The effect of endocytic effectors on β-cleavage and Aβ secretion. N2a cells expressing CFP-swAPP and the dynaminK44A mutant (DynIIDN) or rab4wt constructs were assayed for sAPPβ, Aβ, and full-length APP as described in Materials and Methods. The y axis represents the amount of sAPPβ as a fraction of full-length APP (sAPPβ/APP) (black) or Aβ as a fraction of full-length APP (Aβ/APP) (magenta). The GFP control was normalized to 100%.

Exosomes released from N2a cells contain Aβ peptides. (a) Sucrose gradient fractions of an exosomal preparation from N2a-swAPP cell culture supernatants were immunoblotted with several antibodies. Alix and flotillin-1 mark the exosome-positive fractions (fractions 2–4). Transferrin receptor is excluded from the exosomal fractions and could be detected only in the whole-cell lysates (Cells). Immunoblotting with 6E10 that recognizes the Aβ peptides, full-length APP (FL-APP), β-cleaved C-terminal fragment, and α-cleaved ectodomain reveals that only Aβ peptides fractionate in the exosomal fractions and that full-length APP and other fragments are excluded from the exosomes and could be detected only in the cell lysate (Cells). Note that, in N2a cells, Aβ peptides are efficiently secreted into the medium and, hence, are not detectable at the concentrations of the cell lysates used for blotting. Molecular masses in kilodaltons are indicated to the left of the blots. The densities of the fractions as measured with a refractometer are indicated by the values labeled g/cm3. (b) Exosomes from fractions 3 and 4 of the sucrose gradient were negatively stained with 1% uranyl acetate and immunolabeled with antibodies for the exosomal marker Alix. Exosomes also were immunolabeled for Aβ40 or Aβ42 and cholera toxin B subunit (CTx-B), which binds to the ganglioside GM1.

Involvement of early endosomes in β-cleavage of APP. (a) HeLa cells expressing swAPP were stained for endogenous early endosomal antigen-1 (EEA-1) (green) and the β-cleaved ectodomain (sAPPβ) (red). (b) HeLa cells expressing swAPP were transfected with rab5, rab7, dominant active rab5 mutant (Q79L), or rab11 GFP fusion proteins fixed and stained for the β-cleaved ectodomain (sAPPβ) (red) with ANJJ antibody. (c) HeLa cells expressing the rabGFP constructs, APP, and BACE were crosslinked at 4°C with anti-APP (red) and anti-BACE (blue) antibodies and endocytosed at 37°C. After 5 min, APP and BACE are internalized to rab5–GFP-positive endosomes. For a detailed version, see Fig. 9.

Aβ peptides are localized in MVBs in N2a cells. Immunoelectron microscopy on anti-Aβ-stained cryosections of N2a cells shows that Aβ localizes to MVBs.

Exosomal proteins are enriched in human amyloid plaques. Paraffin-embedded human autopsy tissues from patients with AD or Parkinson's disease or from controls were studied. We performed either Gallyas silver stain and subsequent immunochemistry for Alix on hippocampal sections (d–f) or immunochemistry with hematoxylin counterstain only (a–c). Alix was stained by indirect alkaline phosphatase method with new fuchsin as the chromogen. (Magnification, ×63.) (a and d) Hippocampus of a normal control brain without amyloid plaques or detection of Alix. (b and e) Hippocampus of a patient with Parkinson's disease displaying slight accumulation of pathological tau-filaments but no amyloid plaques and no Alix. (c and f) Hippocampus of a patient with moderate to advanced Alzheimer's pathology [CERAD (Consortium to Establish a Registry for Alzheimer's Disease) score B] with discrete deposits of Alix within typical neuritic plaques (f) containing amyloid, hyperphosphorylated tau-fibrils, and cellular elements (astrocytes and microglia).