Ine Segers-Nolten

Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio

The amyloid peptides Aβ40 and Aβ42 of Alzheimers disease are thought to contribute differentially to the disease process. Although Aβ42 seems more pathogenic than Aβ40, the reason for this is not well understood. We show here that small alterations in the Aβ42:Aβ40 ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ42:Aβ40 ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.

Inhibition of α-synuclein aggregation by small heat shock proteins

The fibrillization of α‐synuclein (α‐syn) is a key event in the pathogenesis of α‐synucleinopathies. Mutant α‐syn (A53T, A30P, or E46K), each linked to familial Parkinsons disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α‐syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α‐syn accumulates intracellularly, molecular chaperones like sHsps may regulate α‐syn folding and aggregation. Therefore, we investigated if the sHsps αB‐crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α‐syn and affect α‐syn aggregation. We demonstrate that all sHsps bind to the various α‐syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α‐syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild‐type and mutant α‐syn. In conclusion, sHsps may regulate α‐syn aggregation and, therefore, optimization of the interaction between sHsps and α‐syn may be an interesting target for therapeutic intervention in the pathogenesis of α‐synucleinopathies. Proteins 2011;

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.

Concentration Dependence of α-Synuclein Fibril Length Assessed by Quantitative Atomic Force Microscopy and Statistical-Mechanical Theory

The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human alpha-synuclein on the mean length of mature alpha-synuclein fibrils, which are associated with Parkinsons disease. We determine that the critical initial concentration, below which low-molecular-weight species dominate and above which fibrils are the dominant species, lies at approximately 15 muM, in good agreement with earlier measurements using biochemical methods. In the concentration regime where fibrils dominate, we find that their mean length increases with initial concentration. These results correspond well to the qualitative predictions of a recent statistical-mechanical model of amyloid fibril formation. In addition, good quantitative agreement of the statistical-mechanical model with the measured mean fibril length as a function of initial protein concentration, as well as with the fibril length distributions for several protein concentrations, is found for reasonable values of the relevant model parameters. The comparison between theory and experiment yields, for the first time to our knowledge, an estimate of the magnitude of the free energies associated with the intermolecular interactions that govern alpha-synuclein fibril formation.

Molecular composition of sub-stoichiometrically labeled α-synuclein oligomers determined by single-molecule photobleaching

Bleaching proteins: Single-molecule photobleaching approaches and sub-stoichiometric labeling with fluorophores give insight into the number of monomers that form a specific alpha-synuclein oligomer. The results show that this alpha-synuclein oligomer is present as a single, well-defined species consisting of 31 monomers.

Single-Molecule FRET Reveals Structural Heterogeneity of SDS-Bound α-Synuclein

SDS‐concentration‐dependent α‐synuclein structure: Upon interaction with SDS, αSyn folds into a structure with two antiparallel α‐helices. We show from single‐molecule FRET that αSynn adopts this conformation in an all‐or‐none fashion below the SDS critical micelle concentration. Population of the folded species is directly coupled to an increase in α‐helix content; this suggests that the entire N terminus is involved in the transaction.

Intracellular Reactions in Single Human Granulocytes upon Phorbol Myristate Acetate Activation using Confocal Raman Microspectroscopy

We have obtained new evidence for the occurrence of intracellular NADPH-oxidase activity in neutrophilic and eosinophilic granulocytes upon stimulation with phorbol myristate acetate (PMA). PMA activation leads to a partial translocation of cytochrome b(558) from the membranes of the specific granules to the plasma membrane. It was suggested that NADPH-oxidase activity only takes place in the plasma membrane, leading to an extracellular release of oxygen metabolites because cellular self-destruction can be avoided in this way. The effects of PMA activation were indirectly studied in recent experiments employing scavengers of extracellular superoxide anion and hydrogen peroxide, and support for intracellular NADPH-oxidase activity was obtained. In this paper we use Raman microspectroscopy as a direct method to study intracellular molecular reactions that result from cellular triggering by PMA. The molecular specificity of this microscopic method enables us to show that intracellular reduction of both myeloperoxidase (MPO) and cytochrome b(558) occurs in neutrophilic granulocytes. Control measurements with cytochrome b(558)-deficient neutrophilic granulocytes did not show a reduction of intracellular MPO. This is direct support for the occurrence of intracellular NADPH-oxidase activity in organelles that must be in close contact with the azurophilic granules that contain MPO. Furthermore, a comparison was made with chemical reactions occurring in eosinophilic granulocytes after activation with PMA. Moreover, in these cells an intracellular reduction of eosinophil peroxidase was observed.

Carotenoid levels in human lymphocytes, measured by Raman microspectroscopy

Carotenoid levels in lymphocytes obtained from peripheral blood of healthy people have been investigated by Raman microspectroscopy. We observed that carotenoids are concentrated in so-called “Gall bodies”. The level of carotenoids in living human lymphocytes was found to be age-dependent and to decrease with age. We performed carotenoid uptake experiments using liposomes containing beta-carotene (egg phosphatidylcholine and egg phosphatidylglycerol, 10/1 molar ratio). We observed that beta-carotene is rapidly taken up in vitro, and transported to the Gall body. From these findings we conclude that Raman microspectroscopy is a sensitive method to determine carotenoid levels in single living cells.

Solution conditions define morphological homogeneity of α-synuclein fibrils

The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10mM Tris-HCl, 10mM NaCl and 0.1mM EDTA at pH7.4 display a narrow distribution of periodicities with an average value of 102±6nm for the A140C mutant and 107±9nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.

Conformational Compatibility Is Essential for Heterologous Aggregation of α-Synuclein

Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all proteins are known to cross-seed aggregation. Cross-seeding has been proposed to be restricted either because of differences in the protein sequences or because of conformations between the seeds and the soluble monomers. Here, we examine cross-seeding efficiency between three α-synuclein sequences, wild-type, A30P, and A53T, each varying in only one or two amino acids but forming morphologically distinct fibrils. Results from bulk Thioflavin-T measurements, monomer incorporation quantification, single fibril fluorescence microscopy, and atomic force microscopy show that under the given solution conditions conformity between the conformation of seeds and monomers is essential for seed elongation. Moreover, elongation characteristics of the seeds are defined by the type of seed.