Ergothioneine-Mediated Neuroprotection of Human iPSC-Derived Dopaminergic Neurons

Cell death involving oxidative stress and mitochondrial dysfunction is a major cause of dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson’s disease patients. Ergothioneine (ET), a natural dietary compound, has been shown to have cytoprotective functions, but neuroprotective actions against PD have not been well established. 6-Hydroxydopamine (6-OHDA) is a widely used neurotoxin to simulate the degeneration of dopaminergic (DA) neurons in Parkinson’s disease. In this study, we investigated the protective effect of ET on 6-OHDA treated iPSC-derived dopaminergic neurons (iDAs) and further confirmed the protective effects in 6-OHDA-treated human neuroblastoma SH-SY5Y cells. In 6-OHDA-treated cells, decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial reactive oxygen species (mROS), reduced cellular ATP levels, and increased total protein carbonylation levels were observed. 6-OHDA treatment also significantly decreased tyrosine hydroxylase levels. These effects were significantly decreased when ET was present. Verapamil hydrochloride (VHCL), a non-specific inhibitor of the ET transporter OCTN1 abrogated ET’s cytoprotective effects, indicative of an intracellular action. These results suggest that ET could be a potential therapeutic for Parkinson’s disease.

Discovery of potential mitochondrial protectants that could cross the BBB could be highly valuable, given the role of mitochondrial dysfunction in PD [11][12][13][14][15]41,42].Yuzawa et al. (2024) [43] reported the protective effect of ET against 6-OHDA neurotoxicity in immortalised mouse hypothalamic cells (GT-17) through reduction in endoplasmic reticulum stress response.Thus far, however, it is not known if ET could protect against damage using cell models more relevant to human PD, such as human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons.Dopaminergic cultures produced from hiPSCs have been demonstrated to replicate the development of the midbrain, and Parkinsonian cellular networks can be formed from neurons that survive and integrate [44].6-hydroxydopamine (6-OHDA) has been used to induce degeneration of dopaminergic neurons in cellular models of PD [45,46] and in vivo in rodents [47].6-OHDA enters dopaminergic neurons via dopamine transporters (DAT), leading to mitochondrial dysfunction, increased oxidative damage and cell death [48,49].In this study, we sought to determine whether ET could protect against 6-OHDA-induced cell death and mitochondrial damage in tyrosine hydroxylase positive (TH+) cells in vitro.TH is a marker for dopamine-containing neurons [50].Human iPSC-derived dopaminergic neurons and human-derived neuroblastoma SH-SY5Y cells were chosen as they are widely used as a cellular model for the investigation of neuronal differentiation and neuroprotection events, including PD [44,[51][52][53].

Cell Culture
To make a 2 mM stock solution, 1 mg of 6-OHDA was dissolved in 2 mL of 0.1% ascorbic acid solution in ice-cold MiliQ water.The solution was filtered and can be stored at −20 • C for up to 1 week.SH-SY5Y cells were cultured in a 5% CO 2 humidified incubator at 37 • C in high-glucose Dulbecco's modified Eagle medium (DMEM-H) with 10% fetal bovine serum (FBS) including glutamine and sodium pyruvate, and 1% penicillinstreptomycin.Cells were then cultured in either 6-well plates (qPCR/Immunofluorescence), 12-well plates (Flow cytometry/ATP assay), or 96-well plates (MTT assay) for 24 h and at 80-90% confluency prior to treatments.

Cellular ET Uptake and Liquid Chromatography Mass Spectrometry
ET was quantified as previously described [24].Cells were washed thrice with ice-cold PBS (Thermo Fisher Scientific) before the addition of methanol spiked with a deuterated internal standard (ISTD) ET-d9.Next, samples were centrifuged at 20,000× g at 4 • C for 10 min, the supernatants collected in glass vials, and the contents evaporated at 37 • C under a N 2 stream.Glass vials containing the sample residues were reconstituted in pure methanol and ET levels were analysed by liquid chromatography mass spectrometry (LC-MS/MS), using an Agilent 1290 UPLC system coupled with an Agilent 6460 ESI mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).Samples were kept at 10 • C in the autosampler.2 µL of the processed samples was injected into a Cogent Diamond-Hydride column (4 µm, 150 × 2.1 mm, 100 Å; MicroSolv Technology Corporation, Leland, NC, USA) maintained at 30 • C. Solvent A was acetonitrile in 0.1% formic acid, and Solvent B was 0.1% formic acid in ultrapure water.Chromatography was carried out at a flow rate of 0.5 mL/min using the following gradient: 1 min of 20% solvent B, followed by a 3 min gradient increase in solvent B to 40% to elute ET.The retention time for ET is 4.2 min.
Mass spectrometry was carried out using the positive ion, electrospray ionisation mode, with multiple reaction monitoring (MRM) for quantification of specific target ions.Capillary voltage was set at 3200 V, and the gas temperature was kept at 350 • C. Nitrogen sheath gas pressure for nebulising the sample was at 50 psi, and the gas flow was set at 12 L/min.Ultra-high purity nitrogen was used as the collision gas.Precursor to product ion transitions and fragmentor voltages (V)/collision energies (eV) for each compound were as follows: ET; 230.1 → 186, 103 V/9 eV and ET-d9; 239.1 → 195.1, 98 V/9 eV.

MTT Assay
A MTT Assay Kit (Sigma) was used to detect the impact of 6-OHDA on cellular metabolic activity.iDA cultures (D22) or SH-SY5Y cells were passaged and counted using a haemocytometer.One thousand cells were seeded per well in a 96-well plate and allowed to differentiate until day 40 prior to treatments.15 µM 6-OHDA was found to induce 60-70% loss of cellular metabolic activity based on preliminary experiments.1 mg of MTT was added into 1 mL of medium, which was incubated for 4 h at 37 • C. The medium in the plate was discarded after 4 h, and 200 µL DMSO (dimethylsulphoxide) was added to each well before being shaken for one minute for dissolution.Absorbance at 570 nm based on cellular dehydrogenase activity was measured in a microplate reader (Tecan, Switzerland).The different experimental conditions were normalised to control absorbance.Experiments were carried out three times.

Quantitative RT-PCR
TRIzol Reagent (Invitrogen TM ) was used for RNA extraction as per the manufacturer's instructions.cDNA was produced from reverse transcription of 1000 ng of RNA (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Waltham, MA, USA).Reverse transcription was performed in a T-Personal Thermocycler (Biometra, Germany) with conditions of 25 • C for 10 min, 37 • C for 30 min, followed by 85 • C for 5 min.qPCR was carried out to quantify OCT4 (octamer-binding transcription factor 4), NANOG (NANOG homeobox), MAP2 (microtubule associated protein 2), NeuN (neuronal nuclei), NeuroD1 (neurogenic differentiation factor 1), BDNF, FOXA2 (foxhead box protein A2) and DAT (dopamine transporter) mRNA expression, using SYBR Green Gene Expression Assay Probes (Applied Biosystems, USA) and SYBR Green Universal PCR Master Mix (Applied Biosystems, USA).GAPDH was used as the housekeeping gene.The RT-qPCR was performed in a 7500 Real-time PCR System (Applied Biosystems, USA) with conditions of 95 • C for 10 min, followed by 95 • C for 15 s and 60 • C for 1 min, for 40 cycles.Subsequently, the relative mRNA expression for the respective genes of interest was quantitated via the comparative CT (∆∆CT) method.Primer sequences can be found in Supplementary Table S1.

Flow Cytometry
Flow cytometry measurements were performed using a CytoFlex LX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN, USA), using 10 6 cells per sample for analysis with 10,000 events per sample recorded.The FL1 channel was used to quantify cell death (propidium iodide, Ex/Em = 535/615 nm), free intracellular calcium (Fluo-4, Ex/Em = 488/525 nm), mitochondrial membrane potential (tetramethylrhodamine, methyl ester (TMRM), Ex/Em = 555/575 nm), mitochondrial ROS (MitoSOX, Ex/Em = 510/580 nm), and mitochondria (MitoTracker green Ex/Em = 490/526 nm).iDAs were cultured on coverslips washed with PBS before being stained with either propidium iodide, MitoSOX, or TMRM for 30 min.Cells were washed with PBS before 15 min fixation in 3.7% paraformaldehyde.Cells were permeabilised in perm buffer (Miltenyi Biotec) before blocking and primary antibody incubation of tyrosine hydroxylase that was FITC conjugated (130-120-350) (Miltenyi Biotec).Cells were then washed with PBS before analysis using flow cytometry.For flow cytometry data acquisition, fluorescent signals were measured on a logarithmic scale of four decades of log.Raw data were processed using FlowJo version 10.5.3.

ATP Assay
A commercially available ATP determination kit (Invitrogen TM ) was used to study ATP levels in cells.Treated cells (iDA or SH-SY5Y cells) were collected and counted using a haemocytometer to ensure all samples contain equal number of cells (10 6 cells).Upon centrifugation and removal of supernatant, 1 mL of boiling ultrapure water from an Arium pro ® ultrapure system was added into the cell pellet and incubated in a water bath for 10 min at 100 • C [55].Samples were then cooled on ice for 30 s and supernatant utilised for ATP assay as per the manufacturer's instructions.Luminescence readings of the samples were performed using Synergy H1 Microplate Reader (BioTek, Winooski, VT, USA).For all experiments, ATP standard curves were run in the range of 0.2 to 1.4 µM.

Western Blot
Cells were lysed with ProteoExtract ® Native Membrane Protein Extraction Kit (Millipore) for OCTN1 expression, performed as per the manufacturer's instructions.Supernatant was collected after centrifugation at 13,000× g at 4 • C for 20 min.Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Sigma Aldrich).The samples were loaded and separated on precast SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA).Proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad) in transfer buffer containing 48 mmol/L Tris-HCl, 39 mmol/L glycine, 0.037% SDS, and 20% methanol at 4 • C for 1 h.Blocking was done in 2.5% non-fat milk for 1 h at room temperature.Binding of OCTN1 primary goat anti-OCTN1 (sc-19819) (Santa Cruz, CA, USA) antibody at 1:200 dilution, overnight at 4 • C, was followed by incubation with secondary horseradish peroxidase-conjugated IgG in 2.5% non-fat milk for 1 h at room temperature.Anti-mouse IgG HRP were obtained from Sigma Aldrich and dilution of 1:5000 was used.The blots were visualised with SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) using iBright Imaging systems (Invitrogen TM ).The blots were processed using open access software platform FIJI version 1.53r (ImageJ).

Protein Carbonylation Assay
Cells were lysed with Pierce ® RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) for protein carbonylation assay.Supernatant was collected after centrifugation at 13,000× g at 4 • C for 20 min.Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Sigma Aldrich).OxyBlot Protein Oxidation Detection Kit (Chemicon/Millipore, Temecula, CA, USA) was utilised.The samples were derivatised and prepared as per the manufacturer's instructions prior to being loaded and separated on precast SDS-polyacrylamide gels (Bio-Rad).Proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad) in transfer buffer containing 48 mmol/L Tris-HCl, 39 mmol/L glycine, 0.037% SDS, and 20% methanol at 4 • C for 1 h.Blocking, incubation with 2,4-Dinitrophenylhydrazine (DNPH) antibody and secondary antibody, respectively, were also performed as specified by the manufacturer.Binding of primary antibodies was followed by incubation with secondary horseradish peroxidase (HRP) conjugated IgG in 2.5% non-fat milk for 1 h at room temperature.Anti-mouse IgG HRP was obtained from Sigma Aldrich, and a dilution of 1:5000 was used.The blots were visualised with SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) using iBright Imaging systems (Invitrogen TM ).The blots were processed using open access software platform FIJI (ImageJ).

Tyrosine Hydroxylase Protein Assay
Flow cytometry was employed to assess tyrosine hydroxylase (TH) protein expression in SH-SY5Y cells.Cells were first detached, and cell pellets were washed with PBS.Cells were incubated for 30 min with 3.7% paraformaldehyde before the permeabilisation step for another 30 min.Blocking was done with 5% BSA before 30 min incubation with FITC-conjugated TH antibody and APC-conjugated Tau antibody (130-119-363) (Miltenyi Biotec).The tau neuronal marker serves as the housekeeping protein.Flow cytometry measurements were performed on a Cytoflex LX flow cytometer (Beckman Coulter Life Sciences), using 1,000,000 cells per sample for analysis.In total, 10,000 events per sample were recorded.Fluorescent signals were measured on a logarithmic scale of four decades of log.Raw data were processed using FlowJo version 10.5.3.

Dopamine Detection ELISA Kit
Dopamine levels were quantified using a commercially available dopamine detection kit (Novus Biologicals, Littleton, CO, USA).The colorimetric assay is a 90 min, singlewash sandwich ELISA designed for quantitative measurement of dopamine.Treated D40 iDA cultures in 12-well plates have 500 µL of total conditioned media collected for dopamine analysis.The same treated cells (with conditioned media removed) were lysed and protein concentration was quantified using a Pierce BCA protein assay kit.Conditioned media from treated cells were collected and centrifuged to remove cellular debris.Protein concentrations in all conditioned media were normalised based on the protein concentration of lysed cells using sample dilution buffer.Next, 50 µL of diluted sample (normalised) and 50 µL of antibody cocktail were added to each well, followed by 1 h incubation on a shaker at 400 rpm.After 1 h, the 3,3 ′ ,5,5 ′ -Tetramethylbenzidine (TMB) solution provided was added before the addition of stop solution as per the manufacturer's instructions.Dopamine standards were prepared as well.Absorbance readings at 450 nm of the samples were performed using Synergy H1 Microplate Reader (BioTek, USA).For all experiments, dopamine standard curves were run in the range of 2.19 to 140 ng/mL.

Statistical Analysis
Data are shown as mean ± SD (standard deviation).All analysis was performed using GraphPad prism 9.0 software.Comparisons were conducted by one-way ANOVAs, with Bonferroni correction for multiple comparison tests.p < 0.05 was regarded as statistically significant.

Generation of hiPSC-Derived Day 40 Dopaminergic Neurons
We differentiated human induced pluripotent stem cells (hiPSCs; BJ and GM23720) and human embryonic stem cells (hESC; H9) to investigate the protective effect of ET on 6-OHDA-treated induced human dopaminergic neurons, a model highly relevant to human PD.We adopted a 40-day differentiation protocol, using various small molecules and growth factors (Figure 1A).Results show a significant decrease in the pluripotency markers OCT4 and NANOG (Supplementary Figure S1A) but significant increases in the neuronal markers MAP2, NeuN, BDNF and NeuroD1 (Supplementary Figure S1B) and the dopaminergic-specific markers FOXA2 and DAT (Supplementary Figure S1C).Protein expression of neuronal and dopaminergic markers was also performed using confocal imaging, which showed that our day 40 iDAs express neuronal proteins of NeuN and TUBB3, and dopaminergic neuron-specific marker TH (Figure 1B).Neuronal yield and dopaminergic yield were found to be approximately 80% (Figure 1C) and 40% (Figure 1D), respectively, which is consistent with other studies [56,57].MTT assay showed that 6-OHDA induced a 70-80% loss of metabolic activity in iDA cultures (Figure 2D).6-OHDA was also found to induce neuronal degeneration observed under the microscope (Figure 3A).Degenerating neurons appear fragmented with neurite degeneration, similar to other in vitro neuronal degeneration phenotypes [58].6-OHDA treatment led to a significant increase in non-viable cells.iDA cultures express the OCTN1 transporter that mediates cellular ET uptake (Figure 2A).Protection of iDA cultures by ET against loss of viability induced by 6-OHDA is concentration dependent (Figure 2B). 1 mM of ET was used in subsequent experiments as it is similar to achievable cellular levels [59,60].The non-specific inhibitor of OCTN1, verapamil hydrochloride (VHCL), greatly reduced intracellular ET levels (Figure 2C).6-OHDA resulted in a higher proportion of non-viable cells in TH-positive (TH+) iDAs than TH-negative (TH−) non-iDAs (Figure 3C).In both TH+ and TH-populations, ET significantly ameliorated the 6-OHDA-induced increase in non-viable cells (except GM23720 non-iDAs; p = 0.233) (Figure 3C).6-OHDA treatment also caused a decrease in intracellular ATP levels, while co-treatment with ET was able to attenuate the decrease (Figure 4A).Dopamine secretion assay shows that 6-OHDA caused a significant reduction in the amount of dopamine secreted by day 40 iDA cultures, while co-treatment with ET was able to attenuate the decrease (Figure 4B).We also investigated the effects of 6-OHDA on mitochondrial function, as measured by the TMRM (mitochondrial membrane potential-MMP) and MitoSOX (mitochondrial ROS) probes.Murphy et al. [61] discussed the pros and cons of this method, and results must always be interpreted with caution, e.g., they can be affected by changes in mitochondrial size, shape, and membrane potential.From the results, we found that 6-OHDA caused a significant decrease in MMP (Figures 4C,D and 5A) and an apparent significant increase in mROS levels ( Figures 4E,F and 5B) in both iDAs and non-iDAs.The effect of 6-OHDA on mitochondrial membrane potential and mitochondrial ROS levels was more significant in iDAs than non-iDAs (Figure 5A,B).VHCL abrogated the effects of ET.

ET Protects
pression of neuronal and dopaminergic markers was also performed using confocal imaging, which showed that our day 40 iDAs express neuronal proteins of NeuN and TUBB3, and dopaminergic neuron-specific marker TH (Figure 1B).Neuronal yield and dopaminergic yield were found to be approximately 80% (Figure 1C) and 40% (Figure 1D), respectively, which is consistent with other studies [56,57].

ET Uptake Also Protects TH+ SH-SY5Y Neuron-like Cells against 6-OHDA Neurotoxicity
To verify if ET can protect other TH+ cells from the neurotoxicity of 6-OHDA, the SH-SY5Y human neuroblastoma cell line, a well-accepted model in PD studies [51], was also used.SH-SY5Y cells also express TH and the OCTN1 transporter (Figure 6A); uptake of ET was abolished with coincubation of VHCL (Figure 6B).6-OHDA-treated SH-SY5Y cells revealed a 60-70% loss of metabolic activity (Figure 6C) and a significant increase in non-viable cells (Figure 6D).6-OHDA also significantly affected cellular metabolism as evidenced by the reduction in intracellular ATP levels (Figure 7A), increased intracellular

ET Uptake Also Protects TH+ SH-SY5Y Neuron-like Cells against 6-OHDA Neurotoxicity
To verify if ET can protect other TH+ cells from the neurotoxicity of 6-OHDA, the SH-SY5Y human neuroblastoma cell line, a well-accepted model in PD studies [51], was also used.SH-SY5Y cells also express TH and the OCTN1 transporter (Figure 6A); uptake of ET was abolished with coincubation of VHCL (Figure 6B).6-OHDA-treated SH-SY5Y cells revealed a 60-70% loss of metabolic activity (Figure 6C) and a significant increase in non-viable cells (Figure 6D).6-OHDA also significantly affected cellular metabolism as evi-denced by the reduction in intracellular ATP levels (Figure 7A), increased intracellular free calcium (Figure 7B), decreased mitochondrial membrane potential (MMP) (Figure 8E), and increased mitochondrial ROS (mROS) levels (Figure 8C,D), as measured by the MitoSOX probe.Since heightened levels of ROS can promote oxidative protein damage, as revealed by protein carbonyl formation [62], and increased brain protein carbonyls is a feature of PD [8], we performed a protein carbonyl assay by immunoblotting.6-OHDA treatment led to a significant increase in oxidised proteins (approximately 3.5-fold) as compared to the control (Figure 8A,B).Tyrosine hydroxylase (TH) was also investigated.Using flow cytometry, 6-OHDA treatment significantly reduced TH protein levels as compared to the control (Figure 7C), and 6-OHDA decreased dopamine secretion in day 40 iDAs.Tau protein, which is also implicated in PD [63], did not appear to have its levels affected by either 6-OHDA or ET treatment (Figure 7D).Co-treatment with ET was able to attenuate any changes induced by 6-OHDA.The addition of VHCL tended to abrogate (note p values on the figure) the effects of ET, although the results were not always significant (p > 0.05).These findings are consistent with our results involving D40 iDAs.
Antioxidants 2024, 13, x FOR PEER REVIEW 13 of 21 free calcium (Figure 7B), decreased mitochondrial membrane potential (MMP) (Figure 8E), and increased mitochondrial ROS (mROS) levels (Figure 8C,D), as measured by the MitoSOX probe.Since heightened levels of ROS can promote oxidative protein damage, as revealed by protein carbonyl formation [62], and increased brain protein carbonyls is a feature of PD [8], we performed a protein carbonyl assay by immunoblotting.6-OHDA treatment led to a significant increase in oxidised proteins (approximately 3.5-fold) as compared to the control (Figure 8A,B).Tyrosine hydroxylase (TH) was also investigated.Using flow cytometry, 6-OHDA treatment significantly reduced TH protein levels as compared to the control (Figure 7C), and 6-OHDA decreased dopamine secretion in day 40 iDAs.Tau protein, which is also implicated in PD [63], did not appear to have its levels affected by either 6-OHDA or ET treatment (Figure 7D).Co-treatment with ET was able to attenuate any changes induced by 6-OHDA.The addition of VHCL tended to abrogate (note p values on the figure) the effects of ET, although the results were not always significant (p > 0.05).These findings are consistent with our results involving D40 iDAs.

Discussion
In this study, we investigated the effect of ET on cell death caused by 6-OHDA in an iPSC model of PD. 6-OHDA-induced oxidative stress occurs as early as after 4 h of incubation, but cell death occurs within 24 h in rat [64], mouse [46], and human [65]

Discussion
In this study, we investigated the effect of ET on cell death caused by 6-OHDA in an iPSC model of PD. 6-OHDA-induced oxidative stress occurs as early as after 4 h of incubation, but cell death occurs within 24 h in rat [64], mouse [46], and human [65] dopaminergic cells.A concentration-dependent decrease in cellular metabolic activity with 60-70% loss of activity was observed at 15 µM 6-OHDA concentration with 24 h incubation.This concentration of 6-OHDA was selected for further experiments, while 1 mM of ET was used on the basis of preliminary data [59,60].Dose-dependent ET protection of D40 iDA cultures against 6-OHDA is presented in Figure 2B.Ergothioneine (ET) is a cytoprotective compound that accumulates at high levels in tissues during times of oxidative damage [22,66].LC-MS analysis confirmed that verapamil hydrochloride (VHCL), a non-specific inhibitor of OCTN1, reduced intracellular ET levels (Figures 2C and 6B) and decreased the protective effect of ET.This demonstrated that the protective effects of ET observed in our experiments are largely or entirely dependent on cellular uptake of ET and not through, perhaps, extracellular neutralisation of 6-OHDA by direct reaction of ET with it or its oxidation products.Previous studies have found that OCTN1 is important for cellular uptake of ET in endothelial cells [67] and that OCTN1 levels could be elevated in response to tissue injury, e.g., in fatty liver [68] or kidney disease [69].To demonstrate that OCTN1 is present in the cells, Western blot (Supplementary Figure S2) and fluorescence imaging (Figures 2A and 6A) were carried out, but it should be noted that there is considerable variability in the specificity of commercial antibodies for OCTN1; nevertheless, our direct measurements of ET showed that it entered the cells and VHCL prevented that.Tyrosine hydroxylase (an enzyme involved in the production of dopamine) was expressed in both D40 iDAs (Figures 1B and 2A) and SH-SY5Y cells (Figure 6A).Results showed that ET could protect against 6-OHDA-induced degeneration of both human iDAs and SH-SY5Y cells.VHCL abolished the protective effect of ET on cell viability.
Mitochondrial dysfunction was also ameliorated by ET in 6-OHDA treated iDA (TH+) and non-iDA neurons (TH-).Mitochondrial dysfunction is characterised by a decrease in ∆Ψm, reduced ATP generation, and an increase in mitochondrial ROS (mROS) production [70].6-OHDA is thought to induce mitochondrial dysfunction in part through the inhibition of complex I of the mitochondrial electron transport chain, which contributes to a decreased ∆Ψm, reduced ATP generation, increase in ROS production, and apoptotic cell death [48].Similar to other studies [71,72], we found that 6-OHDA induced mitochondrial dysfunction as evidenced by a reduction of ∆Ψm, decreased cellular ATP levels and increased mROS levels.ET ameliorated the effects of 6-OHDA with a smaller decrease in ∆Ψm and cellular ATP levels and a lower increase in mROS levels.VHCL inhibited the protective effect of ET on mitochondrial dysfunction.Our results add to previous findings that ET has neuroprotective functions [21,26,28,43] which could potentially aid in the prevention and treatment of PD.We would also like to acknowledge the paper that was published by Yuzawa et al. (2024) [43] while we were preparing this manuscript.Yuzawa et al. reported the protective effect of ET against 6-OHDA neurotoxin in immortalised mouse hypothalamic cells (GT-17), a cell line perhaps less relevant to PD.Nevertheless, their data further illustrate the potential protective effect of ET against neurodegeneration.
The protective effect of ET against 6-OHDA was also observed in TH+ SHSY5Y cells.Besides loss of cell viability, treatment of SH-SY5Y cells with 15 µM 6-OHDA resulted in a significant increase in intracellular free calcium levels.Increases in intracellular free calcium levels are known to induce mitochondrial oxidative stress-mediated apoptosis [73].ET reduced the 6-OHDA-induced increase in intracellular calcium levels.The reduced intracellular calcium levels could be due to an effect of ET as a cytoprotectant or chelator of divalent metal ions, including Ca 2+ [74].Our results demonstrate that ET could aid in the maintenance of intracellular calcium homeostasis.Treatment of SH-SY5Y cells with 15 µM 6-OHDA also resulted in a significant increase in total carbonylated proteins.Protein carbonylation is increased in the brains of PD patients [8,75] and in cells that are undergoing oxidative stress [76].Oxidised proteins contribute to increased ER stress and increased unfolded protein response (UPR) [77] and can result in cellular apoptosis [78,79].6-OHDA was found to induce an increase in carbonylated proteins in SH-SY5Y cells [80], PC12 cells [81], and rats [82].Results indicate a protective effect of ET against protein carbonyl formation.Moreover, ET was able to ameliorate the 6-OHDA-induced decrease in tyrosine hydroxylase levels.Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine production, and low dopamine levels play a key role in PD pathogenesis [83,84].Similar to our study on SH-SY5Y cells, 6-OHDA treated rats were found to have reduced tyrosine hydroxylase [85].The effect of ET on restoring TH levels could be clinically relevant since PD is caused by loss of dopamine-producing neurons.The pathology of PD involves mitochondrial dysfunction, oxidative stress, neuroinflammation, and aberrant protein homeostasis [86].Besides the protective effect of ET as an anti-oxidant and improving cellular bioenergetics, ET could have cytoprotective effects through its influence on neurogenesis [87], potential epigenetic modifications [88], and regulation of sirtuins, a family of NAD + -dependent deacetylases [89,90].Our work demonstrates mitochondrial protection by ET.Moreover, effects of ET on neuroinflammation and aberrant protein homeostasis contributing to PD can be further studied.
In summary, our findings demonstrate that ET can protect human iDA and non-iDA neurons against a 6-OHDA-induced increase in cell death and metabolic dysfunction.Results also suggest that ET has disease-modifying potential by increasing dopamine levels secreted by iDA neurons.Protective effects of ET were abrogated when cell cultures were cotreated with VHCL, a non-specific inhibitor of OCTN1, the ET transporter, suggesting that ET uptake into cells is necessary for protection against 6-OHDA.Results in SH-SY5Y cells also demonstrated the protective effects of ET against a 6-OHDA-induced increase in intracellular free calcium, increase in total carbonylated proteins, and reduction in tyrosine hydroxylase levels.In this paper, we have also shown the protective effects of ET against mitochondrial dysfunction.Uptake of ET into mitochondria in vivo due to conflicting data with regard to the localisation of OCTN1 on mitochondria is unclear [39,91,92].Further studies also need to be carried out to validate the protective effect of ET in animal models of PD.
Day 40 iDA Cultures against 6-OHDA-Induced Increase in mROS, Loss of MMP, Reduction in ATP Levels, and Loss of Dopamine Secretion

Figure 1 .
Figure 1.Derivation of day 40 dopaminergic neurons and quality control analysis.(A) Schematic representation of differentiation protocol to derive day-40-induced dopaminergic neurons from hiPSCs (BJ and GM23720) and hESC (H9).Scale bar= 100 µm (B) Confocal imaging of neuronal and dopaminergic markers.DAPI stains the nucleus.Beta-Tubulin III and NeuN are neuronal markers.Tyrosine hydroxylase (TH) is a dopaminergic marker.Scale bar = 100 µm; Scale bar for zoomed in figures = 20 µm (C,D) ImageJ analysis of confocal images of TH and NeuN to quantify the percentage of neurons and dopaminergic neurons present in the culture.Data are represented as mean ± SD (n = 3).Data were analysed by unpaired Student's t-test.*** p ≤ 0.001.

Figure 4 .
Figure 4. Effect of ET on intracellular ATP levels and dopamine secretion in 6-OHDA-treated iDA cultures; TH+ Day 40 iDAs were also analysed for mitochondrial ROS and mitochondrial membrane potential levels.(A) ATP assay showing relative changes in intracellular ATP levels.Higher absorbance values denote greater quantities of ATP in the cell lysates of the treatment group.(B) Dopamine secretion assay showing the changes in the amounts of dopamine being secreted by the day 40 iDAs in each treatment group.Higher absorbance values denote greater quantities of dopamine.(C) Scatter plot of flow cytometry data (TH-TMRM co-staining) of the day 40 iDAs from the different treatment groups.(D) Bar chart of flow cytometry data for TH and TMRM co-staining to show relative changes in the different treatment groups.(E) Scatter plot of flow cytometry data (TH-MitoSOX co-staining) of the day 40 iDAs from the different treatment groups.(F) Bar chart of flow cytometry data for TH and MitoSOX co-staining to show relative changes in the different treatment groups.(A,B,D,F) Data are represented as mean ± SD (n = 3).(Control: no treatment; 6OHDA: 15 μM 6-hydroxydopamine; ET: 1 mM ergothioneine; VHCL: 100 μM verapamil hydrochloride).Data were analysed by one-way ANOVA with Bonferroni's multiple comparison post hoc test.* p ≤ 0.05.** p ≤ 0.01.**** p ≤ 0.0001.

Figure 4 .
Figure 4. Effect of ET on intracellular ATP levels and dopamine secretion in 6-OHDA-treated iDA cultures; TH+ Day 40 iDAs were also analysed for mitochondrial ROS and mitochondrial membrane potential levels.(A) ATP assay showing relative changes in intracellular ATP levels.Higher absorbance values denote greater quantities of ATP in the cell lysates of the treatment group.(B) Dopamine secretion assay showing the changes in the amounts of dopamine being secreted by the day 40 iDAs in each treatment group.Higher absorbance values denote greater quantities of dopamine.(C) Scatter plot of flow cytometry data (TH-TMRM co-staining) of the day 40 iDAs from the different treatment groups.(D) Bar chart of flow cytometry data for TH and TMRM co-staining to show relative changes in the different treatment groups.(E) Scatter plot of flow cytometry data (TH-MitoSOX co-staining) of the day 40 iDAs from the different treatment groups.(F) Bar chart of flow cytometry data for TH and MitoSOX co-staining to show relative changes in the different treatment groups.(A,B,D,F) Data are represented as mean ± SD (n = 3).(Control: no treatment; 6OHDA: 15 µM 6-hydroxydopamine; ET: 1 mM ergothioneine; VHCL: 100 µM verapamil hydrochloride).Data were analysed by one-way ANOVA with Bonferroni's multiple comparison post hoc test.* p ≤ 0.05.** p ≤ 0.01.**** p ≤ 0.0001.

Figure 6 .
Figure 6.Intracellular uptake of ET, mediated through OCTN1 transporters, also protects TH+ SH-SY5Y cultures against 6-OHDA-induced cell death.(A) Confocal imaging showing expression of TH and OCTN1 in SH-SY5Y cells.Images are representative of three independent experiments (n = 3).

Figure 6 .
Figure 6.Intracellular uptake of ET, mediated through OCTN1 transporters, also protects TH+ SH-SY5Y cultures against 6-OHDA-induced cell death.(A) Confocal imaging showing expression of TH and OCTN1 in SH-SY5Y cells.Images are representative of three independent experiments (n = 3).

Figure 7 .
Figure 7. Effect of ET on cellular metabolism and tyrosine hydroxylase levels in 6-OHDA-treated SH-SY5Y cells.(A) ATP assay showing relative levels in intracellular ATP as compared to the control group.(B) Fluo-4 AM assay measures intracellular free calcium levels.Data are represented as mean ± SD (n = 4).(C) Flow cytometry analysis to quantify relative amounts of tyrosine hydroxylase (TH) protein expression as compared to control.(D) Flow cytometry analysis to quantify relative tau neuronal protein expression among the treatment groups as compared to control.(A-D) (Control: no treatment; 6OHDA: 15 μM 6-hydroxydopamine; ET: 1 mM ergothioneine; VHCL: 100 μM verapamil hydrochloride).Data were analysed by one-way ANOVA with Bonferroni's multiple comparison post hoc test.Unless stated in figures, ns: non-significant, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 7 .
Figure 7. Effect of ET on cellular metabolism and tyrosine hydroxylase levels in 6-OHDA-treated SH-SY5Y cells.(A) ATP assay showing relative levels in intracellular ATP as compared to the control group.(B) Fluo-4 AM assay measures intracellular free calcium levels.Data are represented as mean ± SD (n = 4).(C) Flow cytometry analysis to quantify relative amounts of tyrosine hydroxylase (TH) protein expression as compared to control.(D) Flow cytometry analysis to quantify relative tau neuronal protein expression among the treatment groups as compared to control.(A-D) (Control: no treatment; 6OHDA: 15 µM 6-hydroxydopamine; ET: 1 mM ergothioneine; VHCL: 100 µM verapamil hydrochloride).Data were analysed by one-way ANOVA with Bonferroni's multiple comparison post hoc test.Unless stated in figures, ns: non-significant, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.