Trafficking of Cell-Surface β-Amyloid Precursor Protein: Evidence that a Sorting Intermediate Participates in Synaptic Vesicle Recycling

MATERIALS AND METHODS

Antibodies. Monoclonal antibodies 5A3 and 1G7 (Koo and Squazzo, 1994) recognize nonoverlapping epitopes in the midregion of APP and were used in combination (designated 5A3,1G7) for APP trafficking studies (Yamazaki et al., 1995). Polyclonal antibody CT15 recognizes the 15 C-terminal residues in the cytoplasmic tail of APP (Sisodia et al., 1993). 5A3,1G7 and CT15 are specific for APP and do not cross-react with the related protein APLP-2 (see Slunt et al., 1994). Polyclonal antibody 863 was generated against a bacterial fusion protein encompassing ∼280 amino acid residues in the midregion of APP (from XhoI to BglII restriction sites); the transmembrane and cytoplasmic domains are excluded from this construct. Polyclonal antibody Syt_lum–Abs (Matteoli et al., 1992) (a gift from Dr. P. De Camilli) recognizes the small intraluminal domain of rat synaptotagmin I. Monoclonal anti-synaptotagmin I (gift from Dr. R. Jahn) recognizes the large cytoplasmic domain of synaptotagmin I. Additional monoclonal antibodies include anti-clathrin heavy chain (clone X22, a gift from Dr. F. Brodsky), anti-SV2 (a gift from Dr. K. Buckley), anti-transferrin receptor (a gift from Dr. I. Trowbridge), anti-70 kDa subunit of V-ATPase from bovine brain (clone 3.2-F1, a gift from Dr. M. Forgac), anti-murine H-2 complex cell-surface antigen (a gift from Dr. R. Melvold), anti-synaptophysin (clone SY38, Boehringer Mannheim, Indianapolis, IN), anti-MAP-2 (clone HM-2, Sigma, St. Louis, MO), and anti-α-tubulin (clone DM-1A, Sigma). Polyclonal antibodies to the 70 and 60 kDa subunits of V-ATPase were obtained from Dr. B. Bowman. Antibodies to rab3a, 5a, and 8 were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Cell culture. Primary cerebellar macroneurons were prepared from embryonic day 14 rat using the protocol described for hippocampal neurons by Goslin and Banker (1991). Briefly, cells from the dissected cerebellar anlage (Altman and Bayer, 1978, 1985) were dissociated by trypsinization (15 min at 37°C) followed by trituration with flame-polished Pasteur pipettes. The cells were plated at a density of 150,000 or 450,000 per 60 mm tissue culture dish on polylysine-coated (1 mg/ml) glass coverslips in MEM supplemented with 10% horse serum. After 3–4 hr to allow attachment of neurons, the coverslips were transferred to tissue culture dishes containing glial cell monolayers and serum-free neuronal medium consisting of MEM containing the N2 supplements of Bottenstein and Sato (1979), 0.1% ovalbumin, and 0.01% pyruvate. Glial cultures for conditioning of neuronal medium were prepared as described 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) was established at day 5–6 in culture.

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). Whole-brain and synaptosomal (see below) coated vesicles were incubated with low-ionic-strength buffer containing 5 mm Tris, pH 8.5, 150 mm sucrose, and 0.5 mm EGTA to dissociate clathrin coats (designated “stripped vesicles”), as described (Forgac and Cantley, 1984) before analysis by SDS-PAGE.

Clathrin-coated vesicles were additionally purified from hypotonically lysed synaptosomes (designated “synaptosomal coated vesicles”) using the Maycox et al. (1992) protocol adapted for bovine brain. Briefly, synaptosomes were prepared from cerebral cortical tissue (obtained from whole calf brains) by differential centrifugation followed by Ficoll gradient centrifugation, washed with tartrate buffer to remove bound vesicular material, and lysed by hypotonic shock. The synaptosomal lysate was quickly returned to isotonicity by the addition of 1/10 volume of 10× buffer A (Forgac and Berne, 1986) and subfractionated by equilibrium centrifugation in linear Ficoll/D₂O gradients as described (Forgac and Cantley, 1984). Synaptosomal clathrin-coated vesicles were collected as a small but distinct band from the linear gradients, resuspended in buffer A, homogenized with a Dounce glass–glass homogenizer, and snap-frozen for storage at −80°C.

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) sample buffer, and heated at 85°C for 10 min to dissociate bound proteins. One-half of the resuspended immunobeads fraction was used for analysis by SDS-PAGE and immunoblotting.

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 msucrose, 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 × gfor 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) with minor modifications. The cerebral hemispheres from two adult rat brains were used. Ten fractions were obtained from the linear sucrose gradients. Controlled-pore glass chromatography was not performed.

Immunoisolation of synaptic vesicles with anti-synaptophysin beads was carried out as follows. Crude synaptosomes (P2 fraction) obtained as described (Huttner et al., 1983) were resuspended in ice-cold homogenization buffer (see above) and lysed by hypotonic shock. HEPES/NaOH was immediately added to a final concentration of 7.5 mm, pH 7.4, and the suspension was kept on ice for 30 min. NaCl (150 mm final concentration) was added to restore isotonicity, and the lysate was cleared by centrifugation (11,000 × g for 12 min). The resulting supernatant (S3 fraction) was used as the starting material for immunoisolation. Anti-synaptophysin beads were prepared as follows. Magnetic beads coated with sheep-anti-mouse IgG (Dynal, Great Neck, NY) were incubated with monoclonal anti-synaptophysin (2 mg antibody/mg beads) in PBS overnight at 4°C. The beads were washed three times and resuspended in 150 mm NaCl, 10 mm HEPES, pH 7.4, and 1 mm EDTA (resuspension buffer). Anti-synaptophysin beads (2 mg of bound antibody) were incubated with the S3 fraction (0.5 mg total protein) for 2 hr at 4°C with end-to-end rotation. After the incubation, the beads were retrieved with a magnetic rack (Dynal), washed three times for 10 min each with resuspension buffer, and resuspended in Laemmli (1970) sample buffer. The supernatant from the immunoisolation was centrifuged for 30 min at 97,000 rpm in an AT4 rotor, and the resultant pellet was resuspended in sample buffer. Control beads were prepared by incubation with an equivalent amount of nonimmune mouse IgG.

Immunocytochemistry. Antibodies 5A3,1G7 and Syt_lum–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 0.12m sucrose. All steps were carried out at room temperature. The cells were then washed with PBS, quenched with 0.1 mglycine (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). For labeling of the Golgi compartment, fixed, permeabilized cells were incubated for 1 hr with fluorescein-conjugated lentil lectin (10 μg/ml, Vector Labs). Cells were viewed with a Zeiss axiophot microscope equipped with epifluorescence optics and photographed with T-MAX 400 film (Eastman Kodak, Rochester, NY).

Other methods. SDS-PAGE was performed using the method of Laemmli (1970). Immunoblotting was performed as described by Towbin et al. (1979) using an enhanced alkaline phosphatase detection kit purchased from BioRad (Hercules, CA). For immunoblotting of subcellular fractions from rat brain, an enhanced chemiluminescence kit purchased from Amersham (Arlington Heights, IL) was used. For reprobing of immunoblots, antibodies bound to the nitrocellulose membrane were removed by stripping with 62.5 mm Tris, pH 6.8, 2% SDS, and 100 mm β-mercaptoethanol for 30 min at 70°C. Membranes were rinsed twice in PBS then incubated with a second primary antibody. Protein concentration was determined using the method ofBradford (1976).