N-acetylcysteine increases dopamine release and prevents the deleterious effects of 6-OHDA on the expression of VMAT2, α-synuclein, and tyrosine hydroxylase

ABSTRACT Objectives Current treatments for Parkinson’s disease using pharmacological approaches alleviate motor symptoms but do not prevent neuronal loss or dysregulation of dopamine neurotransmission. In this article, we have explored the molecular mechanisms underlying the neuroprotective effect of the antioxidant N-acetylcysteine (NAC) on the damaged dopamine system. Methods SH-SY5Y cells were differentiated towards a dopaminergic phenotype and exposed to 6-hydroxydopamine (6-OHDA) to establish an in vitro model of Parkinson’s disease. We examined the potential of NAC to restore the pathological effects of 6-OHDA on cell survival, dopamine synthesis as well as on key proteins regulating dopamine metabolism. Specifically, we evaluated gene- and protein expression of tyrosine hydroxylase (TH), vesicle monoamine transporter 2 (VMAT2), and α-synuclein, by using qPCR and Western blot techniques. Moreover, we quantified the effect of NAC on total dopamine levels using a dopamine ELISA assay. Results Our results indicate that NAC has a neuroprotective role in SH-SY5Y cells exposed to 6-OHDA by maintaining cell proliferation and decreasing apoptosis. Additionally, we demonstrated that NAC treatment increases dopamine release and protects SH-SY5Y cells against 6-OHDA dysregulations on the proteins TH, VMAT2, and α-synuclein. Conclusions Our findings contribute to the validation of compounds capable to restore dopamine homeostasis and shed light on the metabolic pathways that could be targeted to normalize dopamine turnover. Furthermore, our results highlight the effectiveness of the antioxidant NAC in the prevention of dopaminergic neurodegeneration in the present model. Abbreviations DAT, dopamine transporter; 6-OHDA, 6-hydroxydopamine; NAC, N-acetylcysteine; PARP, poly (ADP-ribose) polymerase; RA; retinoic acid; ROS, reactive oxygen species; TH, tyrosine hydroxylase; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; VMAT2, vesicle monoamine transporter 2.


Introduction
Parkinson's disease is a neurodegenerative disorder characterized by dopaminergic cell loss in the midbrain substantia nigra nuclei.The etiology of the disease is thought to be multifactorial and oxidative stress has been proposed as one of the causes triggering dopamine-cell degeneration [1].
Dopamine is synthetized in the cytosol by the rate limiting enzyme tyrosine hydroxylase (TH) and then is immediately transported into synaptic vesicles by the vesicle monoamine transporter 2 (VMAT2).Sequestration of dopamine into vesicles is crucial since dopamine can undergo autoxidation in the cytosol and form dopamine-derived quinones which are highly reactive and can lead to oxidative stress and cellular death [2].Defects in dopamine transport by VMAT2 have been observed in Parkinson's disease and suggested as one of the causes increasing cytosolic dopamine levels and formation of excessive reactive oxygen species (ROS) [3].VMAT2 deficient mice present Parkinson's disease symptoms [2,4,5] whereas overexpression of VMAT2 has been shown to increase sequestration of intracellular dopamine and to be neuroprotective [4].Another protein regulating synaptic transmission and mediating vesicle trafficking is α-synuclein [6,7].Under pathological conditions, α-synuclein can aggregate and accumulate in the cells in the form of Lewy bodies, a pathological hallmark in both Parkinson's disease and dementia with Lewy bodies [8].Exposure to parkinsonism-inducing neurotoxins such as paraquat or 1-metyl-4-fenyl-1,2,3,6-tetrahydropyridin (MPTP) increases expression of αsynuclein and this up-regulation has been suggested to be beneficial and part of a neuroprotective response against oxidative stress [9,10].On the other hand, mice overexpressing α-synuclein display impaired dopamine metabolism and significant dopamine cell death [11,12].
Therefore, the exact role of α-synuclein in degeneration is still under debate.Some therapeutic strategies have aimed to restore levels of VMAT2 or α-synuclein [4,13,14].Still, to date there are no available treatments to hinder dopaminergic cell degeneration and/or restore dopamine homeostasis in Parkinson's disease.
The antioxidant N-acetylcysteine (NAC) has emerged as a promising treatment in different neurological diseases [15][16][17][18].In Parkinsonian patients, NAC has shown to be able to cross the blood-brain-barrier, restore brain glutathione levels [19], and to have a beneficial impact on the nigrostriatal dopaminergic system [17,20].In cultured cells, NAC has shown to improve cell survival of human embryonic stem cell (hESC)-derived midbrain dopamine neurons [21] and prevent dopamine-induced apoptosis [22].In animal models of Parkinson's disease, NAC has also showed to be beneficial [23,24].Previously, our group has demonstrated that NAC treatment has an effect in the brain of hemiparkinsonian rats by recovering affected metabolic pathways, replenishing glutathione levels, increasing TH expression, and modulating dopamine transporter (DAT) availability [25,26].Like glutathione, NAC appears to play an important role in maintaining homeostasis of many important cellular functions, including gene and protein expression, cell proliferation, or apoptosis.Additionally, it has been suggested that NAC might interact with cysteine residues in proteins critically involved in dopamine homeostasis modulating the essential metabolic pathways where these proteins are involved.Thus, proposing a potential beneficial role of NAC in the cysteinome deregulation in Parkinson's disease [27,28].
The neurotoxin 6-OHDA is a hydroxylated analogue of dopamine widely used to generate Parkinson's models both in vivo and in vitro due to its ability to enter dopaminergic cells via DAT.Once in the cells, 6-OHDA, in addition to inhibition of the mitochondrial respiratory complex I [29], undergoes autooxidation resulting in toxic derivates that trigger oxidative stress, autophagy, and activation of apoptotic cascades [30][31][32][33].6-OHDA has also been identified in the brains and urine of Parkinson's disease patients [34,35] and reported to be endogenously produced from L-DOPA [36], a dopamine precursor that remains the current standard for symptomatic treatment of Parkinson's disease.
NAC has been shown to interact with 6-OHDA preventing the formation of 6-OHDA toxic derivates and reducing the neurotoxic effect [32].It has also been suggested that NAC, besides being an effective ROS scavenger and glutathione replenisher, could interact with proteins involved in apoptosis and prevent cellular death caused by 6-OHDA exposure [30].
The SH-SY5Y cell line represents an ideal model to test new drugs and therapies in Parkinson's disease.Due to their catecholaminergic phenotype as well as to their ability to synthesize dopamine [37], this cell line has been widely used as an in vitro model for studying dopaminergic cells and for creating neurotoxin-induced models to study Parkinson's disease.Moreover, SH-SY5Y are chromosomally stable and easy to culture, unlike induced pluripotent stem cells (iPSCs) or neuroepithelial stem cells (NESCs).
In this study, SH-SY5Y cells differentiated towards a dopaminergic phenotype were used as an in vitro model of Parkinson's disease to investigate the toxicity of 6-OHDA on the expression of TH, α-synuclein, and VMAT2.In addition, we investigated the potential neuroprotective effect of NAC on the neurotoxicity of 6-OHDA towards these proteins.

Cell culturing and differentiation
SH-SY5Y cells (ATCC; CRL-2266) were maintained in T75 flasks in Minimum Essential Medium alpha (MEM-α; ThermoFisher; #41061029) supplemented with 10% fetal bovine serum (ThermoFisher; 15140122), and 1% penicillin-streptomycin. Dulbecco's Modified Eagle's Medium (DMEM), or a combination of DMEM/F12 medium, is also commonly used for culturing SH-SY5Y cells, but to our experience the cells were more viable in MEM-α without the addition of phenol red.Medium was renewed every 4 days and cells were split using 0.01% Trypsin-EDTA once they had reached 70-80% confluency.Cells were grown for no more than 15 passages to prevent the loss of neuronal markers.
For cell differentiation into a dopaminergic phenotype, we utilized a protocol described by [38].In short, 10 μM all-trans-Retinoic acid (RA; Sigma Aldrich; #R2625) or 80 nM 12-O-tetradecanoyl-phorbol-13acetate (TPA; Sigma Aldrich; #P1585) was added to culture medium for 6 days with medium exchange after 3 days (starting with 150 000 cells/well in a 6-well plate).A combination of the two drugs was also tested, i.e.RA for 3 days and TPA for another 3 days.The expression levels of TH and DAT were determined using real-time qPCR and Western blotting.The concentration of fetal bovine serum in culture did not significantly alter the expression of these two markers (data not shown).

Measurement of proliferation rate and metabolic activity
SH-SY5Y cells were seeded in a 96-well plate (50 000 cells/well) in normal growth medium (as described above) and left for 2 days to fully recover.The cells were then incubated with different concentrations of NAC (1.25-20 mM) and 6-OHDA (10-100 μM) for 24 h.In experiments where a combination of the two drugs was tested, NAC was added to the culture medium 1 h prior to exposing the cells to 6-OHDA.After 24 h of 6-OHDA exposure, cells were incubated with either Bromodeoxyuridine (BrdU) to measure cell proliferation, or Resazurin to measure metabolic activity (see details below).

Cell proliferation enzyme-linked immunoassay
The proliferation rate was measured using Bromodeoxyuridine (BrdU) assay (Roche; #11647229001).BrdU labeling solution was added to the culture wells to a final concentration of 10 μmol/L and incubated for 2 h at 37°C.The cells were fixed for 30 min at room temperature and treated with anti-BrdU-peroxidase working solution for 60 min.The antibody conjugate was then removed, and the cells were washed three times in phosphate-buffered-saline. For color development, substrate solution was added for 5 min.The absorbance was measured using a spectrophotometer set at 370 nm (reference wavelength 492 nm).

Mitochondrial metabolic activity assay
Mitochondrial function was measured using Resazurin according to the manufacturer's instructions (R&D Systems; #AR002).Cells were incubated with 10 μl Resazurin per 100 μl of cell culture medium for 4 h at 37°C.The fluorescence was read using 544 nm excitation and 590 nm emission wavelengths.

Caspase 3/7 activity assay
Caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay (Promega; #G8090).SH-SY5Y cells were cultured in a 96-well plate (50 000 cells/well) in 100 μl of normal growth medium.Three experimental groups were included: control (i.e.non-treated cells); cells treated with 25 μM 6-OHDA; and cells pre-treated with 1.25 mM NAC for 1 h before the addition of 25 μM 6-OHDA.Cells were incubated with 6-OHDA for 24 h. 100 μl of Caspase-Glo Reagent was then added to each well for 60 min.Luminescence was measured in a plate-reading luminometer.

Dopamine ELISA
SH-SY5Y cells (150 000 cells/well in a 6 well-plate) were differentiated into a dopaminergic phenotype using 80 nM TPA for 6 days, as previously described (3 + 3 days with medium renewal in between).To induce depolarization and dopamine release, cells were treated with KCl (50 mM) for 15 min.The culture medium was then collected, and dopamine concentrations were immediately measured using a dopamine ELISA kit (Abnova; #KA3838), following the manufacturer's protocol.

Statistical analysis
All experiments were performed at least three times using three technical replicates per group.Results are presented as mean ± standard deviation.Statistical analyses were performed using GraphPad Prism 9.4.1 San Diego, California, USA.Statistical differences were analyzed using one-or two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test.For pair comparisons, Student's t-test was used.Results were considered statistically significant when p < 0.05.

Effects of NAC and 6-OHDA on cell proliferation
Different concentrations of NAC (Figure 1a,b) and 6-OHDA (Figure 1c,d) were independently tested in SH-SY5Y cells to study the respective dose effect on cell proliferation (Fig. a, c) and mitochondrial activity (Fig. b, d) and to determine the optimal concentrations of these two compounds for further experiments.One-way ANOVA followed by Bonferroni multiple comparison test showed that concentrations of NAC equal to or higher than 5 mM were cytotoxic to human SH-SY5Y cells and significantly reduced the proliferation rate (Figure 1a).Mitochondrial viability was stable at all concentrations of NAC except at the highest concentration tested (20 mM) where it significantly decreased mitochondrial function.When testing 6-OHDA, concentrations between 10 and 100 μM significantly decreased the proliferation rate (Figure 1c), and similar results were obtained in the mitochondrial viability test (Figure 1d).

NAC prevents 6-OHDA-mediated cell death
The combined effect of 25 μM 6-OHDA and different concentrations of NAC was tested in SH-SY5Y cells (Figure 1e) by one-way ANOVA followed by Bonferroni multiple comparison test.The results indicated that 6-OHDA-induced cell death was prevented by preincubation with 0.15-1.25 mM NAC in a concentration-dependent manner.1.25 mM NAC completely abolished the cytotoxic effects induced by 6-OHDA.Lower concentrations of NAC (0.15-0.6 mM) were less effective but still had a significant impact on cell survival.

NAC protects against 6-OHDA-induced apoptosis
SH-SY5Y cells treated with 25 μM 6-OHDA presented higher caspase 3/7 activity compared with control cells.On the other hand, when cells were treated with 1.25 mM NAC, apoptosis was reduced, and caspase activity recovered to almost control levels.Cells that were only incubated with NAC (without 6-OHDA) presented lower caspase activity than control cells, indicating that NAC per se arrests apoptosis in SH-SY5Y (Figure 2a).Additionally, SH-SY5Y cells exposed to 10-25 μM 6-OHDA expressed higher levels of the apoptotic marker cleaved PARP compared with control cells (Figure 2b).Cells treated with 1.25 mM NAC and 6-OHDA had similar levels of cleaved PARP as control cells, i.e. were completely saved from 6-OHDA-mediated cytotoxicity.NAC displayed antiapoptotic properties down to 0.3 mM but was not markedly effective at 0.15 mM.Cells treated with NAC alone did not express cleaved PARP, i.e. were not stressed by the treatment.In all cases where NAC was utilized, except for the lowest concentration 0.15 mM, the expression levels of the uncleaved version of PARP, known to work as a DNA repair protein, seemed to be increased compared with control.

TPA increases TH expression and dopamine levels in SH-SY5Y cells
TH and DAT mRNA (Figure 3a,b) and protein levels (Figure 3c) were measured in SH-SY5Y cells differentiated with either 10 μM retinoic acid, 80 nM TPA, or as a combined treatment for 6 days.Statistical significance was calculated using one-way ANOVA followed by Bonferroni multiple comparison test and revealed that cells treated with TPA adopted a dopaminergic-like phenotype with a significantly higher expression of TH mRNA and protein (Figure 3a, c respectively).The expression of DAT mRNA was low and remained constant, i.e. was not affected by any of the treatments (Figure 3b), however the expression of DAT protein in cells treated with TPA alone was the highest of all treatments (Figure 3c).Student's t-test demonstrated that dopamine release levels were significantly higher in TPA-differentiated cells compared to undifferentiated cells (Figure 3d).

NAC prevents 6-OHDA deleterious effects on TH, VMAT2, and α-synuclein expression
Two-way ANOVA followed by Bonferroni multiple comparison test indicated that TPA-differentiated SH-SY5Y cells incubated with only 6-OHDA presented significantly lower mRNA expression levels of TH (Figure 4a) and VMAT2 (Figure 4b) whereas levels of αsynuclein were higher than control cells (Figure 4c).These changes were restored if cells were treated with NAC (6-OHDA+NAC).NAC alone did not have an effect on the mRNA expression of these three proteins.Due to the low mRNA expression levels of DAT in these cells (CT-values above 35), no significant differences could be observed (Figure 4d).Qualitative protein expression analyses by Western blot corresponded well with the mRNA expression levels (Figure 4e).

NAC prevents 6-OHDA induced dopamine cell loss and affects dopamine synthesis and release in TPA-differentiated SH-SY5Y cells
Microscopy showed that TPA-differentiated SH-SY5Y cells (control) degenerated in the presence of 6-OHDA but not when they were treated with NAC   NAC prevents 6-OHDA-induced dysregulation on the dopamine system.TH and VMAT2 mRNA expression was significantly decreased in the presence of 6-OHDA but was preserved in experimental groups treated with NAC (a-b).αsynuclein mRNA expression was significantly increased in the presence of 6-OHDA but was partially restored after treatment with NAC (c).DAT mRNA levels were low and no significant differences were observed (d).Analyses of protein expression by Western blot (total protein quantification relative to actin) corroborated this data (e).**p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant, two-way ANOVA followed by Bonferroni post hoc correction.
confirming the neuroprotective effect of NAC against 6-OHDA (Figure 5a).Dopamine release significantly increased when cells were incubated with NAC alone or in combination with 6-OHDA (6-OHDA+NAC) compared with control cells (Figure 5b), thus indicating a direct effect of NAC on dopamine release.

Discussion
In this study, we have investigated the beneficial effects of NAC on SH-SY5Y dopaminergic cells and its neuroprotective role against 6-OHDA.Our results demonstrate that NAC has a neuroprotective role in SH-SY5Y cells exposed to 6-OHDA by maintaining cell proliferation and decreasing apoptosis.In TPA-differentiated cells, which present a more dopaminergic phenotype, we observed that 6-OHDA exerts pathological dysregulations on the expression of TH, VMAT2, and α-synuclein.Specifically, 6-OHDA decreased mRNA and protein levels of TH and VMAT2 and increased those of αsynuclein.Down-regulation of TH and VMAT2 is an indication of reduced dopamine synthesis and storage.On the other hand, upregulation of αsynuclein might be seen as beneficial and neuroprotective against oxidative insults [6].Previously, it had been reported that undifferentiated SH-SY5Y cells exposed to 6-OHDA presented decreased VMAT2 and increased phospho-129-α-synuclein protein levels [39].However, our current study goes beyond these results and demonstrates that these changes occur already at the gene regulatory level.Furthermore, we demonstrate that these pathological changes can be prevented by the antioxidant NAC.Treatment with NAC completely hampered 6-OHDA-induced down-regulation of VMAT2 and TH, indicating that dopamine synthesis and compartmentalization are maintained at normal at levels when treating with this antioxidant.Regarding α-synuclein, although expression levels significantly decreased when giving NAC, they did not reach those of the control cells.Perhaps, as mentioned earlier, the neurotoxic effect of 6-OHDA is not completely neutralized by NAC, or possibly, certain up-regulation of α-synuclein levels is still beneficial and neuroprotective [6].
NAC alone did not have a direct effect on TH, VMAT2, or α-synuclein mRNA or protein expression, suggesting that NAC, rather than acting directly on the modulation of these proteins, actuates by indirectly preventing 6-OHDA action mechanisms.It has been documented that NAC is an effective antioxidant against 6-OHDA neurotoxicity [40].However, we can see that in some cases even in the presence of NAC, the neurotoxin is still able to cause certain toxicity.This is for instance demonstrated by the only partial recovery of α-synuclein expression after given NAC and 6-OHDA (Figure 4c) or in the dopamine ELISA assay when dopamine levels in the 6-OHDA+NAC group were not as high as when cells were treated with NAC alone (Figure 5b).
An important and novel finding in this study is that NAC alone had a direct effect on dopamine release.Extracellular dopamine was measured after inducing its intracellular release by KCl in TPAdifferentiated cells.Cells treated with NAC alone had the highest dopamine release levels compared with control (non-treated cells).Additionally, NAC partially restored the dopamine levels in the 6-OHDA+NAC group, thus indicating a direct effect of the antioxidant on the releasable pool of dopamine.Since NAC alone did not have a measurable effect on TH or VMAT2 the increase in dopamine release cannot be justified by a higher production or vesicle storage of the neurotransmitter.As previously suggested, NAC can prevent spontaneous oxidation of cytosolic dopamine [41] and formation of dopamine-derived quinones [42].Therefore, if NAC could stabilize dopamine by preventing the formation of toxic dopaminequinones it will in turn increase the availability of dopamine for being internalized in vesicles and eventually released.Additionally, by preventing the formation of cytotoxic dopamine-derivates, the burden of oxidative stress it will significantly decrease in these cells.This is for instance supported by the lower apoptotic activity that we observed in cells treated with only NAC, compared with control cells without antioxidant treatment.
A possible limitation of this study is the in vitro nature of our results which might not entirely represent the complex environment and dynamics of the brain, especially when it comes to pharmacokinetics.In addition, although NAC treatment is considered to be safe and without obvious side effects, there are risks associated with NAC overdoses [43] or its derivate glutathione [44].Thus, pinpointing the importance of optimizing the correct doses for treatment of neurological diseases.In our experiments, concentrations of NAC above 5 mM were cytotoxic.
In summary, our investigations validate the use of NAC as a possible disease-modifying treatment for Parkinson's disease and contribute to the uncovering of potential therapeutic targets for this disorder.We believe that the primary beneficial impact of NAC on SH-SY5Y cells exposed to 6-OHDA relies on its antioxidant properties, which prevent the formation of ROS and toxic dopamine derivates, reducing the vulnerability of dopaminergic cells to oxidative insults and rescuing dopamine neurotransmission.
Given the significant role of oxidative stress in Parkinson's disease and the endogenous occurrence of 6-OHDA in individuals with Parkinson's disease, our findings offer insights into the key proteins that are involved in the pathogenesis of Parkinson's disease.

Conclusions
This study investigated the beneficial effect of the antioxidant NAC on a damaged dopaminergic system.We demonstrated that in SH-SY5Y cells exposed to 6-OHDA, NAC-treatment reduces neurotoxicity and alterations in proteins involved in dopamine metabolism.
Our results represent an advancement in the field of neuroprotective therapy for Parkinson's disease and contribute to the discovery of potential compounds able to effectively restore dopamine homeostasis.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Dose-response effects of NAC and 6-OHDA on SH-SY5Y cells.Effects and toxicity of NAC (a-b) and 6-OHDA (c-d) on the proliferation rate (a, c) and mitochondrial metabolic activity (b, d) in SH-SY5Y cells.Cells exposed to 25 μM 6-OHDA were completely saved from cell death when treated with 1.25 mM NAC and the protective effect was reduced with decreasing concentrations of NAC (e).**p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant, one-way ANOVA followed by Bonferroni post hoc correction.

Figure 2 .
Figure 2. NAC inhibits 6-OHDA-induced apoptosis.Caspase 3/7 activity measurements demonstrated the anti-apoptotic role of NAC in control and 6-OHDA-induced SH-SY5Y cells (a).Levels of PARP, a DNA repair protein, and its cleaved form which is used as an indicator of apoptosis (b).Cleaved PARP increased when cells were treated with either 10 or 25 μM 6-OHDA.1.25 mM NAC effectively inhibited 6-OHDA-mediated (25 μM) apoptosis in SH-SY5Y cells, i.e. reduced the expression of cleaved PARP to control levels.However, when cells were treated with lower concentrations of NAC, the expression of cleaved PARP decreased accordingly, corroborating the anti-apoptotic effect of NAC.Treatment with NAC alone displayed no cytotoxic effects.****p < 0.0001, ns = not significant, two-way ANOVA followed by Bonferroni post hoc correction.

Figure 3 .
Figure 3. Expression of TH and DAT in differentiated SH-SY5Y cells.Three different protocols were utilized to differentiate SH-SY5Y cells into a dopaminergic-like phenotype, i.e. to boost the cells TH expression: retinoic acid (RA) alone; 12-O-tetradecanoyl-phorbol -13-acetate (TPA) alone; or a combination of the two drugs.The gene and protein expression levels of TH and DAT under each protocol were studied (a and b respectively).Cells treated with TPA alone had a fourfold increased expression of TH mRNA (CT values of around 30) compared to cells grown in normal growth medium (a).TPA-treated cells also had an increased expression of TH protein compared to control cells (c).TPA-differentiated cells produced higher dopamine levels compared to control (non-differentiated) cells (d).**p < 0.01, ns = not significant, one-way ANOVA followed by Bonferroni post hoc correction.****p < 0.0001, Student's t-test.

Figure 4 .
Figure 4. NAC prevents 6-OHDA-induced dysregulation on the dopamine system.TH and VMAT2 mRNA expression was significantly decreased in the presence of 6-OHDA but was preserved in experimental groups treated with NAC (a-b).αsynuclein mRNA expression was significantly increased in the presence of 6-OHDA but was partially restored after treatment with NAC (c).DAT mRNA levels were low and no significant differences were observed (d).Analyses of protein expression by Western blot (total protein quantification relative to actin) corroborated this data (e).**p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant, two-way ANOVA followed by Bonferroni post hoc correction.

Figure 5 .
Figure 5. NAC prevents cell degeneration and affects dopamine synthesis and release.Light microscopic images of TPAdifferentiated cells alone (control); in the presence of 25 μM 6-OHDA; 1.25 mM NAC; or a combination of the two drugs indicated that cells treated with 6-OHDA were apoptotic and displayed a different morphology.NAC treatment prevented apoptosis (a).Dopamine release after stimulation with KCl significantly increased in both NAC treatment alone or in combination with 6-OHDA (b).*p < 0.05, **p < 0.01, ****p < 0.0001, two-way ANOVA followed by Bonferroni post hoc correction.