Acceleration of α-Synuclein Aggregation by Exosomes

Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of α-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed α-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard α-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how α-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets.     

Multi-Pronged Interactions Underlie Inhibition of α-Synuclein Aggregation by β-Synuclein

The intrinsically disordered protein β-synuclein is known to inhibit the aggregation of its intrinsically disordered homolog, α-synuclein, which is implicated in Parkinson's disease. While β-synuclein itself does not form fibrils at the cytoplasmic pH?7.4, alteration of pH and other environmental perturbations are known to induce its fibrilization. However, the sequence and structural determinants of β-synuclein inhibition and self-aggregation are not well understood. We have utilized a series of domain-swapped chimeras of α-synuclein and β-synuclein to probe the relative contributions of the N-terminal, C-terminal, and the central non-amyloid-β component domains to the inhibition of α-synuclein aggregation. Changes in the rates of α-synuclein fibril formation in the presence of the chimeras indicate that the non-amyloid-β component domain is the primary determinant of self-association leading to fibril formation, while the N- and C-terminal domains play critical roles in the fibril inhibition process. Our data provide evidence that all three domains of β-synuclein together contribute to providing effective inhibition, and support a model of transient, multi-pronged interactions between IDP chains in both processes. Inclusion of such multi-site inhibitory interactions spread over the length of synuclein chains may be critical for the development of therapeutics that are designed to mimic the inhibitory effects of β-synuclein.     

Femtosecond Hydration Map of Intrinsically Disordered α-Synuclein

Protein hydration water plays a fundamentally important role in protein folding, binding, assembly, and function. Little is known about the hydration water in intrinsically disordered proteins that challenge the conventional sequence-structure-function paradigm. Here, by combining experiments and simulations, we show the existence of dynamical heterogeneity of hydration water in an intrinsically disordered presynaptic protein, namely α-synuclein, implicated in Parkinson’s disease. We took advantage of nonoccurrence of cysteine in the sequence and incorporated a number of cysteine residues at the N-terminal segment, the central amyloidogenic nonamyloid-β component (NAC) domain, and the C-terminal end of α-synuclein. We then labeled these cysteine variants using environment-sensitive thiol-active fluorophore and monitored the solvation dynamics using femtosecond time-resolved fluorescence. The site-specific femtosecond time-resolved experiments allowed us to construct the hydration map of α-synuclein. Our results show the presence of three dynamically distinct types of water: bulk, hydration, and confined water. The amyloidogenic NAC domain contains dynamically restrained water molecules that are strikingly different from the water molecules present in the other two domains. Atomistic molecular dynamics simulations revealed longer residence times for water molecules near the NAC domain and supported our experimental observations. Additionally, our simulations allowed us to decipher the molecular origin of the dynamical heterogeneity of water in α-synuclein. These simulations captured the quasi-bound water molecules within the NAC domain originating from a complex interplay between the local chain compaction and the sequence composition. Our findings from this synergistic experimental simulation approach suggest longer trapping of interfacial water molecules near the amyloidogenic hotspot that triggers the pathological conversion into amyloids via chain sequestration, chain desolvation, and entropic liberation of ordered water molecules.     

Tryptophan Fluorescence Reveals Structural Features of α-Synuclein Oligomers

Oligomeric α-synuclein (αS) is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric αS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric αS and the structural basis for oligomer-lipid interactions. Several single Trp mutants of αS were used to gain site-specific information about the microenvironments of monomeric αS, oligomeric αS and lipid-bound oligomeric αS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for αS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric αS, the N-terminus is critical in determining oligomer-lipid binding.     

Cellular Uptake of α-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of α-Synuclein and Mediated via Fcγ Receptors

Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson’s disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen–antibody complexes.     

Modulation of α-Synuclein Aggregation by Dopamine: A Review

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized by (1) the selective loss of dopaminergic neurons in the substantia nigra and (2) the deposition of misfolded α-synuclein (α-syn) as amyloid fibrils in the intracellular Lewy bodies in various region of the brain. Current thinking suggests that an interaction between α-syn and dopamine (DA) leads to the selective death of neuronal cells and the accumulation of misfolded α-syn. However, the exact mechanism by which this occurs is not fully defined. DA oxidation could play a key role is the pathogenesis of PD by causing oxidative stress, mitochondria dysfunction and impairment of protein metabolism. Here, we review the literature on the role of DA and its oxidative intermediates in modulating the aggregation pathways of α-syn.     

Formation of Toxic Oligomeric α-Synuclein Species in Living Cells

Background

Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinson''s disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells.

Methodology/Principal Findings

Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein.

Conclusions/Significance

This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.     

Evidence of Native α-Synuclein Conformers in the Human Brain

α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.     

SDS-Induced Fibrillation of α-Synuclein: An Alternative Fibrillation Pathway

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of α-synuclein (αSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per αSN molecule, which is also the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of α-helical structure. In this fibrillogenic complex, 4 αSN molecules initially associate with 40-50 SDS molecules to form a shared micelle that gradually grows in size. The complex initially exhibits a mixture of random coil and α-helix, but incubation results in a structural conversion into β-sheet structure and concomitant formation of thioflavin-T-binding fibrils over a period of several hours. Based on small-angle X-ray scattering, the aggregates elongate as a beads-on-a-string structure in which individual units of ellipsoidal SDS-αSN are bridged by strings of the protein, so that aggregates nucleate around the surface of protein-stabilized micelles. Thus, fibrillation in this case occurs by a process of continuous accretion rather than by the rate-limiting accumulation of a distinct nucleus. The morphology of the SDS-induced fibrils does not exhibit the classical rod-like structures formed by αSN when aggregated by agitation in the absence of SDS. The SDS-induced fibrils have a flexible worm-like appearance, which can be converted into classical straight fibrils by continuous agitation. SDS-induced fibrillation represents an alternative and highly reproducible mechanism for fibrillation where protein association is driven by the formation of shared micelles, which subsequently allows the formation of β-sheet structures that presumably link individual micelles. This illustrates that protein fibrillation may occur by remarkably different mechanisms, testifying to the versatility of this process.     

Regulation of Weibel-Palade Body Exocytosis by α-Synuclein in Endothelial Cells

α-Synuclein is a small presynaptic protein implicated in the pathogenesis of Parkinson disease. Nevertheless, its physiological roles and mechanisms remain incompletely understood. α-Synuclein is not only expressed in neurons but also in the vascular endothelium, which contains intracellular granules called Weibel-Palade bodies (WPBs) that contain a number of chemokines, adhesive molecules, and inflammatory cytokines. This study explored whether the exocytosis of WPB is regulated by α-synuclein. Phorbol 12-myristate 13-acetate-, thrombin-, or forskolin-induced von Willebrand factor release or translocation of P-selectin from endothelial cells were inhibited by α- and β-synuclein but not γ-synuclein. Three point mutants (A30P, A53T, and E46K) found in familial Parkinson disease also inhibited WPB exocytosis similar to that of wild-type α-synuclein. Furthermore, the negative regulation of WPB exocytosis required the N terminus or the nonamyloid β-component of Alzheimer disease amyloid region of α-synuclein, but not the C-terminal acidic tail, and α-synuclein affected WPB exocytosis through interference with RalA activation by enhancing the interaction of RalGDS-β-arrestin complexes. Immuno-EM analysis revealed that α-synuclein was localized close to WPBs. These findings imply that α-synuclein plays as a negative regulator in WPB exocytosis in endothelial cells.     

Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells

Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson’s disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments.     

Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrP^C-overexpressing mice. In addition, α-synuclein binds strongly on PrP^C-expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.     

Dynamical Behavior of Human α-Synuclein Studied by Quasielastic Neutron Scattering

α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson''s disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D₂O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable.     

The Central Theme of Parkinson’s Disease: α-Synuclein

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, defined by the presence of resting tremor, muscular rigidity, bradykinesia, and postural instability. PD is characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. The neuropathological hallmark of the disease is the presence of intracytoplasmic inclusions, called Lewy bodies (LBs) and Lewy neurites (LNs), containing α-synuclein, a small protein which is widely expressed in the brain. The α-synuclein gene, SNCA, is located on chromosome 4q22.1; SNCA-linked PD shows an autosomal dominant inheritance pattern with a relatively early onset age, and it usually progresses rapidly. Three missense mutations, A53T, A30P, and E46K, in addition to gene multiplications of the SNCA have been described so far. Although it is clear that LBs and LNs contain mainly the α-synuclein protein, the mechanism(s) which leads α-synuclein to accumulate needs to be elucidated. The primary question in the molecular pathology of PD is how wild-type α-synuclein aggregates in PD, and which interacting partner(s) plays role(s) in the aggregation process. It is known that dopamine synthesis is a stressfull event, and α-synuclein expression somehow affects the dopamine synthesis. The aberrant interactions of α-synuclein with the proteins in the dopamine synthesis pathway may cause disturbances in cellular mechanisms. The normal physiological folding state of α-synuclein is also important for the understanding of pathological aggregates. Recent studies on the α-synuclein protein and genome-wide association studies of the α-synuclein gene show that PD has a strong genetic component, and both familial and idiopathic PD have a common denominator, α-synuclein, at the molecular level. It is clear that the disease process in Parkinson’s disease, as in other neurodegenerative disorders, is very complicated; there can be several different molecular pathways which are responsible for diverse and possibly also unrelated functions inside the neuron, playing roles in PD pathogenesis.     

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

The aggregation of α-synuclein (α-Syn) is linked to Parkinson’s disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways.