Metformin Attenuates Manganese-Induced Oxidative Stress in N27-A Dopaminergic Neuronal Cells

Abstract

Metformin is an anti-diabetic drug that exerts protective effects against neurodegenerative diseases. In this study, we investigated the protective effects of metformin against manganese (Mn)-induced cytotoxicity associated with Parkinson’s disease-like symptoms in N27-A dopaminergic (DA) cells. Metformin (0.1–1 mM) suppressed Mn (0.4 mM)-induced cell death in a concentration-dependent manner. Metformin pretreatment effectively suppressed the Mn-mediated increase in the levels of oxidative stress markers, such as reactive oxygen species (ROS) and thiobarbituric acid reactive substances. Moreover, metformin restored the levels of the antioxidants, superoxide dismutase, intracellular glutathione, and glutathione peroxidase, which were reduced by Mn. Metformin (0.5 mM) significantly attenuated the decrease in sirtuin-1 (SIRT1) and peroxisome proliferator activated receptor gamma coactivator-1 alpha levels, which were increased by Mn (0.4 mM). In addition, metformin inhibited the expression of microRNA-34a, which directly targeted SIRT1. Metformin also inhibited the loss of Mn-induced mitochondrial membrane potential (ΔΨ_m) and activation of the apoptosis marker, caspase-3. Furthermore, metformin-mediated inhibition of ROS generation and caspase-3 activation, recovery of ΔΨm, and restoration of cell viability were partially reversed by the SIRT1 inhibitor, Ex527. These results suggest that metformin may protects against Mn-induced DA neuronal cell death mediated by oxidative stress and mitochondrial dysfunction possibly via the regulation of SIRT1 pathway.

INTRODUCTION

Parkinson’s disease (PD) is generally caused by external environmental factors.¹⁾ Various metals, such as iron, copper, zinc, aluminum, and manganese (Mn), have been implicated in PD. Although Mn is an essential trace element required for various physiological and metabolic processes, prolonged exposure to high levels of Mn can lead to manganism, a Parkinson’s-like syndrome.²⁾ Although idiopathic PD and manganism share many overlapping symptoms, manganism is characterized by neurological and intellectual loss, with symptoms observed at an earlier age compared to PD.³⁾ Mn is a widely used metal that causes manganism in miners and industrial welders exposed to Mn fumes. Farmers exposed to the fungicides, Maneb and Mancozeb, which are pesticides containing Mn, can also be affected by manganism.⁴⁾ Mn readily crosses the blood–brain barrier (BBB) and accumulates in the brain, primarily in the striatum, globus pallidus, subthalamic nucleus, and substantia nigra (SN) at high levels.⁵⁾ Particularly, Mn accumulation leads to significant dopaminergic (DA) neuronal loss in SN.⁶⁾ Although several studies have demonstrated the toxicity of Mn to neurons and glia, the detailed mechanisms of Mn-induced DA neuronal damage directly linked to manganism remain ambiguous.

Metformin is an inexpensive and effective drug that is widely used for the treatment of type 2 diabetes.⁷⁾ Metformin, a synthetic dimethyl biguanide, acts on the liver, skeletal muscle, and adipose tissue to exert a glucose-lowering effect by inhibiting gluconeogenesis and promoting peripheral glucose uptake.⁸⁾ Recently, metformin has received increasing attention for its effects on metabolic imbalances, including several neurodegenerative diseases.^9,10) Type 2 diabetes may be associated with PD as insulin resistance has been observed in several neurodegenerative diseases, including PD.¹¹⁾ Moreover, type 2 diabetes exacerbates the symptoms of PD.¹²⁾ In addition to its anti-diabetic effects, metformin may exert neuroprotective effects by rapidly crossing the BBB and slow aging by regulating the mitochondrial metabolism.^13,14) Metformin reduces inflammation and autophagy, which are age-related conditions, and α-synuclein phosphorylation and aggregation, which are associated with PD pathogenesis.¹⁵⁾ Metformin also exerts cytoprotective effects against 1-methyl-4-phenylpyridinium (MPP⁺) and rotenone, which cause PD symptoms, in SH-SY5Y, a commonly used neuronal cell model of PD.^16,17) However, the specific effects of metformin on Mn-induced DA cell damage remain unknown. Therefore, in this study, we investigated the protective effects of metformin against Mn-induced N27-A DA cell death. For the first time, we demonstrated that metformin protected N27-A cells by attenuating oxidative stress and mitochondrial dysfunction via the regulation of sirtuin-1 (SIRT1) pathway.

MATERIALS AND METHODS

Cell Culture and Treatment

Rat DA neuronal cell line, N27-A (obtained from Sigma-Aldrich, St. Louis, MO, U.S.A.), was cultured as previously described.¹⁸⁾ Cells were maintained in the Roswell Park Memorial Institute-1640 medium with 10% fetal bovine serum (FBS) (Gibco/Invitrogen, Carlsbad, CA, U.S.A.), 2 mM L-glutamine, and 1% penicillin–streptomycin at 37 °C and 5% CO₂ in a humidified incubator. N27-A cells were seeded at a density of 5 × 10⁴ cells/mL. One day before chemical treatment, the medium was changed to a 0.5% FBS medium to reduce the serum effect. Metformin pretreatment was performed 1 h before MnCl₂ treatment, and MnCl₂ was added for 24 h. To prevent any direct interaction between metformin and MnCl₂, the culture medium was replaced with a fresh low-serum culture medium after metformin pretreatment. Cell viability was measured using the PrestoBlue assay kit (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.), according to the manufacturer’s instructions. Briefly, PrestoBlue reagent was added to the cultured cells, and the amount of fluorescence at 560 nm (excitation)/590 nm (emission) was determined after 1 h at 37 °C. For the overexpression and inhibition of microRNA (miR)-34a, miR-34a mimic or miR-34a inhibitor (MedChemExpress, Monmouth Junction, NJ, U.S.A.), and the miR mimic negative control (MedChemExpress) were transiently transfected into cells using Lipofectamine 3000 (Invitrogen). The cells were harvested 48 h after transfection and used for subsequent assays. To silencing SIRT1, N27A cells were transiently transfected with SIRT1 small interfering RNA (siRNA) duplex (Sc-108043; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) or nonspecific (NC) scrambled siRNA (Sc-37007; Santa Cruz Biotechnology). Cells were transfected for 24 h, and then subjected to various treatments.

Oxidative Stress Assay

Reactive oxygen species (ROS) levels were measured using 2′-7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA). Then, oxidation of non-fluorescent H₂DCF-DA to highly fluorescent dichlorofluorescein (DCF) via intracellular ROS generation was analyzed. N27-A cells were treated with metformin and MnCl₂ for to 24 h. Cells were washed with Hank’s balanced salt solution (HBSS) and incubated with 10 µM H₂DCF-DA at 37 °C for 30 min. Then, the cells were rinsed twice with HBSS, and the fluorescence of DCF was measured at 485 nm (excitation) and 535 nm (emission). Intracellular levels of superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPX), and thiobarbituric acid reactive substances (TBARS) were determined using test kits for SOD (EIASODC; Thermo Fisher Scientific Inc.), GSH (EIAGSHC; Thermo Fisher Scientific Inc.), GPX (703102; Cayman Chemical), and TBARS (ab118970; Abcam), respectively. Then, SOD activity was measured at 450 nm and expressed as U/mg protein. GSH level was measured at 405 nm and expressed as µmol/mg protein. GPX level was measured at 340 nm and expressed as U/mg protein. TBARS content was measured at 532 nm and expressed as µmol/mg protein. The TBARS assay, measuring the lipid peroxidation products, is unsuitable for assessing lipid peroxidation under in vivo conditions, but is adaptable to various cell culture experiments and in vitro conditions due to its good reproducibility and cost effectiveness.

Quantitative (q)RT-PCR

To analyze miR-34a expression, total RNA was extracted using the TRIzol reagent (Invitrogen), reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific Inc.), amplified, and quantified using SYBR Green (Applied Biosystems, Foster, CA, U.S.A.). Relative expression of the target gene was calculated using the 2^−ΔΔCT method, and U6 small nuclear RNA (U6) was used as an internal control. Primers were used as follows: miR-34a (forward: 5′-TCTGTCTCTCTTGGCAGTGTCTT-3′ and reverse: 5′-CTCGCTTCGGCAGCACA-3′), and U6 (forward: 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse: 5′-ACGCTTCACGAATTTGCGTGTC-3′). A 10-min reaction at 95 °C was followed by 40 cycles of thermocycling with 15 s at 95 °C and 30 s at 60 °C.

Western Blotting Analysis

After culturing N27-A cells under different experimental conditions, nuclear extracts were prepared using the Nuclear Extraction kit (Cayman Chemical, Ann Arbor, MI, U.S.A.) containing protease and phosphatase inhibitors. Whole cell protein extracts were prepared using EzRIPA Lysis kit (ATTO Co., LTd., Tokyo, Japan) containing RIPA lysis buffer, protease inhibitors, and phosphatase inhibitors. Equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked with TBST buffer containing 5% non-fat milk (20 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) for 2 h at room temperature, followed by incubation with the anti-SIRT1 (ab110304; 1 : 1000 dilution; Abcam, Cambridge, U.K.), anti-peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α; ab191838; 1 : 2000 dilution; Abcam), anti-Lamin B1 (Sc-377000; 1 : 1000 dilution; Santa Cruz Biotechnology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-365062; 1 : 1000 dilution; Santa Cruz Biotechnology) primary antibodies at 4 °C overnight. After incubation with horseradish peroxidase-conjugated secondary antibodies (1 : 2000 dilution) for 2 h at room temperature, protein expression was detected using an enhanced chemiluminescence reagent (GE Healthcare, Pittsburgh, PA, U.S.A.). Band density was analyzed using the ImageJ software (National Institute of Health, Bethesda, MD, U.S.A.).

Measurement of SIRT1 Activity

SIRT1 activity was measured using the SIRT1 Direct Fluorescent Assay Kit (Cayman Chemical), according to the manufacturer’s instructions. Briefly, assays were performed by incubating the nuclear extract and SIRT1 substrate with oxidized form of nicotinamide adenine dinucleotide (NAD⁺) for 45 min at room temperature. SIRT1 activity was measured at 360 nm (excitation) and 465 nm (emission) using a fluorescence microplate reader (Tecan Infinite F200; Tecan, Grödig, Austria) after stopping the reaction with the stop/developing solution.

Measurement of Caspase-3 Activity

Caspase-3 activity was measured using a colorimetric assay with the specific substrate, Ac-DEVD-pNA (Sigma-Aldrich). Briefly, cells were lysed with a cold lysis buffer (50 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4, 0.1% CHAPS, 1 mM dithiothreitol, and 0.1 mM ethylenediaminetetraacetic acid (EDTA)), incubated on ice for 5 min, and separated via centrifugation at 10000 × g and 4 °C for 10 min. To 10 µg of the prepared protein sample, 200 µM Ac-DEVD-pNA was added and reacted for 1 h at 25 °C. The formation of pNA was measured at 405 nm using a microplate reader (VersaMax; Molecular Devices, Sunnyvale, CA, U.S.A.).

Mitochondrial Membrane Potential Assay

Next, mitochondrial membrane potential (ΔΨm) was measured using JC-1, a fluorescent dye widely used to determine the changes in mitochondrial permeability. JC-1 forms red fluorescent J-aggregates under normal cellular conditions but is mostly present in a green fluorescent monomeric form under damaged cellular conditions. Therefore, the red-to-green fluorescence ratio indicates the degree of damage to the mitochondrial membrane. After treatment under various conditions, cells were incubated with JC-1 (5 µg/mL) for 15 min at 37 °C in the dark. After washing thrice with phosphate-buffered saline, the fluorescence intensity was measured using a fluorescence plate reader. Red light was measured at 535 nm (excitation)/590 nm (emission), and green light was measured at 485 nm (excitation)/535 nm (emission). Results were determined as the ratio of red-to-green fluorescence and expressed as a percentage of the untreated control.

Statistical Analyses

All experiments were repeated at least three times. Data are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analyses were conducted using SPSS (version 27.0; SPSS Inc., Armonk, NY, U.S.A.). Differences among individual groups were analyzed using one-way ANOVA, followed by Tukey’s honestly significant difference (HSD) post-hoc test. A one-sample t test was used for comparisons of two groups. Results were considered statistically significant at a significance level of 2α = 0.01.

RESULTS

Protective Effects of Metformin against Mn-Induced Neurotoxicity

First, we assessed the effect of metformin on Mn-induced loss of N27-A cell viability using the PrestoBlue assay. As shown in Fig. 1A, treatment with Mn (0.2–0.8 mM) for 24 h caused cell death in a dose-dependent manner, with 0.4 mM Mn inducing approximately 49% cell death. Therefore, 0.4 mM Mn was selected for subsequent experiments. Metformin (0.1–1 mM) exhibited no cytotoxicity in 24 h treatment experiments, with no difference from the untreated control (Fig. 1A). To determine the effect of metformin on Mn-induced DA cell death, N27-A cells were pretreated with 0.1–1 mM metformin for 1 h, followed by treatment with 0.4 mM Mn for 24 h. As shown in Fig. 1B, Mn-induced cell loss was attenuated by metformin in a concentration-dependent manner, with 0.5 mM metformin pretreatment providing the most significant protection (91.4% viability).

Fig. 1. Effect of Metformin on Manganese (Mn)-Induced Cytotoxicity in N27-A Dopaminergic Cells

Cell viability was quantified using the PrestoBlue assay, as described in the Materials and Methods section. (A) Cells were treated with the indicated concentrations of metformin or MnCl₂ for 24 h and assayed for cell viability. Statistical significance among groups was determined using one-way ANOVA with Tukey’s post-hoc analysis. Data are represented as the mean ± standard error of the mean (S.E.M.) [for drug effect, F (7,64) = 10.13, p < 0.01; n = 9 per treatment group]. (B) After pretreatment with various concentrations of metformin (0.1, 0.25, 0.5, and 1 mM) for 1 h, cells were treated for 24 h with MnCl₂ (0.4 mM). Cell viability was expressed as the mean ± S.E.M. [for drug effect, F (5,48) = 6.71, p < 0.01; n = 9 per each group). Abbreviation: Con, untreated control.

Metformin Alleviates Mn-Induced Oxidative Stress in N27-A Cells

To determine the involvement of oxidative stress in Mn-induced DA cell cytotoxicity and the possible mechanisms of metformin neuroprotection, we measured the changes in oxidative stress markers and key enzymes of the antioxidant defense system. Intracellular ROS production was also analyzed. As shown in Fig. 2A, in cells treated with Mn (0.4 mM) alone, intracellular ROS increased in a time-dependent manner at various time points (0.5–24 h), with a peak at 16 h of treatment (3.25-fold that of the control). However, pretreatment with metformin (0.5 mM) effectively decreased Mn-induced ROS generation (1.69-fold that of the control group). Subsequently, levels of TBARS, the indicator of lipid peroxidation, were measured. As shown in Fig. 2B, Mn treatment (0.4 mM) for 16 h increased the levels of TBARS, an oxidative damage marker, by approximately 2.52-fold. However, metformin pretreatment inhibited the Mn-induced increase in TBARS levels, which were almost close to control levels. Moreover, 0.4 mM Mn significantly decreased the levels of the major protective antioxidants, SOD, GSH, and GPX, to 0.38-, 0.47-, and 0.53-fold those of the control, respectively, after 16 h of treatment. However, pretreatment with metformin restored the Mn-induced decreased antioxidant levels to almost control levels in N27-A cells (Fig. 2B).

Fig. 2. Effect of Metformin on Mn-Induced Oxidative Stress

N27-A cells were treated with metformin (0.5 mM) for 1 h, followed by MnCl₂ (0.4 mM) treatment for a maximum of 24 h. (A) Reactive oxygen species (ROS) generation was detected using 2′-7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) as a fluorescence probe. The statistical analyses between two groups (control vs. MnCl₂ or MnCl₂ vs. metformin + MnCl₂) in each time points were performed using one-sample t test (n = 6 per treatment group). (B) Levels of superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPX), and thiobarbituric acid reactive substances (TBARS) were measured using commercially available test kits. Data are represented as the mean ± S.E.M. (n = 6 per each treatment group). Statistically significant differences between the control and other individual groups were determined using one-way ANOVA, followed by Tukey’s post-hoc test [SOD, F (3,20) = 40.38, p < 0.009; GSH, F (3,20) = 15.43, p < 0.004; GPX, F (3,20) = 29.19, p < 0.001; TBARS, F (3,20) = 21.56, p < 0.01].

Effects of Metformin on the Expression Levels of SIRT1 and PGC-1α in N27-A Cells

In the pathogenesis of PD, SIRT1 and its downstream co-transcription factor, PGC-1α, play important roles in DA neuronal survival.^19,20) To investigate the involvement of SIRT1 and PGC-1α proteins in Mn-induced DA cell death and metformin-mediated protection, we determined the expression levels of these proteins via Western blotting analysis. As shown in Fig. 3A, Mn treatment significantly decreased the expression levels of SIRT1 (59.10% of that of the control) and PGC-1α (45.67% of that of the control), whereas metformin pretreatment prior to Mn treatment restored them to almost control levels. Compared to the control, metformin slightly increased the SIRT1 and PGC-1α expression levels. Consistent with the protein expression, SIRT1 activity was significantly decreased by Mn treatment and restored to nearly normal levels by metformin pretreatment (Fig. 3B).

Fig. 3. Effect of Metformin on the Modulation of Sirtuin-1 (SIRT1)/Peroxisome Proliferator Activated Receptor Gamma Coactivator-1 Alpha (PGC-1α) Pathway in N27-A Cells

Cells were pretreated with metformin (0.5 mM) for 1 h and then treated with MnCl₂ (0.4 mM) for 12 h. (A) Protein levels of SIRT1 and PGC-1α in nuclei were evaluated via Western blotting analysis. Expression level of lamin B1 was used for normalization. Protein bands were quantified using densitometry and expressed as the ratio to the untreated control. (B) SIRT1 activity was evaluated using the SIRT1 Direct Fluorescent assay kit, as described in the Materials and Methods section. (C) Expression levels of microRNA (miR)-34a were determined using qRT-PCR and normalized to that of U6 small nuclear RNA. (D) N27-A cells were transfected with the miR negative control (miR-NC), miR-34a mimic, and miR-34a inhibitor, and SIRT1 activity was measured. Data are represented mean ± S.E.M. (n = 3 each treatment group). The difference analyses of compared groups were performed by t test.

Effects of Metformin and Mn on miR-34a Expression Levels

miRNAs regulate the expression of several genes. miR-34a directly targets and regulates the expression of SIRT1.²¹⁾ As metformin and Mn regulate the expression of SIRT1 (Fig. 3A), we assessed whether miR-34a, which represses the gene expression of SIRT1, is also modulated by metformin or Mn. As shown in Fig. 3C, qRT-PCR analysis revealed the downregulation of miR-34a expression by metformin (0.69-fold that of the control), and its significant upregulation by Mn (1.96-fold that of the control). However, metformin pretreatment restored miR-34a expression to almost normal levels (1.14-fold that of the control). Notably, SIRT1 activity was decreased by the miR-34a mimic but increased by the miR-34a inhibitor. However, transfection with miR-NC had no effect on SIRT1 activity, similar to the control (Fig. 3D). Similar to SIRT1 activity, expression of PGC-1α, a downstream molecule of SIRT1, was also modulated by miR-34a (data not shown).

Involvement of SIRT1 in Mn-Induced Cytotoxicity and Protective Effects of Metformin

To evaluate the significance of Mn-induced downregulation and metformin-mediated restoration of SIRT1 levels, a pharmacological approach was used with Ex527, a specific SIRT1 inhibitor. As shown in Fig. 4A, 1 h pretreatment of N27-A cells with Ex527 (10 µM) enhanced Mn-induced ROS generation and caspase-3 activation. Furthermore, Ex527 pretreatment partially offset the metformin-mediated suppression of ROS generation and caspase-3 activation (Fig. 4A). As ROS generation and caspase-3-mediated neuronal apoptosis are closely associated with the loss of ΔΨm, we evaluated the changes in ΔΨm using the mitochondria-specific fluorescent dye, JC-1. As shown in Fig. 4B, Mn treatment reduced ΔΨm to 70.01% of that of the control. Although ΔΨm loss was restored to 97.12% of that of the control by metformin, pretreatment with the SIRT1 inhibitor, Ex527, attenuated the effect of metformin on ΔΨm (85.79% of that of the control). Similar to ΔΨm changes, Mn-induced loss of cell viability (approximately 50% cell viability) was further enhanced by Ex527 pretreatment (36.94% of that of the control). Moreover, Ex527 pretreatment partially attenuated the effect of metformin on cell viability (Fig. 4B). To avoid the off-target effects of the pharmacological inhibitor Ex527, we repeated some similar experiments with siRNA targeting SIRT1 specifically. We found that SIRT1 expression was significantly reduced by up to 18.3% when knocked down by SIRT1-specific siRNA (Fig. 4C). As shown in Figs. 4D and E, metformin attenuated Mn-induced ROS generation and cell death in N27-A cells transfected with NC-siRNA, but the protective benefits of metformin were reduced and the Mn-induced effects were enhanced in cells transfected with SIRT1 siRNA, similar to the results of the experiments with Ex527. These results suggest that metformin exerts a protective effect against Mn-induced toxicity by mediating SIRT1 expression in N27-A cells.

Fig. 4. Inhibition of SIRT1 Enhanced the Effects of Mn and Partly Reversed the Effects of Metformin on Reactive Oxygen Species (ROS) Generation, Caspase-3 Activation, Mitochondrial Membrane Potential (MMP), and Cell Viability

(A, B) N27-A cells were pretreated with the SIRT1 inhibitor, Ex527 (10 µM), for 1 h, followed by MnCl₂ (0.4 mM) treatment with or without metformin (0.5 mM). (A) ROS generation and caspase-3 activation were measured and expressed as the relative fold-changes. (B) MMP and cell viability were determined and expressed as the relative percentage changes. ROS generation and MMP were determined at 16 h using the fluorescence-detecting probes, H₂DCF-DA and JC-1, respectively. Cell viability and caspase-3 activity were measured at 24 h using PrestoBlue assay and the specific substrate, Ac-DEVD-pNA, respectively, as described in the Materials and Methods section. Data are represented as the mean ± S.E.M. (n = 9 per treatment group). Statistical analyses between untreated control and other experimental groups were performed by one-way ANOVA, followed by Tukey’s post-hoc test [ROS, F (6,56) = 97.41, p < 0.001; Caspase 3, F (6,56) = 113.73, p < 0.007; MMP, F (6,56) = 47.74, p < 0.001; Cell viability, F (6,56) = 40.94, p < 0.003]. (C) N27-A cells were transiently transfected with nonspecific scrambled siRNA (NC-siRNA) or SIRT1-siRNA for 24 h, and then Western blot analysis was carried out for SIRT1 and GAPDH expression. Data represent mean ± S.E.M. (n = 3 per group). Student’s t test was used for comparison between the indicated groups. (D) Cells were transfected with NC-siRNA or SIRT1-siRNA for 24 h and then treated with metformin (0.5 mM for 1 h) with or without MnCl₂ (0.4 mM, 16 h) for measurement of ROS generation. Data represent mean ± S.E.M. (n = 4 per group) with student’s t test. (E) N27-A cells transfected with siRNAs and exposed to metformin (0.5 mM, 1 h) and/or MnCl₂ (0.4 mM, 24 h) were analyzed and the cell viability was determined by using PrestoBlue assay. Statistically significant was determined by ANOVA, followed by Tukey’s post-hoc test [n = 6, F (6,40) = 20.69, p < 0.004].

DISCUSSION

N27-A cells, clonal derivatives of N27 cells, are widely used as DA neuronal cells and have been modified to stably express DA properties. N27-A cells express close to 100% of the DA markers, tyrosine hydroxylase, dopamine transporter, and Tuj1. They also highly express the DA neuron transcription factors, Nurr1, En1, FoxA2, Pitx3, and the monoamine transporter, VMAT2, but do not express dopamine-β-hydroxylase, the enzyme that converts dopamine to norepinephrine. Hence, N27-A cells are the most suitable cell models for PD research. Therefore, in this study, we adopted N27-A cells as a model system to investigate the effect of metformin on Mn cytotoxicity, which causes PD symptoms.

This study provides strong evidence that metformin directly prevents Mn-induced DA cell death. PD syndrome goes through a prodromal phase for many years before diagnosis. The prodromal period is the beginning of the onset of neurodegeneration and is the ideal time for repair or protection. Neuroinflammation and mitochondrial dysfunction are observed in the prodromal phase of PD.²²⁾ Therefore, administering appropriate neuroprotective drugs during the prodromal period is the most efficient way to treat and delay PD. As PD is a brain disease, candidate drugs must have the ability to cross the BBB, be safe, have no side effects, and exert good neuroprotective effects. Designing new drugs that meet these requirements is time consuming and expensive; therefore, repurposing existing drugs is preferable. Although no drugs can prevent or completely reverse neurodegeneration, anti-diabetic drugs, among other BBB-passable drugs, may be safe and effective in treating PD syndrome.²³⁾ Previous reports have suggested an association between type 2 diabetes and PD.¹²⁾ Several anti-diabetic drugs protect against the progression of PD. The anti-diabetic drug, thiazolidinedione, reduced the risk of PD in patients with diabetes and reduced neuroinflammation and neurodegeneration in animal studies.^24,25) Another anti-diabetic drug, exenatide, reduces DA cell death and improves the motor and cognitive functions.²⁶⁾ Metformin, the most potent anti-diabetic drug, reduced DA neuronal damage in PD models induced by various PD-related toxins, such as MPP⁺, 6-hydroxydopamine, and rotenone.^16,17,27) However, the efficacy of metformin against Mn-induced PD remains unknown.

In this study, Mn-treated N27-A DA neuronal cells were used as a PD model to evaluate the neuroprotective effects of metformin. Mn directly affects neurons and causes mitochondrial dysfunction, oxidative stress, and apoptosis in various cell types.^28,29) Similarly, in this study, Mn (0.4 mM) increased ROS levels in a time-dependent manner, increased the levels of TBARS (a marker of lipid peroxidation), and significantly decreased the levels of intracellular antioxidant enzymes SOD, GSH, and GPX (Fig. 2). However, pretreatment with metformin (0.5 mM) significantly inhibited these changes (Fig. 2). These antioxidant effects of metformin are probably due to its some degree of direct ROS scavenging ability and its mechanism of increasing the expression levels of several genes in mitochondrial complexes that can reduce ROS generation.^30,31) However, it is important to note that metformin’s ROS-scavenging ability may not be as potent as some dedicated antioxidants, and its primary mechanisms of action are often attributed to other pathways, such as PGC-1α regulation.

Metformin alters the mitochondrial respiratory chain to reduce ROS generation.³²⁾ In particular, the mechanism by which metformin protects against MPTP (in vivo model) and MPP⁺ (in vitro model) in mice and SH-SY5Y cells is the upregulation of PGC-1α, which is involved in mitochondrial biogenesis.³³⁾ PGC-1α protects dopamine neurons in animal models of PD, but its expression is reduced in the brains of patients with PD.³⁴⁾ In addition, SIRT1, an upstream protein of PGC-1α, regulates PGC-1α to enhance its antioxidant capacity under oxidative stress conditions.^35,36) Activation of the SIRT1/PGC-1α pathway upregulates the anti-apoptotic protein, Bcl2, but downregulates the proapoptotic protein, Bax.

Here, we found that SIRT1 and PGC-1α protein levels were significantly decreased by Mn, but metformin pretreatment maintained their expression at nearly normal levels (Fig. 3A). In particular, SIRT1 protein activity and expression were severely reduced by Mn; however, these were restored to normal levels by metformin pretreatment (Fig. 3B). These findings suggest that metformin possibly prevents Mn-induced cytotoxicity in N27-A DA cells by directly regulating SIRT1. Nevertheless, the relationship between metformin, ROS, and SIRT1 is complex and interconnected, and further research is needed to fully elucidate the precise mechanisms and outcomes of this interplay.

SIRT1 is a nuclear protein that is activated to protect cells against harmful stimuli, such as oxidative stress, and its activation exerts various beneficial effects in neurodegenerative diseases, such as PD.^37,38) Activation of SIRT1 is regulated at the gene expression level by miRNAs, such as miR-34a.³⁹⁾ Consistently, SIRT1 activity was significantly inhibited by miR-34a in this study (Fig. 3D). Interestingly, the mRNA expression of miR-34a was regulated by both Mn and Metformin (Fig. 3C). Therefore, changes in SIRT1 induced by Mn and metformin may occur via the regulation of miR-34a.

SIRT1 is highly expressed in neurons and microglia in the human brain and exerts neuroprotective effects by enhancing the mitochondrial function in conjunction with its antioxidant, anti-inflammatory, and anti-apoptotic effects.^40–42) Metformin mitigates tissue damage in the body by modulating SIRT1 expression.^43,44) However, the role of SIRT1 in the efficacy of metformin against Mn-induced DA neuronal cytotoxicity has not yet been investigated. In this study, metformin effectively attenuated Mn-induced ROS generation, caspase 3 activation, MMP decrease, and cell loss (Fig. 4). However, when SIRT1 activity was attenuated with the specific inhibitor, Ex527 or siRNA, the Mn-induced effects were further increased, and the counteracting effect of metformin on the Mn-induced impact was partially diminished (Fig. 4). These results suggest the key role of SIRT1 in metformin-mediated neuroprotection against Mn-induced DA cell damage. Accumulating evidence suggests the involvement of various neuroprotective signaling processes in SIRT1 expression and regulation.^33,45) Therefore, further studies are necessary to fully elucidate the action mechanisms of metformin and SIRT1 activation in DA cells.

CONCLUSION

In conclusion, this study is the first to demonstrate that metformin protects DA neuronal cells from Mn-induced cytotoxicity. Here, metformin effectively suppressed Mn-induced DA cell death-related events, such as ROS generation, increase in TBARS levels, decrease in SOD, GSH, and GPX levels and MMP, and activation of caspase-3. Importantly, metformin protected N27-A DA cells via the SIRT1/PGC-1α pathway, probably by modulating the expression levels of antioxidants and mitochondria-related proteins. Our findings can aid in the development of new therapeutic strategies for various neurodegenerative diseases, such as manganism and PD, using metformin as a SIRT1 activator.

Acknowledgments

This study was supported by research fund from Chosun University (2023) to Jae-Sung Kim.

Conflict of Interest

The authors declare no conflict of interest.

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