Epitopes from different APP regions do not colocalize. A, Proteolytic processing of APP [modified from ()]. The amyloidogenic (using β-secretase) and non-amyloidogenic (using α-secretase) pathways generate soluble amino-terminal domains (sAPPs), a cytoplasmic carboxy-terminal domain (AICD), and middle region polypeptides, A β and p3, respectively. The CTFs are intermediary cleavage products. Bars below the upper APP diagram show the position of the epitopes recognized by anti-APP antibodies. The anti-pAPP antibody detects the phosphorylated version of the epitope recognized by antibody 2452. The positions of the APP cleavage sites, and of the transmembrane domain, are shown.
B-O, Antibodies to amino- (APP_N; 22C11) and carboxy-terminal (APP_C; 2452 in B, E, and G; AB5352 in J, M, and O) APP epitopes, and to Aβ (4G8) show distinct, mostly non-overlapping labeling patterns in CAD cells (B-D and J-O) and cortical neurons (E-I). Images in E-G and J-O are from double labeling experiments. Micrographs in H and I show distal neurites with their terminals. Micrographs in K and L are from a control immunolabeling experiment where the primary antibodies were omitted. Note that the background labeling due to the secondary antibodies is minimal. Micrographs in M-O are from an immunolabeling experiment of CAD cells that have been fixed and permeabilized at the same time, using a fixative solution without sucrose. Note that the labeling patterns are similar to those where cells were permeabilized after fixation (see B-J). Immunolabeling images with additional anti-APP antibodies, and additional controls, are shown in and . Scale bars: (in B, J, and M) B-D, J-L, and M-O, 50 μm; (in E) E-G, 40 μm; (in H) H and I, 10 μm.
M, Quantitative measurement of the distribution of APP epitopes within CAD cells. The graph shows, for each of the three epitopes (as detected with the antibodies 22C11, 4G8, and 2452), the percentages that localized to the cell body, neurites (without terminal), and neurite terminals. Error bars indicate -1 SEM.

Antibodies used in this study detect full-length APP, cleaved APP fragments, and the phosphorylated, Thr⁶⁶⁸-containing APP epitope. A, C, D, Immunoblots of CAD cell (A and C) and rat brain (D) extracts probed with antibodies to Aβ (4G8) and APP amino-terminal epitopes (APP_N; 22C11). A, Note that the anti-Aβ antibody detects full-length APP and CTFs, and that these protein bands are not detected in the presence of excess, competing Aβ peptide (Control). The (*) marks a smear on the blot. Although the conditions of this blot are not adequate to detect the small Aβ peptides, we previously showed by Western blotting that CAD cells contain both monomeric and oligomeric forms of Aβ (). C, The 22C11 antibody stains a broad region corresponding to full-length APP and sAPPs, which are resolved at lower exposure of the blot (right lane). D, No non-specific binding of antibody 22C11 in rat brain extract. Lanes contain increasing protein loads, from right to left. Note that even at very high protein loads, the antibody only detects a broad band corresponding to the full-length APP and sAPPs. Molecular weight markers are in kDa.
B, The anti-pAPP antibody does not recognize the non-phosphorylated APP epitope. Gels were loaded with in vitro phosphorylated (right lanes) and non-phosphorylated (left lanes) GST fusion proteins of the cytoplasmic domain of APP [GST-(p)APP_C]. Antibodies to phosphorylated Thr (pThr) and to pAPP detect the phosphorylated GSTAPP_C fusion protein (GST-pAPP_C, right lanes). The antibodies do not detect the non-phosphorylated fusion protein (GST-APP_C), which was subjected to phosphorylation conditions in the absence of the kinase (left lanes; see Materials and Methods). Both phosphorylated and non-phosphorylated fusion proteins were detected by the antibody to the APP carboxy-terminal domain (AB5352; bottom blots).

Epitopes from different regions of APP are confined to distinct vesicle populations in CAD cell neurites. A-F, Immunolabeling of neurites. The antibodies detect epitopes in the carboxy-terminal (APP_C; 2452 and pAPP_C), amino-terminal (APP_N; 22C11), and Aβ region (4G8) of APP. Thr⁶⁶⁸-phosphorylated epitopes (pAPP_C) were detected with antibody 44-336Z. The plot in F shows the fluorescence distribution of APP_N and pAPP_C within the neurite shown in E. Inverted, grayscale images (shown below the plot) were used to generate intensity plots that correspond to the distribution of fluorescent particles. Note that there is minimal overlap between the distributions of the peaks in the two channels (compare with ). The images in B are from a control immunolabeling experiment where the primary antibodies were omitted. The upper image is reproduced at increased contrast in the middle panel. Note that even at extremely enhanced contrast (middle image), the background labeling due to the secondary antibodies is low, and cannot be confused with the specific, particulate labeling seen with anti-APP antibodies. The lower image in B and the upper image in E are phase contrast micrographs. C and D, Specific detection of Aβ and APP_C by immunocytochemistry. CAD cells were immunolabeled with a mixture of antibodies 4G8 and 2452 in the presence of excess Aβ peptide (C) or a polypeptide encompassing the entire APP cytoplasmic domain (D). Note that the immunolabeling is abolished only by the corresponding polypeptide. Arrows indicate the neurite segments that are reproduced at higher magnification in the bottom images. Images from the same double labeling experiment are marked by a side bar. Labeling with the anti-pAPP antibody was prevented by incubation with excess, phosphorylated, but not non-phosphorylated polypeptide corresponding to the APP cytoplasmic domain [data shown in (, )]. Scale bars: A and B, 10 μm; (in D) C and D, 20 μm (bottom images, 10 μm); (in E) E and F, 5 μm.
G, APP_N shows a continuous, and pAPP_C a discontinuous distribution along CAD cell neurites. The gray scale images shown in E were converted to masks using ImageJ software. Only the indicated segment of the neurite is shown. Note that pAPP_C-, but not APP_N-containing vesicles appear clustered.
H, The neurite images shown in E (middle two images) are reproduced as grayscale images that have been inverted and divided into four segments. Each pair of segments has been adjusted for contrast and brightness, to allow clear visualization of the fluorescent particles. Note that APP_N and pAPP_C are localized to different vesicles along the neurite.
I, Surface plot analysis of fluorescence distribution of APP_N and pAPP_C within the proximal section (marked with *) of the neurite shown in H. Note that almost all fluorescent spots corresponding to APP_N labeling distribute along a single longitudinal line (shown in green), roughly corresponding to the central, longitudinal axis of the neurite. By contrast, pAPP_C fluorescent particles are broadly distributed through the entire width of the neurite, with large deviations from the central axis. This indicates that the two epitopes have different distribution patterns. The ratio between the length (X-axis) and width (Y-axis) of the neurite segment has been altered to allow for better resolution of the bell-shaped, fluorescent particles.

APP carboxy-terminal, phospho-epitopes (pAPP_C), but not amino-terminal epitopes (APP_N), localize to peripheral regions of lamellipodia, and to filopodia, and accumulate in advancing protrusions of growth cones. A-C, Immunolabeling of an “exploratory” growth cone. Image A is shown at enhanced contrast in B. Note that the labeling of both APP_N (arrows) and pAPP_C (arrowheads) is particulate. APP_N epitopes clearly align along filamentous tracks, which extend from the central domain (C) into the transition zone (T), but not into the peripheral domain (P) of the lamellipodium. Image C shows the periphery and the three characteristic regions (C, T, P) of the lamellipodium. Note that only pAPP_C localizes to filopodia, at the periphery of the lamellipodium (see also ). Scale bar: (in A), 10 μm.
D-G, pAPP_C accumulates within advancing protrusions (arrows), and mark the direction of turning and advancement of “committed” growth cones. The distribution of microtubules (Tub; anti-tubulin staining) is also shown. Micrograph regions, shown at higher magnification and increased contrast in H-R, are indicated by numbers in F. G is a phase contrast micrograph. Scale bar: (in G), 20 μm.
H and I, Microtubules (Tub) and pAPP_C extend up to the tip of an advancing protrusion (marked by arrow and arrowhead), in the turning growth cone shown in D-G. Scale bar: (in H), 5 μm.
J-R, pAPP_C extends beyond the microtubule tips (marked by arrows) in growth cone filopodia (J-O) and thin, lateral, filopodia-like processes extending from the main process (P-R). Note that pAPP_C vesicles enter filopodia up to their tips (arrowheads). Examples are from the growth cone shown in D-G. L, O, and R are phase contrast micrographs. Scale bars: (in O and R) M-R, 5 μm; (in L) J-L, 10 μm.

APP carboxy-terminal, phospho-epitopes (pAPP_C), but not amino-terminal epitopes (pAPP_N) localize to actin-rich regions of the lamellipodium, filopodia, and filopodia-like lateral processes along CAD cell neurites. A-G, pAPP_C epitopes localize to actin-rich regions of lamellipodia and filopodia in CAD cells. Note that pAPP_C labeling extends into phalloidin-positive regions (Phal) of a lamellipodium (A-D) and filopodia (E and F) of advancing growth cones, and of lateral filopodia-like structures extending from the main process (E-G). The regions between the two arrows in A and E are reproduced at higher contrast in B, C, and G, to allow for better detection of pAPP_C within filopodia. Note that, unlike microtubules (see and ), actin filaments extend to the tip of filopodia, in regions that also contain pAPP_C. D and F are phase contrast micrographs. Scale bars: (in A and F) A, E-G, 10 μm; (in D) B-D, 5 μm.
H-N, Distribution of APP_N epitopes, detected with antibody 22C11. Note that APP_N epitopes do not penetrate into actin-rich regions of lamellipodia and filopodia (K-N). The region between the two arrows in J is reproduced in K.
O-R, Distribution of Aβ epitopes, detected with antibody 4G8. Note that Aβ epitopes occasionally penetrate into the lamellipodium (O-Q) and the filopodia-like extensions along neurites (R). Images in K-R are shown at high contrast. Scale bars: (in J) H-J, 40 μm; K, 10 μm; (in N, Q, R) L-R, 20 μm.

Detection of APP in cryosections from R1.40 mouse brain. A-C, Antibodies to carboxy- (APP_C) and amino-terminal (APP_N) APP epitopes, and to Aβ show distinct labeling patterns in Purkinje cells of the cerebellum. D, low magnification image of the region that includes the area shown in C. Arrows point to cell bodies. Scale bar: (in A and D), 40 μm.

Localization of APP epitopes at high and low APP expression levels. A and B, Epitopes from different regions of APP do colocalize in CAD cells expressing APP at high levels. Images show a CAD cell transfected with carboxy-terminally tagged APPYFP. The carboxy-terminus was detected via the YFP tag; Aβ was detected with antibody 4G8. Scale bar: (in A), 25 μm.
C-E, Antibodies to pAPP_C and Aβ label the same vesicle population in CAD cells transfected with the phospho-mimetic APP mutant, APP-Thr668Glu, where the constitutively phosphorylated APP is expressed at high level. Scale bar: (in E), 5 μm. F, Intensity plots corresponding to the distribution of pAPP_C and Aβ (shown in C and D). Note that there is extensive overlap between the distributions of the peaks in the two plots (compare with ).
G and H, CAD cells expressing low levels of APP-YFP. The APP carboxy-terminus (visualized via the YFP tag) is detected primarily in the cell soma, similar to the endogenous situation (compare with and ). H is a phase contrast micrograph. Scale bar: (in H), 40 μm.
I and J, CAD cells expressing low levels of exogenous APP. The APP carboxy-terminus (APP_C), but not the amino-terminus (APP_N) is concentrated in the Golgi region. The arrow points to a similar accumulation of APP_C (but not APP_N) in an adjacent cell. Fluorescence intensity plots along the lines are shown in K. Scale bar: (in J), 20 μm.
K, Intensity plots corresponding to the distribution of APP_C and APP_N along the colored lines shown in I and J. Note that the two APP epitopes show different distributions within the cell soma.

Diagram showing three possible trafficking pathways of APP within neurites. In (1), APP is transported as full-length protein. In (2), CTFβ and sAPPβ are transported in separate vesicles. In (3), AICD, Aβ, and sAPPβ are transported in separate vesicles. AICD (or pAICD) may bind either to an anchoring protein (as shown in the diagram), or directly to membrane acidic phospholipids (not shown in the diagram). Only the β-secretase processing pathway is shown. Our data suggest a preponderance of pathways (2) and (3).