Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development

Abstract

Proteolytic processing of the Amyloid Precursor Protein (APP) produces beta-amyloid (Aβ) peptide fragments that accumulate in Alzheimer's Disease (AD), but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2) that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL) that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta), we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate APPL-dependent responses within the nervous system. Lastly, targeted expression of our double-tagged constructs (combined with time-lapse imaging) revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, our results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.

Keywords: APPL; D. melanogaster; M. sexta; amphisome; migration; outgrowth; secretase; transport.

Figure 1

Insect APPL is cleaved by the same secretase classes that process APP. (A) Schematic image of the primary domains shared by human APP₆₉₅ and APPL in Drosophila and Manduca. All APP family members contain similar extracellular domains (E1 and E2) that can interact with potential binding partners; a highly conserved cytoplasmic domain (Go) that directly interacts with the heterotrimeric G protein Goα; and a C-terminal tyrosine-based sorting motif (Y) that interacts with a variety of intracellular adapter and signaling molecules. Drosophila APPL contains larger non-conserved regions on either side of the E2 domain that increase the overall size of the holoprotein, and an Aβ-like domain (dAβ) with neurotoxic activity when cleaved from the holoprotein; the biological activity of this domain in Manduca APPL has not yet been verified. Similar to the cleavage products of APP₆₉₅, processing of insect APPL by α- and β-secretases produces soluble ectodomain fragments (sAPPLs) and short transmembrane C-terminal fragments (CTFs); subsequent cleavage of the CTFs by γ-secretase produces an APPL intracellular domain (AICD), as well the dAβ peptide or a p3-like fragment (not shown). Labeled blue bars indicate the epitopes recognized by antibodies against APPL or APP that were used in this study (as described in the Materials and Methods Section). (B,C) Western blots of lysates prepared from Manduca embryos (65 HPF), 5th instar CNS, Manduca GV-1 cells (which endogenously express APPL), and concentrated medium harvested from the GV-1 cultures. (B) Immunoblotting with anti-cAPPL detects both the mature (black arrow) and immature (open arrow) full-length forms of APPL in all three lysates but not in GV-1 cell medium; a larger band (~165 kDa; open arrowhead) detected in mid-stage embryos might represent an additional post-translational modification that is developmentally regulated (as previously reported; Swanson et al., 2005). (C) Immunoblotting with anti-nAPPL detects the same mature (black arrow) and immature (open arrow) full-length forms of APPL, plus cleaved ectodomain fragments (sAPPLs) that are also present in GV-1 medium (black arrowhead). The relative intensity of this ectodomain band reflects the rapid processing of full-length APPL; sAPPL produced by α- vs. β-secretases were not distinguished in this blot. (D,E) Cross-immunoprecipitation of Manduca embryonic lysates with N- and C-terminal-specific antibodies against APPL. (D) Embryonic lysate (input) that was immunoprecipitated with anti-cAPPL (IP) and immunoblotted with anti-nAPPL. (E) Embryonic lysate (input) that was immunoprecipitated with anti-nAPPL (IP) and immunoblotted with anti-cAPPL; both antibodies recognize mature (black arrow) and immature (open arrow) forms of full-length APPL. (F) Western blot of Manduca embryo lysate (lower portion) labeled with anti-cAPPL reveals two CTFs (black arrows) and a candidate AICD fragment (open arrowhead). (G) Western blot of Manduca embryo lysates treated with different secretase inhibitors; in this shorter exposure (compared to F), neither CTF was detected (black arrows). In lysates of embryos treated with a γ–secretase inhibitor (lane 2), both CTFs were readily detected. Treatment with a combination of α- plus γ-secretase inhibitors reduced the relative abundance of the upper CTF band, whereas treatment with β- plus γ-secretase inhibitors reduced the lower CTF band. Separate band labeled with “Act” indicates anti-actin (~42 kDa) as a loading control. (H) Quantification of CTF abundance in western blots of embryonic lysates (as illustrated in G). Treatment with α- plus γ-secretase inhibitors caused a significant decrease in α-CTF levels (^**p = 0.0002) and a more moderate increase in β-CTF levels (^*p = 0.041). Treatment with β- plus γ-secretase inhibitors caused a significant reduction in β-CTF (^*p = 0.041) but did not affect α-CTF levels (p = 0.101). Relative intensities were normalized against γ-secretase-treated lysates in each immunoblot. N ≥ 10 for each group; histograms show means ± SEM. Statistical comparisons were performed using one-way ANOVA followed by pairwise Student's two-tailed t-tests with the Bonferroni correction to obtain reported p-values. (I) Western blots of head lysates from flies expressing additional APPL in the eye (GMR-GAL4; UAS-Appl), immunoblotted with anti-cAPPL. Lane 1, both α- and β-CTFs (arrows) could be readily detected in GMR>Appl flies. Lane 2, expressing additional Drosophila Presenilin in this line (via UAS-dPsn) reduced α- and β-CTFs (β-CTF was no longer detectable at this exposure). Lane 3, expressing additional dPsn plus Drosophila BACE (via UAS-dPsn + UAS-dBACE) preferentially reduced α-CTF levels (β-CTF was still detectable, compared to lane 2). Lane 4, expressing additional Kuzbanian in this line (via UAS-Kuz) caused a marked increase in α-CTF and a corresponding reduction in β-CTF levels. (J) Western blots of head lysates from flies carrying the eye-specific promoter construct GAL4-GMR, immunoblotted with anti-cAPPL. Lane 1, in flies overexpressing APPL (via UAS-Appl), both α- and β-CTFs (arrows) could be readily detected (as in panel I, lane 1). Lane 2, in GMR-GAL4 control flies, only the α-CTF band was faintly detected at this exposure. Lane 3, both CTFs were reduced in flies lacking one copy of the α-secretase Kuzbanian (kuz/+). Lane 4, co-expressing additional APPL and dBACE caused a preferential increase in β-CTF levels. Separate bands in (I,J) labeled with “Tub” show anti-tubulin (~55 kDa) as a loading control.

Figure 2

APPL trafficking and processing in cultured Manduca neurons corresponds to their stage of outgrowth. Neurons harvested from the CNS of fifth instar larvae were grown as dispersed cultures on glass coverslips for 1–14 days in vitro, then fixed and immunolabeled with a combination of anti-nAPPL (green) and anti-cAPPL (magenta) antibodies. Anti-nAPPL and anti-cAPPL are shown individually as gray scale images but are shown in green and magenta (respectively) in the merged images, whereby co-immunolabeling appears white (right hand column). (A) By Day 1, neurons had begun to extend numerous processes with exploratory growth cones (arrows); full-length APPL (white immunolabeling) accumulated in the leading filopodia, while C-terminal fragments (magenta arrowheads) were relatively more abundant throughout the growing neurites and N-terminal fragments (green arrowheads) were enriched in the cell bodies. APPL holoprotein also accumulated in a population of large perinuclear cytoplasmic vesicles (white arrowhead). (B) Higher magnification view of the neuron shown in (A); numerous small vesicles containing only N-terminal fragments (green arrowheads) or C-terminal fragments (magenta arrowhead) were interspersed among the larger vesicles containing the holoprotein (white arrowheads). (C) By day 3, many neurons had extended primary neurites with enlarged growth cones; as is apparent in the merged image, APPL holoprotein (white immunolabeling) continued to be enriched at the leading edges of the growth cones and in some filopodia (arrows), while numerous smaller vesicles containing either N-terminal or C-terminal fragments were distributed throughout the neuronal somata and processes. (D) By day 8, most neurons were no longer undergoing active outgrowth. Full-length APPL was still abundant within vesicles in their somata (white immunolabeling in the merged image) but was no longer concentrated in the distal tips of their processes (open arrows). By comparison, vesicles containing C-terminal fragments (magenta arrowheads) were diffusely distributed throughout the neurons and their processes, while N-terminal fragments (green arrowheads) remained relatively more abundant in the somata. (E) By 12 days, most neurons were either undergoing retraction (open arrows) or initiating degeneration (not shown). APPL holoprotein was almost completely absent from their distal processes, as were vesicles containing N-terminal fragments, whereas vesicles containing C-terminal fragments (magenta) could still be detected throughout the neurons. These results suggest that full-length APPL is selectively transported to the distal tips of developing neurons during periods of active outgrowth. Scale bar = 2 μm in (B); 10 μm in all other panels.

Figure 3

APPL expression is developmentally regulated by the migratory EP cells within the enteric nervous system (ENS) of Manduca. (A–C) Schematic diagrams of EP cell migration, illustrating the progression of the motile neurons (blue) along the midgut muscle bands (orange). Black arrows indicate the positions of the leading neurons on each band pathway. (A) Embryo at 55 HPF; at this stage, the EP cells form a packet of pre-migratory neurons that have spread bilaterally around the circumference of the foregut, adjacent to the foregut-midgut boundary (FG/MG). Small groups of these neurons extend their leading processes onto each of the eight coalescing muscle bands (“b”) on the midgut (only the four dorsal bands are shown). (B) By 60 HPF, small groups of EP cells have begun to migrate and extend leading processes along the muscle bands while avoiding the adjacent interband regions (“ib”). (C) By 65 HPF, the EP cells have transitioned from active migration to an extended period of outgrowth, during which they elongate fasciculated axons along each band pathway (beyond the field of view in C). Only once axon outgrowth is complete (80 HPF) will the neurons extend terminal synaptic processes onto the interband regions (not shown). (D–F) Staged Manduca embryos fileted to expose the developing ENS and immunolabeled with a combination of anti-nAPPL (green) and anti-cAPPL (magenta) antibodies. (A) Embryo at 55 HPF (compare with A). APPL is robustly expressed by the EP cells but not the adjacent muscle cells of the foregut and midgut. Full-length APPL (white immunolabeling) accumulates in the leading processes of neurons that have contacted adjacent muscle bands (arrowheads), and also in a population of large cytoplasmic vesicles within the pre-migratory neurons. By comparison, C-terminal fragments (magenta) are relatively more abundant within fasciculated axons in the anterior esophageal nerve of the foregut (“en”), while N-terminal fragments (green) are diffusely localized throughout the somata of the pre-migratory EP cells. N-terminal fragments are also highly concentrated in peripheral macrophage hemocytes (“m”) that surveil the developing nervous system and sequester cleaved APPL ectodomains (unpublished observations). (D) Embryo at 60 HPF (compare with B). EP cells that have migrated onto the muscle bands continue to exhibit robust levels of APPL expression, with full-length APPL being concentrated in their leading processes and growth cones (arrowheads). At this stage, the leading neurons on each pathway contain substantially fewer large cytoplasmic vesicles enriched with full-length APPL (white immunolabeling) than trailing/stationary neurons. (E) Embryo at 65 HPF (compare with C); at this stage, the EP cells transition from migration to axon outgrowth. Full-length APPL (white immunolabeling) continues to be concentrated in their leading growth cones (arrowheads). C-terminal APPL fragments (magenta) are noticeably more abundant in their fasciculated axons, while N-terminal fragments (green) are more apparent in their somata (as well as in the peripheral macrophages). Open arrows indicate small subsets of EP cells that occupy foregut nerves between adjacent band pathways and that extend short processes onto the interband musculature. Scale bar = 30 μm.

Figure 4

Full-length APPL traffics into the leading growth cones and elongating axons of the motile EP cells. (A–C) show the developing ENS of fileted embryos at progressive stages of development, double-immunolabeled with antibodies against nAPPL (green) and cAPPL (magenta) epitopes. Arrowheads indicate leading growth cones/processes; arrows indicate leading EP cell bodies. (A) 55 HPF: in EP cells that have begun to migrate, full-length APPL (white immunolabeling) is concentrated in their leading processes rather than their cell bodies. In contrast, trailing neurons that have not begun to migrate (open arrowheads) exhibit numerous large vesicles containing the holoprotein. (B) 58 HPF: in EP cells undergoing active migration (arrows), full-length APPL continues to be concentrated in their leading processes (arrowheads), with relatively few large vesicles in their somata. (C) 65 HPF: once the EP cells have transitioned to axon outgrowth, full-length APPL continues to accumulate in their growth cones that extend posteriorly along the midgut (arrowheads). (D) Magnified view of boxed region in (C), revealing intermingled populations of smaller vesicles containing only nAPPL fragments (green arrowhead), cAPPL fragments (magenta arrowhead), or both epitopes (white arrowhead). Scale bar = 5 μm in (A–C), 1.5 μm in (D).

Figure 5

The cytoplasmic distribution of full-length APPL changes with the motile behavior of developing neurons. Magnified views of EP cells at progressive stages of embryogenesis, double- immunolabeled with antibodies against nAPPL (green) and cAPPL (magenta) epitopes. (A) Trailing EP cells that had not yet begun to migrate (55–58 HPF) or that had transitioned from migration to axon outgrowth (65 HPF). (B) Leading EP cells that were undergoing active locomotion (55–58 HPF) or that had transitioned from migration to axon outgrowth (65 HPF). Arrowheads indicate examples of the large vesicles labeled with both nAPPL and cAPPL antibodies (white immunolabeling); this vesicle population was markedly more abundant in the non-migratory neurons. (C) Quantification of the number of large vesicles (>100 nm) that apparently contain full-length APPL. At 55 and 58 HPF, the number of large vesicles was significantly reduced in leading EP cells, compared to trailing EP cells (^**p = 0.002 and ^***p = 0.001, respectively; ns = not significant). At 65 HPF, there was no significant difference in the number of large perinuclear vesicles in trailing vs. leading EP cells (p = 0.106). Statistical comparisons were performed using pairwise Student's two-tailed t-tests; N ≥ 10 per group; histograms show means ± SD. Scale bar in (A,B) = 5 μm.

Figure 6

Different cleavage fragments of APPL are concentrated within distinct domains of neurons in the developing Manduca CNS. (A) Abdominal ganglion of an embryo (at 60 HPF) that was immunolabeled with a combination of anti-APPL antibodies. As in the EP cells, large cytoplasmic vesicles containing APPL holoprotein were abundant in most neurons (white arrowheads). In addition, anti-nAPPL labeling (green) was more abundant in the neuronal somata (located in the cortical regions of the ganglia), whereas anti-cAPPL (magenta) was more abundant in both the somata and their processes within the central neuropil regions, including prominent fascicles of longitudinal axons. Anti-nAPPL antibodies also immunolabeled peripheral macrophages (“m”) that do not themselves express APPL but rather scavenge cleaved ectodomain fragments released by neurons (unpublished observations). Similarly, an additional population of cells within the ganglia immunolabeled only with anti-nAPPL but not anti-cAPPL (yellow arrowheads). (B) Embryonic brain from the same developmental stage; the large cytoplasmic vesicle population containing APPL holoprotein was apparent in most neuronal somata (white arrowheads), interspersed with smaller vesicles containing either nAPPL or cAPPL fragments. (C) Magnified view of the brain shows neurons with vesicles containing the holoprotein (white arrowheads), intermingled with smaller cells that were labeled only with anti-nAPPL (yellow arrows). Scale bar = 30 μm in (A,B); 7 μm in (C).

Figure 7

APPL holoprotein is concentrated in an amphisome-like compartment in GV1 cells and EP cells. (A,B) Examples of Manduca GV1 cells that were fixed and immunolabeled with a combination of antibodies against different Rab proteins (shown in green) and APPL (only anti-cAPPL is shown; magenta). Both anti-Rab7 (A) and anti-Rab11 (B) co-label a population of large cytoplasmic vesicles containing APPL holoprotein (arrowheads), similar to the vesicles found in developing neurons. (C,D) Examples of migrating EP cells in fixed embryos (60 HPF) that were immunolabeled with the same combinations of antibodies. Both anti-Rab7 (C) and anti-Rab11 (D) co-label the large cytoplasmic vesicles containing APPL holoprotein (arrowheads). Scale bar = 7 μm.

Figure 8

Blocking α-secretase activity increases membrane-associated APPL levels in the EP cells and inhibits their migration. (A) Examples of EP cells in cultured embryos that were treated with different secretase inhibitors and then immunolabeled with a combination of anti-nAPPL (green) and anti-cAPPL antibodies (magenta). (A₁) EP cells in a cultured control preparation. (A₂) EP cells treated with an α-secretase inhibitor showed increased levels of membrane-associated full-length APPL (white). (A₃) EP cells treated with a β-secretase inhibitor showed an increased number of cytoplasmic vesicles containing the holoprotein (arrowheads). (A₄) EP cells treated with a γ-secretase inhibitor showed an apparent increase in C-terminal fragments (magenta). (B) Quantification of the relative amount of membrane-associated APPL (black histograms) in EP cells treated with different secretase inhibitors (normalized to adjacent interband regions in each preparation). Treatment with α-secretase inhibitors caused a noticeable increase in membrane APPL that was significant in a pairwise comparison (^*p < 0.02), but not quite significant after applying the Bonferroni correction for multiple comparisons (p < 0.06). In contrast, none of the other secretase inhibitors affected the relative levels of APP, nor was the intensity of Fas II immunoreactivity altered by any of these treatments (quantified in a separate channel; gray histograms). Statistical comparisons between groups were performed using one-way ANOVA followed by unpaired Student's two-tailed t-tests with the Bonferroni correction to obtain reported p-values. N = 10 per group; histograms show means ± SD. (C) Examples of EP cell migration in cultured embryos (redrawn from camera lucida images of immunolabeled preparations). (C₁) Embryo that was fixed and immunolabeled at experimental onset (57 HPF); at this stage, the pre-migratory EP cells extended short exploratory processes onto the midgut band pathways (“b”) but avoid the adjacent interband regions (“ib”). (C₂) Control preparation that was allowed to develop in culture for 18 h; the EP cells had migrated and extended axons posteriorly along the muscle band pathways (black arrowheads). (C₃) Preparation that was treated with an α-secretase inhibitor; EP cell migration and axon outgrowth were markedly reduced compared to controls, although there was no apparent increase in ectopic migration or neuronal death. (D) Quantification of the extent of EP cell migration (black histograms) and outgrowth (gray histograms) along the midgut band pathways in cultured embryos treated with different secretase inhibitors (distances normalized to controls in each experimental group). Treatment with α-secretase inhibitors caused a significant reduction in both migration (^***p < 0.001) and outgrowth (^**p = 0.002), whereas treatment with β- and γ-secretase inhibitors had no apparent effects on these aspects of EP cell development. Migration and outgrowth distances were normalized to mean values obtained from matched control preparations in each experiment. Statistical comparisons between groups were performed using one-way ANOVA followed by unpaired Student's two-tailed t-tests with the Bonferroni correction to obtain reported p-values. N ≥ 16 per group; histograms show means ± SD. Scale bar in (A) = 5 μm; in (C) = 40 μm.

Figure 9

Expression and processing of fluorescently double-tagged APPL and APP₆₉₅ in Drosophila. (A) Schematic diagram of constructs encoding full-length Drosophila APPL and human APP₆₉₅, in frame with N-terminal enhanced Green Fluorescent Protein (GFP; inserted downstream of their signal sequence) and C-terminal monomeric Red Fluorescent Protein (mRFP). E1 and E2 indicate extracellular protein interaction domains; “ss” indicates signal sequences; TM indicates transmembrane domains (compare with Figure 1). Cleavage sites for α, β-, and γ-secretases are indicated by arrows; note that the relative positions of the α and β cleavage sites in APPL are reversed, compared to APP₆₉₅. (B) Western blot of head lysates from flies expressing double-tagged APPL (APPL-tag) and APP₆₉₅ (APP-tag) under the control of the eye-specific promoter construct GMR-GAL4. Immunoblot was labeled with anti-DsRed (targeting mRFP). The full length, mature forms (black arrows) and partially glycosylated immature forms (open arrows) of both constructs were expressed at similar levels. Smaller bands at ~42 kDa represent CTF fragments generated by α- and β-secretase cleavage (not distinguished in this gel). (C) Western blot of head lysates from the same fly lines immunolabeled with anti-GFP; full-length mature forms (black arrows) and cleaved ectodomain fragments (black arrowheads) of both constructs could be readily detected. The smaller size of cleaved ectodomains from double-tagged APP₆₉₅ reflects the presence of larger intervening sequences between the E1 and E2 domains in Drosophila APPL (illustrated in A). Lower gel shows anti-actin staining (“Act”) as a loading control. (D) Western blot of head lysates from flies expressing either double-tagged APP₆₉₅ (APP-tag) or untagged full-length APP₆₉₅ (APP), labeled with an antibody specific for the N-terminus of human APP. Upper bands (black arrows) indicate full-length, mature holoprotein; lower bands (arrowhead) represent cleaved ectodomain fragments. The larger size of the bands in the APP-tag lane is consistent with the combined molecular weight of APP₆₉₅ plus mRFP and eGFP. Lower gel shows anti-tubulin staining (“Tub”) as a loading control.

Figure 10

Trafficking and processing of double-tagged APPL in cultured Drosophila neurons corresponds to their stage of outgrowth. Neurons harvested from the CNS of Drosophila white pupae expressing fluorescently double-tagged APPL were grown on coverslips for 1–8 days before fixation and imaging. GFP (nAPPL) and mRFP (cAPPL) are shown individually as gray scale images but are shown in green and magenta (respectively) in the merged images, whereby co-immunolabeling appears white (right hand column). (A) In neurons that had recently commenced outgrowth (1 DIV), full-length APPL could be detected in the leading tips of their growth cones (arrows), similar to the distribution of endogenous APPL in cultured Manduca neurons (see Figure 2A). In addition, N-terminal fragments (green) were relatively more abundant in their cell bodies, while C-terminal fragments (magenta) were detected throughout the neurons and their processes. (B) Higher magnification view of the cell body of a cultured neuron at 2 DIV. Distinct vesicle populations could be detected that contained either nAPPL fragments (green arrowheads) or cAPPL fragments (magenta arrowheads), while an additional population of larger vesicles apparently contained the holoprotein (white arrowheads). (C) Higher magnification view of the growth cone of a neuron at 2 DIV. Vesicles containing only nAPPL fragments (green arrowheads) were more concentrated in the peripheral domain, in the vicinity of the holoprotein at the leading margins of the growth cone white arrowheads), while vesicles containing cAPPL fragments (magenta arrowheads) were relatively more abundant within the central domain. (D) Older neuron that was undergoing retraction (at 6 DIV); full-length APPL was no longer detectable within its distal processes (open arrows), and N-terminal fragments were largely confined to the cell body (green arrowheads), leaving a diffuse distribution of C-terminal fragments throughout the neurites (magenta arrowheads). These results suggest that the holoprotein is preferentially transported to the distal processes of developing neurons during periods of active outgrowth. Scale bar = 5 μm in (A,D); 1.5 μm in (B), 1 μm in (C).

Figure 11

Dynamic trafficking of fluorescently double-tagged APPL and its fragments in cultured Drosophila neurons. (A,B) Small clusters of partially dispersed Drosophila neurons expressing fluorescently double-tagged APPL (via elav-GAL4) at 1 and 4 DIV, respectively. Numerous vesicles containing either N-terminal fragments (green), C-terminal fragments (magenta), or the holoprotein (white immunolabeling) could be seen throughout the neurons and their processes. (C) Single frames from a time-lapse of the boxed region in A (See also Supplementary Movie S1). White arrow indicates a vesicle containing the holoprotein undergoing retrograde trafficking toward the soma; white arrowhead indicates another vesicle containing the holoprotein in the same neurite undergoing anterograde transport away from the soma. Magenta arrowhead indicates vesicle in a different neurite containing only C-terminal fragments undergoing retrograde transport. (D) Single frames from a time-lapse movie of the boxed region in B (See also Supplementary Movie S2). Green arrowhead indicates a vesicle containing only N-terminal fragments that was initially transported anterograde to the tip of a growing process (panels 1–5) and then reversed direction (panels 6–10) to undergo retrograde transport back toward the soma (M–Q). Scale bar = 6 μm in (A,B), 2 μm in (C,D). (E) Quantification of the number of vesicles containing either APPL holoprotein (white histograms), N-terminal fragments (green histograms), or C-terminal fragments (magenta histograms) that underwent anterograde vs. retrograde transport in time-lapse movies of neurons shown in (A,B). Frames from movies selected at 1 frame/s (approximate transport rates varied from 0.8 to 1.2 μm/s). In (E), each histogram represents average values calculated for 15–20 cells per group, 4 events per cell.

Figure 12

Double-tagged APP₆₉₅ is trafficked and processed in complex patterns within the larval CNS of Drosophila. Images were collected through the cuticle of living 3rd instar Drosophila larvae expressing fluorescently double-tagged APP₆₉₅. (A) Low-magnification view of abdominal nerves containing motor neurons expressing double-tagged APP₆₉₅ pan-neuronally (via Appl-GAL4). (B) Magnified view of boxed region #1 in A. Intermingled transport vesicles containing only N-terminal fragments (green arrowheads), C-terminal fragments (magenta arrowheads), or holoprotein (white arrowheads) could be detected in the fasciculated axons of an abdominal nerve. (C) Magnified view of boxed region #2 in A, focused on a neuromuscular junction (NMJ). N-terminal fragments tended to concentrate in the most distal domains of the NMJ (green arrowhead), while C-terminal fragments were more abundant in more proximal domains (magenta arrowhead). Full-length APP (white) was also detectable in certain regions of the NMJ (white arrowheads), reminiscent of the distribution of the holoprotein in some growth cone filopodia but not others (Figure 10). (D) Images of neurons within the fused segmental ganglia of the CNS. Different subsets of neurons exhibited markedly different concentrations of N-terminal (green arrowhead) or C-terminal fragments (magenta arrowhead) as well as the holoprotein (white arrowhead). Scale bar = 40 μm in (A); 5 μm in (B,C); 25 μm in (D).

Figure 13

Different CNS neurons process double-tagged APP₆₉₅ in diverse patterns within the CNS of Drosophila. Panels show neurons within the brains of Drosophila adults expressing double-tagged APP₆₉₅ under the control of different GAL4 driver lines. (A) Neurons in the brain of an adult fly expressing double-tagged APP₆₉₅ pan-neuronally (via Appl-GAL4). Intermingled populations of neurons showed enhanced concentrations of either N-terminal fragments (green), C-terminal fragments (magenta), or the holoprotein (white) in their somata. (B) Higher magnification view of the brain in A (arrowheads indicate the same neurons at both magnifications). (C–G) Expression of double-tagged APP₆₉₅ in dopaminergic neurons (via Ddc-GAL4). (C) Low magnification view of the adult brain revealed that different subsets of dopaminergic neurons process APP₆₉₅ in dramatically different patterns, whereby many neurons predominantly accumulated N-terminal fragments in their somata (green); a smaller number predominantly accumulated C-terminal fragments (magenta). White arrowhead indicates larger vesicles containing the holoprotein. (D) Example of two dopaminergic neurons that showed robust accumulation of N-terminal fragments within numerous small vesicles in their somata (green arrowheads), as well as larger vesicles containing the holoprotein (white arrowheads). In contrast, C-terminal fragments were concentrated in more sparse vesicle populations in their primary neurites (magenta arrowheads). (E) Another pair of dopaminergic neurons that exhibited abundant small vesicles containing C-terminal fragments throughout their somata and primary neurites (magenta), interspersed with more variable vesicle populations containing N-terminal fragments or the holoprotein. (F) Two octopaminergic neurons expressing double-tagged APP₆₉₅ (via Tdc-GAL4), exhibiting concentrated N-terminal fragments in their somata (green) and quite variable populations of vesicles with C-terminal fragments (magenta arrowhead). (G) A cluster of cholinergic neurons expressing double-tagged APP₆₉₅ (via ChAT-GAL4), exhibiting numerous small vesicles containing N-terminal fragments (green) and sparse populations of larger vesicles containing C-terminal fragments (magenta). Scale bar = 25 μm in (A,C); 15 μm in (B); 2 μm in (D–G).

Figure 14

In vivo trafficking of fluorescently double-tagged APPL within Drosophila CNS neurons. Two cholinergic neurons expressing double-tagged APPL (via ChAT-GAL4) in the adult fly brain. (A) Low magnification view revealed concentrations of the holoprotein (white) in their somata and populations of vesicle containing N-terminal (green) or C-terminal vesicles (magenta) throughout their primary neurites. (B) Higher magnification view of the two neurons shown in (A); N-terminal fragments in numerous small vesicles (green) were distributed throughout the somata and primary neurites, while larger vesicles contained C-terminal fragments (magenta) or the holoprotein (white). (C) Higher magnification view of boxed region 1 depicted in (A) (shown in all three channels). Numerous small vesicles containing N-terminal fragments of APPL were preferentially localized to microtubule tracks within core domain of the primary neurite, while larger vesicles containing C-terminal fragments (arrowheads) were associated with microtubule tracks throughout the circumference of the neurite. (D) Single frames from a time-lapse movie taken from box 2 in (A) (cAPPL channel only, shown in gray scale); see also Supplementary Movie S4. Arrow and arrowhead indicate two vesicles containing C-terminal fragments undergoing anterograde trafficking (away from the soma). (E) Single frames from a time-lapse movie taken from box 3 in (A) (cAPPL channel only, shown in gray scale). Arrowhead indicates one of several vesicles containing C-terminal fragments undergoing retrograde trafficking (toward the soma). Scale bar = 8 μm in (A); 2 μm in (B–E). Frames from movies selected every 2 s (approximate transport rates are 1.2 μm/s).

Figure 15

In vivo trafficking of fluorescently double-tagged APPL within Drosophila photoreceptors is developmentally regulated. (A) Schematic diagram of the developing eye disc in Drosophila; mf = morphogenetic furrow of the eye disc (boundary of photoreceptor differentiation); pr = newly differentiated photoreceptors that are in the process of extending axons through the optic stalk into the optic lobe of the brain; “o” indicates older photoreceptors; “y” indicates younger photoreceptors. Red box indicates the equivalent region of the immunostained preparation shown (B). (B) Low magnification view of developing photoreceptors in a cultured eye disc from a larva expressing double-tagged APP₆₉₅ (via GMR-GAL4). Younger photoreceptors (“y”; near the morphogenetic furrow) that had recently begun to extend axons were enriched with vesicles containing the holoprotein (white) and nAPP fragments (green), whereas older photoreceptors (“o”) exhibited many more large vesicles containing cAPP fragments (magenta). (C) Higher magnification view of boxed region in (B). N-terminal APP fragments were present in numerous small vesicles throughout the photoreceptors and their axons, while cAPP fragments were concentrated in more dispersed larger vesicles. Photoreceptor somata also exhibited larger cytoplasmic vesicles containing the holoprotein (white arrowheads), similar to other neurons in both Manduca and Drosophila. (D) Higher magnification view of boxed region in (C). Photoreceptor axons contained intermixed populations of vesicles with either nAPP fragments (green arrowhead), cAPP fragments (magenta arrowheads), or the holoprotein (white immunolabeling). (E,F) Single frames from a time-lapse of the preparation shown in (B) (magenta channel only; see Supplementary Movie S5). Vesicles containing cAPP fragments could be seen moving in both apical (E) and basal directions (F) within the elongated photoreceptor cell bodies. Scale bar = 5 μm in (B); 3 μm in (C); 0.75 μm in (D); 8 μm in (E,F). Frames from movies selected every 2 s (approximate transport rates are 2 μm/s).