Amyloid-beta precursor protein

Amyloid-beta precursor protein (APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. It functions as a cell surface receptor and has been implicated as a regulator of synapse formation, neural plasticity, antimicrobial activity, and iron export. It is coded for by the gene APP and regulated by substrate presentation. APP is best known as the precursor molecule whose proteolysis generates amyloid beta (Aβ), a polypeptide containing 37 to 49 amino acid residues, whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. Genetics Amyloid-beta precursor protein is an ancient and highly conserved protein. In humans, the gene APP is located on chromosome 21 and contains 18 exons spanning 290 kilobases. Several alternative splicing isoforms of APP have been observed in humans, ranging in length from 639 to 770 amino acids, with certain isoforms preferentiall

The Gamma-Secretase-Mediated Proteolytic Processing of APP C-Terminal Fragments as a Therapeutic Target for Alzheimer's Disease

Jemila Houacine

Alzheimer's disease (AD) is a devastating neurodegenerative disorder which severely impairs cognitive functions by triggering neuronal cell death and synaptic loss, and finally leads the patients to death. Two main histopathological hallmarks can be found in the brain tissue of AD patients: the amyloid plaques composed of aggregated Amyloid-β peptides (Aβ) and the neurofibrillary tangles (NFTs) constituted of hyperphosphorylated tau protein. To explain the underlying molecular mechanisms of AD pathogenesis, the amyloid cascade hypothesis states that early accumulation of Aβ peptides (especially the Aβ42 specie identified as the most toxic one) triggers their assembly into oligomers and their subsequent extracellular aggregation into dense fibrillar deposits. The oligomeric Aβ assemblies further initiate a succession of neurotoxic events including disruptions at the synaptic level, inflammation, neuritic injury, altered calcium homeostasis, oxidative stress, and altered phosphorylation activity leading to the hyperphosphorylation of tau and its aggregation into NFTs. Finally, this cascade of neuronal deleterious effects induces cognitive dysfuntions leading to dementia. The Aβ peptides originate from the amyloidogenic processing of the amyloid precursor protein (APP), a type I membrane protein that undergoes a first cleavage by β-secretase to liberate the soluble APP domain (sAPPβ) in the extracellular space, and the membrane bound APP-C99 fragment. Then, APP-C99 is processed by the intramembrane aspartyl-protease gamma-secretase (γ-secretase) composed of four subunits (presenilin (PS), nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2)) to release the toxic Aβ peptides in the lumen, and the APP intracellular domain (AICD) in the cytosol. Alternatively, APP can undergo the non-amyloidogenic processing, in which it is first shedded by α-secretase to generate the sAPPα domain and the APP-C83 fragment. The latter is further cleaved by γ-secretase into the non toxic p3 peptide and the AICD. AD is the most frequent form of dementia in elderly humans. Despite this evidence, no treatment is currently available to prevent or cure this disease. Thus, multiple potential drugs are tested in clinical trials or are still being developed in preclinical studies. In this work, a new therapeutic approach to lower Aβ production by specifically targeting the γ-secretase-mediated processing of APP-C99 is presented. Monoclonal antibodies against APP-C99 were generated and characterized following mice immunization with the active recombinant substrate APP-C99. When tested in cells, these antibodies decreased the γ-secretase-dependent processing of APP-C99, thus lowering Aβ production. Furthermore, the intracerebroventricular injection of anti-APP-C99 antibodies in a mouse model of AD decreased total soluble Aβ levels. These results validated this new approach as a potential immunotherapeutic strategy to prevent and/or delay the neurotoxic effects caused by Aβ in AD pathogenesis. Next, the γ-secretase-mediated processing of the APP-carboxy-terminal fragments (APP-CTFs) regulating the toxic versus non-toxic pathways was further investigated with the help of in vitro activity assays. This comparative study of APP-C99 and APP-C83 processing by γ-secretase revealed that both substrates were identically processed by the enzyme, with the same AICDs being produced. In addition, the effects of familial AD (FAD) mutations in the PS subunit of highly pure and homogeneous γ-secretase complexes were also investigated, and showed a drastic loss-of-function phenotype linked to these mutants. Overall, in vitro studies of the APP-CTFs processing by γ-secretase underlined particular functional aspects of this processing, which could be related to the cellular pathways involved in AD pathogenesis such as the competition between the amyloidogenic and non-amyloidogenic pathways and the FAD-linked impaired metabolism of APP.

EPFL2011

Studying membrane-protein interplay by modeling realistic conditions

Martina Audagnotto

Realistic models of cellular environments have long captured the imagination of biologists and physical scientists. More than 20 years ago, Goodshellâs inspiring rendering of cellular environments provided the foundation for the idea of capturing the full complexity of biological cells. Since then, increasingly realistic simulations of subcellular models have appeared, showing that we are rapidly moving towards comprehensive, as well as physically and biologically accurate cellular models. The major challenge in developing such models is the necessity to cover a wide range of scales. Indeed, atomistic details of molecular processes are on the length scale of 0.1 nm, while cellular dimensions range from 300 nm (for the smallest bacterial cells) up to 100 ÎŒm (for large eukaryotic cells). The biological range also encompasses the internal dynamics of individual molecules (from ns to ms) as well as entire biological processes (ms to hours). Although an atomistic representation of an entire cell is conceivable, the cost for achieving biologically significant timescales with most advanced computers is very high or nearly impossible currently. Therefore, the combination of in silico approaches with in vivo and in vitro experimental data, allows gaining further insights into the biophysics of the cell. In this thesis, a multi-scale approach was applied to investigate the interplay between proteins and their respective biological environments. In particular, two specific systems were the objects of my Ph.D. project: (i) the amyloid precursor protein (APP) and its interactions at the synaptic plasma membrane, and (ii) the human acyl-protein thioesterase I (hAPT1) and its role in protein depalmitoylation cycle across the Golgi and plasma membranes. Combining atomistic and coarse-grained molecular dynamics (MD) simulations, the stability of the dimeric conformation of APP in a realistic model of the synaptic plasma membrane was investigated. Within this approach, the stable dimeric conformation of the APP as a function of lipid membrane composition was identified, highlighting the important role of lipids unsaturation in protein stability and function. In detail, this indicated not only the preferential dimerization motif of the protein, but also the possible recruiting mode of cholesterol, which has previously been experimentally observed to correlate with Alzheimerâs disease. Furthermore, the role of the synaptic plasma membrane and its ability to form raft microdomains was explored for the interactions of APP with Î³-secretase, which specific catalytic cleavage is responsible for the production of physiological or toxic amyloid Î² peptides, responsible for the Alzheimerâs disease onset. This same multi-scale approach was applied to understand the role of S-acylation in the trafficking and function of hAPT1. In particular, the combination of experimental techniques (i.e., cell biology, biochemistry, microscopy and structural biology) with molecular modeling and simulation disclosed the substrate/enzyme interaction mode, the enzyme/membrane interface, and the role for hAPT1 for a key post-translational modification as S-palmitoylation. Based on our findings, we provided for the first time an atomistic representation of the hAPT1 enzyme mechanism, suggesting that hAPT1 is active in the monomeric form. In conclusion, these studies demonstrate once more the importance of modeling the molecular details of the biological environment at the most accu

EPFL2017

The metalloprotease ADAMTS4 generates N-truncated A4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease

Mitko Dimitrov, Patrick Fraering, Hermeto Gerber, Sandra Lehmann

Brain accumulation and aggregation of amyloid- (A) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length A peptides (mainly A1-40 and A1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by - and -secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble A peptides are truncated at the N-terminus, with A4-x peptides being particularly abundant. A4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the A peptide sequence, which facilitates A4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of A4-40 but unchanged levels of A1-x peptides. In the 5xFAD mouse model of amyloidosis, A4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4(-/-) knockout background, A4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of A species, but A4-40 peptides were absent in cultures derived from ADAMTS4(-/-) mice indicating that the enzyme was essential for A4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of A4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic A peptides.

SPRINGER2019