Bart van Rooijen

Lipid bilayer disruption by oligomeric α-synuclein depends on bilayer charge and accessibility of the hydrophobic core

Soluble oligomeric aggregates of alpha-synuclein have been implicated to play a central role in the pathogenesis of Parkinsons disease. Disruption and permeabilization of lipid bilayers by alpha-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer-membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged alpha-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer-membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer-membrane interactions.

Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism

Background The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinsons disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory. Methodology/Principal Findings Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general. Conclusions/Significance The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.

Antiparallel Arrangement of the Helices of Vesicle-Bound α-Synuclein

alpha-Synuclein (alphaS) is the main component of Lewy bodies from Parkinsons disease. That alphaS binds to membranes is known, but the conformation it adopts is still unclear. Pulsed EPR on doubly spin-labeled variants of alphaS sheds light on the most likely structure. For alphaS bound to vesicles large enough to accommodate also the extended conformation, an antiparallel helix conformation is found. This suggests that the bent structure shown is the preferred conformation of alphaS on membranes.

Extracting atomic numbers and electron densities from a dual source dual energy CT scanner: experiments and a simulation model.

BACKGROUND AND PURPOSE Dual energy CT (DECT) imaging can provide both the electron density ρ(e) and effective atomic number Z(eff), thus facilitating tissue type identification. This paper investigates the accuracy of a dual source DECT scanner by means of measurements and simulations. Previous simulation work suggested improved Monte Carlo dose calculation accuracy when compared to single energy CT for low energy photon brachytherapy, but lacked validation. As such, we aim to validate our DECT simulation model in this work. MATERIALS AND METHODS A cylindrical phantom containing tissue mimicking inserts was scanned with a second generation dual source scanner (SOMATOM Definition FLASH) to obtain Z(eff) and ρ(e). A model of the scanner was designed in ImaSim, a CT simulation program, and was used to simulate the experiment. RESULTS Accuracy of measured Z(eff) (labelled Z) was found to vary from -10% to 10% from low to high Z tissue substitutes while the accuracy on ρ(e) from DECT was about 2.5%. Our simulation reproduced the experiments within ±5% for both Z and ρ(e). CONCLUSIONS A clinical DECT scanner was able to extract Z and ρ(e) of tissue substitutes. Our simulation tool replicates the experiments within a reasonable accuracy.

Tissue transglutaminase modulates α-synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross‐link formation between protein‐bound glutamine residues and primary amines, with Parkinsons disease‐associated α‐synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild‐type and mutant α‐synucleins using surface plasmon resonance approaches, revealing high‐affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective α‐synuclein cross‐linking, resulting predominantly in intramolecularly cross‐linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/α‐synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all α‐synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal α‐synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β‐sheet‐containing oligomers, the tTG/α‐synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild‐type and disease mutant α‐synuclein variants. We propose that tTG cross‐linking imposes structural constraints on α‐synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross‐linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

Membrane binding of oligomeric α-synuclein depends on bilayer charge and packing

Membrane disruption by oligomeric α‐synuclein (αS) is considered a likely mechanism of cytotoxicity in Parkinsons disease (PD). However, the mechanism of oligomer binding and the relation between binding and membrane disruption is not known. We have visualized αS oligomer‐lipid binding by fluorescence microscopy and have measured membrane disruption using a dye release assay. The data reveal that oligomeric αS selectively binds to membranes containing anionic lipids and preferentially accumulates into liquid disordered (Ld) domains. Furthermore, we show that binding of oligomers to the membrane and disruption of the membrane require different lipid properties. Thus membrane‐bound oligomeric αS does not always cause bilayer disruption.

Spin-Label EPR on α-Synuclein Reveals Differences in the Membrane Binding Affinity of the Two Antiparallel Helices

The putative function of the Parkinsons disease‐related protein α‐Synuclein (αS) is thought to involve membrane binding. Therefore, the interaction of αS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of αS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but αS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative‐charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of αS. Thus, the interaction of αS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of αS to membranes. These findings contribute to our understanding of the details and structural consequences of αS–membrane interactions.

Direct Evidence of Coexisting Horseshoe and Extended Helix Conformations of Membrane-Bound Alpha-Synuclein

IDPs lack a well-defined three-dimensional fold anddisplay remarkable conformational flexibility. This property po-tentially enables them to be promiscuous in their interactionsand to adapt their structure according to the needed function.In the case of aS, the protein is capable of adopting a b-sheetstructure in the amyloid fibrils constituting the Lewy bodiesand an a-helical structure in the membrane bound form. Theexact physiological role of aS has yet to be determined, butmembrane binding seems to be important for its function.

A Stable Lipid-Induced Aggregate of alpha-Synuclein

The Parkinsons disease-related protein alpha-Synuclein (alphaS) is a 140 residue intrinsically disordered protein. Its membrane-binding properties are thought to be relevant for its physiological or pathologic activity. Here, the interaction of alphaS with POPG [1-Palmitoyl-2-Oleoyl-sn-Glycero-3-(Phosphorac-(1-glycerol))] small unilamellar vesicles (SUVs) is investigated by spin-label EPR using double electron-electron resonance (DEER). Intermolecular distances between four single mutants reveal that well-defined aggregates are formed. The data suggest a coexistence of two dimer structures with main interactions in the helix 2, encompassing residues 50-100. Previously, the horseshoe conformation was detected by intramolecular restraints obtained by DEER on alphaS double mutants (Drescher et al. J. Am. Chem. Soc. 2008, 130, 7796). The present study suggests that interdigitation of two monomers in the aggregate fills the void between the two helices of each of the monomers thus providing a rationale for the horseshoe structure. This aggregate is lipid induced and affects the structure of the POPG SUVs, which become leaky and diminish in size upon contact with alphaS suggesting a possible origin of conflicting results in the recent literature (Jao et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (50), 19666; Georgieva et al. J. Am. Chem. Soc. 2008, 130 (39), 12856; Bortolus et al. J. Am. Chem. Soc. 2008, 130, 6690).

Efficacy of Radiation Safety Glasses in Interventional Radiology

PurposeThis study was designed to evaluate the reduction of the eye lens dose when wearing protective eyewear in interventional radiology and to identify conditions that optimize the efficacy of radiation safety glasses.MethodsThe dose reduction provided by different models of radiation safety glasses was measured on an anthropomorphic phantom head. The influence of the orientation of the phantom head on the dose reduction was studied in detail. The dose reduction in interventional radiological practice was assessed by dose measurements on radiologists wearing either leaded or no glasses or using a ceiling suspended screen.ResultsThe different models of radiation safety glasses provided a dose reduction in the range of a factor of 7.9–10.0 for frontal exposure of the phantom. The dose reduction was strongly reduced when the head is turned to the side relative to the irradiated volume. The eye closest to the tube was better protected due to side shielding and eyewear curvature. In clinical practice, the mean dose reduction was a factor of 2.1. Using a ceiling suspended lead glass shield resulted in a mean dose reduction of a factor of 5.7.ConclusionsThe efficacy of radiation protection glasses depends on the orientation of the operator’s head relative to the irradiated volume. Glasses can offer good protection to the eye under clinically relevant conditions. However, the performance in clinical practice in our study was lower than expected. This is likely related to nonoptimized room geometry and training of the staff as well as measurement methodology.