Alzheimer's disease mutations in APP but not γ-secretase modulators affect epsilon-cleavage-dependent AICD production

Pathological amino-acid substitutions in the amyloid precursor protein (APP) and chemical gamma-secretase modulators affect the processing of APP by the gamma-secretase complex and the production of the amyloid-beta peptide A beta 42, the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early-onset familial Alzheimer's disease affect both gamma- and epsilon-cleavage sites, by raising the A beta 42/40 ratio and inhibiting the production of AICD50-99, one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to gamma- secretase modulators, which shift A beta 42 production towards the shorter A beta 38, but unequivocally spare the epsilon-site and APP- and Notch-ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its gamma- site, modifying at the same time, the solvation of the epsilon-site.

Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified gamma-Secretase Complexes

Lorène Aeschbach, Patrick Fraering, Jemila Houacine

Background: Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the c-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (A beta) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Ab production by enhancing the A beta 1-42/A beta 1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing A beta 1-42 production, decreasing A beta 1-40 production, or both. Because it could be explained by the heterogeneity in the composition of gamma-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, Delta E9, or P436Q.

Public Library of Science2012

Perturbations of the Straight Transmembrane alpha-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by gamma-Secretase

Matteo Dal Peraro, Mitko Dimitrov, Patrick Fraering, Thomas Lemmin

Background: Amyloid- neurotoxicity depends on the specificity of the proteolytic cleavage of the amyloid precursor protein (APP) transmembrane domain. Results: The APP transmembrane -helix is straight in a biological membrane bilayer. Conclusion: The flexibility of APP is key for adapting to the lipid environment and modulating proteolytic processing by -secretase. Significance: The dynamic characterization of APP is expected to rationalize the design of -secretase modulators. The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by -secretase of its C-terminal fragment produces amyloid- (A) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight -helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the -secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of A38 compared with A40 peptides, which is reminiscent of the effect of -secretase modulators inhibitors.

American Society for Biochemistry and Molecular Biology2014

Nucleation Dependent Polymerization of Amyloid-ß (Aß) as an Important Determinant of Aß Amyloid Formation and Neurotoxicity

Mohammad Asad Jan Qureshi

Aggregation and fibril formation of amyloid-β (Aβ) peptides play a pivotal role in the pathogenesis of Alzheimer's disease (AD). Aβ peptides, principally comprising of 40 or 42 amino acid residues (Aβ40 and Aβ42), are produced by proteolytic processing of the amyloid precursor protein by β and γ-secretase activity. A vast number of academic and biomedical industry projects are devoted to the discovery and development of therapeutic agents which inhibit the process of Aβ aggregation, and associated neurotoxicity, as potential disease modifying therapies for AD. Despite decades of research, the mechanism of Aβ neurotoxicity leading to development of AD and the identity of toxic Aβ species remain debatable. Nevertheless, independent lines of evidence implicate Aβ oligomers, which are precursors to fibrils, as putative toxic species and promising therapeutic targets. Aβ oligomers are characterized by heterogeneity in size and morphology distribution and have been shown to alter normal neuronal physiology in a manner that implicates their causal association with AD pathogenesis. The primary objective of this thesis work was to elucidate the structural determinants of Aβ neurotoxicity, the relationship of fibril formation process with neurotoxicity and identify neurotoxic Aβ species. Towards achieving these goals, we first developed and optimized reproducible protocols for generating various Aβ species, including monomers, protofibrils and fibrils, and established methods for detailed characterization of their structural properties. To better understand the relationship between Aβ aggregation and neurotoxicity, cell culture assays using primary neurons and neuronal cell lines were developed and used to assess the toxicity of various Aβ aggregates. Using these assays and biochemical tools, we sought to answer some of the key outstanding questions in the field concerning the role of Aβ amyloid formation process in neurodegeneration in AD. What is the nature of neurotoxic Aβ species and how does the process of its formation relate to neurodegeneration in AD? The identification of toxic Aβ species and/or process of its formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in AD, and development of effective diagnostic tools and therapeutic interventions. To achieve this, we successfully isolated protofibrils of distinct sizes and morphology distribution and compared their fibrillization and neurotoxicity to monomeric and fibrillar forms of Aβ. The results from our studies implicate an ongoing Aβ polymerization process, rather than distinct Aβ aggregate states (fibrillar or non-fibrillar), as primary determinant of Aβ neurotoxicity. We showed that crude Aβ42 preparations containing mixtures of monomers and heterogeneous oligomers were significantly more toxic than purified Aβ42 protofibril preparations. Subfractionation of protofibril preparations, to separate different protofibril species, resulted in further attenuation of their toxicity. The slow fibrillization of purified and subfractionated Aβ42 protofibrils was strongly linked to their diminished toxicity; thus, demonstrating that Aβ42 toxicity is not necessarily linked to specific prefibrillar aggregate(s), but rather to the ability of these species to grow and undergo fibril formation in presence of monomeric Aβ42. Consistent with this hypothesis, reintroduction of monomeric Aβ42 into these fractions restored amyloid formation and enhanced their toxicity to comparable levels as observed for the crude preparations. Furthermore, selective removal of monomeric Aβ42 from these preparations, using insulin degrading enzyme (IDE), reversed the toxicity of Aβ42 protofibrils. Together, these findings provide strong evidence thatAβ toxicity is linked to an ongoing Aβ polymerization process and is greatly reduced when the polymerization process is slowed down by selective removal/degradation of monomers. What is the molecular basis underlying the protective role of Aβ40 against Aβ42 amyloid formation and neurotoxicity in AD? The ratio of Aβ40/Aβ42 has been shown to be a critical determinant of neurodegeneration and the distribution of Aβ amyloid pathology in brain (parenchyma vs. vasculature) in AD. To investigate the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Aβ40/Aβ42, we assessed fibrillization in the mixtures of different species (monomers, protofibrils and fibrils) of the two peptides at wide range of ratios and concentrations. We showed that monomeric Aβ40 alters the kinetic stability, solubility, and morphological properties of Aβ42 aggregates and prevents their conversion into mature fibrils.Aβ40, at approximately equimolar ratios (Aβ40/Aβ42∼ 0.5-1), inhibits (>50%) fibril formation by monomeric Aβ42, whereas inhibition of protofibrillar Aβ42 fibrillogenesis is achieved at lower, substoichiometric ratios (Aβ40/Aβ42 ∼ 0.1). Additionally, we demonstrated that monomeric Aβ42 and Aβ40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Aβ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Aβ42 protofibrils selectively seed the fibrillogenesis of monomeric Aβ42 but not monomeric Aβ40. Finally, we also showed that the amyloidogenic propensities of different individual and mixed Aβ species correlate with their relative neuronal toxicities. Together, our results demonstrate that maintaining a delicate balance not only between the monomeric forms of Aβ42 and Aβ40, but also the monomeric and aggregated forms of both peptides is essential for maintaining Aβ solubility and preventing Aβ fibrillogenesis and toxicity. Implications for AD pathogenesis and anti-amyloid therapeutics This results presented in this thesis provide novel insight into the mechanisms of Aβ aggregation, fibrillogenesis and toxicity, and have important implications for understanding the role of Aβ amyloid formation in AD pathogenesis and design of therapeutic strategies. Most importantly, our results linking Aβ toxicity to the growth and fibrillization of Aβ oligomers in presence of Aβ monomers, suggest that anti-Aβ therapeutic strategies, many of which are currently being tested in animal models or in clinical trials in AD patients, hold potential as promising disease modifying interventions. We contemplate that targeting the nucleated polymerization of amyloid forming proteins offers an exciting framework for development of anti-amyloid therapies for a host of neurodegenerative diseases characterized by pathological accumulation of aggregated proteins including AD, Parkinson's disease, and prion diseases.