Gold nanoparticles as a new tool in amyloid studies

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-β 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

The Nt17 Domain and its Helical Conformation Regulate the Aggregation, Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity