The cellular prion protein (PrP) has been shown to act as a receptor for soluble oligomers of amyloid-beta (Aβ), an ~4 kDa amyloidogenic peptide that is found in neuritic plaques that are a pathological hallmark of Alzheimer’s disease (AD). Oligomeric forms of the Aβ peptide are thought to be synaptotoxic, and have been shown to produce PrP-dependent dendritic spine loss, suppression of long term potentiation (LTP), and behavioral changes in mouse models of AD. However, the specific molecular interactions between PrP and Aβ have not been fully characterized. In this work, we conducted a robust examination of the kinetic processes leading to Aβ fibril formation, and present evidence that PrP significantly inhibits Aβ polymerization. Using established mathematical models of polymerization kinetics, we show that inhibition is based on binding between PrP and the ends of Aβ filaments, an interaction that blocks elongation. To support these results, we conducted multiple binding assays to show that PrP binds to monomers of Aβ with low affinity, oligomers with intermediate affinity, and to fibrils with high affinity. These results extend upon previous studies, which have focused only on the interaction between oligomeric Aβ and PrP. To better understand the molecular interactions required for binding and inhibition of polymerization, we performed assays with a series of PrP deletion mutants, which revealed that low-affinity binding to Aβ monomer is dependent on the presence of the C-terminal domain of PrP. This domain is also required for Aβ polymerization inhibition. Based on our results, we propose a model in which the unstructured N-terminal domain of PrP binds to the ends of Aβ fibrils, while the C-terminal domain interrupts the docking of new monomers to fibril ends, in part through competing for similar binding sites. This study provides an important contribution to our understanding of the PrP-Aβ interaction that leads to synaptoxicity.

La prévalence croissante du diabète de type II et les risques cardiovasculaires associés, sont maintenant considérés comme un enjeu majeur de santé publique. L'agrégation du polypeptide amyloïde humain des îlots (hIAPP) est liée à une dégénérescence des cel-lules β pancréatiques et à la pathogénèse du diabète de type II. Le mécanisme de la toxi-cité de hIAPP et la nature des espèces concernées (oligomères et/ou fibres) sont loin d'être élucidés, bien que de récentes études ont montré que les oligomères formés lors des étapes précoces du processus pourraient être les plus toxiques. Très peu de tech-niques permettent à l’heure actuelle de suivre cette oligomérisation en temps réel et d’évaluer des inhibiteurs de ce processus pathologique. Au cours de cette thèse, nous avons exploré la CE et l’IMS-MS comme techniques permettant de suivre l’oligomérisation de hIAPP in vitro en temps réel. Une méthode de CE a été développée, permettant d’évaluer de nouveaux inhibiteurs envers cette oligomérisation. Une méthode d’IMS-MS a également été développée pour décrire les interactions formées entre hIAPP et un inhibiteur.Des inhibiteurs peptidomimétiques ont été rationnellement conçus et syn-thétisés afin de déstabiliser les structures β formées lors de l’oligomérisation de hIAPP. L’évaluation de ces composés a permis de mettre en évidence la relation entre leurs structures et leurs activités inhibitrices. Des études de viabilité cellulaire sont en cours afin d’améliorer la compréhension de l’activité de ces molécules. / The rising prevalence of type II diabetes, and associated adverse cardiovascular risks, is now considered as a major public health challenge. The aggregation of human islet amyloid polypeptide (hIAPP) is linked to beta-cell degeneration and to the pathogenesis of type II diabetes. The mechanism of hIAPP toxicity and the species involved (oligomers and/or fibrils) are far to be elucidated, although recent studies have shown that early formed species could be the most toxic species. Very few techniques are currently available to monitor in real time this oligomerization and to evaluate inhibitors of this pathological process. During this PhD project, we investigated CE and IMS-MS as potential techniques to monitor in vitro and in real time the oligomerization of hIAPP. A CE-UV method has been developed, which allows the activity evaluation of new inhibitors. An IMS-MS method has also been developed to investigate the interactions formed between hIAPP and the inhibitors. Peptidomimetics inhibitors have been rationally designed and synthesized in order to destabilize beta-sheets structures formed during the oligomerization process of hIAPP. The evaluation of those compounds revealed a relation between their structures and their inhibitory activities. Cellular viability tests are on-going to get more insights on those molecules activity.

Alzheimer’s disease (AD) patients could benefit from a more effective treatment than the current FDA-approved options. Because amyloid-beta (Aβ) is thought to play a central role in AD pathogenesis, many experimental drugs attempt to reduce Aβ-induced pathology. Preventing amyloid accumulation may be a more effective strategy than clearing Aβ plaques after they form. If preventing Aβ accumulation can treat or prevent AD, then understanding Aβ primary nucleation may aid rational drug design. This study examines Aβ residue interaction networks and reports network and structural observations that may provide insight into primary nucleation. While many studies identify structural features of Aβ that promote aggregation, this study reports features that may resist primary nucleation by examining Aβ42 studies in more and less polar solvents. In Aβ42 in a less polar solvent (PDB ID: 1IYT), Val24 and Ile31 have higher betweenness and residue centrality values. This may be due to a predicted interaction between Val24 and Ile31. Residues in the central hydrophobic cluster (CHC) of Aβ40 and Aβ42 had significantly higher betweenness values compared to the average betweenness of the structures, highlighting the CHC’s reported role in oligomerization. The predicted interaction between Val24 and Ile31 may reduce the likelihood of primary nucleation of Aβ.

The misfolding of native, cellular prion protein (PrPc) to a conformationally altered pathogenic isoform, designated scrapie PrPsc, is the main molecular process involved in the pathogenesis of prion diseases. Prion diseases are marked by the accumulation of conformationally modified forms of cellular prion protein. An N-terminal portion of the prion protein, PrP (106-128), is a 23-residue peptide fragment and is characterized by an amphipathic structure with two domains: a hydrophilic N-terminal domain and a hydrophobic C-terminal domain. In this study, the aggregation characteristics of the PrP (106-128) peptide were investigated using a combination of biophysical approaches. We investigated the effect of different factors including concentrations, pH, and metal ions, on the aggregation of the peptide. Our results demonstrated that the peptide steadily aggregates at concentrations higher than 25 M. The aggregation propensity and fibril formation is higher at pH 7.4 and pH 8.1, and the aggregation is inhibited at pH lower than 6. Furthermore, our results indicate that the Cu2+ has much less effect on the peptide amyloidogenesis, while Zn2+ has a significant influence on the PrP (106-128) amyloidogenesis. We further presented a systematic analysis of the impact of phospholipid liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-racglycerol) (POPG) in the absence or presence of cholesterol, on the amyloidogenesis of PrP (106-128). The results showed that POPC vesicles does not significantly influence the aggregation kinetics of the peptide. However, the anionic lipid POPG delays the aggregation in a concentration-dependent manner, whereas the addition of POPG with the cholesterol shows fast kinetics of fibrillization, thus reducing the lag time of the aggregation kinetics. We also monitored the effect of cholesterol and its derivatives including cholesterol-SO4 and DC-cholesterol on PrP (106-128) amyloidogenesis. Our results showed that the cholesterol inhibits the peptide aggregation and delays the formation of fibrils in a concentration-dependent manner. Cholesterol-SO4 dramatically facilitates the aggregation at high concentrations but has the potential to slow down the fibrillization at low concentrations, whereas cationic DC-cholesterol vesicles can effectively inhibit peptide fibril formation at high concentrations. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

In recent years super-hydrophobic micro-patterned substrates (SHS) have been successfully used for the suspension of a few biological molecules, allowing the further characterization in a background-free environment by label-free techniques such as Raman spectroscopy, SEM and TEM in one device. This result is due to the combined action of laminar flow and shear stress exerted on the molecules contained in a drop that is spotted on top of the SHS and slowly evaporates. This new method is here proposed for the label-free formation and background-free characterization of amyloid fibers. Amyloids are insoluble aggregates formed by proteins that convert from a misfolded form into highly-organized β-sheet structures that could accumulate in different organs and compromise their normal physiological functions. Known amyloid-related diseases, named amyloidosis, are for instance Alzheimer, Parkinson, and type 2 diabetes. In classical crystallography, the study of the amyloid aggregates structure is often hampered by the laborious and time consuming sample preparation techniques. Therefore the need of a quick reproducible technique, has emerged. The amyloid fibers investigated in this work are derived from a lysozyme protein and a Tau-derived short peptide, both known to be related to two forms of amyloidosis. With this technique we demonstrate that threads of protein fibers are deposited on the substrate helped by the patterning of the SHS and its properties, and by characterizing them with Raman spectroscopy technique we revealed that they are anisotropic structures of amyloid nature. This type of sample preparation technique arises from the effect of the evaporation on the SHS, and opens up new possibilities for the formation of oriented fibers of amyloids and more in general, of proteins, ready for a substrate-free characterization, while classic crystallographic methods could have a limitation.

This research focused on development of nanoparticle- based therapeutics against amyloid fibrils. Amyloid fibrils are associated with various diseases such as Parkinson’s, Huntington’s, mad cow disease, Alzheimer’s, and cataracts. Amyloid fibrils develop when proteins change their shape from a native form to a pathogenic “misfolded” form. The misfolded proteins have the ability to recruit more native proteins into the pathogenic forms, which self-assemble into amyloid fibrils that are hallmarks of the various protein-misfolding diseases listed above. Amyloid fibrils are highly resistant to degradation, which may contribute to the symptoms of amyloid diseases. Synthetic drugs, natural compounds, and antibodies are widely explored for potential to stop pathogenic protein assembly or to promote fibril degradation and clearance, but to date have had little success in relieving symptoms in clinical trials. In this research, I have synthesized fluorine-containing silica nanoparticles (NPs), and tested their fibril-inhibiting activity against amyloid fibrils formed by a non-pathogenic protein, β-lactoglobulin (BLG). These fluoro-silica NPs prevented BLG amyloid formation, whereas non-fluorinated nanoparticle analogs did not inhibit fibrillation under the same reaction conditions. The fluoro-silica NPs interacted with the BLG protein in a manner that prevented the protein from adopting a form that could self-assemble into fibrils. Additional applications of the NPs were explored as small-molecule drug-delivery systems; such that multiple functionalities could be introduced into a single nano- therapeutic.

Indiana University-Purdue University Indianapolis (IUPUI) / Cerebral amyloid angiopathy (CAA) is characterized by the cerebrovascular deposition of amyloid. The mechanisms underlying the contribution of CAA to neurodegeneration are not fully understood. In this dissertation, there are three main chapters. The first chapter investigates existing evidence regarding the amyloid diversity in CAA and its relation to tau pathology and immune response, as well as the possible contribution of molecular and cellular mechanisms, previously associated with parenchymal amyloid in Alzheimer disease (AD) and AD-related dementias, to the pathogenesis of CAA. The second chapter demonstrates differential glial reactivity and activation associated with early-stage CAA in a mouse model of Familial Danish Dementia (FDD), a neurodegenerative disease characterized by vascular accumulation of Danish amyloid (ADan). We show that early-stage CAA is associated with dysregulation in immune response networks and lipid processing, severe astrogliosis with a neurotoxic A1-astrocytic phenotype, characterized by increased expression of Complement Component 3 (C3), and decreased levels of Triggering Receptor Expressed On Myeloid Cells 2 (Trem2) with no significant reactive microgliosis. Our results also indicate how cholesterol accumulation and Apolipoprotein E (ApoE) are associated with vascular amyloid deposits at the early stages of pathology. Furthermore, we demonstrate A1 astrocytic mediation of Trem2 and microglia homeostasis. In the final chapter, we addressed whether inflammatory stimulus of other cell types are capable of inducing a subtype of neurotoxic astrocytes. Here we show a subtype of C3+ neurotoxic astrocyte are induced by activated endothelial cells that is distinct from astrocytes classically activated by microglia. We show that endothelial activated astrocytes have upregulated expression of A1-astrocytic genes and exhibit a distinctive extracellular matrix remodeling profile. Finally, we demonstrate that endothelial activated astrocytes are Decorin-positive and are associated to vascular amyloid deposits but not parenchymal amyloid plaques in mouse models and AD/CAA patients. These findings show the existence of potentially extensive and subtle functional diversity of C3+-reactive astrocytes.

No description available.

The Amyloid Cascade hypothesis provides a molecular-level mechanism for the etiology of Alzheimer’s Disease (AD) and proposes a central role for the genesis and aggregation of Aβ protein. Aβ protein is the product of cleavage of the amyloid precursor protein (APP), a single pass transmembrane protein, by secretases and is found in a variety of isoforms, with longer isoforms being linked to the early onset of AD. The isoform distribution is dependent on membrane environment, mutations, and post-translational modifications. Lipid rafts are characterized by lipids induced into the liquid ordered phase by cholesterol, enhancing membrane thickness and lateral lipid density. Protein preference for rafts can control protein kinetics, and has been implicated in determining whether APP is processed by α– or β-secretase in the plasma membrane. In addition to inducing lipid rafts, cholesterol is hypothesized to directly modulate APP, the C-terminal fragment of APP (C99), and γ-secretase structure and function via direct interaction. To date, the molecular details involved in these fundamental events involved in Aβ genesis have yet to be resolved using experimental approaches, suggesting a critical role for computation. This thesis presents the results of investigations of lipid phase separation and cholesterol and their effects on C99 using molecular dynamics simulation. To gain insight into the nature of lipid rafts, studies characterizing the simulation system sizes required for observation of phase separation, exploring the effect of cholesterol concentration on phase separation and lipid phases, and examining the applicability of different lipid and cholesterol models for the simulation of lipid phases and protein structure were performed. To gain insight into the fundamental properties of C99, studies exploring the structure of full-length C99, the interaction of cholesterol with C99 in various mutational states, the effect of membrane thickness on the C99 extramembrane domains, and the structure of C99 monomer and dimer were performed. Taken together these studies advance our molecular-level understanding of the nature of cholesterol, the role of cholesterol in lipid phase separation, the effect of cholesterol on C99, and the structure of the full-sequence C99 monomer and dimer that play a critical role in the evolution of AD.

Solid-state NMR serves as a powerful method for investigating atomic-level details of insoluble biomolecules, enabling the determination of protein 3D structures and probing molecular motions across a broad range of timescales. In this thesis, I present structural studies on a novel heterotypic and functional amyloid, dynamics studies, and chemical shift anisotropy studies of a microcrystalline protein, ubiquitin. In Chapter 1, I provide a summary of the main interactions in solid-state NMR and discuss relevant pulse sequences employed in this thesis. Chapter 2 briefly explores the characteristic properties of amyloids, highlighting well-studied examples of disease-related and functional amyloids. Special treatments employed in amyloid structure determination using solid-state NMR are also summarized. Chapter 3 presents structural studies on a heterotypic functional amyloid, mcmvM45-hsRIPK3, where M45 is a protein encoded by murine cytomegalovirus (MCMV) and RIPK3 is from humans. Both M45 and RIPK3 belong to a family of RHIM-containing proteins, which are involved in innate immunity and immune response through necroptosis. SSNMR data on various isotopically labeled samples enable the chemical shift assignment for both M45 and RIPK3, providing intra- and inter-molecular contacts. By combining these constraints, we calculate the structure of the hetero-amyloid M45-RIPK3, reporting two structures distinct from RIPK1-RIPK3. In Chapter 4, I measure backbone 15N-13CO order parameters of microcrystalline ubiquitin using DCP-REDOR. Two isotopically labeled samples, 1-13C-glucose and 1,3-13C-glycerol, D₂O labeled, are studied and compared, identifying mobile residues and assessing the effect of isotropic labeling on the measurements of backbone 15N-13Co order parameters. Experimental order parameters are compared with a 1μs MD simulation for insights. Chapter 5 focuses on the chemical shift anisotropy (CSA) of uniformly labeled microcrystalline ubiquitin using a novel pulse sequence allowing the measurement of large CSAs under practical conditions. We explore CSA parameter trends, correlations between isotropic shifts, and hydrogen bond geometries. Comparison with solution-NMR results demonstrates high consistencies with asymmetry parameters (η), providing insights into the motion modes of microcrystalline proteins alongside order parameter measurements. Chapter 6 provides a comprehensive summary of the conclusions drawn from the preceding chapters, while also outlining future directions for each project.