Membrane–Protein–Hydration Interaction of α-Synuclein with Anionic Vesicles Probed via Angle-Resolved Second-Harmonic Scattering

Amyloid formation of the protein α-synuclein promotes neurodegeneration in Parkinson's disease. The normal function of α-synuclein includes synaptic vesicle transport and fusion, and the protein binds strongly to negatively charged vesicles in vitro. Here, we demonstrate that nonresonant angle-resolved second-harmonic scattering detects α-synuclein binding to liposomes through changes in water orientational correlations and can thus be used as a high-accuracy and high-throughput label-free probe of protein-liposome interactions. The obtained results support a binding model in which the N-terminus of α-synuclein adopts an α-helical conformation that lies flat on the vesicle surface while the negatively charged C-terminus remains in solution.

Nigrostriatal overabundance of alpha-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity

Patrick Aebischer, Jean-Charles Bensadoun, Wojciech Janusz Bobela, Marco Cantoni, Özgür Genç, Graham Knott, Safa Mohanna, Ralf Schneggenburger

alpha-Synuclein (alpha-syn) is a presynaptic protein present at most nerve terminals, but its function remains largely unknown. The familial forms of Parkinson's disease associated with multiplications of the alpha-syn gene locus indicate that overabundance of this protein might have a detrimental effect on dopaminergic transmission. To investigate this hypothesis, we use adeno-associated viral (AAV) vectors to overexpress human alpha-syn in the rat substantia nigra. Moderate overexpression of either wild-type (WT) or A30P alpha-syn differs in the motor phenotypes induced, with only the WT form generating hemiparkinsonian impairments. Wild-type alpha-syn causes a reduction of dopamine release in the striatum that exceeds the loss of dopaminergic neurons, axonal fibers, and the reduction in total dopamine. At the ultrastructural level, the reduced dopamine release corresponds to a decreased density of dopaminergic vesicles and synaptic contacts in striatal terminals. Interestingly, the membrane-binding-deficient A30P mutant does neither notably reduce dopamine release nor it cause ultrastructural changes in dopaminergic axons, showing that alpha-syn's membrane-binding properties are critically involved in the presynaptic defects. To further determine if the affinity of the protein for membranes determines the extent of motor defects, we compare three forms of alpha-syn in conditions leading to pronounced degeneration. While membrane-binding alpha-syns (wild-type and A53T) induce severe motor impairments, an N-terminal deleted form with attenuated affinity for membranes is inefficient in inducing motor defects. Overall, these results demonstrate that alpha-syn overabundance is detrimental to dopamine neurotransmission at early stages of the degeneration of nigrostriatal dopaminergic axons.

Springer-Verlag2012

Soft matter probed by nonlinear scattering: self-assembly, interfaces, hydration, and long-range order

Jan Dedic

All organisms are made to a large extent of soft matter - macromolecules such as proteins and polysaccharides, or assemblies of small molecules such as lipids embedded in an aqueous environment. Understanding the role of order in soft matter presents a challenge for experimentalists because such systems are neither completely disordered nor perfectly crystalline. This is especially the case for the aqueous environment itself whose order fluctuates on femto- and pico-second time scales. In this thesis, we attempt to elucidate the presence, extent, changes, and implications of orientational order in several soft matter systems: from polyelectrolyte solutions to lipid membrane interfaces and supramolecular structures. To perform this task, we employ nonlinear optical techniques: second-harmonic and sum-frequency scattering. In the first two chapters, we explore the structure of water in aqueous polyelectrolyte solutions. Polyelectrolytes of high molecular mass are shown to induce orientational order in water that spans hundreds of nanometers. Using an adapted model of second-harmonic scattering from charged particles, we can explain the observed ordering in terms of weakly screened long-range electrostatic interactions that perturb the structure of water via charge-dipole interactions. Upon exchanging ordinary (light) water for heavy water, we observe a significant nuclear quantum effect in the induced order, indicating that bulk water-water correlations are enhanced as well by polyelectrolytes. In the following chapter, we exploit the same nuclear quantum effect in polyelectrolyte solutions to show that the polyelectrolyte-induced orientational order correlates with an anomalous increase in the reduced viscosity. We propose that the viscosity anomaly arises from enhanced order in a solvent which hinders the viscous flow. This observation represents a rare direct link between a microscopic property and a macroscopic observable. In the last two chapters, we change focus from homogeneous solutions to dispersions of liposomes which serve as models of biological membranes. In Chapter 5 we explore the binding of the protein alpha-synuclein to anionic vesicles from the perspective of interfacial water. We show that the adsorption of the protein to a vesicle decreases the order in the interfacial water by 30% but does not affect the surface nor the zeta potential. We propose that the changes in the structure of interfacial water can serve as a label-free probe of protein binding. In the last chapter, we investigate the supramolecular chemistry of inclusion complexes of methyl-Î²-cyclodextrin with various lipids. By combining multiple nonlinear scattering techniques, we show that the complexes self-assemble into micrometer-long directed fibers. Based on measurements of different lipids, we propose that the self-assembly process is driven by hydrogen bonding between the lipid headgroup and cyclodextrin. Lastly, we show that the long-range order of the self-assembled structure is transmitted into the structure of the hydrating water.

EPFL2020

Nigrostriatal overabundance of alpha-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity

Patrick Aebischer, Jean-Charles Bensadoun, Wojciech Janusz Bobela, Marco Cantoni, Özgür Genç, Graham Knott, Safa Mohanna, Ralf Schneggenburger

Springer-Verlag2012

Soft matter probed by nonlinear scattering: self-assembly, interfaces, hydration, and long-range order

Jan Dedic

EPFL2020