Structure-Toxicity Relationship in Intermediate Fibrils from α-Synuclein Condensates.

The aberrant aggregation of α-synuclein (αS) into amyloid fibrils is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson's disease. Although the structural properties of mature amyloids of αS are currently understood, the nature of transient protofilaments and fibrils that appear during αS aggregation remains elusive. Using solid-state nuclear magnetic resonance (ssNMR), cryogenic electron microscopy (cryo-EM), and biophysical methods, we here characterized intermediate amyloid fibrils of αS forming during the aggregation from liquid-like spherical condensates to mature amyloids adopting the structure of pathologically observed aggregates. These transient amyloid intermediates, which induce significant levels of cytotoxicity when incubated with neuronal cells, were found to be stabilized by a small core in an antiparallel ß-sheet conformation, with a disordered N-terminal region of the protein remaining available to mediate membrane binding. In contrast, mature amyloids that subsequently appear during the aggregation showed different structural and biological properties, including low levels of cytotoxicity, a rearranged structured core embedding also the N-terminal region, and a reduced propensity to interact with the membrane. The characterization of these two fibrillar forms of αS, and the use of antibodies and designed mutants, enabled us to clarify the role of critical structural elements endowing intermediate amyloid species with the ability to interact with membranes and induce cytotoxicity.

A microfluidic platform for the controlled synthesis of architecturally complex liquid crystalline nanoparticles.

Soft-matter nanoparticles are of great interest for their applications in biotechnology, therapeutic delivery, and in vivo imaging. Underpinning this is their biocompatibility, potential for selective targeting, attractive pharmacokinetic properties, and amenability to downstream functionalisation. Morphological diversity inherent to soft-matter particles can give rise to enhanced functionality. However, this diversity remains untapped in clinical and industrial settings, and only the simplest of particle architectures [spherical lipid vesicles and lipid/polymer nanoparticles (LNPs)] have been routinely exploited. This is partially due to a lack of appropriate methods for their synthesis. To address this, we have designed a scalable microfluidic hydrodynamic focusing (MHF) technology for the controllable, rapid, and continuous production of lyotropic liquid crystalline (LLC) nanoparticles (both cubosomes and hexosomes), colloidal dispersions of higher-order lipid assemblies with intricate internal structures of 3-D and 2-D symmetry. These particles have been proposed as the next generation of soft-matter nano-carriers, with unique fusogenic and physical properties. Crucially, unlike alternative approaches, our microfluidic method gives control over LLC size, a feature we go on to exploit in a fusogenic study with model cell membranes, where a dependency of fusion on particle diameter is evident. We believe our platform has the potential to serve as a tool for future studies involving non-lamellar soft nanoparticles, and anticipate it allowing for the rapid prototyping of LLC particles of diverse functionality, paving the way toward their eventual wide uptake at an industrial level.

Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

Bacteriophages allow selective depletion of gut bacteria to produce a targeted-bacterium-depleted mouse model.

The gut microbiome is essential for human health. Mouse microbiota models, including gnotobiotic mice, are the most prominent tools to elucidate the functions of gut bacteria. Here, we propose a targeted-bacterium-depleted (TBD) model using lytic bacteriophage to selectively deplete gut bacterium of healthy or otherwise defined mice. These phage-treated animals should have a near-complete spectrum of gut bacteria except for the depleted bacterium. To prove the concept, we employed Escherichia coli-specific phage T7 to repress E. coli in the healthy mice. Our results showed that the E. coli-depleted mice exhibited bravery-like behaviors, correlated to the presence of E. coli rather than the equilibrium among gut bacteria. Thus, we demonstrate that the TBD model is a powerful tool to elucidate the function of a specific bacterial species within a near-intact gut microbiota environment and complements gnotobiotic mice models.

Mechanism of misfolding of the human prion protein revealed by a pathological mutation.

The misfolding and aggregation of the human prion protein (PrP) is associated with transmissible spongiform encephalopathies (TSEs). Intermediate conformations forming during the conversion of the cellular form of PrP into its pathological scrapie conformation are key drivers of the misfolding process. Here, we analyzed the properties of the C-terminal domain of the human PrP (huPrP) and its T183A variant, which is associated with familial forms of TSEs. We show that the mutation significantly enhances the aggregation propensity of huPrP, such as to uniquely induce amyloid formation under physiological conditions by the sole C-terminal domain of the protein. Using NMR spectroscopy, biophysics, and metadynamics simulations, we identified the structural characteristics of the misfolded intermediate promoting the aggregation of T183A huPrP and the nature of the interactions that prevent this species to be populated in the wild-type protein. In support of these conclusions, POM antibodies targeting the regions that promote PrP misfolding were shown to potently suppress the aggregation of this amyloidogenic mutant.

A Role of Cholesterol in Modulating the Binding of α-Synuclein to Synaptic-Like Vesicles.

α-Synuclein (αS) is a presynaptic protein whose aggregation is associated with Parkinson's disease (PD). Although the physiological function of αS is still unclear, several lines of evidence indicate that this protein may play a role in the trafficking of synaptic vesicles (SVs) during neurotransmitter release, a task associated with its ability to bind SVs and promote their clustering. It is therefore crucial to identify the cellular factors that modulate this process. To address this question, using nuclear magnetic resonance (NMR) spectroscopy we have characterized the role of cholesterol, a major component of the membrane of SVs, in the binding of αS with synaptic-like vesicles. Our results indicate that cholesterol can act as a modulator of the overall affinity of αS for SVs by reducing the local affinity of the region spanning residues 65-97 in the non-amyloid-ß component (NAC) of the protein. The increased population of bound states that expose the region 65-97 to the solvent was found to induce stronger vesicle-vesicle interactions by αS. These results provide evidence that cholesterol modulates the clustering of synaptic vesicles induced by (α)S, and supports the role of the disorder-to-order equilibrium of the NAC region in the modulation of the biological properties of the membrane-bound state of αS.

Size dependency of gold nanoparticles interacting with model membranes.

The rapid development of nanotechnology has led to an increase in the number and variety of engineered nanomaterials in the environment. Gold nanoparticles (AuNPs) are an example of a commonly studied nanomaterial whose highly tailorable properties have generated significant interest through a wide range of research fields. In the present work, we characterise the AuNP-lipid membrane interaction by coupling qualitative data with quantitative measurements of the enthalpy change of interaction. We investigate the interactions between citrate-stabilised AuNPs ranging from 5 to 60 nm in diameter and large unilamellar vesicles acting as a model membrane system. Our results reveal the existence of two critical AuNP diameters which determine their fate when in contact with a lipid membrane. The results provide new insights into the size dependent interaction between AuNPs and lipid bilayers which is of direct relevance to nanotoxicology and to the design of NP vectors.

Copper2+ Binding to α-Synuclein. Histidine50 Can Form a Ternary Complex with Cu2+ at the N-Terminus but Not a Macrochelate.

α-Synuclein (αSyn) forms amyloid fibrils in the neurons of Parkinson's disease (PD) patients'. Despite a role for Cu2+ in accelerating αSyn fibril formation, coupled with reports of copper dis-homeostasis in PD, there remain controversies surrounding the coordination geometry of Cu2+ with αSyn. Here we compare visible circular dichroism (CD) spectra of Cu2+ loaded on to full-length αSyn together with four peptides that model aspects of Cu2+ binding to the N-terminus and histidine50 of αSyn. With glycine as a competitive ligand, the affinity of Cu2+ for full-length αSyn is determined to have a conditional dissociation constant, at pH 7.4, of 0.1 nM. A similar affinity of 0.3 nM is determined for the tripeptide Met-Asp-Val(MDV) that mimics the N-terminus of αSyn, while the incorporation of a putative histidine side chain in the N-terminal complex facilitates the formation of a macrochelate with the histidine, which results in an increase in the affinity for Cu2+ to 0.03 nM at pH 7.4. Comparisons of the visible absorbance and CD spectra over a range of pH values also indicates that the MDV tripeptide closely models Cu2+ binding to full-length αSyn and rules out a role for His50 in the primary Cu2+ binding complex of monomeric αSyn. However, there are reports that suggest His50 does form a macrochelate with the N-terminal Cu2+ complex; we reconcile these conflicting observations by identifying a concentration dependence of the interaction. Only at the higher concentrations can the imidazole nitrogen bind to the N-terminal Cu2+ to form a ternary complex rather than via a macrochelate. This work shows even for this intrinsically disordered protein a large macrochelate with Cu2+ is not favored. Understanding Cu2+ coordination to αSyn gives a more complete picture of its place in amyloid assembly and cytotoxicity.

N-Terminally Truncated Amyloid-ß(11-40/42) Cofibrillizes with its Full-Length Counterpart: Implications for Alzheimer's Disease.

Amyloid-ß peptide (Aß) isoforms of different lengths and aggregation propensities coexist in vivo. These different isoforms are able to nucleate or frustrate the assembly of each other. N-terminally truncated Aß(11-40) and Aß(11-42) make up one fifth of plaque load yet nothing is known about their interaction with full-length Aß(1-40/42) . We show that in contrast to C-terminally truncated isoforms, which do not co-fibrillize, deletions of ten residues from the N terminus of Aß have little impact on its ability to co-fibrillize with the full-length counterpart. As a consequence, N-terminally truncated Aß will accelerate fiber formation and co-assemble into short rod-shaped fibers with its full-length Aß counterpart. This has implications for the assembly kinetics, morphology, and toxicity of all Aß isoforms.

Truncated Amyloid-ß(11-40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly.

Alzheimer disease coincides with the formation of extracellular amyloid plaques composed of the amyloid-ß (Aß) peptide. Aß is typically 40 residues long (Aß(1-40)) but can have variable C and N termini. Naturally occurring N-terminally truncated Aß(11-40/42) is found in the cerebrospinal fluid and has a similar abundance to Aß(1-42), constituting one-fifth of the plaque load. Based on its specific N-terminal sequence we hypothesized that truncated Aß(11-40/42) would have an elevated affinity for Cu(2+). Various spectroscopic techniques, complemented with transmission electron microscopy, were used to determine the properties of the Cu(2+)-Aß(11-40/42) interaction and how Cu(2+) influences amyloid fiber formation. We show that Cu(2+)-Aß(11-40) forms a tetragonal complex with a 34 ± 5 fm dissociation constant at pH 7.4. This affinity is 3 orders of magnitude tighter than Cu(2+) binding to Aß(1-40/42) and more than an order of magnitude tighter than that of serum albumin, the extracellular Cu(2+) transport protein. Furthermore, Aß(11-40/42) forms fibers twice as fast as Aß(1-40) with a very different morphology, forming bundles of very short amyloid rods. Substoichiometric Cu(2+) drastically perturbs Aß(11-40/42) assembly, stabilizing much longer fibers. The very tight fm affinity of Cu(2+) for Aß(11-40/42) explains the high levels of Cu(2+) observed in Alzheimer disease plaques.