Cerium Oxide Nanoparticles Rescue α-Synuclein-Induced Toxicity in a Yeast Model of Parkinson's Disease

Abstract

Over the last decades, cerium oxide nanoparticles (CeO₂ NPs) have gained great interest due to their potential applications, mainly in the fields of agriculture and biomedicine. Promising effects of CeO₂ NPs are recently shown in some neurodegenerative diseases, but the mechanism of action of these NPs in Parkinson's disease (PD) remains to be investigated. This issue is addressed in the present study by using a yeast model based on the heterologous expression of the human α-synuclein (α-syn), the major component of Lewy bodies, which represent a neuropathological hallmark of PD. We observed that CeO₂ NPs strongly reduce α-syn-induced toxicity in a dose-dependent manner. This effect is associated with the inhibition of cytoplasmic α-syn foci accumulation, resulting in plasma membrane localization of α-syn after NP treatment. Moreover, CeO₂ NPs counteract the α-syn-induced mitochondrial dysfunction and decrease reactive oxygen species (ROS) production in yeast cells. In vitro binding assay using cell lysates showed that α-syn is adsorbed on the surface of CeO₂ NPs, suggesting that these NPs may act as a strong inhibitor of α-syn toxicity not only acting as a radical scavenger, but through a direct interaction with α-syn in vivo.

Keywords: Parkinson’s disease; amyloid aggregates; cerium oxide nanoparticles; cluster of lipid vesicles; nanoceria; neurodegenerative disease; oligomer detoxification; yeast model; α-synuclein.

Figure 1

CeO₂ NPs strongly increase the viability of yeast cells expressing human α-syn. (a) Serial dilution spot assays (10-fold input cell dilutions ranging from 10¹ to 10⁴) were performed on wild-type (WT) and HiTox (PD) strains. Cells were spotted on agar plates containing glucose (SD; ‘repressing’ conditions) or galactose (SGal; ‘inducing’ conditions) as a carbon source, and growth was assessed after 48 h at 28 °C. The extreme toxicity of α-syn expressed in the PD model under the control of the galactose-inducible promoter is shown in the right panel. (b) Dose-response plot of the protective effects of CeO₂ NPs against α-syn toxicity determined by clonogenic assay. The number of colony-forming units (CFU) developed on agar plates at 28 °C for 72 h were recorded and counted manually. Data are the mean ± SD of three independent experiments performed at least in triplicate. Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. (c) α-Syn-expressing cells were grown with or without ASNPs (50 ng/μL) or CeO₂ NPs (50 ng/μL) for 48 h at 28 °C prior to observation. Large mother cells are indicated with black arrows. Phase-contrast microscopy images were shown. CNT indicates the control (untreated) sample, and scale bars were set at 5 μm.

Figure 2

The interaction between CeO₂ NPs and plasma membranes can promote the beneficial effects of these NPs in yeast. (a) α-Syn-expressing cells were grown with or without ASNPs (100 ng/μL) or CeO₂ NPs (50 ng/μL) for 4 h at 28 °C prior to microscopy observations. CNT indicates the control (untreated) sample and scale bars were set at 5 μm. (b) Flow cytometry (FC) analysis of α-syn-expressing cells grown on SGal medium with or without CeO₂ NPs for different times of incubation (24–48 h). A schematic representation of SSC signal detection by FC was illustrated (right panel) in which an optical detector measures light scatter at a ninety-degree angle relative to the laser beam of FC. (c) Yeast cells untreated (left) or treated (right) with lyticase were exposed to CeO₂ NPs (50 ng/μL) for 4 h at 28 °C and then stained with calcofluor white. Phase contrast (left-side) and fluorescence (right-side) microscopy images were shown. Scale bars were set at 5 μm.

Figure 3

CeO₂ NPs affect α-syn localization and toxic foci accumulation. (a) Microscope observation of α-syn-GFP-expressing cells (HiTox strain) grown with or without CeO₂ NPs (50 ng/µL) for 48 h at 28 °C. For each sample, phase contrast (left) and fluorescence (right) microscopy images were shown. CNT indicates the control (untreated) sample, and scale bars were shown at 5 µm. (b) Yeast cells were treated with different concentrations of CeO₂ NPs (10–100 ng/µL) for 48 h at 28 °C and the percentages of cells with α-syn cytoplasmic foci were quantified relative to the total number of cells. Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test (**, p < 0.01; ****, p < 0.0001). (c) Yeast cells were treated as indicated in (b) and the percentages of cells with α-syn associated to the plasma membrane (PM) were quantified relative to the total number of cells. (d) Representative results of a dot blot analysis performed with an anti-α-syn antibody on whole cell extract samples (10 µg total protein each) derived from untreated (1) or NP-treated samples [CeO₂ NP concentrations: 25 ng/µL (2); 35 ng/µL (3); 50 ng/µL (4)]. Immunoreactivity with the constitutively expressed phoshoglycerate kinase enzyme (Pgk1) served as a loading control.

Figure 4

CeO₂ NPs restore mitochondrial morphology and decrease ROS production in α-syn-expressing cells. (a) HiTox strain transformed with a construct for the expression of mitochondrial-localized RFP was grown in ‘inducing’ medium (SGal) for 24 h with (right) or without (left) CeO₂ NPs (50 ng/µL). For each sample, phase contrast (left-side) and fluorescence (right-side) microscopy images were shown. CNT indicates the control (untreated) sample, and scale bars were set at 5 µm. (b) Yeast cells were grown in SGal medium for 4 h with or without CeO₂ NPs (50 ng/µL). Active mitochondria in α-syn-expressing (GFP-positive) cells were monitored by MitoTracker Deep Red (MTDR) fluorescence using flow cytometer analysis in APC channel. Representative histogram plots were shown, and the percentages of MTDR-positive mitochondria in α-syn-expressing cells were indicated for each sample. (c) After NP exposure for 4 h, the cells were stained with CellROX^® Orange Reagent and visualized with fluorescence microscopy. CellROX-positive cells were scored. A representative microscope image is shown above the graph. Significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; ***, p < 0.001).

Figure 5

α-Syn is adsorbed on the surface of CeO₂ NPs. Yeast protein extracts obtained from HiTox (PD) and WT strains were used for an in vitro NP-protein binding assay. Adsorbed proteins from HiTox and WT strains (PD_(a) and WT_(a), respectively) were recovered from CeO₂ NP surface by a centrifugation-washing procedure and analyzed by dot blot using an anti-α-syn antibody. Aliquots (5 µg) of HiTox and WT protein extracts (PD_(b) and WT_(b), respectively) were used as controls for immunodetection. An anti-Pgk1 antibody was used as loading control for protein extracts. PD_(b), protein extract from the HiTox strain (5 µg); WT_(b), protein extract from the WT strain (5 µg); PD_(a), absorbed proteins from an in vitro binding assay performed with protein extracts from the HiTox strain; WT_(a), absorbed proteins from an in vitro binding assay performed with protein extracts from the WT strain.