Inhibition of amyloid precursor protein processing by beta-secretase through site-directed antibodies

Abstract

Amyloid-beta peptide (AbetaP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of beta- and gamma-secretases. The beta-secretase APP cleaving enzyme (BACE), which generates the N terminus of AbetaP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AbetaP and, subsequently, plaque formation. The search for specific inhibitors of beta-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP-BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP-BACE interaction at the cell surface may affect amyloid-beta production. We present here a unique approach to inhibit AbetaP production by means of antibodies against the beta-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the beta-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AbetaP formation in these cells.

Fig. 1.

Generation and in vitro characterization of mAb BBS1 raised against the β-secretase cleavage site of APP. (A) Immune response in BALB/c mice immunized with MAP expressing eight copies of the partially Swedish mutated BACE cleavage site of APP (MAP-[ISEVKLDA]₈). Sera fractions of immunized mice were analyzed for their anti-MAP-[ISEVKMDA]₈ IgG levels displaying the WT sequence of the β-secretase site of APP. Black bars, first immunization; gray bars, second immunization; white bars, third immunization. Spleens were isolated from mice with the higher titers, and mAb was prepared by the hybridoma technique. (B) mAb BBS1 binding properties. mAb BBS1's ability to bind MAP-[ISEVKMDA]₈ was analyzed by ELISA. Percentage of the maximal antibody binding is presented in different antibody concentrations. (C) Competition between soluble antigen (MAP-[ISEVKMDA]₈) and antigen adsorbed to ELISA plates for mAb BBS1 binding. Continuous line, binding of mAb BBS1 after incubation with increasing concentration of MAP-[ISEVKMDA]₈; dashed line, antibody binding after incubation with nonrelevant antigen. (D) Stability of immunocomplex at various pH values. mAb BBS1's ability to bind its antigen (MAP-[ISEVKMDA]₈) was tested by ELISA after 1 h of incubation at different pH values as described in Materials and Methods. Diamonds, pH effect on mAb BBS1 antigen binding; squares, pH effect on the coated antigen.

Fig. 2.

Antibody ability to bind full-length APP expressed by CHOhAPP751 cells. (A) Antibody ability to bind APP expressed by CHOhAPP751 is demonstrated by Western blot (i) and immunofluorescence (ii). (i) CHOhAPP751 cells were lysed, electrophoresed, and transferred to a nitrocellulose membrane. mAb BBS1 was applied at different concentrations, and mAb AMY-33, which binds the midregion of Aβ, was used as a positive control (left). (ii) Immunolabeling of cells with mAb BBS1. Antibody binding is detected by using a Cy2-conjugated secondary antibody and visualized by using an LSM-510 Zeiss confocal microscope. (Scale bar, 10 μm.) (B) Internalization assay of mAb BBS1. CHOhAPP751 cells were administered with mAb BBS1 in the growing media and incubated with the antibody for 60 min. After incubation with the antibody, cells were fixed and permeabilized, and antibody presence inside the cells was detected by using Cy2 secondary antibody (Left). Cells were counterstained for early endosomes by using rabbit anti-EEA1 after Cy3 secondary antibody (Center), and the superposition is demonstrated in Right. Upper and Lower represent two different fields at different magnitudes. (Scale bar, 10 μm.) Cell labeling was visualized by using an LSM-510 Zeiss confocal microscope.

Fig. 3.

Inhibition of extra- and intracellular AβP accumulation. The ability of mAb BBS1 to interfere with the APP processing and thus reduce AβP levels was examined in the following way: CHOhAPP751 cells were administered with mAb BBS1, anti-APP N-terminal polyclonal antibodies, or chloroquine diluted in sera-free media. Untreated cells were used to measure AβP basal level. (A) Secreted AβP levels were measured from the growing media at different time points (white bars, 3 h; black bars, 9 h; gray bars, 24 h), and the ratio between secreted AβP in each treatment group and the untreated group was calculated and presented in percentage. The experiment was repeated six times for each group. (B) Intracellular AβP measurements were performed after 5 days of incubation. The ratio between the intracellular AβP levels in each treatment group and in the untreated group was calculated and presented in percentage (black bar, untreated cells; white bar, mAb BBS1-treated cells; gray bars, anti-N-terminal antibody and chloroquine-treated cells). The experiment was repeated five times for each group. (C) CTFβ levels were measured after 4 h of metabolic labeling and immunoprecipitation with anti-C-terminal antibody. CTFβ is shown at a molecular mass of 12 kDa in the untreated (–) and mAb BBS1-treated (+) cells, and densitometric analysis estimated a 20% reduction of CTFβ levels in mAb BBS1-treated cells. (D) MTT cell viability assay was performed after 5 days of incubation (black bar, untreated cells; white bar mAb BBS1-treated cells; gray bars, anti-N-terminal antibody and chloroquine-treated cells). (E) The levels of sαAPP released to the cells' media were analyzed after 24 and 48 h by Western blot using mAb 196, which binds Aβ in the N terminus. Soluble αAPP was detected at a molecular mass of ≈100 kDa in the supernatant of mAb BBS1-treated cells (+) as well as in that of untreated cells (–).