αS Oligomers Research Articles

Alpha-synuclein stepwise aggregation reveals features of an early onset mutation in Parkinson\u2019s disease

Amyloid formation is a process involving interconverting protein species and results in toxic oligomers and fibrils. Aggregated alpha-synuclein (αS) participates in neurodegenerative maladies, but a closer understanding of the early αS polymerization stages and polymorphism of heritable αS variants is sparse still. Here, we distinguished αS oligomer and protofibril interconversions in Thioflavin T polymerization reactions. The results support a hypothesis reconciling the nucleation-polymerization and nucleation-conversion-polymerization models to explain the dissimilar behaviors of wild-type and the A53T mutant. Cryo-electron microscopy with a direct detector shows the polymorphic nature of αS fibrils formed by heritable A30P, E46K, and A53T point mutations. By showing that A53T rapidly nucleates competent species, continuously elongates fibrils in the presence of increasing amounts of seeds, and overcomes wild-type surface requirements for growth, our findings place A53T with features that may explain the early onset of familial Parkinson’s disease cases bearing this mutation.

Cardiolipin Promotes Pore-Forming Activity of Alpha-Synuclein Oligomers in Mitochondrial Membranes.

Aggregation of the amyloid-forming α-synuclein (αS) protein is closely associated with the etiology of Parkinson's disease (PD), the most common motor neurodegenerative disorder. Many studies have shown that soluble aggregation intermediates of αS, termed oligomers, permeabilize a variety of phospholipid membranes; thus, membrane disruption may represent a key pathogenic mechanism of αS toxicity. Given the centrality of mitochondrial dysfunction in PD, we therefore probed the formation of ion-permeable pores by αS oligomers in planar lipid bilayers reflecting the complex phospholipid composition of mitochondrial membranes. Using single-channel electrophysiology, we recorded distinct multilevel conductances (100-400 pS) with stepwise current transitions, typical of protein-bound nanopores, in mitochondrial-like membranes. Crucially, we observed that the presence of cardiolipin (CL), the signature phospholipid of mitochondrial membranes, enhanced αS-lipid interaction and the membrane pore-forming activity of αS oligomers. Further, preincubation of isolated mitochondria with a CL-specific dye protected against αS oligomer-induced mitochondrial swelling and release of cytochrome c. Hence, we favor a scenario in which αS oligomers directly porate a local lipid environment rich in CL, for instance outer mitochondrial contact sites or the inner mitochondrial membrane, to induce mitochondrial dysfunction. Pharmacological modulation of αS pore complex formation might thus preserve mitochondrial membrane integrity and alleviate mitochondrial dysfunction in PD.

Tau is required for progressive synaptic and memory deficits in a transgenic mouse model of \u03b1-synucleinopathy

Parkinson’s disease dementia (PDD) and dementia with Lewy bodies (DLB) are clinically and neuropathologically highly related α-synucleinopathies that collectively constitute the second leading cause of neurodegenerative dementias. Genetic and neuropathological studies directly implicate α-synuclein (αS) abnormalities in PDD and DLB pathogenesis. However, it is currently unknown how αS abnormalities contribute to memory loss, particularly since forebrain neuronal loss in PDD and DLB is less severe than in Alzheimer’s disease. Previously, we found that familial Parkinson’s disease-linked human mutant A53T αS causes aberrant localization of the microtubule-associated protein tau to postsynaptic spines in neurons, leading to postsynaptic deficits. Thus, we directly tested if the synaptic and memory deficits in a mouse model of α-synucleinopathy (TgA53T) are mediated by tau. TgA53T mice exhibit progressive memory deficits associated with postsynaptic deficits in the absence of obvious neuropathological and neurodegenerative changes in the hippocampus. Significantly, removal of endogenous mouse tau expression in TgA53T mice (TgA53T/mTau−/−), achieved by mating TgA53T mice to mouse tau-knockout mice, completely ameliorates cognitive dysfunction and concurrent synaptic deficits without affecting αS expression or accumulation of selected toxic αS oligomers. Among the known tau-dependent effects, memory deficits in TgA53T mice were associated with hippocampal circuit remodeling linked to chronic network hyperexcitability. This remodeling was absent in TgA53T/mTau−/− mice, indicating that postsynaptic deficits, aberrant network hyperactivity, and memory deficits are mechanistically linked. Our results directly implicate tau as a mediator of specific human mutant A53T αS-mediated abnormalities related to deficits in hippocampal neurotransmission and suggest a mechanism for memory impairment that occurs as a consequence of synaptic dysfunction rather than synaptic or neuronal loss. We hypothesize that these initial synaptic deficits contribute to network hyperexcitability which, in turn, exacerbate cognitive dysfunction. Our results indicate that these synaptic changes present potential therapeutic targets for amelioration of memory deficits in α-synucleinopathies.

Morphological Evaluation of Meta-stable Oligomers of \u03b1-Synuclein with Small-Angle Neutron Scattering

Amyloidogenesis of α-synuclein (αS) is considered to be a pathological phenomenon related to Parkinson’s disease (PD). As a key component to reveal the fibrillation mechanism and toxicity, we have investigated an oligomeric species of αS capable of exhibiting the unit-assembly process leading to accelerated amyloid fibril formation. These oligomers previously shown to exist in a meta-stable state with mostly disordered structure and unable to seed the fibrillation were converted to either temperature-sensitive self-associative oligomers or NaCl-induced non-fibrillating oligomeric species. Despite their transient and disordered nature, the structural information of meta-stable αS oligomers (Meta-αS-Os) was successfully evaluated with small-angle neutron scattering (SANS) technique. By fitting the neutron scattering data with polydisperse Gaussian Coil (pGC) model, Meta-αS-O was analyzed as a sphere with approximate diameter of 100 Å. Its overall shape altered drastically with subtle changes in temperature between 37 °C and 43 °C, which would be responsible for fibrillar polymorphism. Based on their bifurcating property of Meta-αS-Os leading to either on-pathway or off-pathway species, the oligomers could be suggested as a crucial intermediate responsible for the oligomeric diversification and multiple fibrillation processes. Therefore, Meta-αS-Os could be considered as a principal target to control the amyloidogenesis and its pathogenesis.

Single-Molecule Characterization of the Interactions between Extracellular Chaperones and Toxic α-Synuclein Oligomers

SummaryThe aberrant aggregation of α-synuclein is associated with several human diseases, collectively termed the α-synucleinopathies, which includes Parkinson’s disease. The progression of these diseases is, in part, mediated by extracellular α-synuclein oligomers that may exert effects through several mechanisms, including prion-like transfer, direct cytotoxicity, and pro-inflammatory actions. In this study, we show that two abundant extracellular chaperones, clusterin and α2-macroglobulin, directly bind to exposed hydrophobic regions on the surface of α-synuclein oligomers. Using single-molecule fluorescence techniques, we found that clusterin, unlike α2-macroglobulin, exhibits differential binding to α-synuclein oligomers that may be related to structural differences between two previously described forms of αS oligomers. The binding of both chaperones reduces the ability of the oligomers to permeabilize lipid membranes and prevents an oligomer-induced increase in ROS production in cultured neuronal cells. Taken together, these data suggest a neuroprotective role for extracellular chaperones in suppressing the toxicity associated with α-synuclein oligomers.

Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by β-Synuclein.

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants—A30P and G51D—as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS.

The processes of α-synuclein amyloid protein complexes involved in the pathogenesis of Parkinson's disease

To analyze interactions between α-synuclein (αS) protein and lipids using biophysical methods. Recombinant α-synuclein synthesized in prokaryotic cells was used. To characterize the interaction of αS with negatively charged vesicles of DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine, sodium salt) and DOPG (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt) and the consequences of such interactions on αS amyloid formation, combined circular dichroism, fluorescence and imaging methods in vitro were applied. Lipid head-group chemistry modulates αS interactions and also affects amyloid fiber formation. Pre-formed αS oligomers, typically present in a small amount in the αS starting material, acted as templates for linear growth of anomalous amyloid fibers in the presence of vesicles. At the same time, the remaining αS monomers were restricted from vesicle-mediated nucleation of amyloid fibers. Although not a dominant process in bulk experiments, this hidden αS aggregation pathway may be of importance in vivo.

A structural look at α-synuclein oligomers

Neurodegeneration Fibrillar aggregates of the protein α-synuclein (αS) are the major constituents of Lewy bodies in Parkinson's disease. However, small oligomers that accumulate during the process of fibril formation are thought to cause the neuronal toxicity associated with the onset and progression of Parkinson's disease. Little is known about the detailed structural properties of αS oligomers and the molecular mechanisms that lead to their toxicity. Fusco et al. report the structural characterization of two forms of αS oligomers, which elucidates the fundamental structural elements giving rise to neuronal toxicity. Science , this issue p. [1440][1] [1]: /lookup/doi/10.1126/science.aan6160

Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons

α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where αS species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that αS oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble αS oligomers at the ER/M level precedes neuronal degeneration in a mouse model of α-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of αS species associated with the ER/M membrane. We found that by comparison with non-microsomal associated αS (P10), the ER/M-associated αS pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated αS species isolated from diseased A53T human αS transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated αS species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous αS aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of αS high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of αS HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of αS toxic features.

EGCG-mediated Protection of the Membrane Disruption and Cytotoxicity Caused by the \u2018Active Oligomer\u2019 of \u03b1-Synuclein

(−)-Epigallocatechin gallate (EGCG), the major component of green tea, has been re-evaluated with α-synuclein (αS), a pathological constituent of Parkinson’s disease, to elaborate its therapeutic value. EGCG has been demonstrated to not only induce the off-pathway ‘compact’ oligomers of αS as suggested previously, but also drastically enhance the amyloid fibril formation of αS. Considering that the EGCG-induced amyloid fibrils could be a product of on-pathway SDS-sensitive ‘transient’ oligomers, the polyphenol effect on the transient ‘active’ oligomers (AOs) was investigated. By facilitating the fibril formation and thus eliminating the toxic AOs, EGCG was shown to suppress the membrane disrupting radiating amyloid fibril formation on the surface of liposomal membranes and thus protect the cells which could be readily affected by AOs. Taken together, EGCG has been suggested to exhibit its protective effect against the αS-mediated cytotoxicity by not only producing the off-pathway ‘compact’ oligomers, but also facilitating the conversion of ‘active’ oligomers into amyloid fibrils.

Histones facilitate α-synuclein aggregation during neuronal apoptosis.

Ample in vitro and in vivo experimental evidence supports the hypothesis that intercellular transmission of α-synuclein (αS) is a mechanism underlying the spread of αS pathology in Parkinson's disease and related disorders. What remains unexplained is where and how initial transmissible αS aggregates form. In a previous study, we demonstrated that αS aggregates rapidly form in neurons with impaired nuclear membrane integrity due to the interaction between nuclear proaggregant factor(s) and αS and that such aggregates may serve as a source for αS seeding. In the present study, we identify histones as a potential nuclear proaggregant factor for αS aggregation in both apoptotic neurons and brains with αS pathology. We further demonstrate that histone-induced aggregates contain a range of αS oligomers, including protofibrils and mature fibrils, and that these αS aggregates can seed additional aggregation. Importantly, we demonstrate transmissibility in mouse brains from stereotaxic injection. This study provides new clues to the mechanism underlying initial pathological aggregation of αS in PD and related disorders, and could lead to novel diagnostic and therapeutic approaches.

α-Synuclein Oligomers Stabilize Pre-Existing Defects in Supported Bilayers and Propagate Membrane Damage in a Fractal-Like Pattern.

Phospholipid vesicles are commonly used to get insights into the mechanism by which oligomers of amyloidogenic proteins damage membranes. Oligomers of the protein α-synuclein (αS) are thought to create pores in phospholipid vesicles containing a high amount of anionic phospholipids but fail to damage vesicle membranes at low surface charge densities. The current understanding of how αS oligomers damage the membranes is thus incomplete. This incomplete understanding may, in part, result from the choice of model membrane systems. The use of free-standing membranes such as vesicles may interfere with the unraveling of some damage mechanisms because the line tension at the edge of a membrane defect or pore ensures defect closure. Here, we have used supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPC/POPS) to study the membrane damage caused by αS oligomers. Although αS oligomers were not able to initiate the disruption of POPC/POPS vesicles or intact SLBs, oligomers did stabilize and enlarge pre-existing SLB defects. The increased exposure of lipid acyl chains at the edges of defects very likely facilitates membrane-oligomer interactions, resulting in the growth of fractal domains devoid of lipids. Concomitant with the appearance of the fractal membrane damage patterns, lipids appear in solution, directly implicating αS oligomers in the observed lipid extraction. The growth of the membrane damage patterns is not limited by the binding of lipids to the oligomer. The analysis of the shape and growth of the lipid-free domains suggests the involvement of an oligomer-dependent diffusion-limited extraction mechanism. The observed αS oligomer-induced propagation of membrane defects offers new insights into the mechanisms by which αS oligomers can contribute to the loss in membrane integrity.

Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-synuclein oligomers.

α-Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinson's disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of αS are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.

Phenolic compounds prevent the oligomerization of α-synuclein and reduce synaptic toxicity.

Lewy bodies, mainly composed of α-synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low-order αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo-induced cross-linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N-terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long-term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds on α-synuclein (αS) oligomerization. Phenolic compounds, especially Myricetin (Myr) and Rosmarinic acid (RA), inhibited αS oligomerization and secondary structure conversion. Myr and RA ameliorated αS synaptic toxicity on the experiment of long-term potentiation. Our results suggest that Myr and RA prevent αS aggregation process and reduce the neurotoxicity of αS oligomers. Phenolic compounds are good candidates of disease modifying drugs for α-synucleinopathies.

β-Amyloid and α-synuclein cooperate to block SNARE-dependent vesicle fusion.

Alzheimer's disease (AD) and Parkinson's disease (PD) are caused by β-amyloid (Aβ) and α-synuclein (αS), respectively. Ample evidence suggests that these two pathogenic proteins are closely linked and have a synergistic effect on eliciting neurodegenerative disorders. However, the pathophysiological consequences of Aβ and αS coexistence are still elusive. Here, we show that large-sized αS oligomers, which are normally difficult to form, are readily generated by Aβ42-seeding and that these oligomers efficiently hamper neuronal SNARE-mediated vesicle fusion. The direct binding of the Aβ-seeded αS oligomers to the N-terminal domain of synaptobrevin-2, a vesicular SNARE protein, is responsible for the inhibition of fusion. In contrast, large-sized Aβ42 oligomers (or aggregates) or the products of αS incubated without Aβ42 have no effect on vesicle fusion. These results are confirmed by examining PC12 cell exocytosis. Our results suggest that Aβ and αS cooperate to escalate the production of toxic oligomers, whose main toxicity is the inhibition of vesicle fusion and consequently prompts synaptic dysfunction.

Dopamine-induced α-synuclein oligomers show self- and cross-propagation properties.

Amyloid aggregates of α-synuclein (αS) protein are the predominant species present within the intracellular inclusions called Lewy bodies in Parkinson's disease (PD) patients. Among various aggregates, the low-molecular weight ones broadly ranging between 2 and 30 mers are known to be the primary neurotoxic agents responsible for the impairment of neuronal function. Recent research has indicated that the neurotransmitter dopamine (DA) is one of the key physiological agents promoting and augmenting αS aggregation, which is thought to be a significant event in PD pathologenesis. Specifically, DA is known to induce the formation of soluble oligomers of αS, which in turn are responsible for inducing several important cellular changes leading to cellular toxicity. In this report, we present the generation, isolation, and biophysical characterization of five different dopamine-derived αS oligomers (DSOs) ranging between 3 and 15 mers, corroborating previously published reports. More importantly, we establish that these DSOs are also capable of replication by self-propagation, which leads to the replication of DSOs upon interaction with αS monomers, a process similar to that observed in mammilian prions. In addition, DSOs are also able to cross-propagate amyloid-β (Aβ) aggregates involved in Alzheimer's disease (AD). Interestingly, while self-propagation of DSOs occur with no net gain in protein structure, cross-propagation proceeds with an overall gain in β-sheet conformation. These results implicate the involvement of DSOs in the progression of PD, and, in part, provide a molecular basis for the observed co-existence of AD-like pathology among PD patients.

Effects of antiparkinsonian agents on β-amyloid and α-synuclein oligomer formation in vitro

The aggregation of β-amyloid protein (Aβ) and α-synuclein (αS) are hypothesized to be the key pathogenic event in Alzheimer's disease (AD) and Lewy body diseases (LBD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD and LBD. Here, we examined the effects of antiparkinsonian agents (dopamine, levodopa, trihexyphenidyl, selegiline, zonisamide, bromocriptine, peroxide, ropinirole, pramipexole, and entacapone) on the in vitro oligomer formation of Aβ40, Aβ42, and αS using a method of photo-induced cross-linking of unmodified proteins (PICUP), electron microscopy, and atomic force microscopy. The antiparkinsonian agents except for trihexyphenidyl inhibited both Aβ and αS oligomer formations, and, among them, dopamine, levodopa, pramipexole, and entacapone had the stronger in vitro activity. Circular dichroism and thioflavin T(S) assays showed that secondary structures of Aβ and αS assemblies inhibited by antiparkinsonian agents were statistical coil state and that their seeding activities had disappeared. The antiparkinsonian agents could be potential therapeutic agents to prevent or delay AD and LBD progression.

A conformation-switching fluorescent protein probe for detection of alpha synuclein oligomers

We engineered the structural flexibility of an α synuclein (αS) variant, PG65, together with conformation-sensitive fluorescence to create a molecular probe for rapid, specific and quantitative detection of αS oligomers, which are the major toxic aggregate forms in Parkinson's disease.

Cross‐seeding effects of amyloid β‐protein and α‐synuclein

Amyloid β-protein (Aβ) and α-synuclein (αS) are the primary components of amyloid plaques and Lewy bodies (LBs), respectively. Previous in vitro and in vivo studies have suggested that interactions between Aβ and αS are involved in the pathogenesis of Alzheimer's disease and LB diseases. However, the seeding effects of their aggregates on their aggregation pathways are not completely clear. To investigate the cross-seeding effects of Aβ and αS, we examined how sonicated fibrils or cross-linked oligomers of Aβ40, Aβ42, and αS affected their aggregation pathways using thioflavin T(S) assay and electron microscopy. Fibrils and oligomers of Aβ40, Aβ42, and αS acted as seeds, and affected the aggregation pathways within and among species. The seeding effects of αS fibrils were higher than those of Aβ40 and Aβ42 fibrils in the Aβ40 and Aβ42 aggregation pathways, respectively. We showed that Aβ and αS acted as seeds and affected each other's aggregation pathways in vitro, which may contribute to our understanding of the molecular mechanisms of interactions between Alzheimer's disease and LB diseases pathologies.

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson’s disease. It is currently believed that noncovalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE, and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr–Tyr (dityrosine) dimers through –C–C– bonds acting as covalent bridges, with a predominant involvement of residue Y39. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson's disease.