Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain

Abstract

The mechanisms regulating the outgrowth of neurites during development, as well as after injury, are key to the understanding of the wiring and functioning of the brain under normal and pathological conditions. The amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, its physiological role in the central nervous system is not known. Many physical interactions between APP and intracellular signalling molecules have been described, but their functional relevance remains unclear. We show here that human APP and Drosophila APP-Like (APPL) can induce postdevelopmental axonal arborization, which depends critically on a conserved motif in the C-terminus and requires interaction with the Abelson (Abl) tyrosine kinase. Brain injury induces APPL upregulation in Drosophila neurons, correlating with increased post-traumatic mortality in appl(d) mutant flies. Finally, we also found interactions between APP and the JNK stress kinase cascade. Our findings suggest a role for APP in axonal outgrowth after traumatic brain injury.

Figure 1

APP induces increased axonal arborization in the sLNv. (A) Confocal images of the sLNv in third instar larva show normal morphology when expressing human APP (A′) or Drosophila APPL (A″). (B) Overview images of the adult sLNv, showing the dorsally projecting axons (arrowheads) and terminal arbours (arrows). (C) Detail of the sLNv terminal axonal arbour, which extends further and is more extensively arborized (arrows) when human APP (C′) or Drosophila APPL (C″) is overexpressed. (D, D′) Three-dimensional reconstructions rotated at 40 or 90° angles of the dorsal projections of the sLNv without and with overexpression of APP.

Figure 2

The APP axonal arborization phenotype is quantifiable, dosage dependent and can be induced postdevelopmentally. (A) For quantification of the two-dimensional area of the terminal axonal arbour, the axonal stem was marked with a straight line (red vertical line). Between the points where the two terminal arbours deviate from this line, a horizontal line was drawn (horizontal red line). The outline of the axonal processes dorsal to this line was traced (blue line), and the area inside was measured. The distance between the intersection points of the vertical lines with the commissure served as a measure for brain size (green line). (B) Quantification of the axonal arbour area shows a robust increase, independent of genetic background, when APP is expressed (^***P<0.001 compared to control=yellow bars; error bars represent the 95% confidence interval). (C, D) Modifying the dose of APP expression by varying the temperature or the number of drivers (2X=2 copies of the driver) modifies the effect on the axonal arbour area (^*P<0.05; ^***P<0.001 compared to control=yellow bar; error bars represent the 95% confidence interval). (E) Flies raised at 18°C and switched to 28°C for 10 days as adults show outgrowth and arborization of axons (arrows) in different directions.

Figure 3

The C-terminal domain of APP mediates its effects on axonal arborization. (A) Schematic representation of the truncated APP constructs. The red boxes represent the Aβ domain, which is recognized by the 4G8 antibody. (B) Quantification of axonal arbour areas (^***P<0.001 compared to control=yellow bar; error bars represent the 95% confidence interval). (C) Cell bodies of LNv in which different truncated constructs of APP are expressed show 4G8 immunoreactivity; (C′) is taken at higher laser power in the red channel. (D) Terminal axonal arbours of sLNv expressing full-length APP and APPΔCT show 4G8 immunoreactivity, while sLNv expressing APPΔNT do not. Axonal arborization is increased by expression of APP and APPΔNT, but not by APPΔCT.

Figure 4

The Abl signalling cascade is necessary and sufficient to induce axonal arborization. (A) Amino-acid composition of the APP C-terminus, showing the conserved domains and the amino acids changed or removed in the different constructs. (B–S) Confocal images showing the axonal arbour of sLNv visualized with anti-PDH immunostaining (H–K, P–S) or CD8-GFP coexpression (B–G, L–O). (C, D) Expression of APP forms lacking the PEER or Go-protein-binding domain induces increased axonal outgrowth. (E) Deletion of the YENPTY motif (E) and point mutations in Y682 (F) or Y687 (G) of the APP C-terminus abolish its effect on axonal arborization. (C–G, insets) Cell bodies stained with 4G8 antibody (red). (H–K) The APP phenotype is suppressed by genetic reduction of Abl (J), or by coexpression of a kinase-dead form of Abl (K). (L–O) Expression of wildtype Abl has a small effect on axonal outgrowth (L, arrow), while activated forms of human Abl induce marked increases in axonal arborization (M, N). Expression of kinase-dead Abl has no effect (O). (P) Activated Abl induces axonal arborization in appl^d mutant flies. (Q–S) Genetic reduction of Chic (Q) suppresses the APP phenotype, while genetic reduction of Ena (R) or Tsr (S) does not have an effect. Quantification of all phenotypes is shown in Supplementary Figure S2D.

Figure 5

APPL expression is increased in neurons after traumatic brain injury in Drosophila. (A) Schematic of a Drosophila head showing the points of entry and exit of the insect pin used for inducing brain injury (adapted from Drosophila protocols, CSHL press). (B) Three-dimensional reconstruction of a Drosophila brain showing increased expression of APPL in the damaged optic lobe 48 h after injury. The deduced tract of the insect pin is represented by a white arrow. (C) Schematic of a confocal section of a Drosophila brain half stained with TRITC-Phalloidin (red), anti-Elav (a marker for neuronal nuclei; blue) and anti-APPL (green). The same stainings are used in panels D–L. The brain landmarks used to define the depth of confocal sections in panels D, F–I, L are highlighted. (D) Three-dimensional reconstruction of an undamaged adult brain rotated 90°. (E) Confocal section showing the normal staining pattern in an undamaged adult brain. (F, G) Confocal sections of brains 48 h after trauma, showing increased APPL immunoreactivity surrounding the damaged tissue (arrows). (H) At 4 days after trauma, an increased APPL signal is still observed. (I) At 7 days after trauma, only a weak APPL signal is detectable. (J, K) Higher magnification of (F), showing APPL upregulation in neuronal cell bodies (arrowheads) and axons (arrows) surrounding the damaged tissue (black gap in TRITC-Phalloidin staining). (L) No APPL signal is detected in damaged appl^d brains. (M) Graphic representation of the mortality of wildtype versus appl^d mutant flies with or without brain trauma (^*P<0.05; ^***P<0.001).

Figure 6

APPL and JNK signalling are coactivated after traumatic brain injury in Drosophila. (A–C) Confocal sections of puc-LacZ transgenic Drosophila brains triple labelled for APPL (green), TRITC-Phalloidin (red) and BGal (blue, representing levels of JNK signalling). (A) Low levels of APPL and BGal expression are found in an undamaged brain. (B) Increased APPL and BGal expression are found in traumatized areas 48 h after trauma. (B′) Higher magnification of (B), showing partial colocalization (arrowheads) of APPL and BGal expression around the tissue damage (white line). (C) BGal expression is also increased in damaged appl^d mutant brains. (D) Ectopic activation of JNK signalling by expression of HepCA induces axonal overextension without increased arborization. Inactivation of JNK signalling by expression of BskDN does not affect the normal development of the sLNv axonal arbour, but suppresses APP-induced axonal outgrowth, while it has no effect on Abl-induced axonal arborization. (E) Schematic representation of proposed events following brain injury in Drosophila.