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Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativum L.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10−3 s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2Azz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2Azz measurements can provide an estimate of the optimum storage conditions.

Polyelectrolytes are polymers with potentially charged monomer units. Under the right conditions, oppositely charged polyelectrolytes in solution can interact and form polyelectrolyte complexes (PECs). These PECs have peculiar emergent properties. One of these properties is the ability to accumulate certain molecules in very high concentrations. A comparison can be made between PECs and membraneless-organelles (MLOs), which are droplet-like liquid-in-liquid phases present in our cells that aid the cell in organising its interior milieu through selective partitioning of specific molecules. Similar to PECs, MLOs are also composed op oppositely charged polyelectrolytes. In the case of MLOs, these are typically negatively charged RNA molecules and positively charged intrinsically disordered proteins. Taking inspiration from nature, in this dissertation we present the use of PECs for the partitioning of molecules. We investigated whether it was possible to use PECs to selectively extract a protein from a mixture. Followed by whether a PEC containing a protein could be used as a biocatalytic membrane. In chapters 2 – 4 we investigate various extraction procedures of small molecules and proteins using PECs. We find that various system parameters influence the degree of partitioning of these compounds. This culminates in chapter 4, where we were able to selectively extract lysozyme from lyophilised egg white by choosing a specific PEC composition as determined by the polycation:polyanion ratio. After extraction of lysozyme into the PEC phase and release back into a new aqueous phase, the lysozyme remained enzymatically active. In chapter 5 we produced a PEC membrane containing lysozyme via aqueous phase separation (APS). The resulting biocatalytic membrane gained the enzymatic activity of lysozyme without compromising on membrane properties. The membrane remained biocatalytically active for at least one week.

Of all the diseases that lead to dementia, Alzheimer's disease (AD) is the most common, affecting millions of elderly people worldwide. Although a lot of research has been carried out to understand the cellular and molecular events responsible for brain dysfunction and AD pathogenesis, no effective therapy has been identified yet. In the research described in this doctoral thesis we tried to obtain a more detailed understanding of four early hallmarks of AD: Amyloid-beta (Aβ) aggregation, Aβ accumulation, oxidative stress and neuroinflammation. The thesis starts with an overview on A and its role in Alzheimer’s disease (Chapter 1). Four main sections follow the introduction. The first section consists of a description of chemical compounds designed to inhibit A aggregation and toxicity (Chapter 2 and 3). The second section consists of Chapter 4, which investigates the ability of IDE to cleave different A aggregation species. Next, the third section reviews the role of oxidative stress in AD and the drugs, currently under clinical trials, aiming at relieving this condition in patients (Chapter 5). In the last section, the potential link between neuroinflammation and neurodegeneration in AD is explored. In this context, Chapter 6 investigates the microglial IL1-β secretion and loss of neuronal synchronous activity upon exposure to A and interferon gamma (IFN) while Chapter 7 describes the biophysical interaction between A and IFN. Last, the closing section (chapter 8) consists of concluding remarks and gives some perspectives on promising AD research avenues for the future. Summarizing, we have aimed at gaining better insights into the molecular complexity of AD. This thesis highlights the multifactorial and complex character of AD and suggests that a multi-target approach, most likely, will contribute to the development of an effective AD therapy, halting the progression of this devastating disease.

Alpha-synuclein (αS), an intrinsically disordered protein, is thought to be the major player in synucleinopathies such as Parkinson’s disease. The exact function of αS is not known. In vitro experiments show that αS is disordered in buffer and that in the presence of model lipid membranes it can adopt alpha-helical structure. In cells αS has been shown to colocalize with membranes. Taking into account the membrane bound structure found in vitro, one would expect that αS adopts alpha-helical structure on cellular lipid bilayers. In contrast, NMR and EPR studies indicate that αS remains disordered inside the cell. We address this controversy. We showed that cellular αS is associated with membranes inside neuron like cells. We used ultrasensitive microscopy and photobleaching to quantify the number of αS-GFP on vesicles. We found a number of 70 αS-GFP/vesicle which is an extraordinary high number. To identify if this membrane bound αS adopts α-helical structures, we microinjected cells with small amounts of αS labelled with a FRET pair sensitive to the membrane bound conformation (Fig. 1 top). We use the FRET signal as a readout for protein conformation and observed two significant different conformations of αS in cells, one in the cytoplasm and the other on cellular vesicles (Fig. 1 middle and bottom panels). This clearly shows that there are at least two structurally distinct subensembles of αS inside cells: 1) a disordered form in the cytosol, 2) a membrane associated form. Our data shows that the disordered nature of monomeric αS is not fully preserved in cells.

Alpha synuclein (αS) is a ~14 kDa intrinsically disordered protein with a yet unknown physiological function. The conversion of monomeric αS into amyloid aggregates is believed to play a central role of the pathology of Parkinson’s disease (PD). Despite extensive studies on amyloid formation of αS in bulk solution, the mechanistic details of αS aggregation at biological interfaces like lipid membranes are unclear. Further, it is also unknown how amyloid aggregates that are formed in PD potentiate neuronal cell death. Association with (specific) cellular membranes is believed facilitate amyloid formation, but this hypothesis is clouded by the fact that the physiological function of αS probably also involves association with physiological cell membranes. Therefore, understanding interactions of αS with lipid membranes is critical to uncover its possible functional or pathological role, which we have investigated in this thesis. In particular, we focus on the following questions which form the core of the thesis: * How are physical properties of phospholipid membranes affected by αS binding and aggregation and vice versa? * How do early amyloid aggregates of αS perturb phospholipid membranes? * What is the role of N-terminal acetylation in αS on its membrane binding properties and aggregation propensities? * How do terminal domains in αS affect the morphology of amyloid aggregates? To answer these questions a wide range of biochemical and biophysical techniques like fluorescence confocal microscopy, fluorescence anisotropy, circular dichroism, Fourier transform infra-red spectroscopy and fluorescence recovery after photobleaching were used to probe the interactions of lipid membranes with monomeric αS. Our results show the importance of inter-αS, inter-lipid and αS–lipid interactions that are involved both prior to and post amyloid formation.

This thesis sheds light on the function and dysfunction of the protein α-synuclein (α-S) in the test tube and in cells and ultimately its possible involvement in Parkinson’s disease (PD). Following the introduction in Chapter 1, Chapters 2 and 3 concentrate on the investigation of the interaction between the proteins α-S and actin. In Chapter 2 we determine their dissociation constants with microscale thermophoresis and reveal that α-S has a high affinity for G-actin. The determined interaction strength was even comparable to other G-actin binding proteins. Despite this high binding affinity and the postulated function of α-S as a G-actin regulating protein, the binding of α-S to G-actin has no effect on the actin polymerization kinetics. Instead, α-S seems to bind to and stabilize F-actin structures. Chapter 3 reports on how the formation of G-actin/αS complexes attenuates the lag time of α-S aggregation depending on the actin concentration. Whereas low actin concentrations inhibit the onset of α-S aggregation this effect disappears at higher concentrations. However, although the presence of actin can inhibit the onset of aggregation it cannot prevent it. Chapter 4 draws attention to the intra-cellular occurrence of α-S spots, smaller than the diffraction limit, consisting of many proteins. To study and visualize these diffraction-limited α-S spots, we made use of stimulated emission depletion (STED) microscopy. The colocalization of these α-S clusters with a membrane marker and the appearance of α-S on the surface of giant intracellular vesicles suggest that that the spots are small vesicles and α-S functions as membrane binding protein. Chapter 5 focuses on cell models in which α-S inclusion formation is induced in cells. We describe the resemblance of induced α-S inclusion to LBs. Moreover, we not only observe different morphologies, but also observe that α-S aggregates even occur in functionally different inclusions both in vitro and in vivo. Finally, in chapter 6, we investigate the effect of exogenous α-S on the viability and electrophysiology of neuronal networks. Whereas the viability was unaffected, we noted development of α-S aggregates in cells and a progressive decrease in neuronal connectivity and excitability.

This thesis gives insights into the biophysical mechanisms of α-synuclein (αS) oligomer-membrane interactions. Model membranes of different compositions are used to mimic physiological membranes. We have systematically investigated how αS oligomers bind and permeabilize model membranes and how these activities are modulated by membrane charge and lipid composition. We have addressed oligomer binding to complex model membranes. Our studies show that at least 25% of negatively charged DOPG is necessary for αS oligomer binding. The membranes that contain physiologically relevant cholesterol and sphingomyelin content are not damaged by oligomers. A focus is given on mitochondrial inner membrane and plasma membrane inner leaflet mimics because these two membranes are thought to be the primary sites of αS oligomer-induced damage. Mitochondrial model membranes are more prone to oligomer-induced damage at longer timescales while the more complex plasma membrane model systems do not show a concentration dependent permeabilization on the same time scale. We attribute this result to the complexity of the composition of the model plasma membranes. SAXS data showed that all αS oligomers studied (WT, and disease mutants A30P, E46K, A53T, H50Q and G51D) contain approximately 30 monomers. We have found that the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for WT, H50Q and G51D. However, G51D oligomers were not able to disrupt negatively-charged and physiologically-relevant model membranes. We have proven that the toxicity of the externally added oligomers tested here depend on the availability and toxicity of additional compounds rather than on the oligomers themselves. We wanted to gain more details about toxicity of αS oligomers and we suggested that oligomer-induced hemifusion could be connected to the vesicle docking and fusion problems observed in vivo. Multivalent oligomers may be directly involved in disturbing the well-controlled membrane fusion machinery of the cell. We make the conceptual step of considering αS oligomers as small multivalent biological nanoparticles that cause formation of holes in the membrane or membrane thinning. The observed uncontrolled and efficient hemifusion of vesicles sheds a new light on the possible toxicity of oligomers in Parkinson’s disease.

Parkinson's disease is a neurodegenerative disease common with old age. The neuronal protein α-synuclein is considered to play a pivotal role in the onset and progression of the disease. The protein is the main constituent of the Lewy body, the pathological hallmark of the disease, which are large intracellular inclusion bodies that contain aggregated fibrillar protein. In addition mutations in the gene coding for the protein as well as gene multiplications lead to genetic variants of Parkinson's disease. One of the main questions is how the aggregation of α-synuclein is related to the disease. Recent evidence suggests that oligomeric intermediates in the aggregation process of α-synuclein are potentially the neurotoxic species that underlie the disease. A potential mechanism by which these could be toxic is through interaction and disruption of cellular membranes. In this thesis, we try to elucidate how oligomeric α-synuclein interacts with lipid membranes. This interaction was characterized using a number of biophysical techniques using synthetic lipid bilayer systems and recombinantly expressed α-synuclein. Stable off-pathway α-synuclein oligomers were prepared that bound and permeabilized vesicles composed of negatively charged lipids. The lipid binding is mediated by the positively charged N-terminus of the protein. The oligomers contain structural features that cause them to have a destabilizing effect on lipid membranes. Bilayer permeabilization most likely occurs through the formation of defects in the membrane.