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Previous Article | Next Article
Volume 17, Number 1, Issue of January 1, 1997 pp. 140-151
Copyright ©1997 Society for NeuroscienceTrafficking of Cell-Surface
-Amyloid Precursor Protein: Evidence that a Sorting Intermediate Participates in Synaptic Vesicle Recycling
Numa R. Marquez-Sterling1, Amy C. Y. Lo2, Sangram S. Sisodia2, and Edward H. Koo3 1 Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611, 2 Department of Pathology and the Neuropathology Laboratory, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and 3 Departments of Neurology and Pathology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
We recently demonstrated that the Alzheimer's
Key words: Alzheimer's disease; amyloid precursor; axons; cell surface; cerebellar macroneurons; clathrin-coated vesicles; endocytosis; protein trafficking; synaptic vesicle recycling-amyloid precursor protein (APP) is internalized from the axonal cell surface. In this study, we use biochemical and cell biological methods to characterize endocytotic compartments that participate in trafficking of APP in central neurons. APP is present in presynaptic clathrin-coated vesicles purified from bovine brain, together with the recycling synaptic vesicle integral membrane proteins synaptophysin, synaptotagmin, and SV2. In contrast, APP is largely excluded from synaptic vesicles purified from rat brain. In primary cerebellar macroneurons, cell-surface APP is internalized with recycling synaptic vesicle integral membrane proteins but is subsequently sorted away from synaptic vesicles and transported retrogradely to the neuronal soma. Internalized APP partially co-localizes with rab5a-containing compartments in axons and with V-ATPase-containing compartments in both axons and neuronal soma. These results provide direct biochemical evidence that an obligate sorting compartment participates in the regeneration of synaptic vesicles during exo/endocytotic recycling at nerve terminals but do not preclude concurrent "kiss-and-run" recycling. Moreover, APP is now, to our knowledge, the first demonstrated example of an axonal cell-surface protein that is internalized with recycling synaptic vesicle membrane proteins but is subsequently sorted away from synaptic vesicles.
INTRODUCTION
An invariant feature in the cerebral cortex of patients with Alzheimer's disease is the extraneuronal deposition of the
). APP exists as three principal isoforms derived by alternative splicing; predominant expression of the 695 amino acid residue isoform is seen in neurons (Sisodia et al., 1993
-secretase processing of APP results in the release of a 100-110 kDa soluble N-terminal fragment, with membrane retention of a 10 kDa C-terminal fragment (Sisodia et al., 1990
In addition to processing by the secretory pathway, APP is processed by internalization from the cell surface, apparently by receptor-mediated endocytosis (Haass et al., 1992
Fig. 1. Distribution of APP in clathrin-coated vesicles from bovine brain. Clathrin-coated vesicles (CCV) were purified from homogenates of cerebral cortex (whole-brain), unless otherwise indicated, and incubated with low-ionic strength buffer to dissociate clathrin coats before analysis by SDS-PAGE and immunoblotting (20 µg protein/gel lane). Position and relative mass (in kilodaltons) of protein standards are indicated. A, Mature, full-length APP (APP, inset) is present in whole-brain clathrin-coated vesicles, together with the recycling synaptic vesicle integral membrane proteins SV2 (SV2), synaptotagmin I (p65), and synaptophysin (p38). APP reactivity can be eliminated when anti-APP is applied together with its cognate peptide (APP+pep, inset). H-2, Murine H-2 complex cell-surface antigen (negative control); V-ATPase, 70 kDa subunit of vacuolar ATPase from bovine brain (positive control). Laemmli gel, 4-20% (inset, 6%). B, Rab5a (rab5a, arrow) is present in whole-brain clathrin-coated vesicles, but neither rab3a (rab3a) nor rab8 (rab8) can be detected. Rab5a reactivity can be eliminated when anti-rab5a is applied together with its cognate peptide (rab5a+pep). Laemmli gel, 15%. C, To demonstrate definitively that APP is present in the presynaptic subpopulation, clathrin-coated vesicles were additionally purified from hypotonically lysed synaptosomes (synaptosomal). Full-length APP (APP) is present in whole-brain and synaptosomal clathrin-coated vesicles. Transferrin receptor (Tf-R) can only be detected in whole-brain clathrin-coated vesicles (arrow). Laemmli gel, 6%. D, To demonstrate that APP and recycling synaptic vesicle integral membrane proteins are present in the same subpopulation of clathrin-coated vesicles, whole-brain clathrin-coated vesicles were immunoprecipitated with antibody directed to the cytoplasmic tail of APP (CT15), resuspended, and analyzed by SDS-PAGE and immunoblotting for synaptophysin (top panel). Synaptophysin (p38, arrow) is readily detected when CT15 is used but cannot be detected when antibody directed to the midregion of the extracellular domain of APP (863) or nonimmune IgG (rIgG), respectively, is used. The membrane was subsequently stripped and reprobed for APP (bottom panel) to demonstrate that immunoprecipitation with CT15 selectively enriches for APP-containing clathrin-coated vesicles (APP, arrow). Laemmli gel, 12%.
[View Larger Version of this Image (29K GIF file)]
Fig. 2. Distribution of APP in synaptic vesicles immunoisolated from rat brain synaptosomal lysates (S3). Control (MIgG) or anti-synaptophysin-coated magnetic beads were used for immunoisolation. 2×, Twice the amount of control or anti-synaptophysin beads used relative to 1×. After separation with a magnetic rack, equal amounts of immunobeads (p) and supernatant (s) fractions were analyzed by SDS-PAGE and immunoblotting. The membrane was incubated with anti-synaptophysin to detect synaptic vesicles, then stripped and reprobed with CT15 to detect APP. APP (APP) is not detected in the immunobeads fraction using either amount of anti-synaptophysin beads. No synaptophysin-reactive material (p38) is detected in the immunobeads fraction when control beads are used. Laemmli gel, 8%.
[View Larger Version of this Image (55K GIF file)]
Fig. 3. Distribution of APP in synaptic vesicles purified from rat brain by differential and sucrose gradient centrifugation as described by Huttner et al. (1983)
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Fig. 4. Immature cerebellar macroneuron (day 6) fluorescently labeled for total cellular APP using antibody 5A3,1G7. The somatodendritic domain and proximal axonal shaft are shown in a, and the distal axonal shaft and growth cone of the same neuron are shown in b. Diffuse, punctate immunoreactivity for APP is observed in the somatodendritic and axonal domains, including the axonal growth cone and growth cone filopodia, several of which are indicated (arrowheads). Juxtanuclear enrichment of APP reactivity is observed in the neuronal soma. N, Nuclear region. Scale bar, 10 µm.
[View Larger Version of this Image (38K GIF file)]
Fig. 5. Mature cerebellar macroneurons (day 14) fluorescently labeled for internalized APP (a, c) and either total cellular clathrin (b) or total cellular rab5a (d). Partial co-localization of internalized APP and clathrin or rab5a is observed in axons (arrowheads). Stronger co-localization for rab5a is observed. Scale bars, 10 µm.
[View Larger Version of this Image (41K GIF file)]
Fig. 6. Mature cerebellar macroneurons (day 14) fluorescently labeled for internalized APP (a, c) and total cellular V-ATPase (b, d), the enzyme principally involved in maintaining an acidic luminal state in intracellular compartments. Two adjacent distal axonal segments (including small growth cones) are shown in a and b. Partial co-localization of internalized APP and V-ATPase is observed in axons and neuronal soma (arrowheads). Scale bars, 10 µm.
[View Larger Version of this Image (51K GIF file)]
Fig. 7. Immature cerebellar macroneurons (day 4) fluorescently labeled to detect recycling synaptic vesicle membrane proteins and either total cellular or internalized APP. Cells were incubated with anti-synaptotagmin I as a marker for recycling synaptic vesicle membrane proteins, then fixed, permeabilized, and labeled with 5A3,1G7. Partial co-localization of total cellular APP (a, c) and internalized synaptotagmin (b, d) is observed in axons (a-d, arrowheads; iv indicates four puncta of co-localization in an axonal growth cone). Juxtanuclear reactivity for internalized synaptotagmin is also present, as observed previously in hippocampal neurons (Matteoli et al., 1992
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Fig. 8. Mature cerebellar macroneurons (day 14) fluorescently labeled for internalized APP (a, c) and internalized synaptotagmin (b, d). Strong, partial co-localization of internalized APP and internalized synaptotagmin is observed in axonal varicosities (arrowheads in a, b; two intersecting axonal segments are shown). In contrast, no co-distribution of internalized APP and internalized synaptotagmin is observed in neuronal soma (starred arrowheads in d indicate site of selected puncta of internalized APP marked by arrowheads in c). In neuronal soma, internalized synaptotagmin is largely juxtanuclear, whereas internalized APP is diffusely distributed. n, Nuclear region; ax, axonal segments. Scales bars, 10 µm.
[View Larger Version of this Image (39K GIF file)]
Fig. 9. Mature cerebellar macroneurons (day 14) fluorescently labeled for internalized APP (a, c) and internalized WGA (b, d). No co-distribution of internalized APP and internalized WGA is observed in axons (starred arrowheads in b indicate site of selected puncta of internalized APP marked by arrowheads in a) or in neuronal soma (starred arrowheads in d indicate site of selected puncta of internalized APP marked by arrowheads in c). Scale bars, 10 µm.
[View Larger Version of this Image (60K GIF file)]
Fig. 10. Proposed model for endocytotic trafficking of APP in central neurons (see Discussion for additional details). Full-length APP is internalized from the presynaptic plasmalemma via clathrin-coated vesicles, together with recycling synaptic vesicle integral membrane proteins. Presynaptic clathrin-coated vesicles deliver internalized APP and recycling synaptic vesicle membrane proteins to axonal early endosomes, where sorting takes place. Internalized APP is subsequently delivered by retrograde vesicular transport to the neuronal soma. APP does not appear to use the WGA/lectin internalization pathway for retrograde transport to the neuronal soma, but it is not clear whether APP and lectins (bound to cell-surface glycoproteins) are internalized initially via a common pool of clathrin-coated vesicles., APP;
, synaptotagmin I;
, cell-surface glycoproteins;
, lectins;
, clathrin triskelions; + and
ends of axonal microtubules are indicated.
[View Larger Version of this Image (16K GIF file)]
It is not known whether APP internalization by central neurons results in increased secretion of A
MATERIALS AND METHODS
Antibodies. Monoclonal antibodies 5A3 and 1G7 (Koo and Squazzo, 1994
Cell culture. Primary cerebellar macroneurons were prepared from embryonic day 14 rat using the protocol described for hippocampal neurons by Goslin and Banker (1991)
Immature cerebellar macroneurons were typically used at day 4 in culture (day 0 = day that cultures are prepared); no difference in APP distribution was seen between days 4 and 6 in culture. Mature cerebellar macroneurons were used at day 14 in culture. Polarity for the somatodendritic marker MAP-2 (Caceres et al., 1986
Preparation of vesicular fractions from bovine brain. Clathrin-coated vesicles were prepared from homogenates of bovine cerebral cortex (designated "whole-brain coated vesicles") by differential centrifugation followed by equilibrium centrifugation in linear Ficoll/D2O gradients as described by Forgac and Cantley (1984)
Clathrin-coated vesicles were additionally purified from hypotonically lysed synaptosomes (designated "synaptosomal coated vesicles") using the Maycox et al. (1992)
Immunoprecipitation of whole-brain clathrin-coated vesicles with polyclonal antibody CT15 (directed to the cytoplasmic tail of APP) was carried out as follows. Clathrin coats were dissociated as described above, and the resulting stripped vesicles (0.4 mg total protein) were diluted with Tris-saline buffer (10 mM Tris, pH 8.0, 140 mM NaCl) containing 5 mg/ml BSA to a final volume of 0.5 ml. Stripped vesicles were precleared for 1 hr at 4°C using 30 µl of a 50:50 slurry of protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ) in Tris-saline/BSA and incubated with polyclonal antibody CT15 overnight at 4°C using end-to-end rotation. Overnight incubation with an equivalent amount of polyclonal antibody 863 (directed to the midregion of the extracellular domain of APP) or nonimmune rabbit IgG was used as a negative control. A second aliquot (30 µl) of protein A-Sepharose was added, and the incubation was continued for an additional 2 hr. The immunobeads fraction was obtained by sedimentation, washed five times with Tris-saline/BSA, resuspended in Laemmli (1970)
Preparation of vesicular fractions from rat brain. Rat brain synaptosomes were prepared using the following protocol provided by R. Jahn (personal communication). The cerebral hemispheres from one adult rat brain were homogenized in ice-cold buffer (0.32 M sucrose, 5 mM HEPES, pH 7.4, 1 mM EDTA, containing freshly added 0.5 mM PMSF, 10 µg/ml aprotinin, and 5 µg/ml each leupeptin and pepstatin A) using a glass/Teflon homogenizer (10 strokes at 600 rpm). Remaining steps were carried out at 4°C. The homogenate was centrifuged at 800 × g for 10 min, and the resulting supernatant was centrifuged at 11,000 × g for 12 min. The pellet fraction from the 11,000 × g centrifugation step was resuspended in 4 ml of homogenization buffer and layered onto discontinuous Ficoll gradients (3 ml of 13% Ficoll, 0.75 ml of 9% Ficoll, and 3 ml of 6% Ficoll in homogenization buffer). The Ficoll gradients were centrifuged for 30 min at 65,000 × g (23,000 rpm) in a TH641 rotor. After centrifugation, synaptosomes were collected by pooling the bands present at the 6-9% and 9-12% Ficoll interfaces.
Biochemical purification of synaptic vesicles was carried out using the protocol described by Huttner et al. (1983)
Immunoisolation of synaptic vesicles with anti-synaptophysin beads was carried out as follows. Crude synaptosomes (P2 fraction) obtained as described (Huttner et al., 1983
Immunocytochemistry. Antibodies 5A3,1G7 and Sytlum-Abs were applied to the neuronal culture media for 60 min at 37°C to detect internalized APP and internalized synaptotagmin I, respectively. Mouse or rabbit nonimmune IgG was applied to the culture media (same concentration as used for 5A3,1G7) as a negative internalization control. Rhodamine-conjugated wheat germ agglutinin (WGA, Vector Labs, Burlingame, CA) was applied to the culture media at 10 µg/ml. Goat serum (5%) was added to the culture media to reduce background.
Cultured neurons were fixed for 10 min with 4% formaldehyde (prepared freshly from paraformaldehyde powder) in PBS, pH 7.4, containing 0.12 M sucrose. All steps were carried out at room temperature. The cells were then washed with PBS, quenched with 0.1 M glycine (in PBS), and permeabilized with 0.1% Triton X-100. To detect APP confined to the cell surface, permeabilization with detergent was omitted. Cytoskeletal proteins could not be detected without previous permeabilization with Triton X-100, indicating that cell-surface APP only was detected when detergent was omitted. After blocking with 5% goat serum for 20 min, cells were incubated with additional primary antibodies for 1 hr (where applicable), washed, and incubated with fluorescein- or rhodamine-conjugated secondary antibodies (Boehringer Mannheim). For double-labeling with two monoclonal primary antibodies, the antibodies were applied sequentially and the first antibody was visualized using a large excess of affinity-purified, rhodamine-conjugated secondary antibody as the Fab fragment as described (Yamazaki et al., 1995
Other methods. SDS-PAGE was performed using the method of Laemmli (1970)
RESULTS
Full-length APP is present in presynaptic clathrin-coated vesicles
Nerve terminal (presynaptic) clathrin-coated vesicles are felt to participate in synaptic vesicle recycling (Mundigl and DeCamilli, 1994). APP has been shown previously to be enriched in clathrin-coated vesicles (Nordstedt et al., 1993
As predicted, immunoblotting of purified whole-brain coated vesicles with polyclonal antibody CT15 directed to the cytoplasmic tail of APP demonstrates the presence of full-length APP in this preparation (Fig. 1A, inset). Adsorption of CT15 with its cognate peptide results in elimination of APP signal. In addition, the synaptic vesicle integral membrane proteins synaptophysin, synaptotagmin, and SV2 can be detected in this preparation (Fig. 1A). The small GTP-binding protein rab5a can also be detected in this preparation, but rab3a and rab8 cannot be detected (Fig. 1B). Adsorption of anti-rab5a with its cognate peptide results in elimination of rab5a signal.
The precise fraction, albeit small, of whole-brain coated vesicles that is derived from sites other than nerve terminals (e.g., neuronal soma or glia) is not known. To demonstrate unequivocably that APP is present in the presynaptic subpopulation, coated vesicles were additionally purified by equilibrium centrifugation from hypotonic lysates of bovine brain synaptosomes ("synaptosomal" coated vesicles) and immunoblotted for APP. Full-length APP can be detected in synaptosomal as well as whole-brain coated vesicles (Fig. 1C). In contrast, transferrin receptor, an endocytotic marker for the somatodendritic domain (Cameron et al., 1991
To demonstrate that APP and recycling synaptic vesicle integral membrane proteins are endocytosed via a common pool of clathrin-coated vesicles, purified whole-brain coated vesicles were immunoprecipitated with polyclonal antibody CT15 (directed to the cytoplasmic tail of APP) and immunoblotted for synaptophysin. Synaptophysin is readily detected in coated vesicles immunoprecipitated with CT15 but cannot be detected when an equivalent amount of polyclonal antibody 863 (directed to the midregion of the extracellular domain of APP) or nonimmune IgG is used (Fig. 1D, top panel). Reprobing for APP (Fig. 1D, bottom panel) demonstrates that immunoprecipitation with CT15 selectively enriches for APP-containing coated vesicles. APP is largely excluded from synaptic vesicles
The above results indicate that APP is enriched in presynaptic clathrin-coated vesicles. To determine whether APP is specifically excluded from synaptic vesicles, the latter were purified from rat cerebral cortex by magnetic bead immunoisolation and immunoblotted for APP. Both APP and synaptic vesicles are selectively enriched in synaptosomes prepared from rat cerebral cortex (data not shown). Synaptosomes were then lysed by hypotonic shock and incubated with magnetic beads coated with anti-synaptophysin (anti-synaptophysin beads) (Fig. 2) to immunoisolate synaptic vesicles. After immunoisolation, equal portions of immunobeads ("pellet") and supernatant fractions were analyzed by SDS-PAGE and immunoblotting. Approximately 25% of starting synaptophysin-reactive material is recovered with the anti-synaptophysin beads and doubles when twice the amount of beads is used (Fig. 2, top panel, compare lanes 6 and 8.). All of the APP immunoreactivity, however, remains in the supernatant fraction (i.e., is not precipitated with the anti-synaptophysin beads). No synaptophysin-reactive material is recovered when nonimmune beads are used.
Synaptic vesicles were also purified from rat cerebral cortex using the method of Huttner et al. (1983) APP is internalized with recycling synaptic vesicle membrane proteins then sorted away from synaptic vesicles in cerebellar macroneurons
To identify compartments that participate in trafficking of neuronal cell-surface APP, immunofluorescent labeling of primary cerebellar macroneurons with monoclonal antibodies 5A3,1G7 (Koo and Squazzo, 1994
Antibodies 5A3,1G7 were then added to the macroneuronal culture medium for 60 min at 37°C, and the cells were subsequently fixed, permeabilized, and incubated with fluorescent secondary antibody to detect APP internalized from the neuronal cell surface. Incubation with antibodies directed to the ectodomain of APP has been used successfully in both neuronal (Yamazaki et al., 1995
To confirm our finding that cell-surface APP is internalized together with recycling synaptic vesicle membrane proteins (see first part of Results), polyclonal antibody Sytlum-Abs (Matteoli et al., 1992
To determine whether neuronal cell-surface APP and lectins (bound to cell-surface glycoproteins) use a common internalization pathway, WGA was added to the macroneuronal culture medium for 60 min at 37°C. Internalized WGA appears as diffuse, strong puncta and includes large vesicular profiles in the axonal and somatodendritic domains of immature (data not shown) and mature (Fig. 9b,d) neurons. When both WGA and 5A3,1G7 are added to the culture medium, no co-distribution of internalized lectin and internalized APP is observed either in axons or in neuronal soma.
DISCUSSION
We reported previously (Yamazaki et al., 1995
Neurons are believed to maintain distinct subpopulations of clathrin-coated vesicles and early endosomes in their axonal and somatodendritic domains (Parton and Dotti, 1993
We demonstrate further that full-length APP is endocytosed together with recycling synaptic vesicle membrane proteins in primary cerebellar macroneurons but is subsequently sorted away from synaptic vesicles. Endocytosis of APP and recycling synaptic vesicle membrane proteins occurs predominantly from nerve terminals, as is evident in axonal growth cones in immature neurons and in axonal varicosities (Fletcher et al., 1991
Our results provide additional biochemical evidence that an obligate sorting compartment, presumably the axonal early endosome, participates in the regeneration of synaptic vesicles during exo/endocytotic recycling at nerve terminals (see Mundigl and DeCamilli, 1994). Biochemical evidence for a sorting endosomal intermediate in the synaptic vesicle recycling pathway also derives from the finding by Fischer von Mollard et al. (1994)
On the basis of these as well as previous (Yamazaki et al., 1995
The relationship between neuronal APP endocytosis and the molecular pathogenesis of Alzheimer's disease remains unclear. Internalization of cell-surface APP contributes to the extracellular release of A
FOOTNOTES
Received Aug. 2, 1996; revised Oct. 15, 1996; accepted Oct. 22, 1996.
This work was supported by National Institutes of Health Grants K08 AG00681 (N.M.-S.) and AG12376 (E.K.), the State of Illinois Department of Public Health (N.M.-S.), and the Paul Beeson Physician Faculty Scholars in Aging Research Program (E.K.). We thank the following investigators for providing antibodies, without which these studies could not have been accomplished: Dr. B. Bowman, Dr. F. Brodsky, Dr. K. Buckley, Dr. P. De Camilli, Dr. M. Forgac, Dr. R. Jahn, Dr. R. Melvold, and Dr. I. Trowbridge. We thank Dr. K. Buckley for helpful discussions and for critical review of this manuscript. We thank Dr. M. Forgac for providing bovine whole-brain clathrin-coated vesicles. We thank Dr. R. Jahn for providing the rat brain synaptosomal preparation protocol, for assistance with the synaptophysin immunoisolation, and for helpful discussions.
Correspondence should be addressed to Dr. Numa R. Marquez-Sterling, Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611.
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