-lobe loss with no brains showing defects (<i>n</i> = 31). (C) Morphologically aberrant αβ neurons in <i>dsh¹/Y</i>;;<i>dsh-DshGFP-Y473F/+</i> adult brains. Reintroduction of a Tyrosine 473 phospho-mutant form of Dsh in a <i>dsh¹</i> mutant background fails to rescue the β-lobe loss as indicated by the arrow (<i>n</i> = 41). (D–E) Expression pattern of Dsh-GFP in ;<i>dsh-DshGFP-wt/TM6b</i> and of Dsh-Y473F-GFP in ;<i>dsh-DshGFP-Y473F/+</i>. Immuno-fluorescence analysis of adult brains using anti-GFP (green) and anti-FasII (D′, E′ in magenta) antibodies (scale bar, 50 µm). All images are <i>z</i>-projections of two confocal sections (0.9 µm steps). The D″ and E″ panels show the merge of the FasII and GFP channel for the indicated samples.</p

APPL is a novel modulator of Wnt-PCP signaling required for axon guidance.

<p>Schematic representation of the proposed model. APPL is a novel regulator of Wnt-PCP pathway. In the presence of Wnt signaling, Fz binds Dsh, which needs to be phosphorylated by Abelson kinase to correctly relocalize to the membrane. We propose that Appl is part of the membrane complex formed by the core PCP proteins Fz1 and Vang. In turn, Appl recruits Abelson kinase to the complex and positively modulates Dsh phosphorylation. The subsequent activation of the signaling is required for MB β-axon growth.</p