Sodium Propionate Protects Dopaminergic Neurons Against Mitochondrial Toxin–Induced Oxidative Stress In Vitro

Abstract

Identifying a metabolic rescue for mitochondrial toxins induced neurodegeneration is a promising therapeutic target. Dopaminergic neurons are high energy dependent neurons, owing to their metabolic functions, and this makes them vulnerable in conditions of bioenergetic failure and mitochondrial dysfunction. In this study, we explored the protective potential of sodium propionate, a short-chain fatty acid and metabolic precursor of succinate, against mitochondrial toxin-induced neurotoxicity in MN9D dopaminergic cells. Cells were treated with 200 µM sodium propionate after exposure to 1.5 µM rotenone or 10 µM antimycin A, and cell viability, intracellular ATP levels, reactive oxygen species (ROS) generation, and dopaminergic markers were assessed. Our results show that sodium propionate significantly attenuates mitochondrial toxin-induced loss of cell viability and ATP depletion while reducing oxidative stress and preserving the expression of enzymes involved in catecholamine biosynthesis pathway, including tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH). These findings suggest that sodium propionate confers functional protection to dopaminergic neurons under mitochondrial toxin stress. Sodium propionate is proposed to act as a metabolic precursor to succinyl-CoA, thereby replenishing tricarboxylic acid cycle intermediates and supporting cellular metabolic homeostasis. Under Complex I inhibition (rotenone) and complex III inhibition (antimycin A), sodium propionate treatment was associated with preservation of cellular ATP levels. Across conditions, sodium propionate treatment was associated with improved cell viability, reduced oxidative stress associated signals, and preservation of dopaminergic function. Together, these data indicate that sodium propionate supports dopaminergic neuronal resilience through toxin-dependent metabolic and cellular stress modulating effects.

1. Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta, leading to striatal dopamine depletion and the hallmark motor symptoms of bradykinesia, rigidity, and resting tremor [1]. While the precise etiology of PD remains incompletely understood, a growing body of evidence implicates mitochondrial dysfunction, oxidative stress, and impaired energy metabolism as central contributors to disease onset and progression [2].

Among the various mitochondrial perturbations associated with PD, the inhibition of the electron transport chain (ETC) has emerged as a key pathological mechanism [3]. Environmental toxins such as rotenone and antimycin A, known inhibitors of Complex I and Complex III respectively, have been widely used to model PD in vitro and in vivo due to their ability to induce dopaminergic neurodegeneration through mitochondrial impairment, ATP depletion, and excessive reactive oxygen species (ROS) production [4,5,6]. Our previous study demonstrated that short-term exposure of MN9D dopaminergic cells to these toxins results in profound energetic depletion, oxidative stress, altered dopamine metabolism, and apoptotic cell death, thereby validating the utility of this model for probing early mechanistic events in PD pathogenesis [5].

Despite strong evidence linking mitochondrial dysfunction to dopaminergic vulnerability, no current therapeutic strategies directly address the underlying metabolic deficits. One emerging approach involves supporting mitochondrial bioenergetics by providing TCA cycle intermediates or their precursors. Succinate, a key TCA cycle intermediate and substrate for Complex II, can bolster mitochondrial respiration when Complex I activity is impaired [7,8,9]. However, electron flow through Complex II still requires functional Complex III; therefore, succinate cannot restore oxidative phosphorylation when Complex III is fully inhibited. These mechanistic constraints highlight the need for metabolic interventions capable of supporting cellular energy homeostasis through pathways beyond direct ETC restoration.

However, direct administration of succinate is limited by its inability to efficiently cross the blood brain barrier owing to its high polarity, lack of well-established transporter, poor cellular uptake and metabolic instability [10]. Propionate, a naturally occurring short-chain fatty acid, is metabolized to propionyl-CoA and subsequently to succinyl-CoA, replenishing TCA cycle intermediates and enhancing metabolic flexibility. In addition to its anaplerotic potential, propionate engages receptor-mediated and transcriptional pathways including GPR41/43 activation, histone deacetylase inhibition, and Nrf2 signaling which can reduce oxidative stress and modulate inflammatory responses [11,12,13,14]. Emerging evidence also supports the concept of hormesis, whereby low concentrations of certain metabolic substrates or nutrients trigger adaptive cellular stress responses that enhance resistance to oxidative injury. In this context, short-chain fatty acids such as propionate can been described as potential “hormetic nutrients,” capable of activating cytoprotective pathways including Nrf2-mediated antioxidant signaling and downstream targets such as NQO1. Through this adaptive mechanism, sodium propionate may promote redox homeostasis and improve cellular resilience to oxidative stress, processes that are particularly relevant to neurodegenerative disorders characterized by mitochondrial dysfunction [15,16,17]. These combined metabolic and antioxidant properties make sodium propionate (SP) an effective candidate for mitigating mitochondrial and oxidative damage in dopaminergic cells. SP readily crosses the blood–brain barrier, is efficiently metabolized within mitochondria, and has a well-established safety profile as a food additive. Although propionate has been associated with neuroprotective, anti-inflammatory, and antioxidant actions, its ability to modulate bioenergetics in dopaminergic models of mitochondrial dysfunction remains poorly investigated.

In this study, we investigated whether sodium propionate can mitigate the cellular consequences of mitochondrial Complex I and Complex III inhibition in dopaminergic neurons. Using MN9D cells, we examined the effects of SP on cell viability, ATP levels, dopamine production, and oxidative stress–associated signals following exposure to rotenone or antimycin A. By characterizing the functional outcomes of SP treatment under defined mitochondrial stress conditions, this study aims to clarify the context-dependent metabolic and protective effects of sodium propionate in dopaminergic neurotoxicity.

2. Results

2.1. Sodium Propionate Restored Energy Production and Catecholamine Biosynthesis in MN9D Cells

As shown in Figure 1, both ATP and dopamine levels were restored by sodium propionate (SP) treatment following exposure to mitochondrial inhibitors. MN9D dopaminergic cells were first exposed to either rotenone (1.5 µM) or antimycin A (10 µM) for 1 h in KRB–HEPES buffer (pH 7.5) and then treated with SP (200 µM). ATP production was measured using a combined colorimetric/fluorometric assay, and dopamine production was measured by HPLC with electrochemical detection. co-treatment with toxins and SP significantly increased ATP and Dopamine levels in MN9D cells. Thus, SP effectively preserved both ATP levels and catecholamine biosynthesis in MN9D neurons exposed to toxins.

2.2. Tyrosine Hydroxylase Expression After SP Treatment

To determine whether the observed restoration of dopamine production correlated with changes at the enzymatic level, Tyrosine Hydroxylase (TH) expression was examined by Western blotting (Figure 2A,B). MN9D dopaminergic cells were first exposed to either rotenone (1.5 µM) or antimycin A (10 µM) for 1 h in KRB–HEPES buffer (pH 7.5) and then treated with SP (200 µM). While TH expression after exposure to Antimycin A was not altered by SP co-treatment, quantification of the bands from the Rotenone-treated group showed reduced TH expression which was restored by treatment with SP (Figure 2A,B).

2.3. Dopamine β-Hydroxylase Regulation by SP

The effect of SP on Dopamine β-Hydroxylase (DBH) expression after exposure to mitochondrial toxins was evaluated in MN9D cells treated the same way as the cells evaluated for TH expression. DBH protein expression was then evaluated by Western blot analysis (Figure 3A). Quantitative analysis of the Western blot images showed reduced DBH levels in Rotenone as well as in the Antimycin A + SP-treated group (Figure 3B).

2.4. Dopa Decarboxylase (DDC) Expression in MN9D Cells After SP Treatment

MN9D dopaminergic cells were first treated with either rotenone (1.5 µM) or antimycin A (10 µM) for 1 h in KRB–HEPES buffer (pH 7.5) and then treated with SP (200 µM), DDC level was then observed using Western blot (Figure 4A). Quantitative analysis of the observed Western blot bands showed that DDC expression was increased in toxins group after treatment with SP (Figure 4B).

2.5. SP Treatment Ameliorates Oxidative Stress and Cytotoxicity of Mitochondrial Toxins in MN9D Cells

To assess the ameliorative effect of SP on cytotoxicity of mitochondrial toxins, we evaluated cell viability in MN9D dopaminergic neurons using the MTT assay. MN9D cells were treated with Control (KRB-HEPES pH 7.5 buffer), Rotenone (1.5 µM), Antimycin A (10 µM), SP (200 µM), Rotenone (1.5 µM) and SP (200 µM), Antimycin A (10 µM) and SP (200 µM) for 1 h. Toxins exposure significantly reduced cell viability in MN9D cells, SP significantly restored cell viability in both groups after exposure to mitochondrial toxins (Figure 5A).

Similarly, MN9D cells showed increased ROS production in toxins treated group, treatment with SP after exposure to toxins significantly reduced ROS levels in these neuronal cells (Figure 5B and Figure 6).

2.6. SP Treatment Reduced Dopamine Autoxidation in MN9D Cells

Fontana–Masson staining showed that exposure to toxins induced increased dopamine autoxidation product deposition in MN9D cells (Figure 7). Cells treated with rotenone or antimycin A exhibited dark cytoplasmic pigment granules, consistent with enhanced dopamine oxidation. Co-treatment with SP markedly reduced this pigmentation, revealing lighter cytoplasm and clearer nuclear morphology. These findings indicate that SP reduced the oxidative polymerization of catecholamines into dopamine oxidation product under conditions of mitochondrial dysfunction.

2.7. Western Blot Analysis of Effect of SP on GSTM2 Expression

Glutathione S-transferase Mu 2 (GSTM2), a detoxifying enzyme linked to oxidative stress control, was analyzed to evaluate cellular defense responses in MN9D dopaminergic neurons following SP treatment after toxins exposure. GSTM2 level was reduced in Rotenone +SP treated group, on the other hand, SP treatment increased GSTM2 level in MN9D cells after exposure to Antimycin A (Figure 8A,B).

2.8. Effect of SP Treatment on Activation of Antioxidant Response in Dopaminergic Neurons Following Exposure to Toxins

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a key oxidative stress response enzyme, its expression was measured in MN9D cells as an indicator of cellular antioxidant adaptation (Figure 9). NQO1 levels were significantly elevated in SP-treated groups following rotenone exposure compared with cells exposed to rotenone alone, implying that SP activates endogenous antioxidant defenses in rotenone treated dopaminergic neurons.

3. Discussion

This study demonstrates, for the first time, that sodium propionate (SP) confers significant functional protection to dopaminergic MN9D cells exposed to mitochondrial toxins, as evidenced by improved cell viability, partial preservation of ATP levels, reduced oxidative stress levels, and maintenance of dopamine production. Rotenone and antimycin A are well-established inhibitors of Complex I and Complex III of the electron transport chain (ETC), respectively [5,6,18,19,20,21]. They disrupt electron flow, decrease ATP synthesis, and enhance electron leakage leading to ROS overproduction, hallmarks that have been established as key features of Parkinson’s disease (PD) pathology [22]. Consistent with prior reports, exposure to these toxins in our model resulted in energetic impairment, oxidative stress associated cellular damage, and dopaminergic dysfunction. Earlier reports have shown that providing TCA cycle intermediates or their precursors such as dimethyl succinate or α-ketoglutarate can restore respiration and reduce oxidative damage under mitochondrial stress conditions [23,24]. In the present study, SP treatment following toxin exposure was associated with partial preservation of ATP levels, particularly under Complex I inhibition, and with improved dopamine production under both toxin conditions. Importantly, these findings do not demonstrate restoration of oxidative phosphorylation or direct rescue of mitochondrial electron transport. Rather, they indicate that SP supports cellular metabolic homeostasis under defined mitochondrial stress conditions.

Propionate is metabolized to propionyl-CoA and subsequently to succinyl-CoA, thereby replenishing TCA cycle intermediates and contributing to metabolic flexibility. Under Complex I inhibition, maintenance of ATP levels in SP-treated cells may reflect support of residual mitochondrial metabolism or compensatory cellular pathways. Under Complex III inhibition, where classical electron transport is blocked, ATP preservation is more likely attributable to non-mitochondrial processes such as substrate-level phosphorylation or enhanced cell survival rather than restoration of mitochondrial respiration. Accordingly, SP’s effects in this study are interpreted as context- and toxin-dependent metabolic support rather than bioenergetic rescue.

In our study, we observed that treatment of MN9D cells with SP after exposure to Rotenone and Antimycin A restored ATP production in these cells, with about 2-fold restoration in ROT + SP treated cells (Figure 1A). Considering the importance of energy in catecholamine biosynthesis [25,26], we explored the effect of SP on Dopamine production in cells exposed to both rotenone and Antimycin A. We observed that dopamine production in both toxins treated groups was increased after treatment with SP (Figure 1B). The variation in dopamine followed a pattern similar to the energy production change after co-administration of toxins and SP, further confirming the high metabolic dependence of these cells. Our prior study has established that the decrease in dopamine production in toxins treated group is a result of energy depletion and not a result of depletion in enzymes involved in catecholamine biosynthesis pathway [5]. This was also assessed in this study. We found that SP treatment had no significant effect on tyrosine hydroxylase production (the rate limiting enzyme in catecholamine metabolism) following exposure to toxins (Figure 2). On the other hand, DBH and DDC expression were increased in SP treated group following toxins exposure (Figure 3 and Figure 4). These results confirm that restoration of Dopamine production after treatment with SP was a function of energy restoration and not enzyme production.

In addition to rescuing energy metabolism, SP significantly reduced ROS accumulation in toxin-treated MN9D cells (Figure 5B and Figure 6). Prior findings that short-chain fatty acids (SCFAs) possess inherent antioxidants and anti-inflammatory properties, partly mediated through activation of G-protein-coupled receptors (GPCRs) such as GPR41 and GPR43 and modulation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways [27]. While our findings indicate attenuation of toxin-induced cellular stress, the present study does not identify the biochemical mechanisms underlying this effect. No direct measurements of antioxidant capacity, redox metabolites, or signaling pathway activation were performed; therefore, ROS modulation is interpreted as a functional outcome rather than evidence of specific antioxidant pathway engagement.

Also, SP significantly increased cell viability in toxins treated MN9D cells (Figure 5A). These evidence further support SP’s ability to restore dysfunctional features observed in MN9D cells following exposure to mitochondrial toxins.

Our prior study [5] also established that dopamine oxidation product accumulates under conditions of oxidative stress in MN9D cells exposed to toxins. Fontana–Masson staining revealed an increase in dark, insoluble pigment in toxin-exposed MN9D cells that was reduced by SP co-treatment (Figure 7). While immortalized cell lines such as MN9D cells do not produce true human neuromelanin, the dark insoluble pigment revealed by Fontana–Masson staining is a dopamine autooxidation product just like neuromelanin and could be used as a neuromelanin representative invitro. Neuromelanin has been implicated both as a protective factor against oxidative stress and as a potential source of neurotoxicity when it accumulates excessively [28]. Its buildup in response to oxidative stress supports earlier studies that highlight its neuroprotective role. By chelating metals such as iron, neuromelanin can limit iron-driven ROS generation; however, excessive accumulation may, in turn, aggravate neuronal damage [28]. Our results show that dopamine oxidation product levels reduced significantly in MN9D cells treated with SP after exposure to toxins (Figure 7).

Expression of the detoxification-associated enzymes GSTM2 and NQO1 was modulated in a toxin-dependent manner following SP treatment. GSTM2 was significantly increased in rotenone-treated cells (Figure 8), whereas NQO1 exhibited modest changes under antimycin A exposure (Figure 9). These enzymes play distinct but complementary roles in dopaminergic redox homeostasis, with GSTM2 involved in glutathione-dependent detoxification and regulation of dopamine-derived quinones, and NQO1 contributing to quinone reduction and redox buffering. The differential regulation of GSTM2 and NQO1 likely reflects engagement of distinct cellular detoxification strategies in response to the specific oxidative and metabolic stress imposed by Complex I versus Complex III inhibition. Importantly, while these changes are modest in magnitude, they are consistent with adaptive modulation of stress-response pathways rather than random variability. At the same time, these proteins are not presented as mechanistic drivers of ATP preservation or ROS modulation, but as biologically relevant indicators of how dopaminergic cells adjust detoxification capacity under toxin stress in the presence of SP.

Dopaminergic neurons are particularly vulnerable to metabolic and oxidative stress due to high cytosolic dopamine turnover and limited intrinsic antioxidant capacity [25]. The present findings suggest that sodium propionate enhances dopaminergic neuronal resilience under mitochondrial toxin stress through toxins-dependent metabolic and cellular protective effects. While these results support further investigation of propionate-based interventions, additional studies incorporating direct measurements of mitochondrial respiration, glycolytic flux, and redox signaling will be required to define the precise mechanisms involved.

4. Materials and Methods

4.1. Chemicals and Reagents

All reagents and solvents were obtained from commercial suppliers and used at the highest purity grade available. A HEPES-buffered Krebs–Ringer solution (KRB–HEPES) was prepared containing 125 mM NaCl, 5.34 mM KCl, 0.81 mM MgSO₄, 1.3 mM CaCl₂, 0.77 mM NaH₂PO₄, 25 mM HEPES, and 5.55 mM glucose. The pH of the buffer was adjusted to 7.4 before use. Dulbecco’s Modified Eagle Medium (DMEM) was obtained from thermofisher scientific, Waltham, MA, USA (Catalog #12-100-061).

Stock solutions of rotenone, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and antimycin A were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C until use. Stock solutions of sodium propionate was dissolved in KRB-HEPES buffer and stored at −20 °C until use. In all assays, DMSO concentration was maintained below 0.05% (v/v) to avoid solvent-induced toxicity. Working dilutions were freshly prepared in KRB–HEPES buffer (pH 7.5). Control samples were treated with buffer only. The Fontana–Masson staining kit was purchased from Sigma-Aldrich, St. Louis, MO, USA (Catalog #HT200).

4.2. Cell Lines

The mouse dopaminergic hybridoma cell line MN9D was kindly given by Dr. Alfred Heller (University of Chicago).

4.3. Instrumentation

Fluorescence imaging for DCFH-DA-stained cells was performed using a Nikon ECLIPSE Ti inverted fluorescence microscope equipped with a 40× fluor objective (Nikon Instruments Inc., Melville, NY, USA) and recorded at excitation/emission wavelengths of 488/524 nm under identical imaging conditions across all treatment groups.

4.4. Cell Culture

MN9D cells were maintained in 100 mm Falcon tissue culture dishes containing DMEM supplemented with 10% fetal bovine serum (FBS) and 4.5 g/L glucose. Cultures were incubated at 37 °C in a humidified atmosphere with 7% CO₂. For experiments, cells were seeded in 12-well plates, 96-well plates, or glass-bottom dishes, and grown to approximately 70–80% confluency prior to treatment.

4.5. Cell Treatment Concentrations

The concentration of rotenone used in this study was selected based on our previously published dose response analysis in MN9D dopaminergic cells, which identified this dose as sufficient to induce mitochondrial dysfunction and oxidative stress without excessive nonspecific cytotoxicity [5]. The antimycin A concentration was chosen based on previous literature demonstrating reliable Complex III inhibition and ROS generation at comparable concentrations in cell-based models [29]. Sodium propionate concentrations were initially screened in preliminary experiments to identify a well-tolerated and biologically effective working dose. Based on these assessments, 200 µM sodium propionate was selected for subsequent experiments as it consistently produced measurable metabolic and redox effects without evidence of cytotoxicity.

4.6. Measurement of Cell Viability

Viable MN9D cells were measured using MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Cells were seeded in 96-well plates and cultured to 70–80% confluence, then exposed to various toxin concentrations in KRB–HEPES buffer for 12 h at 37 °C. Afterwards, 10 µL of 5 mg/mL MTT was added per well and incubated for 2 h. The resulting formazan crystals were dissolved in 210 µL of a solubilizing solution (50% dimethylformamide, 20% SDS) and incubated for 4 h at 37 °C. Absorbance was read at 570 nm (reference 650 nm), and viability was expressed as the percentage of control (untreated) cells.

4.7. Measurement of Intracellular ATP Levels

ATP levels were measured using the Tribioscience ATP Colorimetric and Fluorometric Assay Kit, Tribioscience, Sunnyvale, CA, USA(Catalog #TBS2010), which relies on ATP-dependent phosphorylation of glycerol to generate glycerol-3-phosphate, detected at 570 nm. Cells grown in 12-well plates were treated with toxins for 1 h, lysed with 100 µL of assay buffer, and transferred to 96-well plates. A 90 µL aliquot of ATP reaction mix was added to each well, and absorbance at 570 nm was measured. ATP concentrations were calculated using a standard curve prepared with ATP standards provided by the manufacturer.

4.8. Measurement of Catecholamine Levels

MN9D cells were exposed to toxins for 1 h at 37 °C in KRB–HEPES buffer. Cells were collected in 1 mL cold buffer, and 50 µL aliquots were saved for protein quantification. The remaining suspension was centrifuged (6000 rpm, 3 min), and pellets were resuspended in 75 µL of 0.1 M HClO₄. Following centrifugation at 13,200 rpm for 10 min, supernatants were analyzed for DOPA and dopamine via high-performance liquid chromatography with electrochemical detection (HPLC-EC) [18]. Quantitation was based on calibration curves generated using standard compounds, and results were normalized to protein content (nmol/mg protein).

4.9. Measurement of Reactive Oxygen Species (ROS)

Intracellular ROS generation was assessed using the DCFH-DA fluorescence method. Cells in 12-well plates were preloaded with 10 µM DCFH-DA in KRB–HEPES for 1 h, washed, and subsequently treated with toxins for 1 h at 37 °C. Afterward, cells were lysed in 0.1 M Tris–HCl buffer (pH 7.5) containing 1% Triton X-100, and lysates were centrifuged to remove debris. Fluorescence of the oxidized product (DCF) was measured at Ex/Em = 504/526 nm and normalized to protein content. Live-cell ROS imaging was performed under a fluorescence microscope at Ex/Em = 488/524 nm using identical exposure settings.

4.10. Measurement of Dopamine Oxidation Levels

Cells were harvested from 6-well plates, rinsed with KRB–HEPES (pH 7.5), and centrifuged at 900 rpm for 2 min. The resulting pellet was washed, centrifuged at 10,000× g for 15 min, and resuspended in 1.5 mL of buffer containing 5 mg/mL SDS, 75 mM Tris (pH 7.5), and 0.4 mg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA #P2308). Samples were sonicated and incubated for 3 h at 37 °C with shaking, followed by centrifugation at 10,000× g for 30 min. The pellet was sequentially washed with 0.9% NaCl and ultrapure water, then dissolved in 1 mL of 1 M NaOH at 80 °C for 1 h. The supernatant’s absorbance at 420 nm was recorded, and dopamine oxidation product concentration was derived from a standard curve generated using melanin standards (Sigma-Aldrich, St. Louis, MO, USA #M0418). Protein levels were quantified by the Bradford assay, and data were normalized to protein level.

4.11. Fontana Masson Staining

Dopamine oxidation level in MN9D cells was visualized using the Sigma-Aldrich Fontana–Masson staining kit according to the manufacturer’s guidelines. Cells were cultured on microscope plates, treated with toxins for 1 h, and stained sequentially with ammoniacal silver, gold chloride, and sodium thiosulfate solutions. After thorough washing, cells were counterstained with Nuclear Fast Red, rinsed, and imaged using a bright-field microscope (Nikon Instruments Inc., Melville, NY, USA).

4.12. Western Blot Analysis

Protein expression of tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), dopamine β-hydroxylase (DBH), glutathione S-transferase Mu 2 (GSTM2), and NADPH quinone oxidoreductase 1 (NQO1) was assessed by Western blotting. MN9D cells were grown to 70–80% confluence and treated with toxins for 1 h. Following PBS washes, cells were lysed in buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich; Thermo Fisher). Lysates were incubated on ice for 30 min, centrifuged at 13,200 rpm for 10 min, and the supernatants collected. Protein concentrations were measured using the Bradford colorimetric assay (Bio-Rad, Hercules, CA, USA).

Equal protein amounts (100 µg) were denatured in Laemmli buffer, separated by 8.5% SDS-PAGE, and transferred to 0.2 µm PVDF membranes (Bio-Rad). Following protein transfer, membranes were blocked in TBST (TBS containing 0.1% Tween-20) with 5% nonfat milk for 1 h at room temperature, then incubated overnight at 4 °C with the appropriate primary antibodies, and mouse anti-β-actin (1:5000, Invitrogen, Carlsbad, CA, USA). After TBST washes, membranes were treated with HRP-conjugated secondary antibodies (Bio-Rad, 1:5000). Bands were visualized using an HRP substrate (Bio-Rad) and captured on a Gel Logic 100 imaging system (Bio-Rad, Hercules, CA, USA). Band intensities were quantified to compare relative protein expression across treatments.

4.13. Protein Determination

Bradford assay was used to measure protein content in cell lysates. Bradford reagent was added to 50 µL of sample, followed by a 10-min incubation at room temperature. The absorbance of the sample was then measured at 595 nm and protein concentrations were determined from a standard curve prepared with BSA.

4.14. Data Analyses

Observed data were analysed using Graphpad prism 10. For cell viability assay, absorbance values were expressed as a percentage calculated relative to control samples. Data represents the mean ± standard deviation (SD), and each group had three to eight replicates. To account for cell density differences, measurements were normalized to the protein content of each sample. Ordinary one-way ANOVA was used to determine statistical difference.

4.15. Technical Statement

Owing to their high toxicity and potential health risks, rotenone and antimycin A were handled following established safety guidelines and with extreme care.

5. Conclusions and Future Perspective

Our findings suggest that SP confers meaningful protection to dopaminergic MN9D cells exposed to mitochondrial toxins. SP treatment preserved ATP levels, preserved dopamine synthesis, reduced ROS accumulation, and decreased dopamine-derived pigment formation in rotenone and antimycin treated cells, indicating that SP supports cellular metabolic homeostasis and stress resilience in dopaminergic neurons subjected to mitochondrial impairment.

SP is metabolized to succinyl-CoA, thereby replenishing tricarboxylic acid cycle intermediates and contributing to metabolic flexibility under conditions of ETC disruption. While the present study does not demonstrate restoration of oxidative phosphorylation, particularly under complete Complex III inhibition, the observed preservation of cellular energy status and dopaminergic function suggests that SP supports neuronal viability through toxin-dependent metabolic and cellular protective effects. These findings highlight the potential of metabolic substrates such as SP to mitigate the downstream consequences of mitochondrial dysfunction that are relevant to Parkinson’s disease and related neurodegenerative disorders.

Our study was conducted in vitro in an immortalized dopaminergic cell line and therefore may not fully recapitulate the complexity of primary neurons or in vivo Parkinsonian pathology, further work is required to define mechanistic implications of SP treatment. Future studies should include direct measures of mitochondrial respiration and glycolytic flux. Also, in vivo studies using animal models of PD (e.g., rotenone, MPTP, or α-synuclein overexpression models) are further required to evaluate SP’s neuroprotective efficacy, pharmacokinetics, and potential to ameliorate behavioral deficits.