A modified Stokes-Einstein equation for Aβ aggregation

<p>Abstract</p> <p>Background</p> <p>In all amyloid diseases, protein aggregates have been implicated fully or partly, in the etiology of the disease. Due to their significance in human pathologies, there have been unprecedented efforts towards physiochemical understanding of aggregation and amyloid formation over the last two decades. An important relation from which hydrodynamic radii of the aggregate is routinely measured is the classic Stokes-Einstein equation. Here, we report a modification in the classical Stokes-Einstein equation using a mixture theory approach, in order to accommodate the changes in viscosity of the solvent due to the changes in solute size and shape, to implement a more realistic model for A<it>β</it> aggregation involved in Alzheimer’s disease. Specifically, we have focused on validating this model in protofibrill lateral association reactions along the aggregation pathway, which has been experimentally well characterized.</p> <p>Results</p> <p>The modified Stokes-Einstein equation incorporates an effective viscosity for the mixture consisting of the macromolecules and solvent where the lateral association reaction occurs. This effective viscosity is modeled as a function of the volume fractions of the different species of molecules. The novelty of our model is that in addition to the volume fractions, it incorporates previously published reports on the dimensions of the protofibrils and their aggregates to formulate a more appropriate shape rather than mere spheres. The net result is that the diffusion coefficient which is inversely proportional to the viscosity of the system is now dependent on the concentration of the different molecules as well as their proper shapes. Comparison with experiments for variations in diffusion coefficients over time reveals very similar trends.</p> <p>Conclusions</p> <p>We argue that the standard Stokes-Einstein’s equation is insufficient to understand the temporal variations in diffusion when trying to understand the aggregation behavior of A<it>β</it>42 proteins. Our modifications also involve inclusion of improved shape factors of molecules and more appropriate viscosities. The modification we are reporting is not only useful in A<it>β</it> aggregation but also will be important for accurate measurements in all protein aggregation systems.</p

Characterization of Beta-amyloid Peptide Aggregation and Acceleration with Non-fibrillar Forming Peptide-based Mediators

Protein aggregation occurs under many circumstances, from the dynamic assembly of tubulin to form microtubules, the aggregation of actin into filaments, as well as plaque formation by amyloid precipitation. One important requirement in studying the mechanism of amyloid aggregation is the ability to monitor the growth kinetics over a wide range in size scales (10 nm to microns) with time that spans microseconds to seconds. Understanding the mechanisms of the aggregation may then lead to improved design of drugs to help control or suppress the aggregation process. In this dissertation, the physical characterization of the â-amyloid peptide and its interaction with áá-amino acid peptide-based mediators was investigated from the early, rapidly evolving stage to the later, slowly diffusing peptide stage by the application of fluorescence photobleaching recovery (FPR). The diffusion rates of â-amyloid peptide and â-amyloid assemblies under the effects of variables including concentration of â-amyloid, blocker peptide, ionic strength, pH, time and temperature were accessible by this method. In some instances, dynamic light scattering (DLS), which acquire signals greater than 10 decades of lag times, without requiring a dye label was used for comparison and to account for the limitations of any given approach. Attachment of 5-carboxyfluorescein did not affect the integrity of the protein and the measured diffusion coefficients were similar to those measured by diffusion ordered spectroscopy (DOSY) and from theoretical expectations. FPR proved more sensitive than DLS for detection of low oligomer aggregates of the â-amyloid peptide coexisting with much larger fibrils. We were able to reverse the conformation of the peptide from the low oligomeric state to the aggregated state under neutral and acidic pH conditions and confirmed that the peptide growth increased with increasing ionic strength. The interaction of â-amyloid peptide with membranes results in several membrane-perturbing effects which may play a pivotal role in the pathogenic cascade leading to Alzheimer’s disease. Model phospholipid bilayer membranes consisting of 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) and 1-Oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] dodecanoyl]-sn-Glycero-3-Phosphocholine (18:1-12:0 NBD PC) were prepared on mica. FPR proved to be a useful technique for obtaining information of the nature of membrane fluidity upon interaction with the â-amyloid peptide

Stilbenes: Therapeutic Interventions Targeting Amyloid β Protein Aggregation In Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common form of dementia and accounts for 60-80 % of all dementia cases. In the United States, AD is ranked as the 6th leading cause of death and it is the only one among the top 10 that cannot be prevented, treated or even slowed. All FDA approved drugs focus on attenuating the symptoms for a limited time by regulating neurotransmitters without any intervention with the underlying disease process. A major player in the initiation and development of this debilitating disorder is the misfolding and subsequent aggregating of amyloid-β (Aβ) peptide. This aggregation process converts non-toxic functional Aβ peptides into a spectrum of neurotoxic Aβ aggregated species, such as, oligomers, soluble aggregates and fibrils. These aggregated forms are believed to be the source of inflammation and oxidative stress that cause neuron death and the loss of synaptic functions. Therefore, small molecules that could intervene with the aggregation of Aβ peptides may present an effective AD therapeutic prevention and treatment. The work presented here examined the intervening effects of eight stilbenes, such as resveratrol and piceatannol, on multiple mechanistic steps of Aβ aggregation. Results identified trans-piceatannol and cis-piceatannol as the most potent compounds that could affect distinct aggregation pathways, specifically, modifying the conformation of Aβ aggregates, as well as, reducing soluble aggregate growth rate. The pronounced change in Aβ aggregate conformation might be related to a change in cytotoxicity. Their superior effects could be attributed to the presence of catechol moiety. The change in Aβ aggregate conformation induced by trans-piceatannol substantially reduced the Aβ aggregates binding to lipid bilayers, which plays a vital role in the induction of neurotoxicity. Other stilbenes, including resveratrol, also altered the morphology of Aβ aggregates but they exhibited only moderate interfering effects on Aβ aggregation pathways. Together, this study provides insight into the effective interventions of piceatannol on Aβ aggregation and proposes this natural compound as a novel promising small molecule for the prevention and treatment of AD

Investigation of Amyloid β Oligomer Dissociation Mechanisms by Single Molecule Fluorescence Techniques

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis

Pathogenic A&#946; A2V versus protective A&#946; A2T mutation : early stage aggregation and membrane interaction

We investigated the effects of punctual A-to-V and A-to-T mutations in the amyloid precursor protein APP, corresponding to position 2 of A\u3b21\u201342. Those mutations had opposite effects on the onset and progression of Alzheimer disease, the former inducing early AD pathology and the latter protecting against the onset of the disease. We applied Static and Dynamic Light Scattering and Circular Dichroism, to study the different mutants in the early stages of the aggregation process, essential for the disease. Comparative results showed that the aggregation pathways differ in the kinetics and extent of the process, in the size of the aggregates and in the evolution of the secondary structure, resulting in fibrils of different morphology, as seen by AFM. Mutated peptides had comparable toxic effects on N2a cells. Moreover, as assessed by X-ray scattering, all of them displayed disordering effects on the internal structure of mixed phospholipids-gangliosides model membranes

On the Kinetics of Protein Misfolding and Aggregation

Protein (mis)folding into highly ordered, fibrillar structures, amyloid fibrils, is a hallmark of several, mainly neurodegenerative, disorders. The mechanism of this supra-molecular self-assembly reaction, as well as its relationship to protein folding are not well understood. In particular, the molecular origin of the metastability of the soluble state of proteins with respect to the aggregated states has not been clearly established. In this dissertation, it is demonstrated, that highly accurate kinetic experiments, using a novel biosensing method, can yield fundamental insight into the dynamics of proteins in the region of the free energy landscape corresponding to protein aggregation. First, a section on Method development describes the extension and elaboration of the previously established kinetic assay relying on quartz crystal microbalance measurements for the study of amyloid fibril elongation (Chapter 3). This methodology is then applied in order to study in great detail the origin of the various contributions to the free energy barriers separating the soluble state of a protein from its aggregated state. In particular, the relative importance of residual structure, hydrophobicity (Chapter 4) and electrostatic interactions (Chapter 5) for the total free energy of activation are discussed. In the last part of this thesis (Chapter 6), it is demonstrated that this biosensing method can also be used to study the binding of small molecules to amyloid fibrils, a very useful feature in the framework of the quest for potential inhibitors of amyloid formation. In addition, it is shown that Thioflavin T, to-date the most frequently employed fluorescent label molecule for bulk solution kinetic studies, can in the presence of potential amyloid inhibitor candidates be highly unreliable as a means to quantify the effect of the inhibitor on amyloid formation kinetics. In summary, the work in this thesis contributes to both the fundamental and the applied aspects of the field of protein aggregation.Engineering and Physical Sciences Research Counci

The good, the bad, and the tiny: A simple, mechanistic-probabilistic model of virus-nutrient colimitation in microbes

For phytoplankton and other microbes, nutrient receptors are often the passages through which viruses invade. This presents a bottom-up vs. top-down, co-limitation scenario; how do these would-be-hosts balance minimizing viral susceptibility with maximizing uptake of limiting nutrient(s)? This question has been addressed in the biological literature on evolutionary timescales for populations, but a shorter timescale, mechanistic perspective is lacking, and marine viral literature suggests the strong influence of additional factors, e.g. host size; while the literature on both nutrient uptake and host-virus interactions is expansive, their intersection, of ubiquitous relevance to marine environments, is understudied. I present a simple, mechanistic model from first principles to analyze the effect of this co-limitation scenario on individual growth, which suggests that in environments with high risk of viral invasion or spatial/temporal heterogeneity, an individual host’s growth rate may be optimized with respect to receptor coverage, producing top-down selective pressure on short timescales. The model has general applicability, is suggestive of hypotheses for empirical exploration, and can be extended to theoretical studies of more complex behaviors and systems

Development of NMR tools to investigate aggregation phenomena

Chemistry and biology are full of examples of aggregation phenomena; from the useful applications in drug molecule stabilisation and tissue engineering, to the negative effects of causing diseases such as Alzheimer's and Parkinson's. NMR is a useful tool for probing aggregation as it can provide detail at the atomic scale; however, the molecular size of the aggregates can lead to poor resolution and spectral broadening, issues which require some development to solve. The research detailed in this thesis aims to develop NMR tools to investigate aggregation phenomena from two angles; firstly, to directly monitor aggregation using diffusion-ordered NMR spectroscopy and secondly to probe the effects of aggregates on biomimetic constructs using NMR techniques. To achieve the first aim, diffusion-ordered NMR Spectroscopy in conjunction with a size-exclusion chromatography stationary phase was developed for the purpose of monitoring aggregation in a time-resolved manner. The size-exclusion stationary phases, commonly used to separate molecules of different sizes, such as proteins from salts, in liquid chromatography methods, provide an additional perturbation of the diffusion profile of molecules of different sizes when added to NMR samples for diffusion-ordered spectroscopy analysis. The development of this method and a selection of applications are detailed within two chapters of the thesis. These studies have been published in Journal of Magnetic Resonancei and Journal of Physical Chemistry Cii. A complementary study of the interactions between aggregates and biological structures such as biomimetic cell membranes using NMR methods such as time-resolved 31P NMR and the implementation of paramagnetic shift reagents is discussed in the final two chapters. Changes in chemical shift caused by additional interactions between the shift reagent and the solvent are fully investigated and the method is applied to the study of membrane permeation by amyloid-β oligomers

The good, the bad, and the tiny : a simple, mechanistic-probabilistic model of virus-nutrient colimitation in microbes

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 10 (2015): e0143299, doi:10.1371/journal.pone.0143299.For phytoplankton and other microbes, nutrient receptors are often the passages through which viruses invade. This presents a bottom-up vs. top-down, co-limitation scenario; how do these would-be-hosts balance minimizing viral susceptibility with maximizing uptake of limiting nutrient(s)? This question has been addressed in the biological literature on evolutionary timescales for populations, but a shorter timescale, mechanistic perspective is lacking, and marine viral literature suggests the strong influence of additional factors, e.g. host size; while the literature on both nutrient uptake and host-virus interactions is expansive, their intersection, of ubiquitous relevance to marine environments, is understudied. I present a simple, mechanistic model from first principles to analyze the effect of this co-limitation scenario on individual growth, which suggests that in environments with high risk of viral invasion or spatial/temporal heterogeneity, an individual host’s growth rate may be optimized with respect to receptor coverage, producing top-down selective pressure on short timescales. The model has general applicability, is suggestive of hypotheses for empirical exploration, and can be extended to theoretical studies of more complex behaviors and systems.This work was supported by the Massachusetts Institute of Technology Charles Vest Presidential Fellowship