Gold nanoparticles as a new tool in amyloid studies

Urszula Beata Cendrowska
EPFL, 2020
EPFL thesis

Abstract

The misfolding and self-assembly of proteins into fibrils is a hallmark of several neurodegenerative and systemic diseases. These disease-associated proteins have the propensity to form fibrils with a cross-Î² sheet structure, called amyloids. Amyloids can assemble into structures of various morphologies and increasing evidence suggests, that this polymorphism gives rise to distinct toxicity and pathology spreading patterns. This was observed for fibrils formed under different conditions or derived from different diseased tissues. Validation of this hypothesis remains challenging due to the laborious methods that are employed to enable the direct visualization and analysis of amyloid fibrils polymorphism in hydrated and complex biological samples. This thesis presents a novel microscopy method for assessing amyloid fibrils polymorphism using small gold nanoparticles (~3 nm diameter) coated with a mixture of 1-octanethiol and 11-mercapto-1-undecanesulfonate. These nanoparticles efficiently label amyloid fibrils that were produced in vitro as well as fibrils that were derived from tissues of human patients (ex vivo), that suffered from diseases linked to amyloid aggregates. Decoration with gold nanoparticles enabled the rapid visualization of the labelled contours of the amyloid fibrils that were imaged using Cryogenic transmission electron microscopy (cryo TEM). This method rendered the structural analysis of the fibrils relatively easy. Due to the labelling of the fibrils, we were able to observe a wide range of the morphologically polymorphic amyloids for in vitro derived samples. Moreover, comparative analysis of these fibrils with ex vivo derived amyloids, revealed significant differences in the morphological homogeneity. Our results showed an unexpected uniformity of the ex vivo derived fibrils, highlighting the influence of the milieu on the aggregation of amyloid fibrils (Chapter 4 and 5). The nanoparticles were also used as a probe to analyze amyloid fibrils surfaces. For example, we showed that sequence modification influences the efficiency of amyloid fibril decoration with our nanomaterial. In Chapter 3, we present that two types of fibrils and their truncated versions, were differently labelled due to the modification of their charged domains. This shows, that we can assess the changes in the primary structure of the amyloidogenic protein by monitoring the interactions between the nanoparticles and the surface of amyloids. Furthermore, results presented in Chapter 6 show that the decoration of the amyloids is possible in various types of biological milieu, including cerebrospinal fluids (CSF) and cell lysates. This finding suggests that our gold nanomaterial can potentially be used for targeted amyloid decoration in more complex systems, such as cells or tissues. The results presented in this thesis show that gold nanoparticles may represent a novel tool for rapid determination of the polymorphism of in vitro and ex vivo derived amyloid fibrils. We demonstrate and validate the advantages of our technique such as simplicity, quick data acquisition and analysis, stability of the labelling and high efficiency of decoration in the complex samples. This can be of high value for the studies of amyloid related diseases as this method allows for relatively easy screen of fibrils derived from the patientsâ tissue and recognition of polymorphic species.

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https://infoscience.epfl.ch/record/277679?ln=en

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Urszula Beata Cendrowska
EPFL, 2020
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/277679?ln=en

About this result

Related concepts (25)

Related concepts

Related publications (7)

Related publications

Unraveling the complexity of amyloid polymorphism using gold nanoparticles and cryo-EM

Nadine Aït Bouziad, Urszula Beata Cendrowska, Anass Chiki, Zekiye Pelin Güven, Paulo Henrique Jacob Silva, Hilal Lashuel, Marie Mueller, Francesco Stellacci, Sophie Vieweg

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

NATL ACAD SCIENCES2020

Discriminative adsorption of amphiphilic monolayer protected gold nanoparticles on amyloid fibers

Paulo Henrique Jacob Silva

This thesis presents a systematic study of how different types monolayer-protected AuNPs interact with amyloid fibers. We report a class of amphiphilic gold nanoparticles capable of adsorbing onto specific surface features on these types of protein fibers. A common disease-associated protein fold is the amyloid state: it is characterized by a cross-beta sheet structure that forces proteins and peptides into a fibrillar state, commonly found in illnesses such as Alzheimerâs disease, Parkinsonâs disease among many others. Amyloid diseases are typically chronic, correlated with ageing and have posed several challenges: the exact structure of the fibers is difficult to determine and their etiologic role is often unclear. This thesis shows, for the first time, that amphiphilic monolayer-protected AuNPs can discriminatively adsorb onto surface features of amyloid fibers made of A1-40 and -synuclein in vitro and that hydrophobicity determines adsorption onto Tau fibers. Given an amyloid fiber that adopts a twisted ribbon morphology, AuNPs protected by a mixture of sulfonated and hydrophobic thiolate molecules adsorb onto specific features on the surface of the fiber, leaving other interfaces uncovered. This generates a novel supra-molecular assembly that directly interfaces an engineered nanomaterial with a biological structure, without using antibodies. Experiments and calculations demonstrated the importance of nanoparticle size and ligand-shell composition: a size cut-off around 4 nm was observed and other types of water soluble nanoparticles did not adsorb discriminatively. Small amphiphilic AuNPs act as surfactants and probably adsorb onto solvent-exposed beta sheets and small amyloidogenic oligomers. The results presented in this thesis provide a systematic framework to understand the interaction between nanoparticles and amyloid fibers. The particles can, moreover, become useful markers for amyloid research and possibly a cross-instrumental probe to reconcile spectroscopic and imaging techniques to help molecular structure determination. During this work, the synthesis and purification of large amounts of sulfonated thiolate molecules was systematized to generate libraries of differently coated water soluble gold nanoparticles (AuNPs). This helped elucidate how amphiphilic AuNPs fuse with lipid bilayers.

EPFL2016

Loss of mitochondrial function and proteostasis typify aging and age-associated degenerative disorders such as Alzheimer's disease and muscle aging. To date, no cure or preventive measure is available to manage these conditions. Alterations of cellular proteostasis, such as accumulation of misfolded or aggregated proteins, can directly affect mitochondrial homeostasis leading to activation of specific mitochondrial stress pathways capable of maintaining mitochondrial function. However, the contribution of these pathways in age-associated diseases characterized by proteotoxic aggregates is largely unknown. My thesis aims to understand the connection between cellular and mitochondrial proteostasis with a particular focus on mitochondrial stress pathways involved in the response to proteotoxic stress. To this end, I performed 2 studies: Reduction of Amyloid-beta proteotoxicity through activation of mitochondrial quality control: Accumulation of Amyloid-beta (Aß) peptidic aggregates, generated by the miscleavage of amyloid precursor protein (APP), is often associated with mitochondrial dysfunction. However, it is largely unknown how mitochondria react to these proteotoxic insults. We identified a cross-species conserved mitochondrial stress response, from nematodes to humans, activated during Aß proteotoxic stress. This response entails key mitochondrial quality control pathways such as the mitochondrial unfolded protein response (UPRmt) and mitophagy. Importantly, activation of these pathways through administration of mitochondrial regulators Doxycycline and the NAD+ booster nicotinamide riboside (NR), was sufficient to reduce Aß accumulation and proteotoxicity in nematodes and human cell lines. Moreover, boosting mitochondrial quality control in vivo in a mouse model of Alzheimer's disease with the compound NR led to a reduction of brain Aß plaques and preservation of cognitive function. Skeletal muscle aging is characterized by amyloid-like protein aggregates: Skeletal muscle aging is characterized by accumulation of dysfunctional mitochondria and misfolded proteins. However, the connection between these two hallmarks of muscle aging remains elusive. We found that aged skeletal muscles present a similar perturbation of mitochondrial and APP processing pathways when compared to muscles from inclusion body myositis patients (IBM), a disease characterized by intracellular protein deposits containing also Aß aggregates. In line with this, we identified amyloid-like aggregates in aged muscle closely resembling the ones observed during IBM with concomitant mitochondrial dysfunction. Strategies aiming at restoring mitochondrial homeostasis led to activation of the mitochondrial quality control accompanied by a reduction of amyloid-like aggregates in vitro and in vivo in mammalian and nematode systems. In summary, our work demonstrated that amyloidosis characterizes muscle, and possibly brain, aging in a similar fashion to what is observed in muscle and brain tissues from IBM and Alzheimer's patients, respectively. Importantly, restoring mitochondrial homeostasis through activation of mitochondrial quality control is sufficient to alleviate protein aggregation and improve tissue function in aging, IBM and Alzheimer's disease models.

EPFL2021