Genetic analysis reveals that amyloid precursor protein and death receptor 6 function in the same pathway to control axonal pruning independent of β-secretase

Abstract

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that β-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that β-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning.

Keywords: degeneration; pruning.

Figure 1.

APP KO mice have impaired pruning of retinocollicular axons. A–C, Dorsal view of the SC at P6 from wild-type (WT; A) and APP KO (B, C) mice with a focal injection of the lipophilic axon tracer DiI into peripheral temporal retina. A, In WT mice, the DiI-labeled temporal RGC axons form a dense, nearly mature TZ in the topographically appropriate anterior (A, arrowheads) SC. A small number of short, simple axon segments persist just outside of the TZ (arrow) at this stage. B, C, In APP KO mice, the labeled temporal RGC axons form a TZ in the appropriate position and of the appropriate size. However, many long, branched RGC axon segments and remedial arbors persist in ectopic positions far outside the TZ (arrows). D–F, Vibratome sections (100 μm sagittal) through the SC of P6 WT (D) and APP KO (E, F) mice. D, In WT mice, significantly fewer axon segments persist outside of the TZ (as determined by axon presence in 100 μm² domains throughout SC, grid; chart), and those that are present are near the TZ (arrowhead). E, F, In contrast, in APP KO mice, RGC axon segments have not been eliminated (arrowheads) from areas far posterior (P) to the TZ, both medial (M) and lateral (L) to it (F, bottom, section from the lateral edge of the case in B). In APP KO mice, axon segments persist in a significantly greater number of 100 μm² domains outside of the dense TZ compared with WT (D; 69% increase, **p < 0.01; Student's t test), a defect in pruning in APP KO mice that is even more pronounced at locations more distant from the TZ (***p < 0.001; n = 10 for WT and APP KO). Scale bar, 250 μm.

Figure 2.

shRNA knock down of APP in retina impairs pruning of RGC axons in the SC. A, Representative Western blot (top) and quantification (bottom) of APP and actin protein from HEK cells cotransfected with an APP-expressing plasmid and an shRNA plasmid against APP or LacZ or untransfected (UT), as indicated, n = 3 repeat experiments. B, The right eyes of E13.5 embryos were co-electroporated with GFP expression plasmid and shRNA against LacZ or APP and RGC axon projections in the SC were examined at P6. At P1, all axons labeled in this way project to the posterior (P) end of the SC (i.e., the PSC; Simon et al., 2012; Fig. 7 A,B). As shown in the diagram (top), by P8, axons have pruned back so that they now only occupy the TZ. The next two panels show representative maximum intensity projection images of GFP-positive axons within the SC at P6 in WT mice coexpressing GFP and shRNA against LacZ (shLacZ) or APP (shAPP). Note that by P6 the pruning of axons in the PSC in shLacZ is largely complete. C, Representative images of the PSC in WT mice expressing shRNA against LacZ or APP. D, Quantification of the number of GFP-positive axons in the PSC, the spread of GFP labeling along the anterior–posterior axis of the SC as an estimate of the size of the TZ, and the number of GFP-positive axons in the PSC normalized to TZ size in mice expressing shLacZ or shAPP. **p < 0.01; n/s, not significant (p > 0.1).

Figure 3.

shRNA knock down of DR6 in the retina impairs pruning of RGC axons in the SC. A, Representative Western blot (top) and quantification (bottom) of DR6 and actin protein from HEK cells cotransfected with a DR6-expressing plasmid and an shRNA plasmid against DR6 or LacZ, or untransfected (UT), as indicated. n = 3 repeat experiments. **p < 0.01. B, C, RGCs of E13.5 embryos were coelectroporated in utero with GFP expression plasmid and shRNA against LacZ or DR6. B, Representative maximum intensity projection images of GFP-positive axons within the SC at P6 in WT mice coexpressing GFP and shRNA against LacZ or DR6. C, Representative images of the PSC in WT mice expressing shRNA against LacZ or DR6. D, Quantification of the number of GFP-positive axons in the PSC, the spread of GFP labeling along the anterior–posterior axis of the SC as an estimate of the size of the TZ, and the number of GFP-positive axons in the PSC normalized to TZ size in mice expressing shLacZ. *p < 0.05; n/s, not significant (p > 0.1).

Figure 4.

shRNA knock down of APP in the retina of DR6 knock-out animals does not impair pruning of RGC axons in the SC beyond what is observed in the DR6 knock-out alone. RGCs of E13 DR6 KO embryos were coelectroporated in utero with GFP expression plasmid and shRNA against LacZ or APP. A, Representative maximum intensity projection images of GFP-positive axons within the SC at P6 in DR6 KO mice coexpressing GFP alone or an shRNA against LacZ or DR6. B, Representative images of the PSC in DR6 KO mice expressing GFP alone or shRNA against LacZ or APP. C, Quantification of the number of GFP-positive axons in the PSC, the spread of GFP labeling along the anterior–posterior axis of the SC as an estimate of the size of the TZ, and the number of GFP-positive axons in the PSC normalized to TZ size in mice expressing shLacZ. n/s, not significant (p > 0.05).

Figure 5.

Pruning of RGC axons in the absence of BACE-1. A, Representative maximum intensity images of the SC from P6 WT and Bace-1 KO mice that were electroporated with a GFP expression plasmid at E13. Bace-1 KO mice exhibit an appropriately localized and sized TZ and no distinguishable difference in the degree of RGC axon pruning compared with wild-type littermates. B, Representative images of the PSC in WT and Bace-1 KO mice are shown. C, Quantification of the number of GFP-positive axons that persist in the PSC, the anterior–posterior spread of GFP labeling in the TZ, and the number of GFP-positive axons in the PSC normalized to the size of the TZ in WT and Bace-1 KO mice. **p < 0.01; n/s, not significant (p > 0.1).

Figure 6.

Genetic deletion of DR6 or APP, but not Bace-1, partially protects sensory axons from degeneration after NGF withdrawal in vitro. A, B, DRGs from E13.5 DR6 (A) or APP (B) KO embryos and wild-type (WT) littermates were cultured for 7 d in the presence of NGF (25 ng/ml; +NGF) and then deprived of NGF by exchanging culture media containing NGF with media lacking NGF and containing a function-blocking anti-NGF antibody (25 μg/ml; −NGF). After 36 h of NGF deprivation, cultures were fixed and axon degeneration was visualized by fluorescence microscopy using an antibody against βIII-tubulin (Tuj1). Representative images are shown for WT and KO axons cultured under various conditions. C, D, Axon degeneration was scored using a five-point scale and then normalized to the degeneration scored for WT axons under −NGF conditions. DRGs from DR6 (n = 11; C) and APP (n = 20; D) KO embryos showed partial protection from axon degeneration compared with WT littermates. E, To test the specificity of protection against axon degeneration offered by the anti-DR6 and anti-APP antibodies, antibodies were included in media at the time of NGF deprivation. Antibodies were used at 20 μg/ml. The anti-DR6 (3F4) and anti-N-APP antibodies prevented axon degeneration following NGF withdrawal to a greater extent than in the knock-outs, but this enhanced protection was still observed when using DRGs from the respective DR6 or APP KO embryos. One control antibody (IgG1, 20 μg/ml) had no effect on axon degeneration (n = 3), but a second (IgG2, 20 μg/ml) showed significant, though modest, protection (n = 4). F, DRGs from E13.5 Bace-1 KO embryos and WT littermates were cultured for 7 d in presence of NGF (25 ng/ml; +NGF) and then deprived of NGF by exchanging culture media containing NGF with media lacking NGF and containing a function blocking anti-NGF antibody (25 μg/ml; −NGF). G, DRGs from Bace-1 (n = 12) and Bace-1/2 KO embryos (n = 5) do not show any protection from degeneration. Error bars indicate SE. *p < 0.05; **p < 0.01; ***p < 0.001; n/s, nonsignificant (p > 0.05).

Figure 7.

The C-APP domain of APP is required for high-affinity interaction of the APP ectodomain with DR6. A, Schematic diagram of the domain structure of APP, and nomenclature used for AP-fusion proteins. GF-like, Growth factor-like domain; CuBD, copper binding domain; TM, transmembrane domain; Cyto, cytoplasmic domain. Note that the APP ectodomain fragments studies here (sAPPβ, N-APP, sAPP-E2, and C-APP) do not include the Aβ sequence. B, COS-7 cells were transfected with constructs for DCC (control) or full-length DR6 or APP. Two days after transfection, cells were incubated with DCC-AP (control), DR6-AP (aas: 1–349), or sAPPβ-AP (aa 1–596) fusion proteins and tested for binding using an AP substrate. Control (DCC-transfected) cells showed negligible background AP activity and no binding for any of the AP fusions proteins. COS-7 cells expressing DR6 showed a specific interaction with sAPPβ-AP, but not with either DCC-AP or DR6-AP proteins. Further, COS-7 cells expressing APP showed a specific interaction with DR6-AP, but not with either of the DCC-AP or sAPPβ-AP proteins. C, To delineate the minimal region of APP that mediates interaction with DR6, COS-7 cells expressing DCC (control) or DR6 were incubated with N-APP-AP (aa 1–286), sAPP-E2-AP (aa 287–596), or C-APP-AP (aa 287–506) fusion proteins. Only sAPP-E2-AP and C-APP-AP showed specific binding to DR6-expressing cells, whereas no detectable N-APP-AP binding was observed. D, Direct interaction between the ectodomains of DR6 and APP was determined by immunoprecipitation. DR6-Fc and DCC- Fc fusion proteins from HEK293 supernatants were immobilized on protein A/G agarose beads and incubated with HIS-tagged sAPPβ, C-APP, or N-APP proteins. DR6-Fc, but not DCC-Fc, selectively immunoprecipitated both sAPPβ-HIS and C-APP-HIS, but not N-APP-HIS. E, The affinity of the interaction of DR6 and APP was assessed by ELISA. Consistent with the cell-binding and immunoprecipitation data, DR6-Fc shows a specific and high-affinity interaction with wells coated with sAPP-HIS (EC₅₀ = 1.4 nm) or C-APP-HIS (EC₅₀ = 10.1 nm) proteins, but not N-APP-HIS-coated wells.