Small Oligomers Research Articles

Amylin is incorporated into extracellular vesicles in an ESCRT-dependent manner and regulates senescence.

Type 2 diabetes mellitus is a disease which initiates with insulin resistance. Then, pancreatic β cells start to counteract this situation by increasing insulin secretion, which is known as pre-diabetic state. Amylin protein or islet amyloid polypeptide (IAPP), has multiple physiological roles such as the regulation of satiety and avoiding gastric emptying. However, amylin is able to aggregate, forming insoluble structures that affects pancreatic β cell survival. Interestingly, not all the amylin from the different species has this aggregate-prone capacity. There are species, which possesses non-amyloidogenic capacity and does not aggregate such as the rodents. However, there are versions of the protein, for instance from humans and primates, which can aggregate. Previously, we observed that small oligomers could be found in extracellular vesicles (EVs). Now, we have used a pancreatic β cell which overexpresses human amylin (hIAPP) (INS1E-hIAPP) and we have explored the capacity of amylin to be incorporated into EVs and how amylin could affect to different essential signaling pathways such as the mammalian target of rapamycin complex 1, endoplasmic-reticulum stress and senescence. Here, we report that amylin can be incorporated into EVs in an endosomal sorting complexes required for transport (ESCRT)-dependent manner. When we treated the cells with the neutral sphingomyelinase inhibitor, GW4869, one of the pathways for EV biogenesis and under high glucose conditions, there was an increased incorporation of soluble amylin into vesicles. Interestingly in this condition, when we isolated the EVs, we clearly observed that the size of the vesicles was higher, compatible with microvesicles (MVs). Resveratrol increased a pro-senescent phenotype but, it was able to revert either the high glucose or GW4869-associated senescent. In summary, these results indicate that amylin can be recruited in an ESCRT-dependent manner into EVs and, resveratrol presents an important role in inducing senescence in INS1E-hIAPP pancreatic β cells.

Impact of Ligand-Induced Oligomer Dissociation on Enzyme Diffusion, Directly Observed at the Single-Molecule Level.

The existence of the phenomenon of enhanced enzyme diffusion (EED) has been a topic of debate in recent literature. One proposed mechanism to explain the origin of EED is oligomeric enzyme dissociation. We used mass photometry (MP), a label-free single-molecule technique, to investigate the dependence of the oligomeric states of several enzymes on their ligands. The studied enzymes of interest are catalase, aldolase, alkaline phosphatase, and vanillyl-alcohol oxidase (VAO). We compared the ratios of oligomeric states in the presence and absence of the substrate as well as different substrate and inhibitor concentrations. Catalase and aldolase were found to dissociate into smaller oligomers in the presence of their substrates, independently of inhibition, while for alkaline phosphatase and VAO, different behaviors were observed. Thus, we have identified a possible mechanism which explains the previously observed diffusion enhancement in vitro. This enhancement may occur due to the dissociation of oligomers through ligand binding.

SuperResNET: Single molecule network analysis detects changes to clathrin structure by small molecule inhibitors.

Here, we apply SuperResNET network analysis of dSTORM single-molecule localization microscopy (SMLM) to determine how the clathrin endocytosis inhibitors pitstop 2, dynasore and Latrunculin A alter the morphology of clathrin-coated pits. SuperResNET analysis of HeLa and Cos7 cells identifies: small oligomers (Class I); pits and vesicles (Class II); and larger clusters corresponding to fused pits or clathrin plaques (Class III). Pitstop 2 and dynasore induce distinct homogeneous populations of Class II structures in HeLa cells suggesting that they arrest endocytosis at different stages. Inhibition is not via actin depolymerization, as the actin-depolymerizing agent latrunculin A (LatA) induces large, heterogeneous clathrin structures. Ternary analysis of SuperResNET shape features presents a distinct more planar profile for pitstop 2 blobs that align with clathrin pits identified with high-resolution MINFLUX, while control structures resemble MINFLUX clathrin vesicles. SuperResNET analysis therefore shows that pitstop 2 arrests clathrin pit maturation at early stages of pit formation, representing an approach to detect the effect of small molecules on target structures in situ in the cell from SMLM data sets.

Methylaluminoxane Reactivities and Anionic Structures: From Small Oligomers to Large Sheets.

The structure and reactivity of small methylaluminoxane (MAO) species (MeAlO)n(Me3Al)m (n = 1-8) have been investigated using DFT (M06-2X), MP2, and CCSD(T) calculations. This hierarchy of methods reveals that DFT artificially stabilizes structures containing 4-coordinate oxygen atoms while higher-level calculations demonstrate a clear preference for structures with 3-coordinate oxygen and 4-coordinate aluminum centers. Analysis of ionization pathways shows these neutral MAO molecules form anions through either methide or Me2Al+ abstraction, with the latter mechanism dominant for sheet structures (n = 5-8). Smaller species (n = 1-4, m ≥ n) show minimal reactivity toward either ionization pathway. The resulting anions consistently adopt sheet structures with 3-coordinate oxygen and 4-coordinate aluminum at all levels of theory. We introduce a comprehensive stability metric incorporating both ionization energies and neutral precursor stability, which successfully explains the distribution of anions observed in ESI-MS spectra during Me3Al hydrolysis. Extension of this analysis to larger species, including the recently isolated and characterized sheet (MeAlO)26(Me3Al)9 (Luo et al., Science, 2024, 384, 1424-1428), reveals unexpected trends in reactivity.

Benzoylmesaconine alters the native structure and activity of hen egg white lysozyme: revealing possible mechanism of aconitum-induced toxicity.

This study examines the interaction between benzoylmesaconine (BMA) and hen egg white lysozyme (HEWL) under various physiological conditions, aiming to determine how BMA affects the HEWL's structure and function. Several analytical techniques were used, including tryptophan assay, light scattering, thioflavin T (ThT)-binding assay, dynamic light scattering, 8-anilino-1-naphthalenesulfonic acid (ANS)-binding assay, circular dichroism (CD) spectroscopy, enzyme activity assay, and molecular docking. The tryptophan assay displayed a concentration-dependent decrease in tryptophan fluorescence, showing an interaction between BMA and HEWL. Light scattering and ThT-binding assays confirmed increased protein aggregation and amyloid fibril formation, while the ANS-binding assay demonstrated altered exposed hydrophobic regions, implying structural changes. CD spectroscopy showed a reduction in α-helix content, indicating conformational alterations, and enzyme activity assays showed a loss of lytic function due to structural distortion. Finally, molecular docking identified significant bonds and hydrophobic interactions between BMA and HEWL residues. BMA binding induces structural changes in proteins, forming small oligomers and amyloid fibrils that decrease HEWL enzymatic activity and disrupt functional integrity.

Synthetic T-Cell Receptor-like Protein Behaves as a Janus Particle in Solution.

Protein engineering enables the creation of tailor-made proteins for a variety of applications. ImmTACs stand out as promising therapeutics for cancer and other treatments while also presenting unique challenges for stability, formulation, and delivery. We have shown that ImmTACs behave as Janus particles in solution, leading to self-association at low concentrations, even when the average protein-protein interactions suggest that the molecule should be stable. The formation of small but stable oligomers was confirmed by static and dynamic light scattering and analytical ultracentrifugation. Modeling of the structure using AlphaFold leads to a rational explanation for this behavior, consistent with the Janus particle assembly observed for inverse patchy particles.

Markov State Model Approach to Simulate Self-Assembly

Computational modeling of assembly is challenging for many systems, because their timescales can vastly exceed those accessible to simulations. This article describes the multiMSM, which is a general framework that uses Markov state models (MSMs) to enable simulating self-assembly and self-organization of finite-sized structures on timescales that are orders of magnitude longer than those accessible to brute-force dynamics simulations. As with traditional MSM approaches, the method efficiently overcomes free energy barriers and other dynamical bottlenecks. In contrast to previous MSM approaches to simulating assembly, the framework describes simultaneous assembly of many clusters and the consequent depletion of free subunits or other small oligomers. The algorithm accounts for changes in transition rates as concentrations of monomers and intermediates evolve over the course of the reaction. Using two model systems, we show that the multiMSM accurately predicts the concentrations of the full ensemble of intermediates on timescales required to reach equilibrium. Importantly, after constructing a multiMSM for one system concentration, yields at other concentrations can be approximately calculated without any further sampling. This capability allows for orders of magnitude additional speedup. In addition, the method enables highly efficient calculation of quantities such as free energy profiles, nucleation timescales, flux along the ensemble of assembly pathways, and entropy production rates. Identifying contributions of individual transitions to entropy production rates reveals sources of kinetic traps. The method is broadly applicable to systems with equilibrium or nonequilibrium dynamics and is trivially parallelizable and, thus, highly scalable. Published by the American Physical Society 2024

Quantitative single-molecule analysis of assembly and Ca2+-dependent disassembly of synaptotagmin oligomers on lipid bilayers

Synaptotagmin-1 (Syt-1) self-assembles into ring-like oligomers, and genetic and biochemical evidence suggest that oligomerization is needed to clamp synaptic vesicles and stabilize them for Ca2+-evoked release. However, oligomerization has not yet been demonstrated on lipid bilayers or studied in quantitative biophysical terms. Here we utilize single-molecule imaging methods to monitor the assembly and disassembly of oligomeric clusters of Syt-1 on lipid bilayers in real-time. Syt-1 assembled into two distinct classes of oligomers, small (5 ± 2 subunits) and large (15 ± 2 subunits). Each class assembled at a constant kon that was always proportional to its ultimate size, but both classes disassembled at the same unit rate (koff) independent of its size. Both large and small oligomers explosively disassembled when Ca2+ was added. The F349A mutation in the Syt-1 nearly eliminates the large class of oligomers but does not reduce the small class. Altogether, the physical-chemical properties of Syt-1 oligomers meet or exceed the physiologic requirements to function as such a clamp.

Early expression of monomeric and oligomeric alpha-synuclein and reduction of tyrosine hydroxylase following intranigral injection of lipopolysaccharide.

The insoluble tangles of alpha-synuclein (α-syn) protein in the nigrostriatal circuit, characteristic of synucleinopathy, originate from low molecular weight oligomers, whose appearance and dissemination are related to neuroinflammation. These oligomeric forms of α-syn are considered highly cytotoxic but transient, so knowing the timing in which they appear remains challenging. Therefore, this study aimed to analyze the abundance of oligomeric forms of α-syn and tyrosine hydroxylase (TH) between 3 and 7 days after inducing neuroinflammation with lipopolysaccharide (LPS). LPS (2.5µg/2.5 µL) was stereotaxically injected in the substantia nigra (SN) of adult male Wistar rats, which were sacrificed 3, 5 and 7 days after this intervention. The brains were processed for semi quantitative Western blot, along with brains from control and sham animals. Our results show an increased expression of α-syn monomer (15kDa) only 3 days after LPS infusion, and the formation of 50 KDa and 60kDa α-syn oligomers in the SN and striatum (STR) between 3 and 7 days after LPS infusion. Furthermore, the presence of these oligomers was accompanied by a decrease in the expression of nigral TH. These findings highlight the rapidity with which potentially toxic forms of α-syn appear in the nigrostriatal circuit after a neuroinflammatory challenge, in addition to allowing us to identify specific oligomers and a temporal relation with neurodegeneration of TH-positive cells. Knowledge of the timing and location in which these small oligomers appear is essential to developing therapeutic strategies to prevent its formation.

Advanced multiparametric image spectroscopy and super-resolution microscopy reveal a minimal model of CD95 signal initiation.

Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of ~10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (~96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.

O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics.

The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among them, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here, we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72), and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive, while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

Measurement of solubility product reveals the interplay of oligomerization and self-association for defining condensate formation.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation is daunting. Using experiments and computation, we therefore studied a simple model system consisting of polySH3 and polyPRM designed for pentavalent heterotypic binding. We tested whether the peak solubility product, or the product of the dilute phase concentration of each component, is a predictive parameter for the onset of phase separation. Titrating up equal total concentrations of each component showed that the maximum solubility product does approximately coincide with the threshold for phase separation in both experiments and models. However, we found that measurements of dilute phase concentration include small oligomers and monomers; therefore, a quantitative comparison of the experiments and models required inclusion of small oligomers in the model analysis. Even with the inclusion of small polyPRM and polySH3 oligomers, models did not predict experimental results. This led us to perform dynamic light scattering experiments, which revealed homotypic binding of polyPRM. Addition of this interaction to our model recapitulated the experimentally observed asymmetry. Thus, comparing experiments with simulation reveals that the solubility product can be predictive of the interactions underlying phase separation, even if small oligomers and low affinity homotypic interactions complicate the analysis.

Red-Light-Photosensitized Tyrosine 10 Nitration of β-Amyloid1-42 Diverts the Protein from Forming Toxic Aggregates.

Several studies have highlighted the presence of nitration damage following neuroinflammation in Alzheimer's disease (AD). Accordingly, post-transcriptional modifications of β-amyloid (Aβ), including peptide nitration, have been explored as a marker of the disease. However, the implications of Aβ nitration in terms of aggregation propensity and neurotoxicity are still debated. Here, we show new data obtained using a photoactivatable peroxynitrite generator (BPT-NO) to overcome the limitations associated with chemical nitration methods. We found that the photoactivation of BPT-NO with the highly biocompatible red light selectively induces the nitration of tyrosine 10 of freshly solubilized full-length Aβ1-42. Photonitrated Aβ1-42 was, therefore, investigated for aggregation states and functions. It resulted that photonitrated Aβ1-42 did not aggregate into small oligomers but rather self-assembled into large amorphous aggregates. When tested on neuronal-like SH-SY5Y cells and microglial C57BL/6 BV2 cells, photonitrated Aβ1-42 showed to be free of neurotoxicity and able to induce phagocytic microglia cells. We propose that light-controlled nitration of the multiple forms in which Aβ occurs (i.e., monomers, oligomers, fibrils) could be a tool to assess in real-time the impact of tyrosine nitration on the amyloidogenic and toxic properties of Aβ1-42.

Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aβ1-40 by NMR.

This study delves into the early aggregation process of the Aβ1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAβ1-40, a modified Aβ1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aβ1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.

Nanoscale Simulation of Plastic Contaminants Migration in Packaging Materials and Potential Leaching into Model Food Fluids.

Polymers are the most commonly used packaging materials for nutrition and consumer products. The ever-growing concern over pollution and potential environmental contamination generated from single-use packaging materials has raised safety questions. Polymers used in these materials often contain impurities, including unreacted monomers and small oligomers. The characterization of transport properties, including diffusion and leaching of these molecules, is largely hampered by the long timescales involved in shelf life experiments. In this work, we employ atomistic molecular simulation techniques to explore the main mechanisms involved in the bulk and interfacial transport of monomer molecules from three polymers commonly employed as packaging materials: polyamide-6, polycarbonate, and poly(methyl methacrylate). Our simulations showed that both hopping and continuous diffusion play important roles in inbound monomer diffusion and that solvent-polymer compatibility significantly affects monomer leaching. These results provide rationalization for monomer leaching in model food formulations as well as bulky industry-relevant molecules. Through this molecular-scale characterization, we offer insights to aid in the design of polymer/consumer product interfaces with reduced risk of contamination and longer shelf life.

Crystal Violet Selectively Detects Aβ Oligomers but Not Fibrils In Vitro and in Alzheimer's Disease Brain Tissue.

Deposition of extracellular Amyloid Beta (Aβ) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aβ and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aβ42 oligomers (AβOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.

Exploring Structural Insights of Aβ42 and α-Synuclein Monomers and Heterodimer: A Comparative Study Using Implicit and Explicit Solvent Simulations.

Protein misfolding, aggregation, and fibril formation play a central role in the development of severe neurological disorders, including Alzheimer's and Parkinson's diseases. The structural stability of mature fibrils in these diseases is of great importance, as organisms struggle to effectively eliminate amyloid plaques. To address this issue, it is crucial to investigate the early stages of fibril formation when monomers aggregate into small, toxic, and soluble oligomers. However, these structures are inherently disordered, making them challenging to study through experimental approaches. Recently, it has been shown experimentally that amyloid-β 42 (Aβ42) and α-synuclein (α-Syn) can coassemble. This has motivated us to investigate the interaction between their monomers as a first step toward exploring the possibility of forming heterodimeric complexes. In particular, our study involves the utilization of various Amber and CHARMM force-fields, employing both implicit and explicit solvent models in replica exchange and conventional simulation modes. This comprehensive approach allowed us to assess the strengths and weaknesses of these solvent models and force fields in comparison to experimental and theoretical findings, ensuring the highest level of robustness. Our investigations revealed that Aβ42 and α-Syn monomers can indeed form stable heterodimers, and the resulting heterodimeric model exhibits stronger interactions compared to the Aβ42 dimer. The binding of α-Syn to Aβ42 reduces the propensity of Aβ42 to adopt fibril-prone conformations and induces significant changes in its conformational properties. Notably, in AMBER-FB15 and CHARMM36m force fields with the use of explicit solvent, the presence of Aβ42 significantly increases the β-content of α-Syn, consistent with the experiments showing that Aβ42 triggers α-Syn aggregation. Our analysis clearly shows that although the use of implicit solvent resulted in too large compactness of monomeric α-Syn, structural properties of monomeric Aβ42 and the heterodimer were preserved in explicit-solvent simulations. We anticipate that our study sheds light on the interaction between α-Syn and Aβ42 proteins, thus providing the atom-level model required to assess the initial stage of aggregation mechanisms related to Alzheimer's and Parkinson's diseases.

Characterization of α-synuclein oligomers formed in the presence of lipid vesicles

Aggregation of α-synuclein into oligomers and fibrils is associated with numerous neurodegenerative diseases such as Parkinson's disease (PD). Although the identity of the pathogenic species formed during the aggregation process is still under active debate, mounting evidence suggests that small oligomeric species rather than fibrillar aggregates are real toxic species. Isolation and characterization of small oligomers is essential to developing therapeutic strategies to prevent oligomer formation. Preparation of misfolded oligomeric species for biophysical characterization is, however, a great challenge due to their heterogenous, transient nature. Here we report the preparation of toxic and non-toxic α-synuclein oligomeric species formed at different pH values in the presence of lipid vesicles that mimic mitochondria membranes containing cardiolipin. Biophysical characterization of the lipid-induced α-synuclein oligomeric assemblies revealed that α-synuclein oligomers formed at pH 7.4 have higher surface hydrophobicity than the aggregates formed at pH 6.0. In addition, the high-pH oligomers were shown to exhibit higher toxicity than the low-pH aggregates. Structural, dynamic properties of the oligomers were also investigated by using circular dichroism (CD) and NMR spectroscopy. Our CD analyses revealed that the two oligomeric species have distinct molecular conformations, and 2D 1H/15N HSQC NMR experiments suggested that the high-pH oligomers have more extended dynamic regions than the low-pH aggregates. The distinct structural and dynamic properties of the oligomers might be associated with their different cytotoxic properties.

Rational Design of Organic Electrocatalysts for Hydrogen and Oxygen Electrocatalytic Applications.

Efficient electrocatalysts are pivotal for advancing green energy conversion technologies. Organic electrocatalysts, as cost-effective alternatives to noble-metal benchmarks, have garnered attention. However, the understanding of the relationships between their properties and electrocatalytic activities remains ambiguous. Plenty of research articles regarding low-cost organic electrocatalysts started to gain momentum in 2010 and have been flourishing recently though, a review article for both entry-level and experienced researchers in this field is still lacking. This review underscores the urgent need to elucidate the structure-activity relationship and design suitable electrode structures, leveraging the unique features of organic electrocatalysts like controllability and compatibility for real-world applications. Organic electrocatalysts are classified into four groups: small molecules, oligomers, polymers, and frameworks, with specific structural and physicochemical properties serving as activity indicators. To unlock the full potential of organic electrocatalysts, five strategies are discussed: integrated structures, surface property modulation, membrane technologies, electrolyte affinity regulation, and addition of anticorrosion species, all aimed at enhancing charge efficiency, mass transfer, and long-term stability during electrocatalytic reactions. The review offers a comprehensive overview of the current state of organic electrocatalysts and their practical applications, bridging the understanding gap and paving the way for future developments of more efficient green energy conversion technologies.

Solubility of α-synuclein species in the L62 mouse model of synucleinopathy

The accumulation of α-synuclein (α-Syn) into Lewy bodies is a hallmark of synucleinopathies, a group of neurological disorders that include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Small oligomers as well as larger fibrils of α-Syn have been suggested to induce cell toxicity leading to a degenerative loss of neurones. A richer understanding of α-Syn aggregation in disease, however, requires the identification of the different α-Syn species and the characterisation of their biochemical properties. We here aimed at a more in-depth characterisation of the α-Syn transgenic mice, Line 62 (L62), and examined the deposition pattern and solubility of human and murine α-Syn in these mice using immunohistochemical and biochemical methods. Application of multiple antibodies confirmed mAb syn204 as the most discriminatory antibody for human α-Syn in L62. Syn204 revealed an intense and widespread immunohistochemical α-Syn labelling in parietal cortex and hippocampus, and to a lower level in basal forebrain and hindbrain regions. The labelled α-Syn represented somatic inclusions as well as processes and synaptic endings. Biochemical analysis revealed a Triton-resistant human α-Syn pool of large oligomers, a second pool of small oligomers that was not resistant to solubilization with urea/Triton. A third SDS-soluble pool of intermediate sized aggregates containing a mixture of both, human and mouse α-Syn was also present. These data suggest that several pools of α-Syn can exist in neurones, most likely in different cellular compartments. Information about these different pools is important for the development of novel disease modifying therapies aimed at α-Syn.