Endogenous Regulators of Gamma-Secretase and Amyloid-Beta Production, and Engineering of Alzheimer's Disease Therapeutic Tools

Sébastien Mosser
EPFL, 2016
EPFL thesis

Abstract

Alzheimer s disease (AD) is a devastating neurodegenerative disease characterized by strong cognitive impairment and memory loss. These symptoms are caused by neuronal death, induced by two pathological hallmarks: extracellular senile plaques composed of aggregated amyloid-beta (Abeta) peptides, and intracellular neurofibrillary tangles generated by the hyperphosphorylation of the actin-binding protein Tau. Genetic and biochemical analysis resulted in the generation of the so-called amyloid cascade hypothesis, which stipulates that Abeta aggregation precedes neurofibrillary tangles and initiate AD pathogenesis. Abeta originates from the proteolytic cleavage of the amyloid precursor protein (APP) by gamma-secretase, an intramembrane cleaving protease. Thus, pharmacological modulation of gamma-secretase emerged as a promising strategy for the treatment of AD. However, strong side effects, including cognitive function worsening, were reported following clinical inhibition of this enzyme, caused by altered processing of the numerous gamma-secretase substrates. Pharmaceutical industries, alongside with academia, oriented their research toward the discovery of compounds selectively affecting the processing of APP, without altering the cleavage of other substrates. In the first part of this thesis work, we focused our research on endogenous modulation of gamma-secretase, and its consequences on actin cytoskeleton dynamics. We performed mass spectrometric analysis of purified gamma-secretase, and identified the adipocyte plasma membrane associated protein APMAP as a gamma-secretase interacting protein. We showed that APMAP modulates the generation of Abeta peptides without affecting the processing of other gamma-secretase substrates, and controls APP degradation by the lysosomal/autophagic pathway. We also investigated the regulation of gamma-secretase by post translational modifications, and concluded that its enzymatic activity is not affected by its phosphorylation. We then observed the effects of gamma-secretase inhibition on physiological processes, and discovered a gamma-secretase-dependent regulation of the binding of Cofilin to actin filaments. These observations showed a direct link between gamma-secretase and the actin cytoskeleton required for cell motility and neuronal plasticity. In the second part of this work, we aimed at finding a new method for monitoring brain structure alteration and provide new tools for the study of neurodegenerative disease progression in animal models. We performed ex-vivo electrical impedance measurement from intracerebral electrode in mice brain, and succeeded in the visualization of cortical layers as well as brain nuclei. Moreover, we used this method to visualize structural alteration caused by Abeta plaques in an AD mouse model. After imaging, we aimed at repairing neuronal loss causing the structural alterations observed in neurodegenerative diseases. We developed a compressible and injectable extracellular-matrix-like cell culture scaffold (cryogel) to promote neuronal growth in brain lesions. Primary cortical neuron culture on our cryogel scaffold showed good axonal sprouting, not affected by cryogel compression.

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Sébastien Mosser
EPFL, 2016
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/215868?ln=en

About this result

Related concepts (19)

Related concepts

Related publications (10)

Related publications

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.

EPFL2010

Preparation and characterization of toxic Abeta aggregates for structural and functional studies in Alzheimer's disease research

Mohammad Asad Jan Qureshi, Hilal Lashuel

The amyloid cascade hypothesis, supported by strong evidence from genetics, pathology and studies using animal models, implicates amyloid-beta (Abeta) oligomerization and fibrillogenesis as central causative events in the pathogenesis of Alzheimer's disease (AD). Today, significant efforts in academia, biotechnology and the pharmaceutical industry are devoted to identifying the mechanisms by which the process of Abeta aggregation contributes to neurodegeneration in AD and to the identity of the toxic Abeta species. In this paper, we describe methods and detailed protocols for reproducibly preparing Abeta aggregates of defined size distribution and morphology, including monomers, protofibrils and fibrils, using size exclusion chromatography. In addition, we describe detailed biophysical procedures for elucidating the structural features, aggregation kinetics and toxic properties of the different Abeta aggregation states, with special emphasis on protofibrillar intermediates. The information provided by this approach allows for consistent correlation between the properties of the aggregates and their toxicity toward primary neurons and/or cell lines. A better understanding of the molecular and structural basis of Abeta aggregation and toxicity is crucial for the development of effective strategies aimed at prevention and/or treatment of AD. Furthermore, the identification of specific aggregation states, which correlate with neurodegeneration in AD, could lead to the development of diagnostic tools to detect and monitor disease progression. The procedures described can be performed in as little as 1 day, or may take longer, depending on the exact toxicity assays used.

Nature Publishing Group2010

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

EPFL2010

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

Jemila Houacine

EPFL2011