Corresponding author: Yongchul Jang Tel +82-2-410-6838 Fax +82-2-410-6945 E-mail: INTRODUCTION
Parkinson’s disease (PD) is a progressive neurodegenerative disorder and the second most prevalent neurological disease following Alzheimer’s disease. With the rapid expansion of the aging population, the global burden of PD is expected to increase substantially; however, disease-modifying therapies capable of halting or reversing neurodegeneration remain unavailable. The pathological hallmark of PD is the selective and progressive degeneration of dopaminergic neurons within the substantia nigra, leading to cardinal motor symptoms such as bradykinesia, rigidity, resting tremor, and postural instability [1]. Although the etiology of dopaminergic neuronal loss is multifactorial and not fully understood, accumulating evidence implicates mitochondrial dysfunction as a central contributor to PD pathogenesis [2].
Mitochondria are multifunctional organelles essential for cellular energy metabolism, redox homeostasis, and regulation of apoptotic signaling. Postmortem analyses of PD patient brains consistently reveal mitochondrial abnormalities, including impaired oxidative phosphorylation and excessive oxidative stress, which are closely associated with disease progression [3].
Beyond their metabolic role, mitochondria are highly dynamic organelles that continuously undergo fusion, fission, mitophagy, and biogenesis—processes collectively referred to as mitochondrial quality control. This tightly regulated network is critical for maintaining mitochondrial integrity and neuronal survival under physiological conditions [4]. Disruption of mitochondrial quality control leads to the accumulation of dysfunctional mitochondria, compromised ATP production, and activation of cell death pathways, thereby accelerating neurodegeneration. Consequently, restoration of mitochondrial homeostasis has emerged as a promising therapeutic target in PD.
Exercise is increasingly recognized as a potent non-pharmacological intervention capable of modifying the trajectory of neurodegenerative diseases. Among various exercise modalities, aerobic exercise has been extensively investigated and shown to improve motor function, balance, and gait performance in patients with PD [5]. Regular aerobic exercise enhances neuroplasticity by upregulating brain-derived neurotrophic factor (BDNF) expression, improving dopaminergic neurotransmission, and promoting neuronal survival [6].
Moreover, evidence from animal studies demonstrates that aerobic exercise attenuates oxidative stress and neuroinflammatory responses, thereby suppressing dopaminergic neuronal degeneration [7,8].
In contrast, resistance exercise has traditionally been emphasized for its peripheral benefits, including stimulation of muscle protein synthesis, prevention of sarcopenia, and improvement of neuromuscular function [9]. However, emerging evidence suggests that resistance exercise also exerts central effects on brain function and neural plasticity [10]. Clinical studies in patients with PD have demonstrated that resistance training improves muscle strength, balance, gait speed, and functional mobility, indicating meaningful benefits for motor performance [11].
Furthermore, preclinical studies have shown that resistance exercise attenuates neuroinflammatory signaling and reduces dopaminergic neuronal loss in experimental models of PD [12]. Despite these promising observations, the molecular mechanisms by which resistance exercise confers neuroprotection, particularly with respect to mitochondrial regulation in the substantia nigra, remain largely unexplored.
Therefore, the present study aimed to examine the effects of 1-methyl-4-phenylpyridinium (MPP+) on mitochondrial alterations and apoptotic cell death in dopaminergic SH-SY5Y cells, and to determine whether a 12-week resistance exercise intervention modulates mitochondrial dynamics, mitophagy, and biogenesis in the substantia nigra of a PD animal model.
METHODS
1. Materials
1) Cell culture and treatment
Human neuroblastoma SH-SY5Y cells were used in this study and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics–antimycotics. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. When cells reached approximately 80–90% confluency, they were passaged using trypsin–EDTA. For MPP+ treatment, cells were seeded at a density of 1×105 cells/mL and allowed to adhere for 24 hours. Cells were then treated with 1 mM MPP+ in DMEM containing 10% FBS for an additional 24 hours.
2) Animals
Eight-week-old male C57BL/6J mice were used for all animal experiments. Mice were housed under controlled environmental conditions (12 hours light/dark cycle, temperature 22±2°C, relative humidity 50%) with ad libitum access to standard chow and water. Animals were randomly assigned to three experimental groups: (1) control group (CON, n=8), receiving saline injections; (2) MPTP-treated group (MPTP, n=8); and (3) MPTP-treated plus body-weight resistance exercise group (MPTP+BR, n=8). All experimental procedures were approved by the Institutional Animal Care and Use Committee of K University (KNSU-IACUC-2022-03).
2. Methods
1) Cell viability assay
Cell viability following MPP+ treatment was assessed using the Cell Viability Image Kit (Invitrogen, R37609). After treatment, NucBlue Live reagent (Hoechst 33342) and NucGreen Dead reagent were added to the culture medium (two drops each), followed by incubation at 37°C for 15 minutes. Fluorescent images were obtained using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Live and dead cells were manually counted and calculated to obtain the percentage of cell death.
2) MPTP-induced mouse model of Parkinson’s disease and Resistance exercise protocol
The Parkinson’s disease mouse model was induced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a dose of 20 mg/kg, administered twice per week for 5 weeks (a total of 10 injections). Control mice received equivalent volumes of saline following the same injection schedule. Following MPTP administration, mice in the resistance exercise group (MPTP+BR) performed a body-weight resistance exercise protocol designed to accommodate the motor impairments associated with PD. Exercise was conducted using a custom-built ladder apparatus (128×24 ×2 cm) positioned at an 80° incline. The training protocol was performed for 12 weeks, with a progressive increase in the number of sets over time. The detailed exercise protocol is presented in Table 1.
3) Motor function test
Motor coordination and balance were evaluated using the rotarod test after completion of the 12-week resistance exercise intervention. Mice were placed on a rotating rod apparatus (JD-A-07RA5, Jeung Do Bio & Plant Co., Ltd., Korea), and the latency to fall was recorded. Prior to testing, mice were habituated to the apparatus at a constant speed of 10 rpm for 120 seconds. The test protocol consisted of accelerating rotation from 5 to 40 rpm, with incremental increases every 30 seconds. Each mouse underwent two test trials, and the average latency was used for statistical analysis. The maximum test duration was set at 300 seconds.
4) Tissue preparation
Twenty-four hours after completion of the resistance exercise protocol, mice were euthanized by cervical dislocation. Brains were rapidly removed, and the substantia nigra was carefully dissected. Tissue samples were immediately frozen and stored at −80°C until needed.
5) Western blotting
Proteins were extracted from cells and brain tissue (substantia nigra) using RIPA lysis buffer (Protein Extraction Solution-RIPA; ELPIS Biotech, Korea). Protein concentrations were determined using the Bradford assay. Normalized protein (cell: 10 μg, tissue: 30 μg) was separated by 10–12% SDS–PAGE and transferred onto PVDF membranes (Amersham, Arlington Heights, IL, USA). Membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C. After washing three times with TBS-T (10 minutes each), membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse; Santa Cruz Biotechnology) diluted 1:5,000 for 1 hour at room temperature. Immunoreactive bands were visualized using Western Blotting Luminol Reagent (SC-2048, Santa Cruz Biotechnology, USA) and detected with a Molecular Imager ChemiDoc XRS system (Bio-Rad, USA). Band intensities were quantified using Quantity One 1D Analysis Software (Bio-Rad, USA).
3. Statistical analysis
All data are presented as mean ±standard error of the mean (SEM) and were analyzed using SPSS Statistics version 20.0 (IBM, USA). Differences between groups in cell experiments were evaluated using an unpaired t-test. For animal experiments, group differences were analyzed by one-way ANOVA, followed by a Bonferroni post hoc test when statistical significance was found to identify group differences. Statistical significance was set at p<.05.
RESULTS
1. Effects of MPP+ on mitochondrial complexes and cell viability in SH-SY5Y cells
To examine a potential relationship between dopaminergic neuronal death and mitochondrial dysfunction, SH-SY5Y cells were exposed to 1 mM MPP+ for 24 hours. Western blot analysis revealed that MPP+ treatment significantly reduced the protein expression levels of mitochondrial respiratory chain complexes I–V compared with the control group (p =.001, p =.001, p =.001, p =.001, respectively; Fig. 1B–E). Consistent with these mitochondrial alterations, cell viability analysis demonstrated a significant increase in cell death following MPP+ exposure (p=.001; Fig. 1G). These findings indicate that MPP+-induced dopaminergic cytotoxicity is closely associated with the impairment of mitochondrial function.
2. Resistance exercise improves motor performance and tyrosine hydroxylase expression
The impairment of motor function, including resting rigidity and postural instability, is a major symptom in PD. To evaluate whether resistance exercise mitigates MPTP-induced motor dysfunction, we examined motor performance via the rotarod test after 12 weeks of exercise intervention (Fig. 2A). The MPTP treated group exhibited a marked reduction in rotarod performance compared with the control group (p =.001). In contrast, mice subjected to resistance exercise following MPTP administration (MPTP+BR) showed a significant improvement in motor performance compared with the MPTP group (p =.038; Fig. 2B). Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, was analyzed as an indirect marker of dopaminergic neuronal integrity. TH levels were significantly reduced in the MPTP group compared with controls (p =.009), whereas resistance exercise significantly restored TH expression in the MPTP+BR group relative to the MPTP group (p =.025; Fig. 2D).
3. Resistance exercise modulates mitochondrial fusion and fission protein expression
Given that mitochondrial dysfunction in PD is closely associated with disrupted mitochondrial dynamics, we examined the effects of resistance exercise on mitochondrial fusion and fission proteins. We found that the levels of the mitochondrial fusion–related proteins mitofusin 1 (MFN1), and optic atrophy 1 (OPA1) were significantly decreased in the MPTP group compared with the control group (p =.031 and p =.006, respectively). Notably, resistance exercise significantly increased MFN1 and OPA1 expression in the MPTP+BR group relative to the MPTP group (p =.047 and p =.007, respectively; Fig. 3B and C). In contrast, analysis of the mitochondrial fission–related proteins FIS1 and dynamin-related protein (DRP1) revealed significant upregulation in the MPTP group compared with the control group (p =.041 and p =.005, respectively). Similarly, the MPTP+BR group also exhibited elevated expression of FIS1 and DRP1 relative to controls (p =.039 and p =.048, respectively), with no significant differences observed when compared with the MPTP group (Fig. 3E and F).
4. Effects of resistance exercise on mitophagy and biogenesis-related proteins
To further elucidate whether resistance exercise modulates mitochondrial quality control, we assessed the expression of proteins involved in mitophagy and mitochondrial biogenesis. PINK1, a key regulator of mitophagy initiation, was significantly increased in the MPTP+BR group compared with both the control and MPTP groups (p=.035 and p =.015, respectively; Fig. 4B). In contrast, PARKIN protein levels did not differ significantly among groups (p =.811; Fig. 4C). Interestingly, the expression of PGC-1α, a key transcriptional regulator of mitochondrial biogenesis, remained unchanged following either MPTP treatment or resistance exercise intervention (p =.928; Fig. 4E).
DISCUSSION
PD is a progressive neurodegenerative disorder characterized by the selective degeneration of dopaminergic neurons in the substantia nigra. To elucidate the pathogenic mechanisms underlying PD, a wide range of cellular and animal models have been developed and extensively utilized. Among these, the MPTP model is one of the most well-established experimental paradigms, as it selectively induces dopaminergic neuronal degeneration in the substantia nigra [13]. MPTP readily penetrates the blood–brain barrier and is metabolized by astrocytes into its neurotoxic metabolite, MPP+. The generated MPP+ is selectively taken up by dopaminergic neurons via the dopamine transporter, where it inhibits mitochondrial complex I, resulting in impaired ATP production, increased oxidative stress, and subsequent neuronal cell death. These pathological processes closely recapitulate key features of PD, including nigrostriatal dopaminergic degeneration and motor dysfunction, thereby supporting the MPTP model as a reliable experimental system for studying PD pathophysiology.
In the present study, we utilized both MPP+-treated SH-SY5Y cells and an MPTP-induced PD mouse model to examine whether resistance exercise exerts neuroprotective effects through the modulation of mitochondrial quality control. Our in vitro results showed that MPP+ exposure markedly reduced the protein expression of mitochondrial electron transport chain complexes I–V and significantly decreased cell viability in SH-SY5Y cells. These findings highlight the close link between mitochondrial dysfunction and dopaminergic neuronal vulnerability, emphasizing that the maintenance of mitochondrial integrity is essential for neuronal survival. Moreover, these cellular observations provide a mechanistic rationale for investigating how exercise-based interventions may mitigate mitochondrial impairment in PD.
MPTP administration is well known to induce motor dysfunction in experimental models of PD. Consistent with previous reports, we assessed motor coordination and balance using the rotarod test and observed a significant impairment in motor performance in the MPTP-treated group compared with controls. In contrast, mice subjected to resistance exercise following MPTP treatment exhibited a marked improvement in motor performance, indicating that resistance exercise effectively attenuated MPTP-induced motor deficits. These findings are in line with prior studies demonstrating the beneficial effects of endurance exercise on motor function in PD models [14,15], and further suggest that resistance exercise may also serve as a viable and effective intervention for alleviating motor dysfunction associated with PD.
To determine whether the observed improvements in motor performance were associated with changes in dopaminergic neuronal integrity, we examined TH protein expression in the substantia nigra. Notably, resistance exercise significantly restored the MPTP-induced reduction in TH expression. Together, these results indicate that resistance exercise not only improves motor function but may also confer neuroprotective effects by preserving dopaminergic neurons.
PD is characterized by mitochondrial dysfunction, which is closely associated with an imbalance between mitochondrial fusion and fission processes [16]. Mitochondrial fusion plays a critical role in restoring damaged mitochondria and maintaining intracellular energy homeostasis, with MFN1 and OPA1 serving as key regulatory proteins in this process [17]. MFN1 mediates fusion of the outer mitochondrial membrane, whereas OPA1 regulates inner membrane fusion, together promoting mitochondrial network connectivity and functional efficiency. Consequently, reductions in these fusion-related proteins can lead to mitochondrial fragmentation, impaired bioenergetics, and cellular dysfunction. In the present study, MPTP administration resulted in a significant decrease in MFN1 and OPA1 protein levels compared with controls, indicating suppressed mitochondrial fusion and mitochondrial impairment. These findings are consistent with previous reports demonstrating reduced expression of mitochondrial fusion proteins in PD models [18]. Notably, resistance exercise following MPTP treatment significantly restored MFN1 and OPA1 expression compared with the MPTP group. This recovery suggests that resistance exercise may enhance both outer and inner mitochondrial membrane fusion, thereby facilitating the repair of damaged mitochondria and contributing to the preservation of cellular function.
Mitochondrial fission plays a critical role in maintaining mitochondrial integrity by segregating damaged or dysfunctional mitochondria for quality control processes [19]. This process is primarily regulated by fission-related proteins, including fission protein 1 (FIS1) and DRP1, whereas excessive mitochondrial fission has been implicated in mitochondrial dysfunction and the activation of cell death pathways [20]. DRP1 translocates from the cytosol to the mitochondrial outer membrane to initiate membrane constriction, while FIS1 functions as an adaptor protein facilitating DRP1 recruitment and stabilization at the mitochondrial surface. Consistent with previous studies, we found that FIS1 and DRP1 levels were significantly elevated in the MPTP-treated group compared with controls [21]. Notably, increased expression of FIS1 and DRP1 was also observed in the MPTP plus resistance exercise group relative to controls. However, when considered alongside the concurrent upregulation of mitochondrial fusion proteins observed in the exercise group, these findings suggest that resistance exercise may not simply suppress mitochondrial fission but rather contribute to a rebalancing of mitochondrial dynamics. Specifically, resistance exercise appears to promote the restoration of mitochondrial network integrity by enhancing fusion capacity while modulating fission-related signaling, thereby improving overall mitochondrial homeostasis.
Along with alterations in mitochondrial dynamics, the efficient removal of damaged mitochondria and the generation of new mitochondria are essential components of mitochondrial quality control in PD. Among these processes, mitophagy plays a pivotal role in maintaining cellular homeostasis by selectively eliminating dysfunctional mitochondria and has classically been regulated through the PINK1–PARKIN signaling pathway [22]. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane, where it facilitates the recruitment of PARKIN and promotes the targeting of impaired mitochondria to autophagosomes for subsequent degradation. In the present study, PINK1 protein levels were significantly increased in the MPTP plus resistance exercise group compared with both the control and MPTP-treated groups. This observation suggests that resistance exercise may enhance mitochondrial quality control by promoting mitophagy and facilitating the selective clearance of damaged mitochondria. In contrast, PARKIN protein expression did not differ significantly among the experimental groups. Recent evidence indicates that mitophagy can be initiated through multiple PARKIN-independent pathways in addition to the canonical PINK1–PARKIN axis [23,24]. In this context, our findings raise the possibility that resistance exercise may prime mitophagy initiation through PINK1 accumulation, whereas the downstream execution of mitochondrial clearance may be mediated via alternative or compensatory signaling mechanisms. Collectively, these results suggest that resistance exercise regulates mitochondrial quality control through a multifaceted and integrated network, rather than exclusively relying on a single canonical pathway.
Mitochondrial biogenesis is primarily governed by transcriptional regulators, among which PGC-1α is widely recognized as a master regulator essential for the generation of new mitochondria. However, in the present study, PGC-1α protein levels did not differ significantly among the experimental groups. This finding suggests that, under conditions of MPTP-induced neuronal injury, the primary effects of resistance exercise are unlikely to be mediated through the induction of mitochondrial biogenesis. Instead, resistance exercise appears to preferentially promote the restoration of mitochondrial function and the clearance of damaged mitochondrial components. Notably, despite the absence of significant changes in PGC-1α expression, we observed marked alterations in proteins governing mitochondrial fusion and fission. These results indicate that resistance exercise may regulate mitochondrial homeostasis predominantly through modulation of mitochondrial dynamics, thereby facilitating structural repair and optimization of the mitochondrial network rather than stimulating the generation of new mitochondria. Such dynamic remodeling may represent an adaptive strategy to preserve mitochondrial efficiency and maintain cellular energy homeostasis under neurodegenerative stress.
Taken together, our findings demonstrate that resistance exercise exerts beneficial effects on motor performance and dopaminergic neuronal preservation in a PD animal model. Moreover, these neuroprotective effects are accompanied by the restoration of mitochondrial dynamics and the activation of PINK1-mediated mitophagy, collectively supporting a role for resistance exercise as a potent modulator of mitochondrial quality control and neuronal resilience in PD.
CONCLUSION
In conclusion, this study examined the effects of resistance exercise on motor performance, dopaminergic neuronal preservation, and mitochondrial quality control in an MPTP-induced model of Parkinson’s disease. Resistance exercise significantly improved motor function and attenuated dopaminergic neuronal loss, accompanied by the restoration of mitochondrial dynamic balance through the regulation of proteins involved in mitochondrial fusion and fission. In addition, resistance exercise activated PINK1-mediated mitophagy, thereby facilitating the selective removal of damaged mitochondria. Notably, the absence of significant changes in PGC-1α expression suggests that the beneficial effects of resistance exercise are not primarily associated with the induction of mitochondrial biogenesis, but rather with the functional recovery and quality control of existing mitochondria. Collectively, these findings indicate that resistance exercise confers neuroprotective and motor benefits in PD, potentially mediated through improvements in mitochondrial quality control, highlighting mitochondrial regulation as a plausible mechanistic pathway underlying exercise-induced neuroprotection.
PDF Links
PubReader
ePub Link
XML Download
Full text via DOI
Download Citation 
Print 
