Graphene oxide sheets and quantum dots inhibit alpha-synuclein amyloid formation by different mechanisms

Aggregation and amyloid formation of the 140-residue presynaptic and intrinsically disordered protein alpha-synuclein (alpha-syn) is a pathological hallmark of Parkinson\u27s disease (PD). Understanding how alpha-syn forms amyloid fibrils, and investigations of agents that can prevent their formation is therefore important. We demonstrate herein that two types of graphene oxide nanoparticles (sheets and quantum dots) inhibit alpha-syn amyloid formation by different mechanisms mediatedviadifferential interactions with both monomers and fibrils. We have used thioflavin-T fluorescence assays and kinetic analysis, circular dichroism, dynamic light scattering, fluorescence spectroscopy and atomic force microscopy to asses the kinetic nature and efficiency of this inhibitory effect. We show that the two types of graphene oxide nanoparticles alter the morphology of alpha-syn fibrils, disrupting their interfilament assembly and the resulting aggregates therefore consist of single protofilaments. Our results further show that graphene oxide sheets reduce the aggregation rate of alpha-syn primarily by sequestering of monomers, thereby preventing primary nucleation and elongation. Graphene quantum dots, on the other hand, interact less avidly with both monomers and fibrils. Their aggregation inhibitory effect is primarily related to adsorption of aggregated species and reduction of secondary processes, and they can thus not fully prevent aggregation. This fine-tuned and differential effect of graphene nanoparticles on amyloid formation shows that rational design of these nanomaterials has great potential in engineering materials that interact with specific molecular events in the amyloid fibril formation process. The findings also provide new insight into the molecular interplay between amyloidogenic proteins and graphene-based nanomaterials in general, and opens up their potential use as agents to manipulate fibril formation

Redox-Dependent Copper Ion Modulation of Amyloid-β (1-42) Aggregation In Vitro

Plaque deposits composed of amyloid-β (Aβ) fibrils are pathological hallmarks of Alzheimer’s disease (AD). Although copper ion dyshomeostasis is apparent in AD brains and copper ions are found co-deposited with Aβ peptides in patients’ plaques, the molecular effects of copper ion interactions and redox-state dependence on Aβ aggregation remain elusive. By combining biophysical and theoretical approaches, we here show that Cu2+\ua0(oxidized) and Cu+\ua0(reduced) ions have opposite effects on the assembly kinetics of recombinant Aβ(1-42) into amyloid fibrils in vitro. Cu2+\ua0inhibits both the unseeded and seeded aggregation of Aβ(1-42) at pH 8.0. Using mathematical models to fit the kinetic data, we find that Cu2+\ua0prevents fibril elongation. The Cu2+-mediated inhibition of Aβ aggregation shows the largest effect around pH 6.0 but is lost at pH 5.0, which corresponds to the pH in lysosomes. In contrast to Cu2+, Cu+\ua0ion binding mildly catalyzes the Aβ(1-42) aggregation via a mechanism that accelerates primary nucleation, possibly via the formation of Cu+-bridged Aβ(1-42) dimers. Taken together, our study emphasizes redox-dependent copper ion effects on Aβ(1-42) aggregation and thereby provides further knowledge of putative copper-dependent mechanisms resulting in AD

Amyloid formation of fish β-parvalbumin involves primary nucleation triggered by disulfide-bridged protein dimers

Amyloid formation involves the conversion of soluble protein species to an aggregated state. Amyloid fibrils of β-parvalbumin, a protein abundant in fish, act as an allergen but also inhibit the in vitro assembly of the Parkinson protein α-synuclein. However, the intrinsic aggregation mechanism of β-parvalbumin has not yet been elucidated. We performed biophysical experiments in combination with mathematical modeling of aggregation kinetics and discovered that the aggregation of β-parvalbumin is initiated by the formation of dimers stabilized by disulfide bonds and then proceeds via primary nucleation and fibril elongation processes. Dimer formation is accelerated by H2O2 and hindered by reducing agents, resulting in faster and slower aggregation rates, respectively. Purified β-parvalbumin dimers readily assemble into amyloid fibrils with similar morphology as those formed when starting from monomer solutions. Furthermore, addition of preformed dimers accelerates the aggregation reaction of monomers. Aggregation of purified β-parvalbumin dimers follows the same kinetic mechanism as that of monomers, implying that the rate-limiting primary nucleus is larger than a dimer and/or involves structural conversion. Our findings demonstrate a folded protein system in which spontaneously formed intermolecular disulfide bonds initiate amyloid fibril formation by recruitment of monomers. This dimer-induced aggregation mechanism may be of relevance for human amyloid diseases in which oxidative stress is often an associated hallmark

Amyloid formation of bovine insulin is retarded in moderately acidic pH and by addition of short-chain alcohols

Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2-7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin\u27s native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30-35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in alpha-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular

Role of regulatory T cells in acute myeloid leukemia patients undergoing relapse-preventive immunotherapy

Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov ) 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency

Impact of NK cell repertoires on immunotherapy in acute myelod leukemia

Natural killer (NK) cells are lymphocytes endowed with cytotoxicity against aberrant cells, including transformed and virus-infected cells. NK cell function is dictated by a fine-tuned interplay between activating and inhibitory receptors expressed on the NK cell surface. While the different activating receptors interact with unique ligands present on healthy or transformed cells, inhibitory NKG2A and killer immunoglobulin-like receptors (KIRs) invariably recognize HLA class I molecules. The purpose of this thesis was to elucidate how interactions between inhibitory NK cell receptors and HLA class I impact on anti-leukemic functions of NK cells and on NK cell-mediated termination of inflammation. In a phase IV trial, 81 AML patients received histamine dihydrochloride and low-dose interleukin-2 (HDC/IL-2) for the prevention of recurrence of leukemia after the completion of chemotherapy. The trial comprised immunophenotyping of serial blood samples along with KIR/HLA genotyping and assessment of cytomegalovirus (CMV) serostatus. Results from papers I and II imply a beneficial role of NK cell subsets that are less inhibited by HLA while prior CMV infection, which promotes the expression of additional KIRs, impacted negatively on relapse risk and survival. Additionally, a single nucleotide polymorphism in HLA-B that dictates NK cell inhibition to be preferentially mediated by NKG2A impacted positively on outcome in this trial (paper III). The relevance of the interplay between activating and HLA-mediated inhibitory signaling was further illustrated in a non-malignant setting in paper IV, where modulation of NK cell receptor ligands expressed by inflammatory neutrophils was associated with enhanced susceptibility to NK cell cytotoxicity. In conclusion, these studies support i) that low-grade KIR-mediated inhibition of NK cells is relevant for the benefit of relapse-preventive immunotherapy in AML and ii) that NK cells participate in the resolution of inflammation

Development of a Microfluidic Platform for Cell Migration Studies along Gradients

Cell migration is a cellular fate process essential for embryogenesis, tissue regeneration and wound healing. In vivo cell migration is promoted by soluble chemoattrac-tants which stimulate cell movement, a process called chemotaxis. Migration is also controlled by cell surface interactions where focal adhesions are formed between cell integrins and attachment peptides, such as RGD (Arginine-‐Glycine-‐Aspartic acid), in the extra cellular matrix. It has been shown that by varying the spacing of these attachment peptides on a surface, cell attachment, spreading and focal adhesion formation can be controlled. In addition, varying the spacing of attachment peptides is known to influence cell fate processes, such as apoptosis, proliferation and differentiation. The hypothesis that we wish to investigate is that cell migration is affected by the spacing of attachment peptides. The aim of this project was to design and evaluate an experimental system that could monitor cell migration as a function of attachment peptide spacing by simultaneously exposing cells to a chemotactic gradient in the culture media. Therefore, Au nano‐patterned surfaces, with Au nano-‐dots placed in hexagonal pattern with an inter-particle spacing increasing gradient-‐wise from 65 to 85 nm over 6 mm (Spatz group, MPI Intelligent Systems, Stuttgart) were used to control the spacing of a cyclic RGD attachment peptide, which preferentially binds to the Au nano-‐dots. These surfaces were combined with a microfluidic network capable of generating stable concentration gradients of chemoattractants. The microfluidic chip was designed and evaluated with help of a finite element method (Comsol, Stockholm, Sweden) where simulations were performed and concentration gradient formation, velocity, and flow and shear stress profiles were investigated. Several designs were tested and parameters such as placing of inlets and outlets, channel dimensions and connections, were evaluated. Based on the simulation data, the final microfluidic chip was designed, produced and experimen-tally characterized. The design is a diffusion based gradient generator capable of generating a concentration gradient inside a flow-‐free cell culture chamber, ensuring no shear stress induced migration in the main cell culture channel. The microfluidic chip was further combined with the cRGD functionalized surfaces and it was shown that Human Umbilical Vein Endothelial Cells (HUVECs) attach and spread and that they can be cultured in the system for at least five days during static conditions (longest time evaluated). The developed system is able to generate relevant concentration gradients of a chemoattractant factor, the microfluidic channel can be combined with an Au nano-‐dot patterned substrate, and cells can be cultured and monitored in situ, both in live and fixed/stained states, using bright field, phase contrast and fluorescent microscopy. In conclusion, a microfluidic platform was developed in which it is possible to study the effect of attachment peptide spacing during directed cell migration

Simulation of Chemotractant Gradients in Microfluidic Channels to Study Cell Migration Mechanism in silico

Cell migration of endothelial cells along gradients is an important process in vivo and an interesting target for cancer therapeutics. Microfluidics offer very powerful tools to study such migration processes in detail in the lab. In order to optimize microfluidic systems multiphysics simulations are very well suited. In this study, we describe a model to simulate molecular gradients in a diffusion based microfluidic gradient generator and how a cell senses these gradients via cell receptors. The results show the importance of incorporating the binding reaction into the model and that there is a large difference between the molecular gradient in solution and the gradient sensed by the cell. The approach presented here allows capturing of the whole process from gradient formation with a microfluidic channel to binding of molecules to cell receptors and allow a better prediction of parameters for cell experiments

Simulation of Chemotractant Gradients in Microfluidic Channels to Study Cell Migration Mechanism in silico

Cell migration of endothelial cells along gradients is an important process in vivo and an interesting target for cancer therapeutics. Microfluidics offer very powerful tools to study such migration processes in detail in the lab. In order to optimize microfluidic systems multiphysics simulations are very well suited. In this study, we describe a model to simulate molecular gradients in a diffusion based microfluidic gradient generator and how a cell senses these gradients via cell receptors. The results show the importance of incorporating the binding reaction into the model and that there is a large difference between the molecular gradient in solution and the gradient sensed by the cell. The approach presented here allows capturing of the whole process from gradient formation with a microfluidic channel to binding of molecules to cell receptors and allow a better prediction of parameters for cell experiments