# Investigating the impact of Cu2+ on α-synuclein aggregation: A single-molecule approach

Imad Abrao-Nemeir, Saly Charles-Achille, Bastien Cayrol, Sébastien Balme

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Imad Abrao-Nemeir, Saly Charles-Achille, Bastien Cayrol, Sébastien Balme. Investigating the impact of Cu2+ on α-synuclein aggregation: A single-molecule approach. International Journal of Biological Macromolecules, 2025, 307, pp.142161. 10.1016/j.ijbiomac.2025.142161. hal-05081159

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# Investigating the impact of Cu²⁺ on α-synuclein aggregation: A single-molecule approach

Imad Abrao-Nemeir<sup>a</sup>, Saly Charles-Achille<sup>a</sup>, Bastien Cayrol<sup>b</sup>, Sebastien Balme<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Amyloid

Nanopore

Confocal fluorescence spectroscopy

Single molecule

Alpha synuclein

## ABSTRACT

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the abnormal aggregation of α-synuclein. This study investigated the impact of Cu²⁺ ions on α-synuclein aggregation in oligomer distribution using two single-molecule techniques. The aggregation of α-synuclein monomers with and without Cu²⁺ revealed that Cu²⁺ accelerated the formation of ThT-positive β-sheet structured aggregates. Nanopipettes of varying diameters from 0.7 to 134 nm were employed to characterize the oligomers formed during the lag phase, demonstrating that Cu²⁺ generated a wider range of oligomers from 10 nm³ to 20,000 nm³ over time. Confocal fluorescence spectroscopy analysis of ThT-positive fibrils in the plateau phase showed that Cu²⁺ induces larger oligomers and fewer in number. The introduction of preformed seeds to the control and Cu²⁺-containing samples further accelerated the aggregation. The combination of seeds and Cu²⁺ produced structurally distinct oligomers, with seeds catalyzing the formation of small oligomers that detach from the parent fibers and Cu²⁺, promoting the formation of larger oligomers. These results reveals that seeds and Cu²⁺ act synergistically, with two different aggregation pathways coexisting in the early phase, leading to an intermediate composition of fibers and clusters at the end of aggregation.

## 1. Introduction

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders affecting millions of people worldwide [1]. It is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and the hallmark formation of Lewy bodies, proteinaceous inclusions found within affected neurons. Central to this pathology is α-synuclein, a small, intrinsically disordered protein whose abnormal aggregation into toxic oligomers and insoluble fibrils drives disease progression [2,3]. The aggregation of α-synuclein progresses through a complex pathway consisting of a lag phase, growth (or elongation) phase, and plateau phase [4,5]. Initially, in the lag phase, monomeric α-synuclein molecules undergo subtle structural rearrangements and begin to interact, forming small oligomeric nuclei [6]. Molecular events within the lag phase remain poorly understood because of the transient, low-concentration nature of the intermediates involved. As stable and proaggregative nuclei are formed, the aggregation shifts into the growth phase, during which the rapid increase in fibril concentration structured in β-sheets is detectable by classical Thioflavin T (ThT) fluorescence. The aggregation process reaches the plateau phase,

where the fibril formation rate slows down as monomeric and aggregated α-synuclein reach a steady-state equilibrium. At an early stage, the formation of nuclei can follow two mechanisms. Primary nucleation in amyloid aggregation refers to the formation of new aggregates from monomers, whereas secondary nucleation involves the generation of new aggregates on the surface of existing aggregates [7,8]. This process can lead to the production of toxic oligomers, making them key targets for therapeutic development [8]. The chiral specificity of secondary nucleation has been demonstrated, with the process observed only in self-seeding experiments [9]. Direct observations of Aβ42 aggregation have provided mechanistic insights into secondary nucleation, revealing that this is a catalytic multistep process involving the attachment of soluble species to the fibril surface [10]. Several metals ion have been shown to accelerate α-synuclein aggregation such as Cu²⁺ [11]. The latter is an essential cofactor for numerous enzymes such as cytochrome c oxidase. It is also involved in the synthesis of neurotransmitters such as dopamine, whose deficiency is a hallmark of PD. The interaction between Cu²⁺ and α-synuclein is particularly significant because of the high-affinity binding at the N-terminus, which enhances fibrillation kinetics through specific structural interactions [12]. This leads to

<footnote><sup>a</sup> Institut Européen des Membranes, UMR5635 University of Montpellier ENSCM CNRS, Place Eugène Bataillon, 34095 Montpellier cedex 5, France</footnote>

<footnote><sup>b</sup> PHIM Plant Health Institute, Univ Montpellier, INRAE, CIRAD, Institut Agro, IRD, 34000 Montpellier, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: sebastien.balme@umontpellier.fr (S. Balme).</footnote>
cytotoxic fibrils due to the increased β-sheet content [13]. However, the effect of Cu²⁺ on α-synuclein aggregation is complex and not fully understood, as demonstrated by its dual role in promoting the aggregation of fibrillar structures at low concentrations but inhibiting fibrillization at higher concentrations [14]. In addition, Cu²⁺ has been reported to promote the formation of annular α-synuclein oligomers [15] and amyloid pores [16]. Conversely, Cu²⁺ induces conformational changes that stabilize initial β-sheet formation, facilitating subsequent misfolding and aggregation [17]. It could also disrupt fibril elongation, leading to the formation of shortened fibrils instead of the typical elongated structures [18]. Cu²⁺ does not affect the seeded aggregation kinetics, suggesting that it primarily affects secondary nucleation rather than primary nucleation [19]. In summary, despite the evidence to accelerate α-synuclein aggregation [13], the impact of Cu²⁺ on α-synuclein aggregation the does not make consensus remains a challenge.

Here, we address the question about the effect of Cu²⁺ on α-synuclein
aggregation with a specific focus on the evolution of oligomer distri-
bution. Solving this question requires a detailed description of the spe-
cies formed during the lag phase, which remains particularly
challenging to characterize owing to the polymorphic and transient
nature of the oligomers formed. Unlike fibrils found in the plateau
phase, these early stage oligomers are Thioflavin T (ThT)-negative due
to the lack of a mature β-sheet structure. Traditional continuous tech-
niques and static characterization fail to capture early stage oligomers
and their evolution over time, leaving a significant gap in the under-
standing of Cu²⁺-mediated effects on aggregation dynamics and the role
of seeds in the oligomer distribution. To bridge this gap, we propose a
continuous measurement approach to characterize these early oligomers
in real time during the lag phase, using a single nanopore technique
[20,21]. Nanopores are analytical devices for label-free single-molecule
detection of particles ranging from DNA to protein to nanoparticles
[22–25]. Nanopipettes offer a powerful and cost-effective tool for
investigating prion-like aggregation in the early phases, as they allow
precise monitoring of oligomer formation and structural transitions at
the single-molecule level [26,27]. They allow the detection of individual
oligomers for each translocation event, generating unique current

blockades that correlate with the volume of the detected species [28]
(Fig. 1a). Recently, our group reported a method that involves incu-
bating α-synuclein directly in the reservoir of a nanopipette and
detecting the aggregate formed in real time [21]. This method is
particularly useful for capturing transient oligomers in the lag phase,
which are Thioflavin T-negative and thus invisible to traditional
detection methods. To complete the methodology and specifically probe
β-sheet-structured aggregates, confocal fluorescence spectroscopy is
particularly interesting [29,30]. This technique consists to record the
fluorescence emitting photo along the time. As nanopipettes, this single-
molecule technique involves measuring individual fluorescence bursts
corresponding to the passage of one aggregate through the confocal
volume. The analysis of each fluorescence burst allows us to obtain in-
formation on the size of the oligomers and draw a map of individual
object (Fig. 1b). Compared to the usual analysis by fluorescence corre-
lation spectroscopy [31,32] or diffusion light scattering (DLS) [33] that
provides a mean distribution of species and required for DLS a high
concentration of aggregates, this approach provides more precise in-
formation on the heterogeneity of the β-sheet structured oligomer. The
combination of these two single-molecule approaches provides valuable
complementary information that confirms the secondary nucleation
mechanism of αβ-42 [34].

The present study aims to investigate the effect of Cu²⁺ on the evo-
lution of α-synuclein aggregates distribution over the time. To this end,
we will use nanopore sensing to probe all the species independently their
structure produced during the first hours of aggregation. Then, confocal
fluorescence spectroscopy will be used to map the β-sheet-structured
aggregates at the plateau phase (Fig. 1). After discuss the effect of Cu²⁺
on the aggregation of α-synuclein monomers, the same methodology
will be employed to characterize the species formed when preformed
seed are added to monomer. This second part of this study aim to state
about the effect of the Cu²⁺ on secondary nucleation, which is a point
still in debate.

## 2. Materials and method

2.1. Protein solubilization and seed production

α-Synuclein monomers were purchased from ND Biosciences (Laus-
sanne, Switzerland). The solubilization of α-synuclein monomers in 1 ×
PBS at 4 °C was performed according to a previously described protocol
[21]. The concentration of the monomer solution was determined by
measuring absorbance at 280 nm using a JASCO spectrophotometer.
The solution was then aliquoted to a final concentration of 36 µM and
stored at –80 °C until use. To produce seeds used for seeding experi-
ments, wild-type recombinant α-synuclein monomers (stock solution 58
µM in PBS 1×) were diluted to 50 µM in PBS with 75 µM of ThT in low-
binding Eppendorf tubes and incubated at 37 40 °C under shaking 500
rpm to allow aggregation. After 15 days of incubation, when the ThT
curve reached a plateau, the solutions were diluted in PBS to a con-
centration of 2 nM and stored at –80 °C until the seeding experiments.
For microscopy of α-synuclein seeds, samples were deposited onto For-
mvar carbon-coated grids, negatively stained with freshly filtered 2 %
uranyl acetate, and then dried. TEM images were obtained using a JEOL
1400 electron microscope at an accelerating voltage of 80 kV.

## 2.2. Characterization of aggregation kinetics

The recombinant α-synuclein monomers (stock solution 58 µM in
PBS 1×) were diluted to 30 µM in PBS with ThT 45 µM. The kinetics of
aggregation of monomer, monomer + Cu²⁺ equimolar, monomer + seed
10 %, and monomer + Cu²⁺ equimolar + seed 10 % were measured in
tubes at 40 °C with stirring 500 rpm. The fluorescence was recorded
using a Qubit fluorimeter. After 14 days, when a plateau was reached for
all samples, the samples were investigated by confocal fluorescence
spectroscopy.

<figure><img src="image_4.png" /><figcaption>Fig. 1. a) Sketch of nanopipette experiments and current blockade parameters
b) Sketch of the experimental confocal fluorescence spectroscopy and fluorescence burst parameters.</figcaption></figure>
## 2.3. Confocal fluorescence spectroscopy

The confocal fluorescence spectroscopy was done performed using a laboratory-made setup. The excitation was provided by a collimated laser diode module (Thorlabs, model CPS450) with a wavelength of 450 nm, which was focused using a UPlanApo 60×/1.20 objective (Olympus). The emitted fluorescence was separated using a 488 nm laser BrightLine single-edge laser dichroic beam splitter (Di02-R488-25 × 36, Semrock) and collected using a Tau-SPAD (Picoquant) connected to a field-programmable gate array (FPGA) card (EM acquisition card, National Instruments PCI-7830R R Series Multifunction RIO). The emitted photons were binned with a time scale of 10 ms, and the photon trace was analyzed using the custom-made LabVIEW software. To detect fluorescence bursts, a threshold of 4 σ (σ is the standard deviation of the baseline signal) was used. The fluorescence burst was characterized by its intensity relative to the number of photons on the peak, time taken at the peak base, and area, which is the integration of the total photons emitted during the fluorescence burst. Dwell time calibration was performed using fluorescent beads with diameter 0.2 µm (fluoresbruite YG microsphere, polysciences) and 0.03 µm (Latex beads, carboxylate-modified polystyrene, fluorescent yellow-green, Merck (see ref. [34]). The diameter of the beads was controlled using dynamic light scattering (DLS, Nanophox).

## 2.4. Characterization of oligomer in the lag phase by nanopipette

Nanopipettes were produced by pulling them using p-2000 with several pulling parameter programs to obtain various diameters from 7 nm to 134 nm (see Section S1: nanopipette pulling conditions, Table S1). After pulling, the nanopipette was activated using oxygen plasma at 100 W for 4 min (Henneker 100). They were then filled with deionized and degassed water following the method previously reported [35]. The nanopipette conductivity and rectification factor were then measured using a solution of 1 M NaCl with PBS by measuring the resulting IV curve from -100 to +100 mV and from -1 to +1 V, respectively. After finishing the experiment, the nanopipette was filled with a solution of rhodamine to measure the angles using epifluorescence microscopy (Leica DM6000, with objectives 100×, Olympus). The resulting nanopipette conductivity (G) and angles (α) were used to calculate the radius ($r_p$) of the nanopipette using the following equation:

$$r_p = \frac{G}{\kappa\pi\tan(\alpha) + 0.25} \quad (1)$$

where the conductance (κ) of the solution were measured using conductometer (HANNA, Edge EC, HI2003). The incubation and detection of oligomer in situ inside the nanopipette was performed as previously reported by Meyer et al. 2022 [21]. A solution of α-synuclein monomer at 100 nM monomers in PBS 1× pH =7.4, 1 M NaCl, with or without CuCl₂ (100 nM) and/or 200 pM (equivalent monomer) of sonicated seeds is injected into the nanopipette reservoir.

A resistive pulse experiment was conducted to detect and characterize the resulting aggregates every 30 min. It consisted of applying a fixed voltage of +500 mV for 7.5 min across the nanopipette. The current was recorded using an EPC10 Quatro or Duo patch-clamp amplifier (HEKA, Lambrecht, Germany) with a sampling rate of 20 µs. The signal was filtered using a Bessel filter at 10 kHz. The resulting current traces were then analyzed by a custom program created using Labview software (Peak Nano Tool), where the signal was further filtered using a Butterworth filter of 5 kHz (order 3). The baseline fluctuation was corrected using a Savitzky-Golay filter (Order 1). Event detection was performed using 8 σ (σ is the standard deviation of the baseline signal) with the maximal amplitude recorded (ΔI/I₀). The events were characterized by their relative maximum blockade amplitudes (ΔI/I₀) and dwell times (Δt). From the ΔI/I₀ and geometrical parameters of the nanopipette, the volume of each individual oligomer was estimated

following the methods previously reported by Charles-Achille et al. [28] using MATLAB custom scripts (Matlab2021a).

## 3. Results and discussion

First, we investigated the effect of Cu²⁺ ions on α-synuclein aggregation (Fig. 2). To this end, we evaluated the effect of Cu²⁺ on the aggregation kinetics of α-synuclein under two conditions: a control condition containing only monomers (30 µM) and a condition in which Cu²⁺ was added at equimolar concentrations. The formation of ThT-positive β-sheet-structured aggregates was monitored by measuring emitted fluorescence. Under the experimental conditions, the increase in fluorescence for the control condition commenced after 5 d of incubation, whereas in the presence of Cu²⁺, an increase in fluorescence was observed after 2 d of incubation. This result corroborates the promoter effect of Cu²⁺, which has been commonly reported in the literature [36,37]. The second set of experiments was conducted under similar conditions with the addition of 10 % of seeds. Under these conditions, Cu²⁺ acts as a promoter by reducing the lag time (Fig. 2).

### 3.1. Impact of Cu²⁺ on aggregation of α-synuclein monomers

To further evaluate the impact of Cu²⁺ on α-synuclein aggregation, we examined oligomer composition during the initial hours of aggregation and at the plateau phase. The primary advantage of utilizing nanopipettes is their ability to readily adjust their diameter by manipulating the pipette puller parameters and selecting the internal diameter of the quartz capillary. Generally, each nanopipette size had an optimal range for detecting the aggregates, as previously demonstrated. Employing nanopipettes with small diameters allows the detection of small aggregates (< 15 nm in diameter), although they may become obstructed by larger aggregates. Conversely, nanopipettes with a larger diameter (here, larger than 15 nm) are less susceptible to clogging but will only detect larger oligomers, potentially overlooking smaller aggregates. Additionally, because of the limited detectable diameter range of a given nanopipette [28], it was essential to employ a set of nanopipettes with different diameters to draw a comprehensive map of the oligomers. Therefore, we utilized a set of nanopipettes with diameters ranging from 7 nm to 60 nm (see Tables S2 and S3).

The detection of aggregates formed during the initial 8 h (480 min) of aggregation was conducted at 30-min intervals by applying a voltage of 500 mV. Fig. 3a illustrates examples of the current traces obtained under various experimental conditions at different time points. Blockages were detected from the current traces, and their relative amplitudes were measured (Fig. S1). By calculating the nanopore diameter from the conductance measured in 1 M NaCl and the angle measured by fluorescence microscopy, it was possible to estimate the volume of the detected oligomer using the following relationships (see section S4: Calculation of oligomer volume from the relative current blockade distribution and reference [28]). Initially, aggregation of α-synuclein monomers in a solution of NaCl and PBS at pH 7 was investigated. The aggregate volume map is shown in the Fig. 3b. Utilizing a set of nanopipettes with diameters up to 25 nm, a limited number of aggregates were detected during the first few hours of the experiment, with volumes ranging from 15 to 1000 nm³ (Fig. 3c). Oligomers were observed within the first 30 min of the incubation. In experiments conducted with larger-diameter nanopipettes, no events were detected. This did not preclude the formation of larger oligomers; however, under the experimental conditions, their concentration was insufficient for detection. The corresponding diameters ranged from 3 to 12 nm according to the volume distribution of the oligomers. These values were consistent with those reported in the literature [38–40]. Events were observed in five nanopipettes when α-synuclein monomers aggregated with Cu²⁺ at equimolar concentrations, with radii ranging from 7 to 62 nm (Fig. S2 and S3). The primary observation (Fig. 3e and f) was that the frequency of events was significantly higher than that in the control condition (>0.1
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** a) Aggregation kinetics of α-synuclein monomer in microtubes in the absence (red) and in the presence (blue) of Cu²⁺ b) Aggregation kinetics of α-synuclein monomer in microtubes with 10%seeds harvest in the plateau phase (see SI) in the absence (red) and in the presence (blue) of Cu²⁺.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Characterisation of α-synuclein oligomers produced from a α-synuclein monomer only during the lag phase Example of current traces recorded after inclusion of α-synuclein 3 h inside the nanopipette a) without and d) with Cu²⁺. Distribution of α-synuclein oligomer volume detected using nanopipette with diameter below 15 nm b) and >15 nm c) at a function of incubation time. Distribution of α-synuclein oligomer volume detected using nanopipette with diameter below 15 nm e) and >15 nm f) at a function of incubation time.</figcaption></figure>

events/s). Oligomers with volumes between 10 and 100 nm³ were detected during the first 30 min of the incubation. Over time, the volume distribution broadened, ranging from 10 to 500 nm³ between 90 and 240 min of incubation. After 240 min of incubation, oligomers with volumes smaller than 100 nm³ were rarely detected. A population of oligomers with volumes between 500 nm³ and 3000 nm³ (corresponding to diameters of 10 and 18 nm, respectively) emerged after 30 min of

incubation. Over time, the volume distribution of the oligomers expanded to reach 20,000 nm³ (equivalent to a diameter of 34 nm). It is also noteworthy that the frequency of observed events decreased to between 0.05 and 0.1 events/s, which can be attributed to the lower concentration and coefficient of these oligomers. Overall, the median volume increased with the incubation time.

We investigated the composition of the ThT-positive fibrils in the
plateau phase. To accomplish this, we analyzed the samples at T = 13 d using confocal fluorescence spectroscopy. In both cases, fluorescence traces exhibited bursts of fluorescence induced by the passage of β-sheet-structured fibers through the confocal volume (Fig. 4a and b). By plotting the intensity versus the passage time of each detected burst across triplicate experiments, we observed significant dispersion in residence times for both experimental series (Fig. 4c). This observation is consistent with the heterogeneous fibril composition. Notably, for the control sample, 95.5 % of events had residence times of <1 s. In contrast, for the sample incubated with Cu²⁺, 30.5 % of detected bursts had residence times exceeding 1 s (Fig. 4d). Additionally, we observed a significant decrease in the capture rate of the sample containing copper compared to the control (2 ± 1 min⁻¹ versus 10 ± 3 min⁻¹, respectively). These results suggest that during the lag phase, the presence of Cu²⁺ influences aggregation dynamics. To further analyze these bursts, we attempted to estimate the size of the detected aggregates by calibrating the diffusion coefficient (Dt) and friction coefficient (see supporting information of ref. [34]). Based on the residence times and their distribution, we deduced that the control sample was composed of fibrils and clusters with a friction coefficient generally lower than that of a nanoparticle (NP) with a diameter of 200 nm. The addition of copper leads to a heterogeneous cluster composition with friction coefficients similar to those of NPs with diameters of 500 nm to 1 µm. Upon comparing the different oligomers formed during the initial hours (negative ThT) and those released during the plateau phase (14 days), we observed that Cu²⁺ generated more oligomers over time with a wider range of distribution. This diversity of oligomers may partially explain the accelerated onset of the elongation phase, as it implies that the oligomers formed were end-on. Previous research has demonstrated that Cu²⁺ binds tightly to α-synuclein, promoting aggregation by incorporation into amyloid fibers and altering their morphology and chemical properties [41]. It also stabilizes α-synuclein interactions, promotes the formation of annular oligomers, and enhances aggregation by altering protein conformation and increasing β-sheet content [15]. It has been reported that Cu²⁺ strains α-synuclein monomer conformation through macrochelation, disrupting fibril elongation while enhancing nucleation, leading to the formation of shortened α-synuclein [18]. However, analysis of the ThT-positive oligomers sampled in the plateau phase at 14 days demonstrated that the presence of Cu²⁺ induced larger oligomers and consequently fewer in number, given that the experiments were conducted at a constant monomer concentration. These findings are consistent with those obtained through nanopore analysis. Cu²⁺ stabilizes the pro-aggregative β-sheet structure. This phenomenon could potentially result in a reduction in the observed latency phase, as Cu²⁺

was present. Furthermore, it is plausible that these β-sheet-structured
aggregates would rapidly reassemble into clusters due to their abun-
dance and hydrophobicity. At this stage, it is pertinent to consider
whether these clusters are formed by the assembly of small, independent
oligomer units or whether they grow from fibers induced by a secondary
nucleation mechanism.

3.2. Impact of Cu²⁺ ions on the secondary nucleation

Various studies have established that secondary nucleation mecha-
nisms can induce two types of oligomers [34,42]. The first population
adopts a β-sheet conformation and remains attached to a parent fiber,
whereas the second population is released. To address the question of
the impact of Cu²⁺ on the secondary nucleation, we conducted a sub-
sequent series of experiments by introducing 10 % preformed seeds to
both the control (α-synuclein monomer 100 nM) and the sample con-
taining Cu²⁺ (equimolar to the α-synuclein monomer). These seeds were
fibrils obtained after 14 days of aggregation and sonicated to disrupt the
clusters and obtain individual fibril of hundred nm scale (characteriza-
tion is reported in section S5 seed characterization). Monitoring the
aggregation kinetics demonstrated that the addition of seeds to the
control reduced the lag phase to 4 days (Fig. 1). This phenomenon is well
documented and can be attributed to the catalytic role of preformed
fibers through a secondary nucleation mechanism [8,43]. The addition
of Cu²⁺ also accelerated the formation of ThT-positive aggregates, with
a lag phase of less than one day compared to two days without seeds
(Fig. 1a and b respectively).

Utilizing the previously described methodology, we characterized the volumes of aggregates, regardless of their structure, formed during the initial 8 h using a set of nanopipettes with diameters ranging from 7 to 134 nm. As previously, the current blockade (Fig. S7 to S10) were extracted from the recorded current traces (Fig. 5a and d). In experiments containing 10 % seeds, two distinct populations of oligomers were observed after 30 min of incubation (Fig. 5b and c). The first population, with volumes ranging from 10 nm³ to 500 nm³, corresponded to small oligomers with diameters between 3 and 10 nm. The second-largest population, with volumes ranging from 2000 nm³ to 20,000 nm³ (diameters 16 nm to 34 nm), was detected concurrently. Comparing the oligomer distribution (Fig. 2b and c) with that obtained without seeds (control condition), we observed a higher frequency of events and a larger oligomer population. This finding suggests that during the lag phase, the seeds catalyze the formation of small oligomers that detach from the parent fibers. For α-synuclein, these oligomers were larger than those formed without the promotion of secondary nucleation induced by

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Characterization of α-synuclein fibrils produced from a α-synuclein monomer only at the plateau phase. Example of experimental fluorescence traces recorded a) without and b) with Cu²⁺. Map of fluorescence burst parameter recorded from the aggregation performed a) without and b) with Cu²⁺.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Caracterisation of α-synuclein oligomers produced from a α-synuclein monomer with seeds 10 % during the lag phase Example of current traces recorded after incubation of α-synuclein 3 h inside the nanopipette a) without and d) with Cu²⁺. Distribution of α-synuclein oligomer volume detected using nanopipette with diameter below 15 nm b) and >15 nm c) at a function of incubation time. Distribution of α-synuclein oligomer volume detected using nanopipette with diameter below 15 nm e) and >15 nm f) at a function of incubation time.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Characterization of α-synuclein fribrils produced from a α-synuclein monomer with seed 10 % at the plateau phase. Example of experimental fluorescence traces recorded a) without and b) with Cu²⁺. Map of fluorescence burst parameter recorded from the aggregation performed a) without and b) with Cu²⁺.</figcaption></figure>
seeds. This interpretation is consistent with previous studies demon-
strating that secondary nucleation involves the formation of two olig-
omer populations: one that remains on the fiber, while the other that
does not adopt a favorable conformation is released into the solution
[6,34,42]. Notably, the addition of Cu²⁺ significantly altered the size
distribution of oligomers (Fig. 5e and f), with the minimum detected
volume ranging from 100 nm³ to 2000 nm³ (diameters of 7–16 nm) from
the initial minutes of aggregation. In other words, oligomers below 200
nm³ observed for the control with seed or Cu²⁺ alone were not detected.
After 90 min, a second population of larger oligomers (between 2000
nm³ and 40,000 nm³, corresponding to diameters of 16–42 nm) emerged. After 360 min, a population of oligomers between 100 and 2000 nm³ was no longer detected. These results underscore the signifi-
cance of seed addition and thus, secondary nucleation in the aggregation
process. The absence of oligomers with volumes below 100 nm³ suggests
that they may exist at concentrations that are too low to be detected,
whereas they are generally predominant under other conditions.
Therefore, it is plausible that the seeds and Cu²⁺ act synergistically to
produce structurally distinct oligomers.

We analyze now the composition of the ThT-positive fibrils consti-
tuting the plateau phase using confocal fluorescence spectroscopy
(Fig. 6a and b). As in the experiments conducted without the addition of
seeds, the proportion of bursts longer than 1 s and their frequencies were
analyzed (Figs. 6c). The addition of seeds slightly increased the fre-
quency of recorded events (8.4 bursts/min compared to 7 for the ex-
periments without seeds) corresponding with the ratio of fluorescence
bursts longer than 1 s (6.5 ± 2 compared to 4.5 ± 1 for the experiments
without seeds). This result is consistent because the addition of seeds can
modify the final composition of aggregates, particularly because sec-
ondary nucleation induces fiber growth on the parent fiber, thus
explaining the higher proportion of clusters. When Cu²⁺ was added
(Fig. 6d), it was observed that the ratio of long events was 18 %,
compared to 30 % without seeds and 6.5 % with only seeds. In addition,
the frequency of events was approximately 10 bpm. This result dem-
onstrates the role of seeds alongside Cu²⁺ in the production of ThT-
positive oligomers, the size distribution and concentration of which
differ from those in experiments conducted without seeds. The results
obtained from the two single-molecule techniques were combined to
elucidate the role of seeds and Cu²⁺ in alpha-synuclein aggregation. The
addition of seeds to the control accelerated the transition to plateau
phase. At the onset of aggregation, the presence of more numerous and
larger aggregates indicates the catalytic effect of the seeds, similar to
what has been previously reported for Aβ, producing oligomers that are
released into the solution. At the end of the aggregation, the ratio of
large clusters supports the hypothesis that growth on the fibers occurs
during secondary nucleation. The addition of Cu²⁺ affects both the early
and plateau phases of aggregation. In the early phase, a wide range of
aggregates were observed, similar to the experiment conducted without
seeds, and no small aggregates were detected. This suggests a combined
effect of the seeds and Cu²⁺. Given that, in the plateau phase, the cluster
ratio is intermediate between the experiments with only seeds and only
Cu²⁺, it is plausible that both effects coexist independently. In this
scenario, the seeds would catalyze the formation of oligomers with
structures different from those formed by primary nucleation with Cu²⁺.
Thus, two different aggregation pathways coexist in the early phase,
resulting in an intermediate composition of fibers and clusters at the end
of aggregation.

4. Conclusion

This study provides valuable insights into the complex interplay
between Cu²⁺ ions and α-synuclein aggregation, by combining two
single-molecule approaches, nanopipette and confocal fluorescence
spectroscopy techniques. Cu²⁺ accelerates α-synuclein monomer ag-
gregation. It promotes the formation of larger, more diverse oligomers
during early aggregation stages. Cu²⁺ stabilizes the pro-aggregative

β-sheet structure that can explain the observed lag phase decrease. Additionally, the abundance and hydrophobicity of these β-sheet-structured aggregates may cause them to rapidly reassemble into clusters. The combination of Cu²⁺ and seeds leads to a synergistic effect, producing structurally distinct oligomers and altering the size distribution of the aggregates. For the fibrils present in the plateau phase, Cu²⁺ induces the formation of larger, less numerous oligomers, whereas seeds promote the growth of fibers through secondary nucleation. The coexistence of two different aggregation pathways in the presence of both Cu²⁺ and seeds results in an intermediate composition of fibers and clusters at the end of aggregation. These findings contribute to our understanding of the mechanisms underlying α-synuclein aggregation and the role of Cu²⁺ in this process with the access to novel information about the size distribution at an early stage. On the other hand, the methodology that combines the two single-molecule approaches provides complementary information for a better understanding of aggregation process since they allowing continuous measurement. They can be extended to investigate the effects of chemical or physical factors on the aggregation of numerous prion-like protein and peptide with a specific focus on the lag phase.

CRediT authorship contribution statement

Imad Abrao-Nemeir: Investigation, Formal analysis. Saly Charles-Achille: Writing – review & editing, Investigation, Formal analysis.

Bastien Cayrol: Investigation. Sebastien Balme: Writing – original draft, Supervision, Methodology, Investigation, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgements

This study was funded by the Michael J Fox Foundation for Parkinson's Research (grant ID: MJFF-021093).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2025.142161.

Data availability

Data will be made available on request.

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