Making the final cut: pathogenic amyloid-β peptide generation by γ-secretase

Abstract

Alzheimer´s disease (AD) is a devastating neurodegenerative disease of the elderly population. Genetic evidence strongly suggests that aberrant generation and/or clearance of the neurotoxic amyloid-β peptide (Aβ) is triggering the disease. Aβ is generated from the amyloid precursor protein (APP) by the sequential cleavages of β- and γ-secretase. The latter cleavage by γ-secretase, a unique and fascinating four-component protease complex, occurs in the APP transmembrane domain thereby releasing Aβ species of 37-43 amino acids in length including the longer, highly pathogenic peptides Aβ42 and Aβ43. The lack of a precise understanding of Aβ generation as well as of the functions of other γ-secretase substrates has been one factor underlying the disappointing failure of γ-secretase inhibitors in clinical trials, but on the other side also been a major driving force for structural and in depth mechanistic studies on this key AD drug target in the past few years. Here we review recent breakthroughs in our understanding of how the γ-secretase complex recognizes substrates, of how it binds and processes β-secretase cleaved APP into different Aβ species, as well as the progress made on a question of outstanding interest, namely how clinical AD mutations in the catalytic subunit presenilin and the γ-secretase cleavage region of APP lead to relative increases of Aβ42/43. Finally, we discuss how the knowledge emerging from these studies could be used to therapeutically target this enzyme in a safe way.

Keywords: Alzheimer's disease; amyloid β-peptide; presenilin; γ-secretase.

Figure 1. FIGURE 1: APP processing and generation of Aβ.

APP is first cleaved by β-secretase (β) in its ectodomain close to the extracellular/luminal membrane border thereby generating a 99 amino acid C-terminal APP fragment (C99). Consecutive cleavages of C99 by γ-secretase at ε-, ζ-, and γ-sites releases the APP intracellular domain (AICD) into the cytosol and 37-43 amino acid Aβ species into the extracellular space or lumen of secretory pathway organelles. Longer Aβ forms such as in particular Aβ42 are highly aggregation prone and ultimately deposit as plaques in AD patient brains. An alternative cleavage of APP by α-secretase (α) in the Aβ domain prevents the formation of Aβ. Pathogenic APP FAD mutations that have been shown or are likely to cause relative increases in the generation of Aβ42 species are located in the γ-secretase cleavage region of the APP TMD and in the AICD.

Figure 2. FIGURE 2: Structure of γ-secretase.

The atomic resolution structure of γ-secretase (PDB: 5FN3) shows a membrane embedded core containing the catalytic subunit presenilin-1 cleaved into NTF (blue) and CTF (cyan) flanked by the subunits PEN-2 (yellow) and APH-1a (purple), which is covered by the large bilobar extracellular domain of the nicastrin subunit (green). This domain allows nicastrin to serve as gatekeeper controlling substrate access to the active site by excluding proteins with too large (or sterically incompatible) ectodomains. Red spheres depict the active sites aspartate residues.

Figure 3. FIGURE 3: Substrate recruitment of C99 by γ-secretase.

(A) Schematic representation of the most prominent C99 residues interacting with γ-secretase subunits as determined by site-directed photocrosslinking using the unnatural amino acid p-benzoyl-phenylalanine. (B) Model depicting the stepwise translocation of C99 from exosites (purple) in NCT and PEN-2 (stage 1) and the PS1 NTF (stage 2) to the active site (stage 3). (C) Structure of γ-secretase (5FN3) with a co-isolated α-helix (orange), which might represent a substrate-mimic. Red spheres depict the active site aspartate residues. Light pink spheres represent candidate sites for substrate interactions with NCT. Although not visible in this view, L571 is less buried than E333. Numbers indicate TMDs of the PS1 NTF that surround the substrate-mimicking α-helix.

Figure 4. FIGURE 4: Stepwise cleavage model of APP.

(A) The two product lines leading to the generation of Aβ40 and Aβ42 are depicted. In the major product line Aβ40 is generated by consecutive tripeptide-releasing cleavages (green) at the ε-49, ζ-46 and γ-43 sites. In a minor product line, Aβ42 is generated in a similar manner by consecutive cleavages (red) at the ε-48 and ζ-45 sites. (B) Besides γ-byproducts from the two main product lines (green and red) including a hexapeptide resulting from direct cleavage of Aβ49 to Aβ43, multiple additional minor peptides have been identified suggesting multiple product line crossings. The pentapeptide resulting from cleavage of Aβ43 to Aβ38 is indicated in purple. (C) Sequential cleavage continuously requires sterically compatible interactions of P2´ residues with the S2´ enzyme subsite of γ-secretase; major product line shown. The adjacent P4´ residue K53 is additionally presented for the comparison of side chain proportions.

Figure 5. FIGURE 5: FAD mutations in presenilin.

Schematic representation of the nine TMD structure of presenilin in its cleaved form with the NTF (blue) and CTF (cyan). Pathogenic presenilin mutations (http://alzforum.org/mutations/) are found in all TMDs and in some of the HLs. The compared to PS1 less frequent PS2 FAD mutations are represented in italics. The red arrow indicates the site of endoproteolysis.

Figure 6. FIGURE 6: Mechanism of presenilin FAD mutations.

(A) Model depicting impaired processivity in the Aβ40 and Aβ42 product lines causing relative increases in the generation of the longer Aβ species Aβ42 and Aβ43. Structural instability of FAD mutant presenilin leads to impaired processivity manifested by faster dissociation rates and premature release of long Aβ such as Aβ42 from the enzyme. (B) Presenilin FAD mutations cause a mispositioning of C99 as shown by altered interactions of the substrate cleavage domain with the enzyme. Residues that were identified to show increased or decreased interactions with two different PS1 FAD mutants are shown in red and blue, respectively. Yellow asterisk indicates a FAD mutation.

Figure 7. FIGURE 7: Mechanism of APP FAD mutations.(A) Model depicting the shift in the Aβ40 and Aβ42 product lines by APP FAD mutations thereby causing relative increases in the generation of the longer Aβ42 species. Yellow asterisk indicates a FAD mutation.

(B) Product line usage is governed by interactions with the S2´ enzyme subsite of γ-secretase. Sterically demanding aromatic amino acids at the P2´ position of C99 such as in the synthetic M51F mutant clash with the S2´ subsite of the Aβ40-line. (C) Pathway block by a clash of the aromatic P2´ with the S2´ subsite of the Aβ40-line in the FAD-associated I45F mutant causes a shift to the Aβ42-product line. Since this mutant does not alter the initial ε-site cleavage, Aβ42 is most likely generated from Aβ46.

Figure 8. FIGURE 8: Pseudoinhibiton of γ-secretase by semagacestat.

Cleavage of C99 by γ-secretase causes the generation of secreted and intracellular Aβ pools as well as γ-byproducts from carboxy-terminal trimming of the C99 TMD. Unlike TSA-GSIs such as L-685,458, semagacestat causes an intracellular accumulation of long Aβ species and byproducts demonstrating a pseudo-inhibition of γ-secretase by this and related compounds.