Alice Soragni

In vivo demonstration that alpha-synuclein oligomers are toxic.

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.

Mechanism of Membrane Interaction and Disruption by α-Synuclein

Parkinsons disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-β-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.

The Amyloid–Congo Red Interface at Atomic Resolution

Amyloids are b-sheet-rich proteinaceous aggregates that are a pathological hallmark of a number of human diseases. They can replicate by seeded nucleation and propagate as prions, and may even exert important physiological activities. Amyloids are universally defined by their stainability with Congo red and the resulting green birefringence. Yet, remarkably, the binding mechanism, geometry, and fine structure of the Congo red/amyloid complex are not known. By using solid-state NMR spectroscopy we have characterized, at atomic resolution, the binding interface between Congo red and amyloid fibrils formed by the prion domain of the fungal HET-s protein. The dye binds highly sitespecifically by interacting with residues flanking a groove in the vicinity of a b-arc. The three-dimensional (3D) structure of the fibril is strongly conserved upon the binding of Congo red. Remarkably, a single point mutation, designed according to the binding information, provides an artificial amyloid structurally indistinguishable from HET-s but not stainable by Congo red. The methods used require no isotope labeling of the small molecule and can be used to characterize the interaction of a broad range of dyes, drugs, and tracers with amyloids or other insoluble proteins. Congo red (Figure S1 in the Supporting Information) is a small molecule that reacts specifically with amyloids and has been used since the 1920s as the analytical “gold standard” for amyloid characterization and diagnostics. Despite its binding specificity and its antiamyloidogenic properties, the use of Congo red as a molecular amyloid tracer in vivo and as an antiamyloid drug has been hindered by its high toxicity and poor pharmacokinetics. Contradictory reports exist on the nature of the interaction between Congo red and amyloid fibrils, but the following facts are generally agreed upon: Congo red binds roughly stochiometrically, 9] and the bound form displays a characteristic red-shift in its absorbance maximum, and shows dichroism and birefringence. 10] The negatively charged sulfate groups of Congo red are believed to be relevant for binding to two sites separated by a distance of approximately 20 . In the absence of atomic-resolution structure information, the binding mode is still debated. The long axis of the dye has been postulated to orient parallel or perpendicular to the fibril axis, and the dye binds as a monomer, oligomer, or micelle, or intercalated between the b-sheets. Hydrogen bonds and hydrophobic, aromatic, and ionic interactions have been assumed to contribute to the binding of Congo red to amyloid fibrils. Also, different side chains from residues such as histidine, arginine, and lysine have been suspected to promote congophilia. Atomic-resolution structures of amyloids, which have become available recently, enable studies of the interplay between these proteins and their binding partners in detail. HET-s(218–289), the prion-forming domain of the HET-s prion from the filamentous fungus Podospora anserina, is presently one of the structurally most precisely defined amyloids. To identify the protein surface that interacts with binding partners by NMR spectroscopy, we set out to detect characteristic chemical-shift perturbations (CSP) upon binding by comparing two-dimensional (2D) C–C correlation spectra (proton-driven spin diffusion, PDSD) of stained and unstained [C,N]-labeled fibrils. Since CSPs can be allosteric, we also performed polarization-transfer (PT) experiments which directly probe the spatial proximity of ligand and protein by exploiting the pronounced distance dependence (r ) of the dipolar interaction. Previously published PT approches have proven difficult for our system as they require isotope-labeled Congo red. We have therefore devised a method relying on polarization transfer from ligand H atoms to protein C atoms, which uses standard [H,C,N]-isotope-labeled protein but does not require isotopically labeled ligand. In such samples, the protons from Congo red are the unique polarization source in H–C polarization-transfer experiments, and the C signals detected identify residues in spatial proximity to Congo red (< 4 ). For enhanced spectral resolution, the C atoms are detected in a two-dimensional correlation experiment (dipolar recoupling enhanced by amplitude modulation, DREAM). The main results of the NMR experiments are summarized in Figure 1; details are given in Figures S2–S4 in the [*] A. K. Sch tz, A. Soragni, Dr. M. Ernst, Prof. B. H. Meier Physikalische Chemie, ETH Z rich, 8093 Z rich (Switzerland) E-mail: beme@ethz.ch

Solution structure of selenoprotein W and NMR analysis of its interaction with 14-3-3 proteins.

Selenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13 → Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins.

Structural characterization of binding of Cu(II) to Tau protein.

Transition metals have been frequently recognized as risk factors in neurodegenerative disorders, and brain lesions associated with Alzheimers disease are rich in Fe(III), Zn(II), and Cu(II). By using different biophysical techniques (nuclear magnetic resonance, circular dichroism, light scattering, and microcalorimetry), we have structurally characterized the binding of Cu(II) to a 198 amino acid fragment of the protein Tau that can mimic both the aggregation behavior and microtubule binding properties of the full-length protein. We demonstrate that Tau can specifically bind one Cu(II) ion per monomer with a dissociation constant in the micromolar range, an affinity comparable to the binding of Cu(II) to other proteins involved in neurodegenerative diseases. NMR spectroscopy showed that two short stretches of residues, (287)VQSKCGS (293) and (310)YKPVDLSKVTSKCGS (324), are primarily involved in copper binding, in agreement with mutational analysis. According to circular dichroism and NMR spectroscopy, Tau remains largely disordered upon binding to Cu(II), although a limited amount of aggregation is induced.

Infectious and Noninfectious Amyloids of the HET-s(218–289) Prion Have Different NMR Spectra†

The molecular basis for prion infectivity is not yet understood. The NMR spectra of noninfectious and infectious amyloids of the prion-forming domain 218-289 of the fungal prion HET-s are clearly different (see picture) but are indicative for a cross- arrangement in both cases. The fibrils formed at pH 3 are not infectious because their molecular structure apparently differs substantially from that formed at physiological pH.

Structural similarity between the prion domain of HET-s and a homologue can explain amyloid cross-seeding in spite of limited sequence identity.

We describe a distant homologue of the fungal HET-s prion, which is found in the fungus Fusarium graminearum. The domain FgHET-s(218-289), which corresponds to the prion domain in HET-s from Podospora anserina, forms amyloid fibrils in vitro and is able to efficiently cross-seed HET-s(218-289) prion formation. We structurally characterize FgHET-s(218-289), which displays 38% sequence identity with HET-s(218-289). Solid-state NMR and hydrogen/deuterium exchange detected by NMR show that the fold and a number of structural details are very similar for the prion domains of the two proteins. This structural similarity readily explains why cross-seeding occurs here in spite of the sequence divergence.

A Combined Solid‐State NMR and MD Characterization of the Stability and Dynamics of the HET‐s(218‐289) Prion in its Amyloid Conformation

Dynamic and rigid: The prion HET‐s(218–289) consists, in its amyloid form as shown here, of highly ordered and rigid parts and a very dynamic loop, which could be of great importance for fibril formation. Indeed, MD simulations explain the experimental NMR results and describe the dynamics of the salt‐bridge network that stabilizes the amyloid fibril, a feature not easily accessible by experiment.

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinsons disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

Superresolution Imaging of Amyloid Fibrils with Binding-Activated Probes

Protein misfolding into amyloid-like aggregates underlies many neurodegenerative diseases. Thus, insights into the structure and function of these amyloids will provide valuable information on the pathological mechanisms involved and aid in the design of improved drugs for treating amyloid-based disorders. However, determining the structure of endogenous amyloids at high resolution has been difficult. Here we employ binding-activated localization microscopy (BALM) to acquire superresolution images of α-synuclein amyloid fibrils with unprecedented optical resolution. We propose that BALM imaging can be extended to study the structure of other amyloids, for differential diagnosis of amyloid-related diseases and for discovery of drugs that perturb amyloid structure for therapy.