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. 2024 Apr 3;15(7):1484-1500.
doi: 10.1021/acschemneuro.3c00771. Epub 2024 Mar 14.

Influence of Cigarette Aerosol in Alpha-Synuclein Oligomerization and Cell Viability in SH-SY5Y: Implications for Parkinson's Disease

Yu-Xin Shen  1   2   3 , Pe-Shuen Lee  1   2   3 , Ming-Chu Teng  1   2   3 , Jhih-Hong Huang  2   3 , Chia C Wang  2   3 , Hsiu-Fang Fan  1   2   3
Affiliations

Influence of Cigarette Aerosol in Alpha-Synuclein Oligomerization and Cell Viability in SH-SY5Y: Implications for Parkinson's Disease

Yu-Xin Shen et al. ACS Chem Neurosci. .

Abstract

Although cigarette aerosol exposure is associated with various adverse health issues, its impact on Parkinson's disease (PD) remains elusive. Here, we investigated the effect of cigarette aerosol extract (CAE) on SH-SY5Y cells for the first time, both with and without α-synuclein (α-Syn) overexpression. We found that α-Syn aggravates CAE-induced cell death, oxidative stress, and mitochondrial dysfunction. Fluorescence cross-correlation spectroscopy (FCCS) revealed a dual distribution of α-Syn within the cells, with homogeneous regions indicative of monomeric α-Syn and punctated regions, suggesting the formation of oligomers. Moreover, we observed colocalization of α-Syn oligomers with lysosomes along with a reduction in autophagy activity. These findings suggest that α-Syn overexpression exacerbates CAE-induced intracellular cytotoxicity, mitochondrial dysfunction, and autophagy dysregulation, leading to elevated cell mortality. Our findings provide new insights into the pathogenic mechanisms linking exposure to cigarette aerosols with neurodegenerative diseases.

Keywords: Parkinson’s disease; autophagy activity; cigarette aerosol; fluorescence correlation spectroscopy; particulate matter; α-synuclein oligomerization.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Distribution of IC50 values from 3-repeated tests of CAE effects in the SH-SY5Y cell line with or without expressing WT α-Syn. The IC50 values of (i) OP extract and (ii) WP extracts. I∼VI indicate the size of cigarette aerosol obtained with MOUDI listed in Supporting Information Table 1. (B) Influence of cigarette aerosol extraction on intracellular H2O2 generation in the SH-SY5Y cell line with or without expressing WT α-Syn. The influences of cigarette aerosol extraction on (C) mitochondrial membrane potential polarization and (D) ATP generation in the SH-SY5Y cell line with or without expressing WT α-Syn. CCCP: Carbonyl cyanide 3-chlorophenylhydrazone. *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.001). The numbers indicate the number of investigated cells.
Figure 2
Figure 2
Impact of cigarette aerosol OP extraction on (A) caspase-1 and (B) caspase-3 activities and autophagy activities, as assessed through (C) LC3-II immunofluorescence images, (D) quantified LC3-II intensity derived from immunofluorescence images, and (E) autophagy activity assay analysis in SH-SY5Y cells, both with and without the expression of α-Syn. *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.001).
Figure 3
Figure 3
(A) Confocal images of SH-SY5Y cells expressing eGFP-α-Syn and mApple-α-Syn with or without treatment of OP CAE for 24 h. (B) Analysis of the mean diameter of puncta from SH-SY5Y cells expressing eGFP-α-Syn/mutant variants and mApple-α-Syn variants with or without treatment of OP CAE for 24 h. (C) Cross-correlation ratio of eGFP-α-Syn/mutant variants and mApple-α-Syn variants in SH-SY5Y cells with or without treatment of OP CAE for 24 h. (D) eGFP-α-Syn variants and mApple-α-Syn variants in SH-SY5Y cells with or without treatment of OP CAE for 24 h. Each FCS curve was fitted with the one-component 3D free diffusion model to obtain the corresponding diffusion coefficient (listed in Table 1). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.001). The numbers indicate the number of investigated cells.
Figure 4
Figure 4
(A) Confocal images of SH-SY5Y cells expressing eGFP-α-Syn and mApple-α-Syn with or without treatment of WP CAE for 24 h. (B) Analysis of the mean diameter of puncta from SH-SY5Y cells expressing eGFP-α-Syn variants and mApple-α-Syn variants with or without treatment of WP CAE for 24 h. (C) Cross-correlation ratio of eGFP-α-Syn variants and mApple-α-Syn variants in SH-SY5Y cells with or without treatment of WP CAE for 24 h. (D) eGFP-α-Syn variants and mApple-α-Syn variants in SH-SY5Y cells with or without treatment of WP CAE for 24 h. Each FCS curve was fitted with the one-component 3D free diffusion model to obtain the corresponding diffusion coefficient (listed in Table 2). *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.001). The numbers indicate the number of investigated cells.
Figure 5
Figure 5
(A) Confocal images of SH-SY5Y cells expressing eGFP-WT α-Syn (green) and stained with MitoTracker (red) (i) without or (ii) with treatment of OP CAE for 24 h. (B) Confocal images of SH-SY5Y cells expressing eGFP-WT α-Syn (green) and stained with LysoTracker (red) (i) without or (ii) with treatment of cigarette aerosol OP extraction for 24 h. (C) Confocal images of SH-SY5Y cells expressing eGFP (green) and stained with MitoTracker (red) (i) without or (ii) with treatment of OP CAE for 24 h. (D) Confocal images of SH-SY5Y cells expressing eGFP (green) and stained with LysoTracker (red) (i) without or (ii) with treatment of cigarette aerosol OP extraction for 24 h. Quantified analysis of colocalization of eGFP-WT α-Syn and eGFP with (E) mitochondria or (F) lysosomes. Arrows indicate the overlapping region. *, **, and *** represent significant differences (* = p < 0.05), (** = p < 0.01), and (*** = p < 0.001).
Figure 6
Figure 6
Summary mechanism of CAE-induced cell death in SH-SY5Y cells expressing α-Syn (description given in the Discussion section).
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