The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

Abstract

Correct targeting of proteins to axons and dendrites is crucial for neuronal function. We showed previously that axonal accumulation of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) depends on endocytosis (Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. J. Cell Biol. 162:1317-1328). Two endocytosis-dependent pathways to the axon have been proposed: transcytosis and selective retrieval/retention. We show here that axonal accumulation of L1/NgCAM occurs via nondegradative somatodendritic endosomes and subsequent anterograde axonal transport, which is consistent with transcytosis. Additionally, we identify the neuronal-specific endosomal protein NEEP21 (neuron-enriched endosomal protein of 21 kD) as a regulator of L1/NgCAM sorting in somatodendritic endosomes. Down-regulation of NEEP21 leads to missorting of L1/NgCAM to the somatodendritic surface as well as to lysosomes. Importantly, the axonal accumulation of endogenous L1 in young neurons is also sensitive to NEEP21 depletion. We propose that small endosomal carriers derived from somatodendritic recycling endosomes can serve to redistribute a distinct set of membrane proteins from dendrites to axons.

Figure 1.

Colocalization of endocytosed NgCAM with markers for early and late endosomes. (A) NgCAM-expressing neurons were incubated for 20 min with anti-NgCAM antibodies, which were detected after permeabilization with an Alexa 488–goat anti–mouse antibody, whereas EEs were detected with a polyclonal anti-EEA1 antibody (red). Yellow arrowheads indicate examples of colocalizing puncta. (B) NgCAM-expressing neurons were incubated with anti-NgCAM antibody for 30 min at 16°C and washed, and internalized NgCAM antibody was chased at 37°C for 1 h. Surface NgCAM was detected before permeabilization with a cy5–goat anti–mouse secondary antibody (blue). After permeabilization, endocytosed NgCAM antibodies were detected with Alexa 568–goat anti–mouse antibody (red) and LEs/lysosomes were detected with a polyclonal anti-lgp120 antibody (green). A single confocal section is shown. The boxed regions shown in A and B are magnified sections of dendrites for easier comparison of colabeling. (C) Quantification of colocalization of EEA1 and NgCAM endocytosed for 20 min and of lgp120 and NgCAM endocytosed and chased for 60 min. For each puncta, the ratio of intensities of endocytosed NgCAM to either EEA1 (left) or lgp120 (right) was determined as described in Materials and methods. For EEA1 colocalization, 560 endosomes were scored. For lgp120 colocalization, 497 endosomes were scored. (D) Basic organization of endosomes, adapted from fibroblasts. Three distinct pathways are indicated by the arrows: degradative cargo follows the red arrows, somatodendritically recycling cargo follows the green arrows, and transcytosing cargo follows the blue arrows. Cargo enters via several pathways in small carriers vesicles (ECV) that fuse with existing EEs. EEs contain a vacuolar portion as well as tubular extensions. The tubular extensions accumulate recycling cargoes and bud off to transport recycling cargos back to the plasma membrane either directly or via the RE. Endosomal carrier vesicles (ECVs) can be spherical or elongated and serve as transport carriers between compartments. The vacuolar portions of the EE accumulate internal vesicles and mature into MVBs. MVBs are transport carriers that carry cargo to LE/lysosomes (LE/lys) for degradation (red). Some MVBs might not be predegradative but be capable of recycling. Cargo destined for either axons or dendrites are sorted in the RE and transported to their respective final destination from there.

Figure 2.

Ultrastructural identification of NgCAM-containing somatic endosomes. (A) Hippocampal neurons were infected with AdNgCAM for 24 h and subsequently fixed. NgCAM was detected with anti-NgCAM antibodies and 15 nm gold–protein A. NgCAM is found on the plasma membrane, in numerous MVBs (asterisks), and in small profiles of unknown identity (arrowheads). (B–E) NgCAM-expressing neurons were fed with anti-NgCAM antibodies and 3 nm gold-BSA for 15–60 min and then washed and fixed. Endocytosed NgCAM (15 nm gold) is found in MVBs together with varying amounts of 3 nm gold BSA. The labeled MVB in B contains many 3-nm gold particles (appearing as dark, grainy material) as well as large 15-nm gold particles (arrows). A larger magnification of the boxed region is shown in the inset below to highlight the two different sizes of particles. The MVB in C has few 3-nm gold particles, whereas the one in D has virtually none. (E) Occasionally, NgCAM is found in tubules that are devoid of cointernalized BSA-gold and likely represent RE. Bars, 150 nm.

Figure 3.

Endocytosed NgCAM leaves somatic endosomes and recycles preferentially to the axonal plasma membrane. (A) After 20 min of anti-NgCAM antibody loading (t = 0), intracellular endocytosed NgCAM can be visualized after acid stripping of surface-bound antibodies in somatodendritic but also axonal (arrows) endosomes. (B) After 90 min of chase at 37°C (t = 90′), somatodendritic endosomes are less prominently labeled, whereas axonal endosomes are still clearly detected (arrows). (A′ and B′) Surface reappearance of endocytosed NgCAM in the same cells was assayed with an Alexa 647–goat anti–mouse antibody after acid stripping and chase. No surface labeling was detectable at t = 0 (A′), demonstrating the efficiency of the acid strip. After 90 min of chase, axonal surface labeling was apparent (B′), demonstrating recycling of NgCAM to the plasma membrane. (C) The disappearance of somatic endosomal NgCAM was plotted as percentage of fluorescence remaining after acid stripping over time (open diamonds). Recycling of Tf from somatic endosomes was plotted as well (circles). Surface reappearance of endocytosed NgCAM was plotted as percentage of the chase end point of 2.5 h (triangles and broken line). The mean of four independent experiments is shown. SEM is indicated for each time point. (D) Representative intensity line scans are shown for dendrites at t = 0 (top left), axons at t = 0 (top right), dendrites at t = 90 min (bottom left), and axons at t = 90 min (bottom right). Brightly staining endosomes correspond to the peaks on the trace. The ratio of axon/dendrite average puncta intensity for t = 0 and t = 90 min is indicated to the right of the traces. n = 4 independent experiments.

Figure 4.

Live imaging of NgCAM-containing endosomes. (A–C) Neurons (DIV10) transfected with NgCAM were allowed to endocytose Alexa 488–anti-NgCAM antibodies in live imaging chambers for 60 min before washing and imaging at an acquisition speed of 0.5 Hz. Somatodendritic endosomes can be easily visualized (A and A′). See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1). (A and A′) Three frames of video were merged so that frame 1 (0 s) is red, frame 2 (2 s) is green, and frame 3 (6 s) is blue. Nonmoving endosomes therefore appear white. Moving endosomes appear colored (arrowheads). (B and C) Single frames of endocytosed NgCAM, 2 s apart, are shown to illustrate examples of a moving NgCAM-containing tubule (B, arrow), a nonmoving tubule (B, arrowhead), or a moving NgCAM-containing round carrier (C, arrow). The trajectory of the carrier in C is depicted on the graph. The speeds of observed movements ranged from 0.1 to 1.4 μm/s, averaging 0.5 μm/s. (D) Axonal endosomes were imaged live after acid stripping. A lower magnification image of the labeled cell is shown. Axons and dendrites are indicated with arrows and arrowheads, respectively. (D1–D3) Anterograde and retrograde movements of small NgCAM-containing endosomal carriers in the axon are observed (arrows). Three examples are shown in D1–D3, where frame 1 is red, frame 2 is green, and frame 3 is blue. Frames are 10 s apart as indicated (see Video 2). Nonmoving endosomes appear white. Anterograde movement is toward the top. Fig. S2 shows a single channel display. (E) Quantification of the behavior of NgCAM-containing somatodendritic endosomes. Three size classes are distinguishable: large round (apparent diameter, 1–1.3 μm), medium round (apparent diameter, 0.6–0.8 μm), small round (0.2–0.3 μm), and elongated. NgCAM was mostly transported in small round endosomal carriers. n = 232.

Figure 5.

Dual live imaging of internalized Tf and NgCAM. (A) Neurons were transfected with NgCAM and incubated with anti-NgCAM antibodies (red) and FITC-Tf (green) at 16°C, washed and chased at 37°C in the continued presence of Tf for 15 (A, left) or 60 min (A, right), and then fixed. Surface NgCAM (blue) was detected with a cy5–anti–mouse secondary antibody before permeabilization. Yellow arrowheads indicate overlapping puncta. At 60 min of chase, the overlap is frequently not precise, resulting in a traffic light appearance. Single confocal sections are shown. The traffic light pattern is most abundant at late chase times. (B and C) Neurons (DIV12) expressing NgCAM were loaded with Texas red–Tf and Alexa 488–anti-NgCAM antibodies for 1 h and then washed and imaged live every 3 s. Tf (red) and endocytosed NgCAM (green) can be easily detected in somatodendritic endosomes in live neurons. See Video 3 (available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1). (B) Examples of endosome behavior is shown in four consecutive frames 3 s apart: stationary endosomes containing only NgCAM (green), only Tf (red), or both NgCAM and Tf (yellow) are indicated with arrowheads. Motile endosomal carriers are indicated by arrows (red, Tf only; green, NgCAM only). The four compartments marked with symbols were traced over time and their trajectories plotted in the adjacent graph. For the moving compartments, the arrow points at the first frame of the sequence. A proximal dendrite is shown. See Video 4. (C) Consecutive frames of a traffic light endosome are shown. The endosome appeared initially yellow but in subsequent frames appears as one slightly elongated carrier (or two tethered carriers) in which Tf (red) is laterally segregated away from endocytosed NgCAM (green). The field shown is from the soma of the cell. See Video 5. (D) Labeled endosomal puncta were categorized as Tf only, NgCAM only, or both NgCAM and Tf. All labeled structures in each panel were subdivided by shape (round or elongated) and size (large, medium, or small) and by motility (stationary or moving). Structures containing both Tf and NgCAM rarely moved, whereas structures containing Tf only moved occasionally. Structures containing NgCAM only were the most motile. n = 365 endosomes.

Figure 6.

Endocytosed NgCAM traverses the NEEP21-positive EE. (A) Neurons were cotransfected with NgCAM and either dominant-negative Ti-VAMP (left) or anti-sense NEEP21 (right). GFP was expressed as a control. 18 h after transfection, surface NgCAM was detected with immunostaining. Coexpression of AS-NEEP21 but not Ti-VAMP-DN led to a significant decrease in the A/D PI (see Materials and methods). Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. n = 4 independent experiments, scoring 20–25 cells per experiment for each condition. (B) NgCAM-expressing neurons were allowed to endocytose anti-NgCAM antibodies for 20 min before fixation. Endocytosed NgCAM was detected with a red secondary antibody, whereas endogenous NEEP21 was detected with a rabbit anti-NEEP21 antibody (green). Precise overlap of NgCAM and NEEP21 is observed (colocalization appears yellow). Single channels as well as overlaid channels are shown for the boxed area. A single confocal section is shown. Arrowheads indicate dendrites. (C) Extent of overlap was scored for cells loaded with anti-NgCAM antibodies for 20 min without a chase (left; t = 0) and after a 1-h chase (right; t = 60). Extent of overlap was binned into strong colocalization, NEEP only, and NgCAM only. NgCAM showed high colocalization (black) at t = 0, which diminished with time (right), whereas the no colocalization categories NEEP only and NgCAM only increased with time. (D) The EE populations enriched in EEA1 (red) or in NEEP21 (green) show poor colocalization. Single channel panels are shown on the left.

Figure 7.

Down-regulation of NEEP21 leads to missorting of endosomal NgCAM to the somatodendritic surface and to lysosomes. (A) The A/D PI was determined for NgCAMY33A at steady state (left) and for NgCAM for endosomal recycling to the plasma membrane (t = 2.5 h; right) in cells coexpressing GFP as controls (black) or AS-NEEP21 (gray). Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. n = 4 independent experiments scoring 12–17 cells per experiment for each condition. (B) The extent of anti-NgCAM antibody loading at t = 0 into somatic endosomes was quantified in control cells (black) or AS-NEEP-GFP coexpressing neurons (gray). Values were normalized to GFP-expressing control cells (Ab loading). (C) The presence of endocytosed NgCAM in somatic endosomes (soma retention) was quantitated at t = 0 and t = 40 min for cells coexpressing either GFP as control (black) or antisense NEEP21-GFP (AS-NEEP21; gray). Values were normalized to the t = 0 levels for each condition. n = 4 independent experiments, scoring 12–17 cells per experiment for each condition. Error bars indicate SEM. *, statistical significance from controls at P < 0.01; #, statistical significance from controls at P = 0.055 (Mann Whitney U test). (D) The extent of colocalization between the lysosomal lgp120 (blue) and endocytosed NgCAM (90-min chase; red) was visualized for cells coexpressing NgCAM and GFP (top) or NgCAM and AS-NEEP21-GFP (bottom). Single channel and merged images of the soma region are shown separately on the right. In these panels, lgp120 is displayed in aqua. Overlap with the red channel appears white. (E) Quantification of the number of colocalizing puncta from the experiment in D. n = 3 independent experiments scoring 12–17 cells per experiment and condition. Error bars indicate SEM; **, statistical significance from GFP controls at P < 0.001. (F) Localization of internalized Tf (aqua) and endocytosed NgCAM (red; 20-min load at t = 0) in cells coexpressing NgCAM and AS-NEEP21-GFP. The right panel shows the single channel for Tf. Although endocytosed NgCAM can be detected in endosomes along axons (red; left, arrows), no missorting of Tf (aqua) to the axon (arrows) is observed.

Figure 8.

Endogenous L1 mislocalizes to the somatodendritic domain when NEEP21 is down-regulated. (A and B) Dissociated neurons were electroporated with GFP as control (A and A′) or AS-NEEP21-GFP (B and B′) and surface L1 was detected with a polyclonal anti-L1 antibody (red) at DIV3. In GFP-expressing control cells (A and A′), endogenous L1 is highly polarized to the axon (arrows) and only weakly found on dendrites (arrowheads). In AS-NEEP21-GFP–expressing cells (B and B′), endogenous L1 is additionally detected on the soma and dendrites. (C) The A/D PI was determined for endogenous surface L1 for control cells expressing GFP or AS-NEEP21-GFP. n = 50 cells for GFP; n = 63 cells for AS-NEEP21-GFP from two independent experiments. Error bars indicate SEM. **, statistical significance at P < 0.0001. (D) DIV3 neurons were incubated with polyclonal anti-L1 antibody for 20 min and then fixed and stained with secondary antibody after permeabilization. Labeled endosomes can be seen in axons, somata, and dendrites. Two examples are shown.

Figure 9.

Dynamic behavior of NEEP21-GFP and endocytosed NgCAM in endosomes. (A and B) Live imaging of endocytosed NgCAM (red) and NEEP21-GFP (green). A portion of a proximal dendrite is shown (A). Images were captured every 2 s as indicated above each panel. Frames not shown displayed no movements. The trajectories for all twenty frames (0–38 s) are displayed in B for the three compartments marked by arrowheads, arrows, and asterisks in A. The starting point at 0 s is indicated. The NEEP21-containing and the NgCAM-containing compartments (marked with arrows/arrowheads) show movements, whereas the compartment containing both NEEP21 and endocytosed NgCAM (marked with an asterisk) does not move. See Video 6 (available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1). (C) Quantification of all scored endosomes (n = 443) in the 1-min imaging period. (D) Quantification of moving endosomes (n = 79; colors as defined in C). (E) Model of endosomal compartments involved in NgCAM transcytosis. Tf and NgCAM (aquamarine arrows) enter EEA1-positive EEs (orange) in the somatodendritic domain as well as NEEP21-positive EEs (purple), from where they are transported to the somatodendritic RE. Tf (green arrows) but not NgCAM (blue arrows) recycles to the somatodendritic surface from the EE and the RE. NgCAM (blue), however, sorts away from Tf into putative “transcytotic REs” (red) and travels anterogradely up the axon in small carriers. Large stationary endosomes along the axon (blue) accumulate endocytosed NgCAM and might provide intermediary stopover points into which motile carriers fuse and from which new motile carriers are generated. When NEEP21 is down-regulated by antisense, NgCAM is missorted, presumably in the NEEP21 endosome, toward the somatodendritic surface as well as to lysosomes (gray arrows; LE/lys). Tf, however, is not missorted to the axon.