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

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.


■ INTRODUCTION
PM2.5−10 exposure has been reported to increase proinflammatory and cancer biomarker levels in rat brains. 1 Suspended particulate matter (SPM) contributes to neuroinflammation, cerebral vascular damage, impairment of the prefrontal cortex, and reduction in brain volume.Fine and ultrafine particulate matter affects the central nervous system through various pathways including (1) direct entry into the brain through the olfactory pathway; (2) indirect entry into the brain through the bloodstream after reaching the lungs; (3) induction of inflammation in the nasal and lung tissues by SPM, leading to the release of pro-inflammatory cytokines into the bloodstream and subsequent effects on the brain; and (4) activation of the hypothalamic−pituitary−adrenal axis, resulting in increased stress hormone (cortisol) levels in the bloodstream, thereby influencing the brain. 2−12 A singlephoton emission computed tomography study found higher deposition ratios of smaller particles in rat lungs. 13The deposition areas varied according to particle size: 14 particles larger than 10 μm primarily deposited in the nasopharyngeal area, particles between 5 and 10 μm deposited in the bronchial area, and particles smaller than deposited 2.5 μm in the alveolar area. 15Particles smaller than 0.1 μm are more likely to enter the interstitial tissue than those smaller than 2.5 μm. 16,17oreover, research also indicates that 15 nm aerosolized quantum dots, inhaled by mice, accumulate in the olfactory bulb and neuron axons, suggesting that small aerosols can impact the brain. 12xposure to toxic aerosols or SPM, such as those from diesel engine exhaust or urban air pollution, can cause oxidative stress, DNA damage, neuroinflammation, and increased proinflammatory cytokines in rat brains, indicating brain inflammation. 18,19Moreover, a longitudinal study in Ontario from 2001 to 2013 revealed a 4% increase in Parkinson's disease (PD) incidence per 3.8 μg/m 3 increase in PM2.5, 20 and short-term exposure to PM2.5 was reported to worsen symptoms in PD patients in Seoul. 21These studies suggest a strong association between aerosols and PD.Aerosols in cigarette smoke and air pollution share similar composition, including PAHs, heterocyclic compounds, nitrosamines, aromatic amines, aldehydes, phenolic compounds, hydrocarbons, nitroalkanes, metals, and other components, 22−24 indicating a potential link between cigarette smoke aerosols and PD.−30 These observations indicate that the association between cigarette smoke and PD remains inconclusive.
While the link between cigarette smoke and Alzheimer's disease (AD) has been extensively investigated, 31−33 the relationship between cigarette smoke and PD remains unclear.In a previous study, we collected cigarette aerosols in a sizesegregated manner, enabling a comprehensive assessment and comparison of the impacts exerted by cigarette aerosol extract (CAE) on biological systems. 34We used SH-SY5Y cells overexpressing α-synuclein (α-Syn) as a cellular PD model to investigate the effects of CAE exposure, monitoring changes in the oligomeric state of α-Syn using dual-fluorescence crosscorrelation spectroscopy (FCCS).Employing live-cell imaging techniques, we aim to elucidate the potential pathological link between cigarette aerosols and PD progression.
■ RESULTS AND DISCUSSION α-Syn Overexpression Exacerbates CAE-Induced Cell Death, ROS Generation, and Mitochondrial Dysfunction in SH-SY5Y Cells.In our previous study, it has been reported that exposure of CAE decreases cell viability, increases intracellular reactive oxygen species (ROS) accumulation, and causes mitochondrial dysfunction. 34Moreover, the effects were most pronounced in SH-SY5Y among the three cell lines investigated. 34α-Syn is known for its role in the genetics and neuropathology of PD, with its misfolding and aggregation into Lewy bodies being pathological hallmarks of PD. 35−38 Our previous research established a positive correlation between the concentration of intracellular α-Syn and enhanced cytotoxicity and reduced viability in SH-SY5Y cells. 39In this study, we used SH-SY5Y cells transfected with a plasmid to induce overexpression of α-Syn, a widely used cellular model for PD, 40−43 to explore the potential impact of CAE on PD progression.We treated SH-SY5Y cells overexpressing α-Syn with various concentrations of CAE and assessed cell viability after 24 h.A significant decrease in cell viability (Figure S1) and a lower IC 50 value (Figure 1A) were observed in cells overexpressing α-Syn and cocultured with CAE, indicating that α-Syn overexpression aggravates CAE-induced cell death.In contrast, cells overexpressing fluorescence protein and cocultured with CAE showed similar IC 50 values to the control, suggesting that eGFP overexpression does not exacerbate CAE-induced cytotoxicity.Moreover, SH-SY5Y cells overexpressing eGFP-α-Syn and cocultured with CAE had comparable IC 50 values to those overexpressing untagged α-Syn, further confirming the detrimental effect of α-Syn overexpression (Figure S2).
To investigate how α-Syn overexpression might exacerbate CAE-induced oxidative stress, we conducted experiments in SH-SY5Y cells overexpressing α-Syn for 24 h, followed by an 80 min treatment with organic particle (OP)−CAE or watersoluble particle (WP)−CAE to monitor intracellular H 2 O 2 levels.Both α-Syn overexpression and CAE treatment independently increased intracellular H 2 O 2 levels, but their combination led to an even more pronounced increase (Figure Previous studies have shown mitochondrial dysfunction in the substantia nigra of PD patients, characterized by impaired mitochondrial complex I function 44,45 and mitochondrial DNA deletions. 46Our previous experiments demonstrated decreased mitochondrial membrane potential and ATP levels in cells treated with CAE. 34We then assessed whether α-Syn expression exacerbated CAE-induced mitochondrial dysfunction.After 24 h of α-Syn overexpression followed by 150 min of CAE treatment, we observed a significantly pronounced decrease in the mitochondrial membrane potential using the JC-1 probe (Figure 1C).This result suggests that overexpressed α-Syn exacerbates CAE-induced mitochondrial dysfunction in SH-SY5Y cells.
Mitochondria are crucial for cellular energy production, generating adenosine triphosphate (ATP), and play a vital role in regulating the cell fate. 47,48Impairment in mitochondrial function can lead to reduced ATP synthesis, disrupted Ca 2+ homeostasis, and excessive ROS production. 49We assessed cellular ATP levels in SH-SY5Y cells overexpressing α-Syn following CAE treatment and observed a considerably greater reduction in ATP levels than that under control conditions (Figure 1D).These findings indicate that α-Syn overexpression aggravated CAE-induced mitochondrial dysfunction, resulting in a substantial reduction in both mitochondrial membrane potential and cellular ATP levels.

α-Syn Overexpression Exacerbates CAE-Induced Apoptosis and Pyroptosis but Attenuates CAE-Induced
Autophagy Activity in SH-SY5Y Cells.Programmed cell death includes apoptosis, regulated necrosis, and autophagic cell death. 50,51Previous studies have demonstrated that cigarette smoke can induce cell death through various mechanisms such as pyroptosis, 52−55 ferroptosis, 56−58 autophagy, 59 and apoptosis 60−63 in various cell lines.In our prior study, we observed robust induction of caspase-1, caspase-3, and LC3-II activities upon exposure to CAE in multiple cell lines, indicating the activations of pyroptosis, apoptosis, and autophagy. 34In our study, we cocultured SH-SY5Y cells expressing eGFP-α-Syn variants and mApple-α-Syn variants with CAE samples (OP: 20 μg/mL and WP: 200 μg/mL) for 24 h to investigate the effect of CAE treatment.Without CAE treatment, we observed a homogeneous distribution of WT α-Syn variants without significant aggregation (Figures 3A_a and 4A_a), similar to previous findings. 39However, the coexpression of eGFP-α-Syn variants and mApple-α-Syn variants in the presence of CAE led to the formation of punctate structures (OP−CAE: Figures 3A_b and  S3; WP−CAE: Figures 4A_b and S4).This punctate distribution was also observed in SH-SY5Y cells overexpressing untagged WT α-Syn when cocultured with CAE, suggesting that the punctate distribution is not a result of eGFP tagging (Figure S5).Interestingly, SH-SY5Y cells coexpressing eGFP and mApple also displayed similar punctate distributions in the presence of CAE (OP−CAE: Figure 3A_b; WP−CAE: Figure 4A_b).Subsequent imaging analysis revealed that the average diameter of these puncta was around 0.70 μm under all          experimental conditions (OP−CAE: Figure 3B; WP−CAE: Figure 4B).
To further assess the impact of CAE treatment, we conducted repetitive fluorescence correlation spectroscopy (FCS) and FCCS measurements with a 60 s interval (Figures S3,S4).Without CAE treatment, diffusion coefficients for eGFP, eGFP-tagged WT α-Syn, and eGFP-mApple were 39.19 ± 5.10, 23.71 ± 7.97, and 36.10 ± 7.28 μm 2 /s, respectively, with concentrations <0.5 μM (OP−CAE: Figure 3C; WP− CAE: Figure 4C; and Tables 1 and 2), consistent with previously reported values. 39Following OP−CAE treatment, diffusion coefficients of eGFP significantly dropped to 22.46 ± 8.51 μm 2 /s in the homogeneous region and 2.22 ± 2.34 μm 2 /s in the puncta region (Figure 3 and Table 1).−71 To investigate further, a one-component anomalous diffusion model, commonly used to account for environmental crowdedness, intracellular interactions, and polydispersity behaviors of probe molecules, 64−71 was used to analyze FCS curves.Anomalous diffusion modeling revealed increased hindrance, with an average anomalous factor of 0.74 ± 0.11 in the homogeneous region and 0.18 ± 0.14 in the puncta region compared to 0.91 ± 0.07 without CAE treatment (Figure 3 and Table 1).Lower cross-correlation ratios (25.50 ± 7.90 and 21.96 ± 8.53%) after OP−CAE treatment suggested that eGFP remained mostly monomeric, with the observed diffusion changes attributable to environmental factors.Similar results were obtained with the WP−CAE treatment (Figure 4 and Table 2).Next, we examined the influence of the CAE on α-Syn variants.After OP−CAE treatment, eGFP-α-Syn variants in the homogeneous region showed no significant difference in diffusion coefficients compared to eGFP-WT α-Syn without CAE treatment (Figure 3 and Table 1).For eGFP-α-Syn variants in the homogeneous region after OP−CAE treatment, anomalous diffusion modeling revealed increased hindrance (an anomalous factor of 0.75−0.82)and FCCS experiments exhibited lower cross-correlation ratios of 20.91∼30.93%,indicating a monomeric state (Figure 3 and Table 1).In contrast, the puncta region showed a notable decrease in diffusion coefficients for the eGFP-α-Syn variants.Anomalous diffusion modeling suggested increased environmental hindrance with anomalous factors of 0.19−0.46and local transport coefficients of 9.86−16.83μm 2 /s α , and FCCS experiments revealed a higher synchronization ratio of 66.18∼74.76%,indicating the presence of oligomeric forms in this region after CAE treatment (Figure 3 and Table 1).Similar trends were observed with WP−CAE treatment, confirming that both treatments affected the oligomeric distribution of α-Syn in SH-SY5Y cells (Figure 4 and Table 2).

CAE Treatment Leads to the Formation of α-Syn Puncta That Colocalize with Lysosomes in SH-SY5Y
Cells.Following CAE treatment, significant punctal structures were observed.This led us to investigate potential intracellular structures that might overlap with these intracellular oligomeric α-Syn puncta.Initially, we used MitoTracker Deep Red FM, a fluorescent dye for live mitochondria, to assess whether the intracellular oligomeric α-Syn puncta colocalize with mitochondria.SH-SY5Y cells expressing eGFP α-Syn were cocultured with OP−CAE for 24 h, and mitochondrial localization was stained with MitoTracker Deep Red FM.The resulting images showed α-Syn distribution in green and mitochondrial localization in red (Figure 5A).The merged image, appearing yellow, indicated that the α-Syn puncta induced by OP−CAE did not associate with mitochondria (Figure 5A).
Previous study has suggested a significant overlap between α-Syn and LAMP-2 protein, implicating lysosomal degradation of α-Syn. 72Subsequently, we examined whether CAE-induced oligomeric α-Syn puncta colocalize with lysosomes in SH-SY5Y cells.Using LysoTracker Red DND-99, we observed a significant overlap between the oligomeric α-Syn puncta and lysosomes, suggesting an association (Figure 5B).Control experiments with SH-SY5Y cells expressing eGFP and treated with OP−CAE for 24 h showed no significant overlap of eGFP puncta with either mitochondria or lysosomes (Figure 5C,D).This is consistent with the observed differences in the diffusion behavior and oligomeric state within puncta regions between eGFP and eGFP-α-Syn variants after CAE treatment.Quantification of the overlap between intracellular oligomeric α-Syn puncta and mitochondria or lysosomes was performed by using ImageJ software (Figure 5E,F).The analysis revealed an overlap percentage of 8.78 ± 7.70% for mitochondria and 54.38 ± 21.62% for lysosomes.This significant difference suggests that CAE-induced intracellular oligomeric α-Syn puncta are predominantly associated with lysosomes, implying that CAE-induced α-Syn puncta might affect the lysosomedependent regulation mechanism.

■ DISCUSSION
−76 Moreover, α-Syn may bind with the mitochondrial adenine nucleotide translocator, facilitating the opening of membrane transport proteins and causing a decrease in the mitochondrial membrane potential, leading to mitochondrial dysfunction and promoting cell apoptosis. 73,74,77In this study, we investigated the synergetic effects of CAE treatment and α-Syn overexpression in SH-SY5Y cells.We found that α-Syn overexpression exacerbates CAE-induced intracellular cytotoxicity, accumulation of ROS, and mitochondrial dysfunction, resulting in increased cell death.The extent of neuronal cell death in PD patients has been positively correlated with the proportion of activated caspase-3-positive neurons. 78Furthermore, both fibrillar and monomeric forms of α-Syn have been reported to induce complete NLRP3 inflammasome activation, leading to caspase-1 activation and IL-1β production. 79In our study, substantial escalations in both Caspase-1 and Caspase-3 activities were observed in SH-SY5Y cells overexpressing α-Syn and cocultured with CAE.This suggests that α-Syn overexpression might potentiate CAE-induced cell death through the activation of pyroptosis and apoptosis.
In the FCCS experiments, we noted reduced diffusion coefficients for eGFP and eGFP-α-Syn variants in SH-SY5Y cells cocultured with CAE, compared to previous findings. 39oreover, anomalous factors in homogeneously distributed regions ranged from ∼0.74 to 0.83 for eGFP and eGFP-α-Syn variants after CAE treatment (Figures 2 and 3 and Tables 1  and 2), indicating a significant increase in environmental hindrance due to CAE treatment.Previous research has shown that PM 10 exposure in A549 cells increases filamentous actin (F-actin) presence by 50%. 80Nicotine has been reported to stimulate the release of mitogens, such as the platelet-derived growth factor, affecting the cell cytoskeleton. 81Furthermore, volatile organic components in cigarette aerosols, like acrolein and acetate, have been observed to alter the cytoskeleton in human gingival cells. 82Based on these studies, it is reasonable to infer that coculturing SH-SY5Y cells with CAE induces changes in the cell cytoskeleton, potentially leading to the entrapment of eGFP and eGFP-α-Syn variants within specific organelles and a decrease in diffusion coefficients, consistent with our observations here.
Epidemiological investigations have shown an inverse relationship between cigarette smoking and the progression of PD. 83−86 However, conflicting reports suggest that cigarette smoking has either no association 87,88 or is strongly linked with PD-related psychosis 89 and cognitive impairment. 90,91Therefore, the relationship between cigarette smoking and PD remains controversial.In our previous study, we found that eGFP-α-Syn variants formed oligomers without CAE treatment, in line with prior research. 92,93However, when cocultured with CAE, we observed increased FCCS ratios (66.18−74.76%) in puncta regions, resembling those in the positive control group (55.55 ± 5.34%, Table 1, eGFP-mApple without CAE).Conversely, in homogeneously distributed regions, FCCS ratios decreased to about 20.9−30.9%,akin to the negative control group (23.60 ± 5.77%, Table 1, eGFP and mApple without CAE).Lower FCCS ratios in homogeneous regions suggest a shift toward monomeric structures of α-Syn, while higher FCCS ratios in puncta regions indicate α-Syn oligomerization.−96 It is intriguing to explore whether CAE treatment similarly slows down the oligomerization rate of α-Syn in SH-SY5Y cells, resulting in the monomeric form observed here after 24 h of transfection and cotreatment with CAE.Extending the coculture and transfection period to 48 h showed no significant change in FCCS ratios and diffusion coefficients (Figures S6 and S7 and Table 3), suggesting that CAE facilitates the transition of α-Syn into monomeric structures rather than inhibiting oligomerization.Previous research indicates that WT α-Syn exists in a stable tetrameric form, while missense mutations tend to form monomeric structures that trigger pathological aggregation. 97−101 Our results suggest that CAE disrupts the α-Syn oligomeric equilibrium, favoring monomers with hindrance in homogeneous regions and causing oligomer formation in punctate distributions in SH-SY5Y cells.This finding highlights the potential link between CAE and α-Syn pathology.
Significant colocalization between CAE-induced α-Syn oligomeric puncta and lysosomes was observed in SH-SY5Y cells overexpressing α-Syn and cocultured with CAE.This suggests that CAE-induced α-Syn puncta might affect lysosome-dependent regulatory mechanisms.The lysosome plays an important role in mediating the autophagy process to maintain cellular homeostasis. 102−106 Although CAE treatment induces autophagy activity, a decreasing trend in autophagy levels was observed in SH-SY5Y cells overexpressing α-Syn and cocultured with CAE, compared to that in cells treated with CAE alone (Figure 2C−E).Interestingly, the decreased autophagy activity was not observed in SH-SY5Y cells overexpressing eGFP and cocultured with CAE (Figure S8), further confirming α-Syn influence.This suggests a dysregulation of autophagy levels when α-Syn-overexpressing cells are exposed to CAE, highlighting the interplay between α-Syn pathology and autophagy dysregulation.
In conclusion, our study provides compelling evidence that α-Syn overexpression exacerbates CAE-induced intracellular cytotoxicity, leading to the accumulation of ROS and mitochondrial dysfunction.These cellular abnormalities ultimately result in significant cell death through apoptosis and pyroptosis.Moreover, we observe dysregulation of autophagy levels in α-Syn-overexpressing cells exposed to CAE, highlighting the intricate complex interplay between α-Syn pathology and autophagy dysregulation.CAE treatment disrupts the dynamic equilibrium in the oligomeric state of α-Syn variants, shifting native oligomers to monomers suffering significant environmental hindrance in homogeneously distributed regions and causing oligomeric α-Syn variants in punctate distributions colocalized with lysosomes in SH-SY5Y cells.These findings shed light on the molecular mechanisms underlying α-Syn-associated neurodegenerative disorders and the impact of CAE on α-Syn pathology (Figure 6).

■ MATERIALS AND METHODS
DNA Constructs for the Live-Cell Studies.In our study, we used a peGFP-C1 vector to express eGFP in SH-SY5Y cells.The eGFP-α-Syn variants were constructed by ligating α-Syn into the peGFP-C1 vector using XhoI and Hind III restriction sites.Sitedirected mutagenesis was employed to introduce mutations, resulting in the creation of Egfp-A30P, Egfp-E46K, Egfp-H50Q, Egfp-G51D, and Egfp-A53T.The eGFP-N103 construct was similarly generated by ligating N103 into peGFP-C1 using XhoI and Hind III.For the expression of mApple in SH-SY5Y cells, the pmApple-C1 construct was used.Following the same procedures as with eGFP, we constructed mApple-α-Syn, mApple-A30P, mApple-E46K, mApple-H50Q, mApple-G51D, mApple-A53T, mApple-N103, and eGFP-mApple. 39igarette Smoke Aerosol Collection and Preparation of Its Extracts.A locally popular cigarette brand (Long Life Gentle 6, Taiwan), containing 5 mg of tar and 0.4 mg of nicotine per cigarette, was burned in a homemade smoking chamber.The resulting cigarette aerosol was collected using a 10-stage Micro-Orifice Uniform Deposit Impactor (Model 120 MOUDITM II Impactor, USA) at a pump flow rate of 30 m 3 min −1 (GAST, 1023−101Q-SG608X).The collected aerosol samples were categorized based on particle size into different stages.The filters containing aerosol of various diameters were extracted with ddH 2 O and ethyl acetate to obtain water-soluble components (WP) and organic components (OP), respectively. 34he extracted samples were then dried and stored at −80 °C for further experiments on cytotoxicity and toxicology. 34ell Culture and Transfection.SH-SY5Y cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco, 11965−084) supplemented with 1% penicillin/streptomycin (Gibco, 15140−122) and 10% (v/v) fetal bovine serum (FBS, Corning, 35− 010-CV).The cells were maintained in 10 cm culture dishes (α-plus, 16203−1SS) at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO 2 .For assays measuring cell viability, ROS generation, mitochondrial function verification, caspase 1/3 activity, and autophagy activity, SH-SY5Y cells were transfected with a specific amount WT α-Syn for 24 h using TurboFect (Thermo Fisher, R0531), following the vendor's instructions, either in the absence or presence of CAE.For FCS experiments, SH-SY5Y cells were plated at a density of 4.5 × 10 5 cells/plate and transfected with eGFP-tagged WT α-Syn/mutant variants and mApple-tagged WT α-Syn/mutant variants for 24/48 h using TurboFect (Thermo Fisher, R0531), as per the vendor's instructions, in the absence or presence of CAE.Before confocal imaging and FCS measurements, the medium was replaced with phenol red-free DMEM (Gibco, 31053−028).During data acquisition, cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO 2 in a culture dish microincubator (Warner, DH-40iL).
Detection of Intracellular ROS (H 2 O 2 ) Generation.SH-SY5Y cells either expressing or not expressing α-Syn were plated at a density of 3.5 × 10 4 cells/well in 96-well plates (α-plus, 116196−3SB) at 37 °C for 24 h.To measure intracellular hydrogen peroxide (H 2 O 2 ), the OxiVision Green hydrogen peroxide sensor (AAT Bioquest, 11,503) was preincubated with the cells for 1 h before CAE treatment (OP: 55 μg/mL, WP: 1000 μg/mL) for an additional 80 min at 37 °C.The fluorescence intensity inside the cells was measured using the microplate reader (Molecular devices, SpectraMax iD3) with an excitation wavelength of 490 nm and an emission wavelength of 525 nm.
Mitochondrial Membrane Potential Assay.SH-SY5Y cells either expressing or not expressing α-Syn were plated at a density of 3.5 × 10 4 cells/well in 96-well plates (α-plus, 116196−3SB at 37 °C for 24 h.To measure the mitochondrial membrane potential, cells were preincubated with 100 μL of JC-1 (Abcam, ab113850) for 10 min.Subsequently, the cells were washed twice with 1X dilution buffer before CAE treatment (OP: 55 μg/mL, WP: 1000 μg/mL) for a further 150 min at 37 °C.The fluorescence intensity inside the cells was measured using the microplate reader (Molecular devices, SpectraMax iD3) with an excitation wavelength of 475 nm and an emission wavelength of 550 nm for monomeric JC-1 and with an excitation wavelength of 535 nm and an emission wavelength of 590 nm for oligomeric JC-1.
ATP Assay.SH-SY5Y cells, with or without expressing α-Syn, were plated at a density of 3.5 × 10 4 cells/well in 96-well plates (α-plus, 116196−3SB) at 37 °C for 24 h.To measure the ATP level, cells were treated with CAE (OP: 55 μg/mL, WP: 1000 μg/mL) for 150 min at 37 °C.Then, 50 μL of detergent (supplied by vender) was added and shaken for 5 min, followed by the addition of 50 μL of substrate solution (supplied by vender) and further shaking for 5 min.The cells were then left to incubate in the dark for 10 min.Luminescence intensity was measured by using a microplate reader (Molecular devices, SpectraMax iD3).Statistical analysis was performed using a paired t-test to compare the differences between each pair of groups.
Autophagy Activity Detection.SH-SY5Y cells, with or without α-Syn expression, were seeded at a density of 3.5 × 10 4 cells/well in 96-well plates (α-plus, 116196−3SB).To measure autophagy activity, cells were exposed to CAE (OP: 55 μg/mL; WP: 1000 μg/mL) for 4 h at 37 °C.After the treatment, cells were incubated with the detection reagent (supplied by vender) for 30 min.Following the removal of the reagent, 100 μL of 1X assay buffer was added, and fluorescence intensity was measured using a microplate reader (Molecular devices, SpectraMax iD3) with excitation and emission wavelengths of 480 and 530 nm, respectively.A paired t-test was used to analyze the difference between each pair of groups.
Fluorescence Correlation Spectroscopy Experiments.FCS was conducted using a custom confocal system based on a Nikon Ti Eclipse, utilizing 470 nm (Picoquant, LDH−P−C-470M) and 560 nm (Picoquant, LDH-D-TA-560B) lasers to excite the sample.The lasers were directed through a 405/488/561/635 nm dichroic mirror (Semrock, Di01-R405/488/561/635) and focused onto the sample using a Nikon Apochromat 100× objective (NA 1.40, oil).Fluorescence emission was collected through a 405/488/561/635 nm notch filter (Semrock, NF03−405/488/561/635 × 10 −25 ) and recorded by using avalanche photodiodes (Picoquant, MPD-5C5T).FCS curves were generated by acquiring 60 s of fluorescence intensity using Symphotime (Picoquant, SP1 + 2).The curves were fitted using either a one-component three-dimensional (3D) free diffusion model or an anomalous model described in eqs 1 and 2, with specific parameters defined for each experimental condition.Here, V eff denotes the effective excitation volume, characterized by an axial (z 0 ) to lateral (r 0 ) dimension ratio w ( = z 0 /r 0 ), and <C> is the average concentration of the molecules under observation.T R and τ R refer to the triplet state population and its triplet state relaxation time, respectively, while τ is the correlation time and τ D represents the diffusion time.The structure parameter (w) and V eff are determined through calibrating with a standard dye, R6G (D = 414 μm 2 /s). 107he anomalous factor α accounts for the effects of the intracellular environment heterogeneity on diffusion. 69,108,109We used a 470 nm laser at 15 μW excitation power to excite eGFP and eGFP-tagged α-Syn variants and a 561 nm laser at 40 μW excitation power to excite mApple and mApple-tagged α-Syn variants.To minimize photobleaching and triplet-state blinking, the laser power was adjusted to 25−30 μW during the FCCS experiments.
Mitochondrial and Lysosome Tracking.For the visualization of mitochondria or lysosomes, SH-SY5Y cells expressing eGPF-α-Syn were incubated with 5 nM MitoTracker Red (Invitrogen) or 5 nM LysoTracker Red (Invitrogen) at 37 °C for 15 min before image acquisition.Confocal image was performed using a homemade confocal system based on a Nikon Ti eclipse.The 470 nm (Picoquant, LDH−P−C-470M) and 635 nm (COHERENT, 9010346) lasers were used to excite eGFP-α-Syn and MitoTracker or LysoTracker, respectively.The overlap between eGFP-α-Syn and mitochondria or lysosomes was quantified by measuring the percentage surface area covered by eGPF-α-Syn, MitoTracker Red, and LysoTracker Red using ImageJ software.
Statistical Analysis.Data are presented as mean ± SD.Statistical analysis was performed using the unpaired t-test with repeated measures.Significant differences were denoted as *, **, and *** for p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 1 .
Figure 1.(A) Distribution of IC 50 values from 3-repeated tests of CAE effects in the SH-SY5Y cell line with or without expressing WT α-Syn.The IC 50 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 H 2 O 2 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 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 6 .
Figure 6.Summary mechanism of CAE-induced cell death in SH-SY5Y cells expressing α-Syn (description given in the Discussion section).

Equation 1
describes the correlation function for a 3D diffusion model

Table 3 .
Diffusion Coefficients, Concentrations, and Anomalous Parameters of eGFP, mApple, eGFP-WT, and mApple-WT in SH-SY5Y Cells after 48 h of Transfection with OP CAE and WP CAE

■ ASSOCIATED CONTENT * sı Supporting Information The
Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00771.Cell viability of SH-SY5Y cells with or without expression of WT α-Syn after treatment of cigarette aerosol; cell viability of SH-SY5Y cells with or without expression of WT α-Syn, eGFP, and eGFP α-Syn after treatment of OP CAE and WP CAE; FCS curves and normalized FCCS curves SH-SY5Y cells coexpressing eGFP α-Syn variants and mAppple-α variants with or without treatment of OP CAE for 24 h; FCS curves and normalized FCCS curves SH-SY5Y cells coexpressing eGFP α-Syn variants and mAppple-α variants with or without treatment of WP CAE for 24 h; representative immunofluorescence images of SH-SY5Y cells expressing untagged WT α-Syn with or without treatment of CAE for 24 h; confocal images of SH-SY5Y cells expressing eGFP α-Syn and mAppple-α with or without treatment of OP CAE for 48 h, analysis of mean diameter, synchronization ratio, and diffusion coefficient; confocal images of SH-SY5Y cells expressing eGFP α-Syn and mAppple-α with or without treatment of WP CAE for 24 and 48 h, analysis of mean diameter, synchronization ratio, and diffusion coefficient; and impact of OP CAE on autophagy activities in SH-SY5Y cells with and without the expression of mApple-tagged WT α-Syn or mApple (PDF) Department of Chemistry and Aerosol Science Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan; orcid.org/0000-0003-3214-9937;Email: chiawang@mail.nsysu.edu.twHsiu-Fang Fan − Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan; Department of Chemistry and Aerosol Science Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan; orcid.org/0000-0002-4870-351X;Email: bendyfan@imst.nsysu.edu.tw(NSYSU, Taiwan), and Aerosol Science Research Center (NSYSU, Taiwan) grants to H.F.F. and C.C.W.