Modulation of α-Synuclein Oligomer and Aggregate Populations by pH and Metal Ions

TL;DR

This study shows how pH and metal ions influence the formation of α-synuclein structures linked to diseases like Parkinson's.

Contribution

The paper reveals how pH and metal ions dynamically modulate α-synuclein assembly states, offering new insights into disease mechanisms.

Findings

α-synuclein forms different structures at acidic and neutral pH, with distinct assembly patterns.

Metal ions like Fe3+, Cu2+, and Zn2+ strongly influence α-synuclein aggregation in a pH-dependent manner.

Dynamic light scattering reveals pH-dependent redistribution of α-synuclein assembly mass.

Abstract

α-Synuclein (α-syn) aggregation underlies synucleinopathies, yet the physicochemical determinants that govern which assembly states form under defined solution conditions remain incompletely resolved. Here, we examine how pH and metal ions reshape α-syn self-assembly. Across acidic and physiological pH conditions, α-syn populates monomeric, nanoscale oligomeric, and mesoscale aggregate states whose relative abundances evolve over time. Fluorescence microscopy reveals robust mesoscale assembly at pH 5, minimal aggregation at pH 7, and transient assemblies at pH 3, highlighting the limitations of imaging-based detection alone. Therefore, we use dynamic light scattering (DLS) to resolve oligomeric populations and quantify pH-dependent redistribution of assembly mass. Toxicity-mitigating modulators altered α-syn assembly in a strongly pH-dependent manner. Anle138b increased the abundance of oligomeric species at low pH, whereas EGCG produced divergent effects at pH 5 and pH 3. We further examined the effects of metal ions, finding that Fe3+ stabilized higher-order assemblies under acidic conditions, Cu2+ delayed assembly at pH 5 while enhancing aggregation at pH 3, and Zn2+ increased oligomerization primarily at low pH. Overall, these results demonstrate that α-syn assembly is highly sensitive to coupled effects of pH, metal chemistry, and time.

Figures

Full text

1. Introduction

Lewy bodies and Lewy neurites are defining pathological hallmarks of Parkinson’s disease (PD) and dementia with Lewy bodies, with α-synuclein (α-syn) constituting a principal filamentous component of these inclusions [1,2]. Beyond their role as end-stage pathological features, α-syn assemblies participate in a broader disease process that is coupled to proteostasis failure, organelle dysfunction, and intercellular propagation mechanisms that can shape synucleinopathy progression [3,4,5,6,7,8]. A central challenge for the field is that α-syn does not follow a single, invariant aggregation pathway. Instead, it occupies an ensemble of self-assembled states whose populations depend strongly on the chemical environment, making direct comparison across studies difficult when experimental conditions differ [9,10,11].

α-Syn is an intrinsically disordered protein that rapidly interconverts among many conformations rather than adopting a single stable fold [12,13]. This conformational plasticity enables multiple assembly routes and produces oligomeric intermediates that can differ in structure and biological activity even when derived from the same sequence [11,14,15,16]. Consequently, in vitro “aggregation outcomes” should be understood as environment-dependent redistribution among competing assembly states (e.g., monomer/low-order species, nanoscale oligomers, and larger aggregate forms), rather than as a binary presence or absence of fibrils. Importantly, oligomeric α-syn species are increasingly implicated as clinically relevant assemblies in synucleinopathies. Elevated levels of α-syn oligomers have been reported in cerebrospinal fluid from PD patients, and in some studies track with disease severity, supporting their utility as biomarkers and their association with disease state [17]. These observations motivate efforts to understand how solution chemistry and cofactors bias α-syn populations toward or away from oligomeric states, rather than focusing exclusively on fibrillar endpoints [18].

Solution pH is a dominant driver of this redistribution because it reshapes the protein’s net charge and charge patterning and can reorganize long-range intramolecular contacts within the disordered ensemble. Prior studies showed that α-syn aggregate morphology and dye binding depend sensitively on pH and ionic strength [9], and that acidic conditions remodel intramolecular contacts and the collapsed/extended character of the ensemble in ways that can accelerate aggregation [19,20]. At mildly acidic pH (approximately 4–5), α-syn can rapidly yield turbid, dye-bright aggregates with distinct ThT and ANS behavior relative to fibrillar endpoints [9], and solution-condition mapping demonstrates that pH can tune the relative contributions of nucleation, growth, and secondary processes during aggregation [11]. These results collectively highlight that the same protein can populate qualitatively different assembly regimes over a relatively narrow pH range.

Metal ions are a second major class of α-syn modulators with relevance to synucleinopathies. Altered metal levels have been reported in PD tissue [21], and divalent and trivalent metals can influence α-syn conformation, aggregation kinetics, and aggregate structure in vitro and in cellular contexts [10,22]. However, reported metal effects are often concentration- and condition-dependent: Fe^3+^ has been described to accelerate aggregation at low metal-to-protein ratios while inhibiting or altering fibrillation at higher concentrations, Cu^2+^ can enhance or redirect aggregation depending on stoichiometry and environment, and Zn^2+^ has been reported to promote or suppress aggregation in different experimental contexts [23,24,25,26]. Importantly, many of these studies were performed at different pH values and in different buffers, despite pH independently exerting strong effects on α-syn assembly. Because metal speciation, hydrolysis, and chelation by buffer ligands also depend on pH, comparisons across datasets can be confounded when pH and metal chemistry are not considered together.

While many studies have examined the effects of pH, metal ions, or toxicity-mitigating compounds on α-syn aggregation, these variables are typically explored in isolation and under non-matched conditions, limiting cross-study comparison. Here, to address this gap, we perform a controlled, side-by-side survey of α-syn self-assembly sizes using DLS across pH 7, 5, and 3 under a fixed buffer composition. We first tested two aggregation- and toxicity-mitigating compounds, anle138b and epigallocatechin gallate (EGCG). Both compounds reduce α-syn aggregation and/or associated cytotoxicity, while acting through distinct and context-dependent mechanisms rather than simple elimination of higher-order assemblies. We further tested the effect of three metal ions (Fe^3+^, Cu^2+^, and Zn^2+^) at low and high concentrations. pH 7 was chosen to represent near-physiological cytosolic conditions. We chose pH 5 to model mildly acidic intracellular environments, such as endolysosomal compartments and regions of impaired autophagic or vesicular trafficking implicated in α-syn accumulation and pathology [27,28,29]. Although pH 3 is not representative of bulk cytosolic conditions, it provides an experimentally tractable lower-pH boundary that accentuates charge redistribution and intramolecular contact reorganization in α-syn, allowing us to define limiting assembly regimes and to disentangle pH-driven effects from metal- and toxicity mitigating compound-dependent modulation. We integrate fluorescence microscopy and ThT detection of mesoscale assemblies with dynamic light scattering (DLS) to quantify time-dependent shifts in size distributions and to resolve oligomeric populations that are poorly captured by microscopy alone. By explicitly mapping how pH and cofactors jointly redistribute assembly mass across size regimes, this work provides a comparative framework to interpret disparate observations in the α-syn aggregation literature and emphasizes that pH sets a baseline regime that gates the directionality and magnitude of metal- and drug-driven effects.

2. Materials and Methods

2.1. Expression and Purification of α-Syn

Expression and purification of α-syn were performed as previously described [30,31,32]. Briefly, a modified pET-41a plasmid (Novagen, EMD Biosciences, Gibbstown, NJ, USA) encoding α-syn was transformed into E. coli BL21 cells, grown in LB medium at 37 °C, and induced with 1 mM IPTG at an OD_600_ of ~0.5–0.7. Cells were grown for an additional ~3 h, harvested by centrifugation, and resuspended in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 1 mM EDTA. The suspension was subjected to three freeze–thaw cycles (−80 °C to room temperature), followed by sonication and centrifugation. The lysate was heated at 80 °C for 10 min and centrifuged, and the supernatant was purified by Q-Sepharose chromatography (Amersham Biosciences, Amersham, Buckinghamshire UK) using a salt gradient. Fractions containing α-syn were mixed 1:5 (v/v) with 50 mM sodium acetate and incubated with SP-Sepharose beads (Amersham Biosciences) at pH 5.7 for 1 h at 4 °C with stirring, followed by filtration to remove the beads. The supernatant was then incubated with Q-Sepharose beads at pH 4.4 for 1 h at 4 °C with stirring. After removal of the beads, the supernatant was further purified by reverse-phase chromatography using a C4 column (Agilent Technologies, Santa Clara, CA, USA). α-syn-containing fractions were collected, aliquoted, lyophilized, and stored at −80 °C until use.

2.2. α-Syn Sample Preparation

Lyophilized α-syn was dissolved in H_2_O that had been pH-adjusted to 8.0 using 1 N NaOH and centrifuged at 20,000 × g for 10 min. The supernatant was filtered through a 0.02 µm filter prior to use. Protein concentrations were determined by UV absorbance at 280 nm using the extinction coefficient calculated from tyrosine content according to the Edelhoch method [33,34].

2.3. Microscopy of α-Syn Mesoscale Assemblies

Mesoscale α-syn assemblies were imaged by fluorescence microscopy under defined pH conditions. Samples contained α-syn at a final concentration of 20 µM in buffers composed of 25 mM sodium phosphate, 25 mM citrate, and 200 mM NaCl, adjusted to pH 3, 5, or 7. Alexa Fluor 647-labeled α-syn was included at a 1:30 labeled-to-unlabeled ratio. α-Syn was dye-labeled using N-hydroxysuccinimide (NHS) ester chemistries with Alexa Fluor 647 (A647; Invitrogen, Carlsbad, CA, USA). Labeling reactions were conducted at a 1:10 protein:dye ratio. Thioflavin T (ThT) was added to each condition at a final concentration of 3 µM. Samples were transferred onto the coverslip of a μ-Dish (35 mm; ibidi, Martinsried, Germany) and sealed with Parafilm to minimize evaporation. Imaging was performed immediately after sample preparation and again following a 12 and 24 h incubation. For all conditions and at all time points, images were acquired using the Nikon Eclipse Ti2 (Nikon, Tokyo, Japan) equipped with a CSU-W1 Confocal Scanner Unit (Yokogawa, Tokyo, Japan) for spinning disk confocal microscopy using a 40X objective using the GFP and Cy5 fluorescent channels at 10% and 55% laser power, respectively. Images were taken in Z-stacks (40 µm thick at 1 µm/slice). Each condition was prepared and imaged in triplicate, and representative images were selected from reproducible samples. All image processing was performed in parallel using the NIS-Elements software (Nikon) consisting of a max intensity projection and cropping of all images to the same region to allow comparison of fluorescent intensities across samples. Image contrast and brightness adjustments were done equivalently for all images. For ThT fluorescent intensity quantification, only regions with Cy5 and ThT colocalization were considered for analysis.

2.4. Dynamic Light Scattering of α-Syn Assemblies

Dynamic light scattering (DLS) measurements were performed using a DynaPro NanoStar II instrument (Wyatt Technology, Waters Corporation, Santa Barbara, CA, USA) equipped with a 658 nm laser to monitor fluctuations in scattered light intensity arising from Brownian motion of particles in solution. All experiments were conducted using unlabeled α-syn under the same buffer conditions used for fluorescence microscopy, consisting of 25 mM sodium phosphate, 25 mM citrate, and 200 mM NaCl adjusted to pH 3, 5, or 7. α-Syn was present at a final concentration of 20 µM.

Samples were prepared containing α-syn alone or supplemented with either toxicity-mitigating compounds (anle138b and EGCG, each at 0.3 and 5 µM final concentrations, respectively) or metal ions, including Fe^3+^ (FeCl_3_), Cu^2+^ (CuSO_4_), or Zn^2+^ (ZnSO_4_), added at final concentrations of 5 or 50 µM. Each condition was prepared in independent triplicates. Measurements were acquired immediately after sample preparation and again following a 12 and 24 h incubation at room temperature.

Samples were loaded into 5 µL plastic cuvettes (Wyatt Technology, Waters Corporation) and measured at 25 °C. For each sample, four acquisitions were collected at 3 s intervals over a 5 min period. Intensity autocorrelation functions were analyzed using the Dynamics software (Wyatt Technology, Waters Corporation). Diffusion coefficients ( $[eqn]$ ) were extracted from the autocorrelation data and converted to hydrodynamic radii ( $[eqn]$ ) using the Stokes–Einstein equation:

$$[eqn]$$

where k is the Boltzmann constant, T is the absolute temperature, and η is the solution viscosity.

Size distributions were initially obtained as intensity-weighted hydrodynamic radius profiles and subsequently converted to mass-weighted distributions using the instrument’s built-in Mie scattering-based model to more accurately reflect the relative abundance of distinct α-syn assembly populations. Reported values represent averages across independent triplicates.

2.5. Statistical Analysis

All experiments were performed using independent triplicates, as specified for each assay. For fluorescence microscopy experiments, each condition was prepared and imaged in triplicate, images were equivalently cropped and analyzed, and representative images were selected from reproducible samples. For DLS measurements, each condition was prepared and analyzed as described above. Quantitative comparisons of oligomeric α-syn populations were assessed using multiple t-tests.

For all bar graph representations, data are reported as mean ± standard error of the mean (SEM), with error bars denoting SEM. For DLS hydrodynamic radius distribution plots, SEM is shown as shaded regions surrounding the mean distribution. Regardless of the analysis method, significance levels were denoted respectively by *, **, ***, or **** for p < 0.05, <0.01, <0.001, or <0.0001. All graphs were generated and statistical analyses performed using the GraphPad Prism software (version 10.4.2 (534); San Diego, California, USA), and no data points from a particular measurement were excluded.

3. Results

3.1. pH-Dependent Emergence of Distinct α-Synuclein Assembly Regimes

α-Synuclein (α-Syn) is known to populate a wide spectrum of self-assembled states, ranging from monomeric and small multimeric species to higher-order oligomers and large aggregates that differ markedly in size, structure, and biophysical properties [35,36]. Conceptually, these assemblies can be broadly grouped into monomeric or low-order multimeric species with hydrodynamic radii on the order of ~1–10 nm, nanoscale oligomeric intermediates typically reported in the ~50–100 nm range, and much larger mesoscale assemblies extending to micron dimensions (≥ 1000 nm) (Figure 1a). While these boundaries are approximate and ensemble-dependent, prior biophysical and single-molecule studies support the existence of α-syn populations spanning these general size regimes [9,11,14,37,38,39].

Because mesoscale α-syn assemblies are readily detectable by fluorescence microscopy, we first examined how pH influenced the formation of large aggregates under our experimental buffer conditions. α-Syn (20 µM) was imaged in buffers containing 25 mM sodium phosphate, 25 mM citrate, and 200 mM NaCl adjusted to pH 7, 5, or 3 (Figure 1b). At pH 7, no detectable aggregate structures were observed by α-syn-647 fluorescence at either the initial time point, or after 12 or 24 h, consistent with a predominantly monomeric protein population. In contrast, at pH 5, discrete α-syn assemblies were evident immediately after sample preparation and evolved over 24 h into large, extended structures that were strongly ThT-positive, indicating the emergence of amyloid-like order within these mesoscale aggregates (Figure 1b, Supplemental Figure S1a). At pH 3, only faint and poorly resolved α-syn assemblies were observed by microscopy, suggesting the presence of smaller and/or less abundant aggregates that fall near or below the detection threshold of this imaging modality (Figure 1b).

These observations motivated the use of DLS to more comprehensively resolve α-syn assembly states across hydrodynamic radius scales, including oligomeric species not readily captured by fluorescence microscopy. DLS measurements revealed that α-syn assemblies under these buffer conditions consistently partition into three major size classes corresponding to the monomeric/low-order multimeric species, nanoscale oligomers, and mesoscale aggregates described in Figure 1a (Figure 1c, Supplemental Figure S1b). At pH 7, the mass distribution was dominated by the monomeric or multimeric population from 0 to 24 h, with only a minor contribution from larger oligomeric species (Figure 1c, Supplemental Figure S1b). At pH 5, α-syn exhibited a markedly broader distribution, with substantial populations in both the nanoscale oligomeric regime and the mesoscale aggregate range, consistent with the prominent ThT-positive structures observed by microscopy (Figure 1c, Supplemental Figure S1a,b). In contrast, at pH 3, α-syn populated nanoscale oligomers and smaller mesoscale species at early time points; however, within 12 h (Supplemental Figure S1b) these higher-order populations diminished and the mass distribution shifted back toward predominantly monomeric species which were maintained through the 24 h time point (Figure 1c).

Quantitative analysis of the DLS data further highlighted the distinct pH-dependent redistribution of α-syn assemblies. When the total population was expressed as the percentage of mass attributed to oligomeric species, α-syn in pH 5 conditions exhibited the highest oligomeric burden at all time points tested, significantly exceeding the corresponding populations at both pH 7 and 3 at 12 and 24 h time points (Figure 1d, Supplemental Figure S1b). At pH 7, oligomeric species accounted for only a small fraction (~8%) of the total mass, consistent with a largely monomeric ensemble. Although samples at pH 3 remained predominantly monomeric overall, a detectable fraction of oligomeric species was present, particularly at the initial time points (Figure 1d), which rapidly transitioned to a more monomeric population by the 12 h time point (Supplemental Figure S1b), thus indicating a possible transient access to higher-order assemblies under strongly acidic conditions.

3.2. Toxicity-Mitigating Compound Effects on α-Syn Oligomer Populations Depend on pH

Toxicity-mitigating compounds that modulate α-syn aggregation have been widely used as chemical probes to perturb assembly pathways and, in some contexts, reduce α-syn-associated toxicity [40,41,42,43,44]. Anle138b has been described as an oligomer/aggregate modulator with in vivo efficacy in synucleinopathy models and structure-dependent binding to pathological α-syn assemblies [40,41]. EGCG has been shown to remodel α-syn amyloid species and redirect aggregation toward alternative, often less toxic endpoints rather than simply eliminating all higher-order assemblies [42,43,44]. We therefore treated α-syn independently with anle138b and EGCG to determine how pH affects the distribution of oligomeric versus mesoscale populations detected by DLS under otherwise matched buffer conditions.

When 0.3 µM anle138b was added to 20 µM α-syn, the resulting mass-weighted hydrodynamic-radius distributions retained pronounced pH dependence (Figure 2a). At pH 7, anle138b-treated samples remained largely monomer-dominated, with a small nanoscale oligomer peak reproducibly detected and persisting after 12 and 24 h (Figure 2a, Supplemental Figure S2a). At pH 5 and 3, anle138b coincided with an emergence of larger assemblies spanning the nanoscale and mesoscale regimes primarily at the initial time point (Figure 2a). When compared quantitatively relative to the matched no-compound control at the same pH and time point, anle138b significantly increased oligomer populations at pH 5 at the initial time point (Figure 2b). At pH 3, anle138b mildly increased oligomeric populations only after 24 h, consistent with stabilization of higher-order populations under strongly acidic conditions (Figure 2b). At pH 7 and 5, however, oligomeric species remained roughly similar to that of untreated controls at both the 12 and 24 h time points (Figure 2b, Supplemental Figure S2a). Across all pHs tested, ThT staining was largely negative, indicating anle138b reduced amyloid-like fibril oligomer formation (Supplemental Figure S2b).

Addition of 5 µM EGCG increased the oligomeric mass fraction immediately after mixing at pH 5 and 3 when compared to matched controls (Figure 2c,d). Within 12 h, however, this trend was lost at both pH 5 and 3. At pH 5, oligomeric species were significantly decreased (Supplemental Figure S2c). While the reduction in oligomers at pH 5 was maintained at the 24 h time point, interestingly, at pH 3, this pattern was inverted, with a significant increase in oligomer population (Figure 2d). These time-resolved findings support that EGCG initially formed larger oligomeric species in acidic conditions, and that these large oligomers are then reshaped to form ThT negative oligomeric species at both pH 5 and 3 ( Supplemental Figure S2d). Prior work indicates that EGCG engages α-syn through distributed interactions with a strong contribution from the N-terminal region and that its aggregation-modulating activity depends on oxidative chemistry that is sensitive to solution conditions, including pH, providing a plausible chemical basis for the divergent effects observed here at pH 5 versus 3 [42,43,44]. Together, these data show that both anle138b and EGCG alter α-syn oligomerization, but the directionality and magnitude of their effects depend strongly on pH and evolve over time.

3.3. Concentration- and pH-Dependent Modulation of α-Synuclein Assemblies by Fe3+

Transition metal ions have long been implicated in α-syn aggregation, and prior work has shown that ferric iron (Fe^3+^) can exert opposing effects on α-syn assembly depending on metal concentration, accelerating aggregation at low Fe^3+^:protein ratios while inhibiting or altering fibril formation at higher concentrations [10]. This prompted us to examine how Fe^3+^ modulates α-syn oligomerization across pH regimes under matched buffer conditions.

At a low Fe^3+^ concentration (5 µM FeCl_3_ added to 20 µM α-syn), mass-weighted DLS distributions revealed distinct, pH-dependent effects on α-syn assembly (Figure 3a). At pH 7, α-syn remained predominantly monomeric from 0 to 24 h, with a modest increase in oligomeric species appearing at 12 h (Figure 3a, Supplemental Figure S3a). Quantification confirmed a small but significant oligomeric peak at pH 7 at 12 h relative to matched controls, although these assemblies remained a minor population (Supplemental Figure S3b). At pH 5, Fe^3+^ treatment produced nanoscale oligomers at early time points and robust ThT-positive mesoscale assemblies after 12 and 24 h (Figure 3a, Supplemental Figure S3a,b). At pH 3, low Fe^3+^ markedly stabilized mesoscale α-syn assemblies (Figure 3b). Distinct peaks in the mesoscale hydrodynamic radius range were evident at both 0 and 24 h, in contrast to metal-free conditions where such assemblies diminished over time (Figure 3a,b). These data support that low concentrations of Fe^3+^ selectively sustain higher-order α-syn assemblies under strongly acidic conditions.

Increasing the Fe^3+^ concentration to 50 µM produced different outcomes, particularly at pH 7 and 3 (Figure 3c). At pH 7, higher Fe^3+^ led to a modest increase in oligomerization, particularly at the intermediate 12 h time point (Supplemental Figure S3c). Interestingly, DLS analysis at the 24 h time point revealed that the resulting size distributions of oligomer populations were notably broad, lacking sharp discrete peaks observed under other conditions, consistent with a heterogeneous ensemble (Figure 3c). At pH 3, high Fe^3+^ redistributed oligomeric species toward the nanoscale regime rather than the mesoscale range seen at 5 µM Fe^3+^ (Figure 3c). Ultimately, higher Fe^3+^ concentrations resulted in early enhanced oligomerization, which was not maintained after 24 h (Figure 3d).

Across both Fe^3+^ concentrations, pH 5 conditions exhibited a largely depleted monomeric α-syn population immediately upon Fe^3+^ addition (Figure 3a,c), a behavior not observed in the absence of metal. Despite this redistribution away from monomeric species at initial time points, the total oligomeric mass fraction at pH 5 did not differ significantly from matched controls (Figure 3b,d), reflecting rapid conversion into large mesoscale aggregates that dominate the assembly landscape after incubation.

3.4. pH- and Concentration-Dependent Modulation of α-Syn Assemblies by Cu2+

Copper has been implicated in α-syn biology and pathology because of its binding interactions with α-syn and because Cu^2+^ alters protein conformation, oligomerization, and aggregation kinetics in a manner that depends on metal stoichiometry and solution conditions [10,45,46]. Biochemical and structural studies demonstrated that Cu^2+^ binds preferentially to the α-syn N-terminus and can either accelerate oligomer formation or redirect aggregation pathways depending on concentration and environment [10,22,45,46]. We therefore tested Cu^2+^ across pH regimes and concentrations.

At low Cu^2+^ concentration (5 µM CuSO_4_ added to 20 µM α-syn), Cu^2+^ did not increase oligomeric populations compared to controls immediately after mixing at pH 7 and 5. Notably, at pH 5 oligomerization was significantly reduced (Figure 4a,b). This suppression was maintained after 24 h, but remained ThT-positive. At pH 7, however, oligomerization increased after 12 and 24 h, but remained ThT-negative. These results suggest a pH dependent effect of Cu^2+^ on α-syn oligomerization and fibrillation (Figure 4b, Supplemental Figure S4a,b).

At pH 3, Cu^2+^ largely depleted the monomeric α-syn population at 0 h, with the distribution dominated by nanoscale oligomeric species (Figure 4a). After 12 h, monomeric species began to form while some nanoscale oligomers remained (Supplemental Figure S4a). After 24 h, the α-syn populations appeared bifurcated, with some populations fully redistributing into monomeric species while others matured into large mesoscale assemblies (Figure 4a). Notably, such mesoscale aggregates were not observed under metal-free conditions at pH 3. These observations indicate that, under strongly acidic conditions, higher-order α-syn assemblies are not robustly maintained in the absence of Cu^2+^ and may either dissociate back into smaller species or fall below the detection window due to sedimentation or instability. In this context, Cu^2+^ appears to stabilize higher-order assemblies that are otherwise poorly sustained under these solution conditions. Consistent with this redistribution, a pronounced and significant increase in oligomeric mass fraction at pH 3 was found in the presence of 5 µM Cu^2+^ at 0 h, which was then lost after redistribution at the 12 and 24 h time points (Figure 4b, Supplemental Figure S4a).

Increasing the Cu^2+^ concentration to 50 µM largely preserved the trends seen at 5 µM while altering the timing and persistence of oligomerization (Figure 4c). At pH 7, higher Cu^2+^ modestly increased oligomerization, reaching statistical significance only at the initial time point (Figure 4d, Supplemental Figure S4c), suggesting that elevated Cu^2+^ promotes early, but not necessarily stable, oligomer formation. At pH 5, the suppressive effect of Cu^2+^ on oligomeric species was sustained, with a significant decrease in oligomeric abundance observed at 24 h, which remained ThT-positive (Figure 4d, Supplemental Figure S4d), consistent with prolonged inhibition or delay of assembly at higher metal concentration. At pH 3, high Cu^2+^ again promoted the formation of large mesoscale aggregates, particularly at the initial time point (Figure 4c,d).

Overall, Cu^2+^ modulates α-syn self-assembly in a manner that is both pH- and concentration-dependent, with particularly strong and divergent effects across acidic conditions. Cu^2+^ can suppress or delay mesoscale conversion at pH 5 while forming early large assemblies at pH 3, highlighting that solution chemistry modulates the apparent direction of copper effects.

3.5. pH-Dependent Modulation of α-Syn Assemblies by Zn2+

Given the distinct and metal-specific effects observed for Fe^3+^ and Cu^2+^, we next examined Zn^2+^ as an additional modulator of α-syn assembly. Zinc has been reported to influence α-syn oligomerization under certain conditions, although its effects are generally more context-dependent and less strongly concentration-dependent than those of redox-active metals [10,46,47,48,49]. We therefore tested Zn^2+^ across the same pH and concentration regimes to determine whether it reshapes α-syn assembly landscapes in a manner similar to or distinct from iron and copper.

At low Zn^2+^ concentration (5 µM ZnSO_4_ added to 20 µM α-syn), DLS measurements revealed pH-dependent changes in α-syn assembly (Figure 5a). At pH 3, Zn^2+^ robustly increased the abundance of oligomeric species relative to metal-free controls, with distinct oligomeric peaks emerging at all time points (Figure 5a,b, Supplemental Figure S5a). Quantitative analysis confirmed significant increases in oligomeric mass fraction at pH 5 at the initial time point only, with a drastic reduction in oligomeric species at 12 h (Supplemental Figure S5a) and no significant change in oligomer population by 24 h (Figure 5b). As expected, these oligomers formed at pH 5 remained ThT-positive (Supplementary Figure S5b). Interestingly, despite Zn^2+^ and Cu^2+^ sharing the same valence, the lack of suppression that 5 µM Zn^2+^ had on late time point oligomerization at pH 5 is distinctly unique from that of Cu^2+^, suggesting the two metals harbor differential outcomes on α-syn oligomerization at pH 5.

Increasing the Zn^2+^ concentration to 50 µM yielded similar assembly profiles across all pH conditions compared with 5 µM Zn^2+^ (Figure 5c, Supplemental Figure S5c). At pH 3, higher Zn^2+^ again promoted oligomer formation relative to controls, with strong contributions from mesoscale species at pH 3 that persisted after 24 h (Figure 5c,d). At pH 5, oligomeric abundance remained largely unchanged after 24 h despite redistribution within the size distribution (Figure 5c,d). These oligomers likewise remained ThT-positive (Supplemental Figure S5d). Thus, Zn^2+^ exhibited comparatively weak concentration dependence in this condition space, with similar oligomeric populations emerging at both 5 µM and 50 µM.

4. Discussion

α-Syn self-assembly cannot be described by a single aggregation trajectory. Instead, it reflects a balance among competing assembly states whose relative stability is conditioned by solution chemistry, measurement timescale, and the readout used to define “aggregation” (Figure 1a). Across the condition matrix examined here, pH establishes the baseline assembly regime, while toxicity-mitigating compounds and metal ions primarily act by redistributing population mass among monomer/low-order species, nanoscale oligomers, and mesoscale assemblies.

Near-neutral pH favors a predominantly monomeric ensemble with only minor nanoscale oligomer populations, whereas pH 5 conditions robustly support progression toward large, ThT-positive mesoscale assemblies over time. A plausible physicochemical basis is that at pH 5, conditions lie close to the reported isoelectric point of α-syn (~4.7), reducing net charge and electrostatic repulsion and thereby lowering the barrier for intermolecular association [37]. We note that pH 5 does not represent a uniform cytosolic condition in vivo, but instead approximates acidic microenvironments such as endolysosomal compartments or stressed intracellular niches [27,28,29]. Accordingly, the compound- and metal-dependent effects observed at pH 5 should be interpreted as context-dependent behaviors that may be relevant only in specific subcellular environments, and their direct translation to physiological or therapeutic settings should be considered cautiously.

At pH 3, α-syn accesses oligomeric and aggregate states early; however, under our control conditions these higher-order populations are not sustained after 24 h. This seemingly paradoxical trend might be explained by the formation of liquid–liquid phase separated condensates forming at initial time points that later mature into smaller densely packed nanosolid-like structures. Because DLS reports only species present within the scattering volume, an apparent reduction in large species may reflect dissociation into smaller assemblies and/or physical loss of large material from the measurement volume (sedimentation or adsorption). Distinguishing between these mechanisms would require additional experiments beyond DLS. Accordingly, we do not interpret the pH 3 regime as evidence of a strictly reversible dissociation process.

This baseline behavior is broadly consistent with prior solution condition studies demonstrating strong pH-dependent shifts in α-syn aggregate morphology and dye binding [9] and with NMR and replica exchange molecular dynamics simulation analyses showing that acidic pH conditions cause reorganization of long-range contacts and the charge distribution within the disordered ensemble [19,20]. Importantly, our microscopy results emphasize that imaging can under-report populations that remain below the optical detection threshold, whereas DLS resolves size distributions spanning nano- to mesoscale regimes under the same buffer conditions. This highlights why studies that rely on a single endpoint (e.g., ThT-only, turbidity-only, or microscopy-only) can reach seemingly conflicting conclusions when the dominant populated size class differs across conditions.

Toxicity-mitigating compounds further illustrate why “inhibition” is not a single mode of action. EGCG decreases the higher-order burden at pH 5 while increasing oligomeric mass at pH 3, consistent with prior work showing that EGCG can redirect amyloidogenic proteins into remodeled, off-pathway oligomers and can remodel mature fibrils rather than abolishing assembly [42,43,44]. In the pH 5 regime, where mesoscale growth is strongly favored, EGCG appears to divert material away from growth-competent large assemblies toward smaller species. In contrast, in the pH 3 regime, where higher-order populations are otherwise not sustained, EGCG stabilizes higher-order species and increases the late-time oligomer/aggregate burden. Anle138b shows its clearest effect after incubation at pH 3, consistent with its established role as an oligomer/aggregate modulator that targets pathological assemblies and slows disease progression in synucleinopathy models [40,41].

For metal ions, pH-dependent behavior is expected because pH controls both the protonation of α-syn ligands (notably Asp/Glu and His) and metal speciation and complexation by solution components. Under our buffer conditions (phosphate/citrate), pH will influence the fraction of metal present as free aqueous species versus ligand-bound complexes, which can contribute to non-linear pH- and dose-dependent outcomes [50,51,52]. Thus, observed metal effects should be interpreted as emergent consequences of coupled changes in the α-syn ensemble and in metal availability/coordination environment rather than as a single direct-binding interaction in isolation.

Within this coupled framework, Fe^3+^ most strongly stabilizes higher-order assemblies under acidic conditions, in line with earlier reports that iron can accelerate aggregation at low stoichiometry while altering or attenuating fibril formation at higher concentrations [10,23]. Notably, at pH 3 we observe a concentration-dependent shift in the dominant size class: low Fe^3+^ stabilizes mesoscale assemblies, whereas higher Fe^3+^ biases the population toward nanoscale oligomers. This behavior is consistent with Fe^3+^ tuning the balance between growth into fewer large assemblies and partitioning into many smaller assemblies, and it provides a mechanistic context for concentration- and condition-dependent outcomes reported across studies performed under different pH values, buffers, and metal:protein ratios.

Cu^2+^ exhibits divergent effects across acidic regimes where it delays mesoscale conversion at pH 5 and, at initial time points, promotes larger assemblies at pH 3. Copper binding to the α-syn N-terminus and its ability to redirect aggregation pathways have been reported previously [24,45,46]. In a pH 5-regime that already favors rapid large-assembly growth, Cu^2+^ may transiently disrupt or compete with assembly-driving intermolecular contacts, consistent with delayed mesoscale emergence at early time points. In contrast, in the pH 3 regime, Cu^2+^ stabilizes higher-order states that are otherwise not sustained. These observations offer a straightforward reconciliation for why copper is sometimes described as accelerating aggregation and sometimes as suppressing fibrillation. The apparent direction of the effect depends on where the baseline pH places the system within the assembly landscape and on the readout used to define “aggregation.”

Zn^2+^ increases oligomerization most clearly at pH 3 (and to a lesser extent at pH 7) while leaving the strongly aggregation-prone pH 5 regime largely unchanged after 24 h. Prior studies report context-dependent effects of Zn^2+^ on α-syn assembly and structure [40,41,42,43]. Our results are consistent with zinc acting as a comparatively weak modulator in this condition space: it shifts oligomer populations in regimes where assembly is not already maximized (pH 7) or where higher-order states are otherwise unstable (pH 3), but it does not measurably suppress the robust mesoscale growth observed at pH 5 at later time points.

Although the present experiments were conducted in vitro, the redistribution among nanoscale oligomers and larger mesoscale assemblies observed here parallels the growing recognition that α-syn pathology in human synucleinopathies is structurally heterogeneous rather than dominated by a single fibrillar species [53]. Postmortem analyses of PD brain tissue indicate that Lewy pathology contains abundant lipid membranes and crowded organellar material rather than consisting solely of densely packed amyloid fibrils [53]. Complementing this, a cellular model that recapitulates Lewy body-like inclusion formation shows that the process of Lewy body formation drives major cellular dysfunction, consistent with inclusion maturation involving more than just fibril accumulation [54]. In parallel, oligomeric α-synuclein species are implicated as pathogenic assemblies, as stabilized α-syn oligomers exhibit pronounced neurotoxicity in vivo [55]. In this context, our mapping of pH- and metal-dependent shifts between transient oligomeric states and more persistent higher-order assemblies provides a physicochemical framework for understanding how local chemical environments could bias α-syn populations toward distinct pathological assembly states in disease-relevant cellular compartments.

Taken together, Figure 6 can be read as a map of pathways rather than a binary inhibitor/activator summary. pH 5 occupies a robust mesoscale-forming regime in which additives mainly delay or remodel the trajectory (green asterisks), whereas pH 3 is the most tunable regime: multiple perturbations convert transient higher-order populations into persistent nano- and/or mesoscale assemblies (red asterisks), increasing the late-time oligomer/aggregate burden relative to matched pH controls. This regime dependence provides a unifying explanation for apparent inconsistencies across the α-syn aggregation literature and emphasizes that meaningful comparisons require explicit reporting (and, ideally, systematic exploration) of pH, buffer ligands, and metal stoichiometry alongside the chosen aggregation readout.

5. Conclusions

This study provides a systematic, side-by-side dissection of how solution pH and metal chemistry jointly shape α-syn assembly outcomes, addressing a common source of inconsistency across the aggregation literature. Using DLS to resolve distinct α-syn assembly populations, we map coexisting size regimes (monomer/low-order species, nanoscale oligomers, and mesoscale assemblies) and show that pH defines distinct behaviors, including particularly robust ThT-positive mesoscale assembly at pH 5. Toxicity-mitigating compounds (anle138b and EGCG) do not exert uniform inhibition. Instead, they redistribute α-syn populations in pH-dependent ways, stabilizing or remodeling higher-order states depending on the chemical environment. Metal ions likewise exhibit pH- and dose-dependent effects. Fe^3+^ stabilizes higher-order species under acidic conditions with a concentration-dependent shift in dominant size class, Cu^2+^ can delay mesoscale conversion at pH 5 while promoting large assemblies at pH 3, and Zn^2+^ enhances oligomerization under acidic conditions without suppressing pH 5 mesoscale growth at later time points. Collectively, these findings shift interpretation of α-syn aggregation from binary endpoints to environment-dependent population redistribution and provide a quantitative framework for comparing studies.