Mun'delanji C. Vestergaard

Detection of Alzheimer's tau protein using localised surface plasmon resonance-based immunochip.

In this study, we present the detection of tau protein, at room temperature, using a multi-spot localised surface plasmon resonance (LSPR)-based immunochip. To the best of our knowledge, this is the first report of an immunochip for tau protein. The detection method includes fabrication of a gold-capped nanoparticle LSPR chip, formation and functionalisation of a self-assembled monolayer (SAM), immobilisation of a suitable linker, effective blocking of non-specific adsorption, immobilisation of a monoclonal anti-tau antibody (tau-mAb), and finally, the optimum conditions for the immuno-reaction between tau-mAb and the antigen were determined. The method has a high performance, enables detection of tau at 10pg/mL, lower than the cut-off value of 195pg/mL (for AD) for tau protein in cerebral spinal fluid (CSF). Further, we demonstrated selectivity of the technique by showing that the introduction of bovine serum albumin (BSA), perhaps the most abundant protein component in serum and CSF, does not interfere with the detection of tau. This method also offers a potential platform for studying tau interactions with other proteins and/or potential drug candidates and could also be easily adapted for detecting phosphorylated tau and other AD biomarkers.

Membrane fusion and vesicular transformation induced by Alzheimer's amyloid beta.

Amyloid beta (Aβ) peptides, produced through endo-proteolytic cleavage of amyloid precursor protein, are thought to be involved in the death of neural cells in Alzheimers disease (AD). Although the mechanisms are not fully known, it has been suggested that disruption of cellular activity due to Aβ interactions with the cell membrane may be one of the underlying causes. Here in, we have investigated the interaction between Aβ-42 and biomimetic lipid membranes and the resulting perturbations in the lipid vesicles. We have shown that Aβ oligomeric species localized closer to the membrane surface. Localization of the fibrillar species of Aβ-42, although varied, was not as closely associated with the membrane surface. We have demonstrated that the presence of Aβ-42 leads to an increase in membrane surface area, inducing lipid temporal vesicular transformation. Furthermore, we have unequivocally shown that Aβ-peptides mediate membrane fusion. Although membrane fusion induced by Aβ has been hypothesized/proposed, this is the first time it has been visually captured. This fusion may be one of the mechanisms behind the membrane increase in surface area and the resulting vesicular transformation. We have shown that the longer amyloidogenic isoform causes vesicular transformation more readily, and has a higher membrane fusogenic potential than Aβ-40. Although not core to this study, it is hugely interesting to observe the high agreement between membrane dynamics and the reported amyloidogenicity of the peptides and aggregation species opening up the potential role of vesicular dynamics for profiling and biosensing of Aβ-induced neuro-toxicity.

Physicochemical Profiling of Surfactant-Induced Membrane Dynamics in a Cell-Sized Liposome.

We used a cell-sized model system, giant liposomes, to investigate the interaction between lipid membranes and surfactants, and the membrane transformation during the solubilization process was captured in real time. We found that there are four distinct dynamics in surfactant-induced membrane deformation: an episodic increase in the membrane area prior to pore-forming associated shrinkage (Dynamics A), fission into many small liposomes (Dynamics B), the formation of multilamellar vesicles and peeling (Dynamics C), and bursting (Dynamics D). Classification of the diversity of membrane dynamics may contribute to a better understanding of the physicochemical mechanism of membrane solubilization induced by various surfactants.

Selective localization of Alzheimer's amyloid beta in membrane lateral compartments

Model membrane systems revealed that lateral heterogeneity of the membrane mediates the localization of amyloid beta peptides in a peptide aggregation-dependent manner.

Structure-dependent interactions of polyphenols with a biomimetic membrane system.

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.

Electrochemical DNA Biosensors: Protocols for Intercalator-Based Detection of Hybridization in Solution and at the Surface

An electrochemical DNA biosensor is a device that utilizes the inherent ability of two complementary strands of nucleic acids to form a double helix. The specificity of this reaction, namely hybridization, is used in the detection of target DNA sequences with a view toward developing point-of-care devices. Since the early 1990s, great progress has been made in this field, but there are still numerous challenges to overcome. This chapter describes the components of an electrochemical DNA biosensor for researchers new to the field, paying particular attention to intercalator-based DNA biosensors. We will use a well-defined electro-active DNA intercalator Hoechst 33258, as our running example. Two of the most classic DNA sensing methods: solution-based and surface-immobilized methods are discussed, along with guiding notes that would help identify and overcome possible problems in a typical electrochemical DNA biosensor experiment.

Alzheimer's Amyloid beta: Lipid membrane interactions, detected in real-time

There are strong implications that Amyloid beta (Aβ) peptide causes neurotoxicity in Alzheimers disease (AD) through (i) pore formation, (ii) the disruption of ionic channels that could affect calcium homeostasis, and (iii) receptor binding. The actual mechanism(s) remains unclear. In this study, we utilised cell-sized model membranes to observe the stability of lipid vesicles, in real-time, in the presence of Aβ-peptides. Using fluorescence-labelled Aβ-peptides, we imaged the localisation of pre-fibrillar Aβ species on the membrane surface, whereas mature fibrils hardly co-localised with the membrane surface. Further, we investigated the interaction of different oligomeric species of the Aβ peptide with lipid vesicles, observing membrane stability in terms of fluctuations and morphological changes. We observed a few different membrane morphological changes, with oligomeric species inducing a higher level of membrane instability. Interestingly, gramicidin A, a pore-forming peptide, did not induce any membrane transformations. We propose that this membrane transformation may be a different toxicity mechanism, from the proposed pore-formation hypothesis. This ‘toxicity’ mechanism may aid in real-time observation of these morphological understanding the mechanisms behind Aβ-induced neuro-toxicity in Alzheimers.

Effect of phospholipids on conformational structure of bovine pancreatic trypsin inhibitor (BPTI) and its thermolabile mutants

The effects of negatively charged phosphatidylserine-prepared membranes (PS) and neutral phosphatidylcholine-prepared membranes (PC) on the structure of wild-type and mutant bovine pancreatic trypsin inhibitor (BPTI) at neutral pH were investigated. The presence of PC did not have any effect on the protein structure while PS induced a non-native structure in three mutant BPTI proteins. However, the negatively charged membrane did not have any effect on wild-type BPTI. The findings revealed that (i) elimination of some disulphide bonds results in dramatic change in protein structure, and, (ii) that this biochemical interaction is surface-driven and electrostatic interactions may play a very strong role in influencing the fore-stated changes in protein structure. Of further interest were the results obtained from investigating the possible role of PS fluidity and concentration in altering mutant. When the value of Gibbs free-energy change of unfolding (DeltaG(U)) was positive, various non-native structures were formed in a concentration-dependent manner. However, when the value of DeltaG(U) was negative, only two types of non-native structures were formed: one with high beta structure content at low PS fluidity state, and the other with a high alpha-helical content at high PS fluidity state. Our study reveals how particular combinations of phospholipid:protein interactions can induce a protein conformation transition from a native to a non-native one at neutral pH, especially when the native structure is predestabilized by amino acid substitutions. This revelation may open up opportunities to explore alternative ways in which phospholipids may play a role in protein mis-folding and the related pathologies.

Dynamic transformation of a cell-sized liposome containing ganglioside

It is important to understand the physicochemical mechanisms that are responsible for the morphological changes in the cell membrane in the presence of various stimuli such as osmotic pressure. Role of micro domains such as lipid rafts in cellular processes is now beginning to unfold. In this study, we examined how constituent molecules affect the dynamical movement of liposomes. We used cell sized lipid vesicles to enable direct observation of these changes. We observed the effect of ganglioside (GM1) to osmotic stress induced membrane transformation in homogeneous and heterogeneous liposomes. Interestingly, it was observed that for the formation of sphero-stomatocyte there exists particular critical cut-off concentration. Also in the case of heterogeneous liposomes it was observed that at 10% molar ratio of GM1 almost all domains pinched out from the vesicles, forming their own homogeneous liposomes. Incorporation of GM1 into membrane leads to an increase of the line tension. Thus, necessary proteins can find themselves in one common raft and start the corresponding cascade of reactions.