8th European Biophysics congress, August 23 - 27, 2011
NBP will represented by several members: Vinod Subramaniam, Annelies Vandersteen, Anja Stefanovic, Niels Zijlstra & Ellen Hubin
Biophysical consideration of gamma-secretase modulation as potential target for Alzheimer’s disease
The amyloid beta peptide (Ab), implicated in Alzheimer’s disease (AD), is released from the Amyloid Precursor Protein (APP) by secretase-induced cleavage. This process results in the release of a range of Ab peptides varying in length. The brains of AD patients often contain longer Ab peptides while the total concentration of Ab is unaffected. Longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. As Ab is necessary for normal neuronal function, research activities into AD therapeutic development currently explore the possibilities of modulating g-secretase activity to produce short Ab peptides. Whether such an approach effectively ameliorates the toxic effect of Ab has not been explored yet. To answer this question, we studied the impact of heterogeneity in Ab pools in an in vitro biophysical and in cellulo context using MicroElectrode Array to assay the synaptic activity of primary neurons. We show that various lengths of the Ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. We also show that small amounts of less abundant peptides Ab38 and Ab43 induce aggregation and toxicity of Ab40 while the behavior of Ab42 is unaffected.
Mechanistic insights into oligomeric alpha-synuclein/membrane interactions
Anja N. D. Stefanovic, Martin T. Stöckl, Mireille M. A. E. Claessens, Vinod Subramaniam
The amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (aS) with membranes play an important role in Parkinson’s disease. Oligomers are thought to permeabilize membranes and interfere with Ca2+ pathways. Permeabilization by aS requires the presence of negatively charged phospholipids. Whether aS can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored.
Here we report on the binding of aS to giant unilamellar vesicles (GUVs) with physiologically relevant lipid compositions. Comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and GUVs (HPTS efflux assay) show that aS is not able to permeabilize these membranes. The presence of cholesterol has a stabilizing effect on these membrane systems. In agreement with these findings, we do not observe concentration dependent aS toxicity using in vivo MTS assays. However, in the calcein release assay, different aS preparations show differences in kinetics and aS concentrations that cause 100% leakage. These results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific aS species.
Establishing the composition of alpha-synuclein oligomers using single-molecule photobleaching
Niels Zijlstra, Christian Blum, Ine Segers-Nolten, Mireille Claessens, Vinod Subramaniam
Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands
The neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of Parkinson’s disease. Fibrillar aggregates of alpha-synuclein are the main constituents of the Lewy bodies that are found in the brains of Parkinson patients. However, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. Very little is known about the structure and composition of these oligomeric aggregates.
We present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. Our results show that the oligomers have a narrow size distribution, consisting of ~13-20 monomers per oligomer. Fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. In combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer.
Alpha-synuclein oligomers impair membrane integrity - A mechanistic view
One of the most prevalent neurodegenerative diseases is Parkinson’s disease (PD), which is accompanied with the loss of dopaminergic neurons. Although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (aS) is one of the pivotal factors. Previous studies indicate that especially oligomeric forms of aS show a detrimental effect on membrane integrity. As an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death.
We use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. Atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the aS oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. Using an approach based on phospholipid vesicles we were able to observe for the first time that aS oligomers cause an enhanced lipid-flip flop, suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in PD.
Effect of ApoE isoform and lipidation status on proteolytic clearance of the Amyloid-beta peptide
1. Department of Molecular and Cellular Interactions, Flanders Institute for biotechnology (VIB), Brussels, Belgium
2 Jean Jeener NMR Centre, Structural Biology Brussels, Brussels, Belgium
3. Vrije Universiteit Brussel (VUB), Brussels, Belgium
4. MIRA Institute for Biomedical Technology and Technical Medicine, Nanobiophysics Group, University of Twente, Enschede, The Netherlands
Alzheimer’s disease (AD) is the most common type of dementia in the elderly. The most important genetic risk factor identified for AD is the isoform, e2, e3 or e4, of apolipoprotein E (ApoE), a lipid-carrying protein. One hallmark of AD is the accumulation of amyloid-beta peptide (Aβ) in the brain which is thought to result from an imbalance between the production of Ab and its clearance. Previous studies report an important role for ApoE in Ab degradation. We sought to determine the effect of ApoE isoform and lipidation status on the degradation of soluble Aβ by proteinases such as insulin-degrading enzyme and neprilysin. In this study an in vitro Aβ clearance assay based on the competition between Aβ and a fluorogenic peptide substrate is developed to quantify Aβ degradation. To elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the Aβ sequence with the different ApoE isoforms. The results suggest that ApoE influences the rate of Aβ degradation.