Induced Dimerization of the Amyloid Precursor Protein Leads to Decreased Amyloid-β Protein Production

Abstract

The amyloid precursor protein (APP) plays a central role in Alzheimer disease (AD) pathogenesis because sequential cleavages by ␤-and ␥-secretase lead to the generation of the amyloid-␤ (A␤) peptide, a key constituent in the amyloid plaques present in brains of AD individuals. In several studies APP has recently been shown to form homodimers, and this event appears to influence A␤ generation. However, these studies have relied on APP mutations within the A␤ sequence itself that may affect APP processing by interfering with secretase cleavages independent of dimerization. Therefore, the impact of APP dimerization on A␤ production remains unclear. To address this question, we compared the approach of constitutive cysteine-induced APP dimerization with a regulatable dimerization system that does not require the introduction of mutations within the A␤ sequence. To this end we generated an APP chimeric molecule by fusing a domain of the FK506-binding protein (FKBP) to the C terminus of APP. The addition of the synthetic membrane-permeant drug AP20187 induces rapid dimerization of the APP-FKBP chimera. Using this system we were able to induce up to 70% APP dimers. Our results showed that controlled homodimerization of APP-FKBP leads to a 50% reduction in total A␤ levels in transfected N2a cells. Similar results were obtained with the direct precursor of ␤-secretase cleavage, C99/SPA4CT-FKBP. Furthermore, there was no modulation of different A␤ peptide species after APP dimerization in this system. Taken together, our results suggest that APP dimerization can directly affect ␥-secretase processing and that dimerization is not required for A␤ production.The mechanism of ␤-amyloid protein (A␤) 2 generation from the amyloid precursor protein is of major interest in Alzheimer disease research because A␤ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (1, 2). In the amyloidogenic pathway A␤ is released from the amyloid precursor protein (APP) (3) after sequential cleavages by ␤-secretase BACE1 (4 -6) and by the ␥-secretase complex (7,8). BACE1 cleavage releases the large ectodomain of APP while generating the membrane-anchored C-terminal APP fragment (CTF) of 99 amino acids (C99). Cleavage of ␤-CTF by ␥-secretase leads to the secretion of A␤ peptides of various lengths and the release of the APP intracellular domain (AICD) into the cytosol (9 -11). The ␥-secretase complex consists of at least four proteins: presenilin, nicastrin, Aph-I, and Pen-2 (12). Presenilin is thought to be the catalytic subunit of the enzyme complex (13), but how the intramembrane scission is carried out remains to be elucidated. Alternatively, APP can first be cleaved in the non-amyloidogenic pathway by ␣-secretase within the A␤ domain between Lys-16 and 15). This cleavage releases the APP ectodomain (APPs␣) while generating the membrane-bound C-terminal fragment (␣-CTF) of 83 amino acids (C83). The latter can be further processed by the ␥-secretase complex, resulting in the secretion of the sm...

Pharmacology of Heparin and Related Drugs

“…APP and its mammalian homologs, the APP-like proteins, can form intracellular homo-and heterodimers; although APP is monomeric in solution in vitro, it dimerizes in the presence of heparin (Gralle et al, 2006), thus possibly reducing Ab generation (Eggert et al, 2009). The E2 domain of APP-like protein 1 crystallizes as a dimer, and a heparin hexasaccharide soaked into the crystal is bound asymmetrically within the dimer, making different contacts with the two protein monomers; one heparin oligosaccharide is bound to each dimer (3QMK) (Xue et al, 2011).…”

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

“…Surprisingly, A␤ could not be detected for the K28E mutant in cultured cells and the cell-free assay system by SDS-PAGE (data not shown). This may reflect a potential alteration of the folding of Lys-28 mutant A␤ peptides (45), which could possibly have affected their detectability in some of the previous reports (22,46,47 FIGURE 4. Validation of cell-free assay system concerning the cleavage specificity of purified ␥-secretase on mutant APP C-terminal fragment based C100-His 6 substrates.…”

“…This region is known to be critical for A␤ aggregation (19 -21), and aggregation inhibitors interacting with this region also act as GSMs (18). However, a recent study demonstrated that dimerization per se might not be a factor that determines ␥-secretase cleavage specificity (22), and whether GXXXG mutants inhibit dimerization is controversial (23). In addition, the lack of a consistent response to GSMs regarding an inversely correlated production of A␤ 42 and A␤ 38 by PS FAD mutants observed earlier argued against a strict precursor-product relationship between A␤ 42 and A␤ 38 (24,25).…”

PrPCHomodimerization Stimulates the Production of PrPCCleaved Fragments PrPN1 and PrPC1

“…1) (Spencer et al, 1993;Eggert et al, 2009). In contrast to cross-linking antibodies, AP20187 is permeable and is able to target intracellular Fv-PrP.…”