Exercise Mitigates MPTP-Induced Mitochondrial Fragmentation through the Irisin/AMPK/SIRT1 Pathway

doi:10.21203/rs.3.rs-4945713/v1

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Exercise Mitigates MPTP-Induced Mitochondrial Fragmentation through the Irisin/AMPK/SIRT1 Pathway

https://doi.org/10.21203/rs.3.rs-4945713/v1

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Purpose

Mitochondrial dysfunction plays a crucial role in the pathogenesis of Parkinson’s disease (PD), yet therapeutic strategies targeting mitochondrial function remain limited. Exercise has shown neuroprotective benefits in PD, but the underlying mechanisms are not fully understood. This study aimed to investigate how exercise affects MPTP-induced excessive apoptosis and mitochondrial fission in PD mice, with a focus on the irisin/AMPK/SIRT1 pathway.

Methods

Thirty-two male C57BL/6J mice, aged 7–8 weeks, were randomly assigned to control (n = 8) and experimental groups (n = 24). Mice in the experimental groups were administered intraperitoneal injections of MPTP to induce the PD model. Subsequently, the experimental mice were divided into three groups (8 mice in each group): the sedentary group (PD), the group subjected to ten weeks of treadmill exercise (PDEX), and the group receiving both treadmill exercise and irisin antagonist injections (EXRG). Upon completion of the ten-week intervention, behavioral assessments were performed. Following this, the mice were euthanized to collect brain samples and subjected to immunohistochemistry, immunofluorescence, ELISA, and Western blot analyses.

Results

MPTP-treated mice exhibited significant motor dysfunction and dopaminergic neuron loss in the nigrostriatal regions, which were ameliorated by a 10-week exercise intervention. Exercise notably reduced MPTP-induced neuronal apoptosis, as evidenced by decreased cellular fragments and abnormal nuclear morphology, increased Bcl-2 protein levels, and decreased Bax expression. Additionally, exercise mitigated abnormal mitochondrial fission in PD mice, as shown by reduced immunohistochemistry and protein expression of Drp1, Fis1, and MFF. In the substantia nigra of PD mice, the expression levels of irisin, p-AMPK, and SIRT1 were decreased but were elevated following the 10-week exercise intervention. However, blocking the irisin signaling by chronic treatment with cyclo RGDyk potentially counteracted the exercise-induced elevations in p-AMPK and Sirt1 expression. Moreover, the beneficial effects of exercise on neuronal apoptosis and mitochondrial fission were reversed by blocking irisin signaling pathways.

Conclusion

These findings suggest that regular exercise is beneficial in alleviating motor dysfunction in MPTP-treated mice, partly achieved through the preservation of dopaminergic neurons, reduction of excessive neuronal apoptosis, and improvement of normal mitochondrial fission. The excise-associated neuroprotective effects are likely linked to the irisin/AMPK/Sirt1 signalling pathway.

Exercise

Parkinson’s disease

Apoptosis

Mitochondrial dysfunction

Irisin

AMPK

1. Ten weeks of treadmill exercise significantly improved the motor dysfunction, further loss of dopaminergic neurons, excessive apoptosis of neurons, and mitochondrial fragmentation in MPTP-induced Parkinson's disease mice.

2. Irisin is a key muscle factor in the neuroprotective effect of exercise, and AMPK/Sirt1 is involved in this process.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, along with dementia ^1,2. The prevalence and incidence of PD increase exponentially with ageing ^3,4, affecting approximately 10–18 per 100,000 people ⁵. One study proposes that the number of individuals with PD will increase by over 50 per cent by 2030 ¹, placing a substantial financial burden on the community. The hallmark pathological feature of PD is the loss of dopaminergic (DA) neurons in the substantia nigra compacta (SNpc), characterized by the aggregation of α-synuclein (α-syn) in Lewy bodies ⁶. However, current treatments for PD are largely limited, and available drugs cannot prevent the pathological progression and have serious side effects with long-term use ⁷. Therefore, elucidating the specific mechanisms underlying the loss of DA neurons is crucial for developing therapeutic strategies for PD.

Several factors can contribute to the loss of dopaminergic neurons in PD, including mitochondrial dysfunction and neuronal apoptosis ⁸. Apoptosis is a controlled form of cell death that regulates cell numbers and tissue development while eliminating damaged structures ^9,10. In the substantia nigra, dopaminergic neurons typically have a lower baseline mitochondrial content and insufficient potential to maintain their homeostasis ¹¹, implying the vulnerable nature of mitochondrial function in conditions like PD. Apoptosis and mitochondrial dynamics are often interconnected; for instance, high levels of cellular stress leading to apoptosis also result in excessive mitochondrial fission ¹². This process involves several mitochondrial fission-associated proteins, such as Dynamin-related protein 1 (Drp1), fission protein 1 (Fis1), and mitochondrial fission factor (MFF) ¹³. Recent research has highlighted the critical role of Drp1-induced abnormal mitochondrial fission in the apoptosis of dopaminergic neurons in PD. The upregulation of Drp1 triggers excessive mitochondrial fission and dopaminergic neuronal apoptosis linked to PD pathology ^14–18. Conversely, inhibiting Drp1 activity normalizes mitochondrial fission, diminishes neuronal apoptosis, and ultimately ameliorates PD symptoms ^14–18. This underscores the potential of addressing mitochondrial dysfunction as an effective therapeutic approach for PD. Exercise has emerged as a significant neuroprotective approach to PD ¹⁹. For example, Koo et al. ²⁰ demonstrated that regular exercise can delay PD progression by preserving dopaminergic neurons and improving mitochondrial function. Furthermore, Jang et al. ²¹ reported that six weeks of treadmill exercise alleviated MPTP-induced motor impairments in PD mice, reduced abnormal mitochondrial fission, mitigated excessive apoptosis, and promoted neuroprotection against PD. However, the precise molecular mechanisms responsible for the beneficial effects of exercise in PD remain unclear.

The increasing evidence indicates that exercise exerts its positive effects on the body by regulating a variety of cytokines. These cytokines are produced in the muscles, circulate in the bloodstream, and perform diverse functions in different tissues ^22–25. One such cytokine is irisin, which is released by the cleavage of the transmembrane precursor protein, fibronectin type III domain-containing protein 5 (FNDC5), during exercise ²⁶. Exercise serves as a potent trigger for irisin secretion ^27,28. Studies have demonstrated that irisin treatment can enhance motor function and mitigate the degeneration of DA neuron degeneration in PD model functions ^29,30. Furthermore, irisin has the potential to counteract excessive mitochondrial fission by alleviating heightened neuronal apoptosis in PD models, thereby playing a neuroprotective role in PD ³¹. Thus, it is proposed that irisin may represent a crucial target for the amelioration of PD during exercise. However, the precise mechanisms through which irisin influences mitochondrial fission and apoptosis necessitate further investigation.

In addition to irisin, other myokines such as adenosine monophosphate-activated protein kinase (AMPK) ^32,33 and Silent information regulator 1 (Sirt1) ³⁴ also play essential roles in regulating the process of mitochondrial division and influencing mitochondrial kinetic stability ^32,33. For example, AMPK activation has been linked to a potential slowdown in the progression of PD ³⁵, while Sirt1, downstream of AMPK, is crucial in various physiological and pathological processes including energy metabolism and apoptosis ³⁴. Decreased expression of Sirt1 results in increased mitochondrial fission, disrupting mitochondrial dynamics and leading to cellular damage ^32,36. Importantly, these myokines often communicate during exercise³⁷. However, the specific interactions between irisin signaling and the AMPK/Sirt1 pathway during exercise in regulating apoptosis and excessive mitochondrial fission remain unclear. Therefore, the current study hypothesizes that exercise-induced irisin secretion may alleviate PD by mitigating excessive mitochondrial fission and apoptosis through its interaction with the AMPK/Sirt1 signaling pathway.

2.1 Experimental animals and groups

Thirty-two male C57BL/6J mice (20–25 g), aged 7–8 weeks, were obtained from the Yangzhou University Laboratory Animal Center (Yangzhou, Jiangsu, China). The mice were housed in plastic cages with free access to water and food, under environmental conditions of a 12-hour light/dark cycle. The room temperature was maintained at 20–26°C with 50–56% humidity. Mouse bedding was changed regularly during the experimental period, and the condition of the mice and their access to dietary water were monitored daily. The mice were acclimatized to the laboratory environment for four weeks before the start of experimental procedures. Surgical and behavioral measurements were performed in the laboratory of the College of Physical Education, Yangzhou University.

All mice were randomly divided into control (CON) and experimental groups. The experimental group underwent intraperitoneal injection of MPTP. After the MPTP injection was completed and successful modeling was confirmed, the experimental group was further divided into a sedentary PD group, a PDEX group (which underwent ten weeks of treadmill exercise intervention), and an EXRG group (which underwent ten weeks of treadmill exercise intervention along with a tail vein injection of Cyclo RGDyk, an inhibitor of irisin). Each group consisted of 8 mice. This study was conducted under the guidance of the Animal Ethics Committee of Yangzhou University (No. 202303133), and the guidelines for the use of experimental animals were strictly adhered to.

2.2 Drug injection program

Construction of Parkinson's model

Previous studies have demonstrated that the MPTP-induced Parkinson's model can reliably produce damage in the nigrostriatal pathway and replicate the typical symptoms of Parkinson's disease. Therefore, we used MPTP for PD model construction. MPTP (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% saline at a concentration of 3.0 mg/ml, and mice were injected intraperitoneally with 30 mg/kg/day for 5 days. The control group was injected with the same dose of DMSO buffer. Strict precautions were taken during the procedure.

Irisin signaling inhibitor cyclo RGDyk injection

Researchers have shown that the αV/β5 inhibitor Cyclo RGDyk can block irisin-induced signaling³⁸. Therefore, we used Cyclo RGDyk, dissolved in DMSO, and injected it into the tail vein of mice at a dose of 2.5 mg/kg twice a week for 10 weeks.

2.3 Treadmill exercise program

In this study, a 10-week treadmill exercise program was conducted five times per week. During the first week, for acclimatization, the treadmill speed was gradually increased from 8 m/min to 12 m/min, the incline was kept at 0°, and the exercise duration was gradually extended from 10 minutes to 60 minutes. From the second week until the tenth week, the mice were exercised on the treadmill at 12 m/min for 60 minutes on each exercise day^39,40.

2.4 Rotarod test

The rotating rod test is effective in assessing motor function in mice⁴¹. Therefore, we performed the rotarod test on the mice after they underwent intraperitoneal injection of MPTP and at the end of the 10-week treadmill exercise intervention, recording the residence time of the mice on the rotarod. Briefly, before the formal test, mice were trained for three days with a gradual increase in rotarod speed to 20 rpm for 120 seconds. In the formal test, the rotarod speed was gradually increased to 50 rpm. The longest residence time of the mice on the bar was recorded as 300 seconds. The results were determined as the average of three repetitions of the experiment, with an interval of at least 40 minutes between each repetition.

2.5 Immunohistochemistry and immunofluorescence staining

In this study, paraffin-embedded tissue sections (4 µm) of the substantia nigra (SN) and striatum (ST) regions were prepared. For immunohistochemistry, the sections were treated with xylene and different gradients of alcohol, followed by antigen retrieval and endogenous peroxidase blocking to prevent nonspecific binding. We used 5% skimmed milk for 1 hour of blocking treatment, after which the sections were incubated with primary antibodies: TH antibody (1:1000, GB11181, Servicebio, China), Drp1 (1:200, A17069, ABclonal), and Fis1 (1:200, A19666, ABclonal), and refrigerated at 4°C overnight. The next day, the sections were washed three times with PBS, followed by 40 minutes of incubation with the corresponding secondary antibodies. Finally, DAB was used for color development.

For the immunofluorescence experiments, the same steps were performed as described above, but the primary antibody used was Caspase-3 (1:50, sc-56053, Santa, USA). After treatment with its corresponding secondary antibody, DAPI/Hoechst 33258 was added dropwise to visualize the nuclei of the cells.

2.6 Enzyme-linked immunosorbent assay (ELISA)

In this study, serum irisin levels in mice were measured using a highly sensitive monoclonal antibody ELISA kit (JL46388-96T, detection range: 15.6–1000 pg/ml, sensitivity: 7.7 pg/ml). The ELISA kit, sourced from Jianglaibio, China, was selected for its reliability and precision in detecting irisin levels. The entire measurement procedure was meticulously performed according to the manufacturer's standard protocol to ensure accuracy and reproducibility. Samples were collected and processed carefully to prevent any potential contamination or degradation of irisin. This rigorous approach ensured the integrity of the data and the validity of the findings related to serum irisin levels in the experimental mice.

2.7 Western blot analysis

SN tissues were collected and lysed to obtain total protein, and the protein concentration was determined using a BCA assay. The proteins were then separated using gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skimmed milk for 1 hour at room temperature, the membranes were incubated with the following primary antibodies: irisin (1:1500, SRP8039, Merck, China), AMPK (1:1000, AF6195, Beyotime, China), p-AMPK (1:2000, AF5908, Beyotime, China), Sirt1 (1:500, sc-74465, Santa Clara, USA), Drp1 (1:2000, A17069, ABclonal), Fis1 (1:2000, A19666, ABclonal), MFF (1:2000, GB114102, Servicebio), BAX (1:1000, WL01637, Wanlei, China), and Bcl-2 (1:1000, WL01556, Wanlei, China). The membranes were incubated overnight at 4°C on a shaker. The following day, after washing three times with TBST, the membranes were incubated with the corresponding secondary antibodies: goat anti-rabbit IgG (1:200, AS014, ABclonal, USA) or goat anti-mouse IgG (1:200, G1214-100UL, Servicebio, China) for 1-1.5 hours. β-actin (1:50,000, AC048, ABclonal) was used as a loading control. Final imaging was developed and quantified using ImageJ software.

2.8 Statistical analysis

We processed and analyzed the data using GraphPad Prism software (version 9.5.0, CA, USA). Results are expressed as mean ± S.E.M. Data between groups were analyzed using one-way analysis of variance (ANOVA) and t-test methods. A p-value of < 0.05 was considered statistically significant.

3.1 Exercise intervention alleviates MPTP-induced Motor Deficits and restores dopaminergic neuron loss

As previously described, a mouse model of PD was established through intraperitoneal injection of MPTP (Fig. 1A). Following the creation of the PD models, the rotating rod test was utilized to assess the impact of MPTP on motor functions. Results show that the mice treated with MPTP exhibited a significantly reduced duration of stay on the rotating rod compared to the normal controls (Fig. 1D), suggesting impaired motor function in MPTP-induced PD mice.

Immunohistochemical examination of tyrosine hydroxylase (TH) was utilized to evaluate the dopaminergic neuron loss in the substantia nigra (SN) and striatum (ST) regions. It was revealed that a notable decrease in the number of TH-positive neurons was identified in the SN and ST regions within the PD group compared to the Control group (Fig. 1B, C, E, F). Additionally, Nissl staining further confirmed the harmful effects of MPTP, revealing a significant decline in the neuron count in the SN region of the PD group compared to the Control group (Fig. 1G, H). Collectively, these results indicate that the MPTP treatment effectively induces a PD model.

Following a 10-week treadmill exercise regimen, while PD mice exhibited significant motor dysfunction, the PDEX mice demonstrated an enhanced motor performance, as evidenced by a substantial increase in their duration on the rotating rod (Fig. 1I). Moreover, exercise also alleviates dopaminergic neuron loss, which is demonstrated by the greater count of TH-positive cells in the SN and ST compared to the PD mice. Additionally, Nissl staining further identified an increased number of neurons in the SN region of PD mice that underwent 10-week exercise training (Fig. 1J, K, M, N, O). Conclusively, these findings suggest that exercise can alleviate motor deficits and dopaminergic neuron loss in MPTP-induced PD mouse models.

3.2 Exercise reduces excessive apoptosis in the nigrostriatal neurons of mice with PD

Recent studies have highlighted that pathological factors, such as excessive apoptosis, are key mechanisms leading to the loss of dopaminergic neurons in PD ⁴². However, therapeutic strategies are currently lacking. We, therefore, investigated whether 10 weeks of exercise training could ameliorate apoptosis in PD conditions. Considering the pivotal role of caspase-3 in neuronal apoptosis ⁴³, we conducted immunofluorescence experiments on caspase-3 levels to evaluate cell apoptosis. Our findings revealed a considerable rise in the number and area of caspase-3 positive cells in the SN of PD mice compared to the control group. Moreover, there was a notable increase in the number and area of Hoechst 33258-positive cells in the SN of PD mice (Fig. 2A, C), characterized by augmented nuclear debris and intense staining, indicative of heightened apoptosis. However, after 10 weeks of treadmill exercise, there was a significant reduction in abnormal nuclear morphology, suggesting an improvement in hyperapoptosis (Fig. 2A, C). Moreover, our results indicated that, in comparison to the CON group, there was a reduction in the expression of the anti-apoptotic protein Bcl-2 in the PD group, while the expression of the pro-apoptotic protein BAX was elevated. Interestingly, 10 weeks of treadmill exercise down-regulated the expression level of BAX and up-regulated the expression level of Bcl-2 (Fig. 2B, D, E). In conclusion, these findings imply that a 10-week treadmill exercise regimen may counteract excessive apoptosis in PD mice, offering a protective benefit.

3.3 Exercise ameliorates abnormal mitochondrial fragmentation and the expression of key mitochondrial fission factors in the substantia nigra of Parkinson's disease mice

A growing body of evidence indicates that apoptosis is a critical contributor to excessive mitochondrial fission ^14–18. and abnormal mitochondrial fission has been identified as a potential key pathological mechanism in PD ⁴⁴. Therefore, we aimed to investigate changes in mitochondrial fission-related markers in the SN of PD mice. Immunohistochemical analysis showed that the expressions of Drp1 and Fis1, key regulators of mitochondrial fission, were significantly increased in SN of PD mice compared with the CON group. However, a 10-week treadmill exercise regimen effectively mitigated the abnormal fission, significantly decreasing the positive areas of Drp1 and Fis1 (Fig. 3A, B, C, D). In addition, Western Blot results revealed that the expressions of Drp1, Fis1, and MFF proteins, key regulatory factors of mitochondrial fission, were significantly increased in the SN of the PD group compared to the CON group. Importantly, our results further indicated that 10 weeks of treadmill exercise can effectively improve abnormal mitochondrial fission (Fig. 3, E, F, G, H). These results suggest that exercise plays a crucial role in improving the expression of abnormal mitochondrial fission-related markers.

3.4 Exercise enhances AMPK/SIRT1 signaling, however, this effect is inhibited by an irisin receptor blocker

Previous research has indicated that regular exercise can effectively alleviate neuronal apoptosis and abnormal mitochondrial fission, apoptosis, and neuronal damage, thereby playing a protective role in neurodegenerative diseases^45,46. However, the specific signaling pathway by which exercise inhibits abnormal mitochondrial fission in PD remains unclear. Recent studies have identified the muscle factor irisin as an essential mediator of exercise^27,28. Which can alleviate excessive neuronal apoptosis and inhibit excessive mitochondrial fission, potentially serving a neuroprotective function in neurodegenerative conditions³¹. It remains unclear how exercise interacts with irisin to mediate mitochondrial dysfunction in PD conditions. Our results found that irisin expression was significantly decreased in both serum and SN of mice in the PD group compared with the CON group. However, its expression level increased after a ten-week treadmill running (Fig. 4A, B, C).

It is indicated that mitochondrial fission, energy metabolism, and apoptosis are primarily regulated by AMPK and its downstream target, the Sirt1 pathways^35,47. Additionally, irisin is also believed to impact mitochondrial function and energy metabolism^48–50. Therefore, we further investigated the interaction between irisin signaling and the AMPK and Sirt1 pathways in PD mice during exercise.

We found that the expression of P-AMPK as well as downstream Sirt1 was significantly decreased in the SN of the PD group compared to the CON group, whereas its expression level was increased after the 10-week running table exercise intervention (Fig. 4D, E, F). Notably, the exercise-induced phosphorylation of AMPK and the elevation of downstream Sirt1 expression were reversed by inhibiting the irisin signaling after ten weeks of administration of Cyclo RGDyk (Fig. 4D, E, F). These findings suggest that changes in AMPK and downstream Sirt1 may be regulated by irisin signaling and play an important role in PD.

3.5 Blocking the irisin signaling pathway reduces the neuroprotective effects of exercise against excessive apoptosis and abnormal mitochondrial fragmentation

As mentioned above, inhibition of irisin signaling has been shown to reduce the rise in the AMPK phosphorylation of P-AMPK and Sirt1 during exercise. Subsequently, we investigated whether blocking irisin signaling could diminish the beneficial effects of exercise on excessive apoptosis and abnormal mitochondrial fission in PD mice. The findings showed that the administration of cyclo RGDyk led to a significant increase in the presence of caspase-3 and Hoechst 33258 apoptotic cells, accompanied by elevated Drp1 and Fis1 expression in the SN of exercised mice not treated with cyclo RGDyk (Fig. 5A, C, F, G, H, I). Furthermore, there was a significant increase in the expression of the apoptosis-related marker protein BAX and a decrease in the expression of the anti-apoptotic protein Bcl-2 following intervention with cyclo RGDyk. The cyclo RGDyk treatment also increased the expression levels of mitochondrial fission-related marker proteins Drp1, Fis1, and MFF (Fig. 5B, D, E, J, K, L, M). Collectively, these findings underscore the pivotal role of irisin signaling in modulating increased apoptosis and abnormal mitochondrial fission in PD mice improved through exercise.

In this study, we found that regular exercise can have multiple beneficial effects on the MPTP-induced PD mouse model, including slowing down the loss of dopaminergic neurons, ameliorating motor dysfunction, and decreasing excessive apoptosis and abnormal mitochondrial fission. Our findings indicate that irisin plays an important role in exercise-induced positive effects via interacting with the AMPK/Sirt1 signaling pathway. Though previous research has suggested a link between mitochondrial function and exercise benefits, the specific molecular mechanisms underlying these favorable effects on PD are not fully understood ²¹. Our study provides novel experimental evidence that helps address this gap to some extent.

Because PD has a complex pathological mechanism, it is generally considered incurable, and current treatments primarily target improving motor dysfunction and other symptoms without being able to affect the disease's progression. However, our findings showed that exercise intervention can effectively alleviate the persistent loss of DA neurons and improve motor function. Our results align with previous studies that highlight the crucial role of exercise in slowing the progression of PD, such as enhancing movement control and slowing the loss of DA neurons, thereby bringing overall benefits to PD patients ^51,52. The positive impact of exercise on PD is partly linked to the enhancement of mitochondrial dynamics. In PD, excessive mitochondrial fission causes mitochondrial fragmentation, exacerbating mitochondrial dysfunction and ultimately leading to neuronal death ^53–55. Our results showed that MPTP-treated mice had increased levels of Drp1, Fis1, and MFF, key regulators of mitochondrial fission, as well as elevated levels of Caspase-3 and BAX, key regulators of apoptosis, while BCL-2 was significantly decreased. However, the aberrant mitochondrial fission and excessive apoptosis could be ameliorated by ten weeks of treadmill exercise. Our results are consistent with recent findings. For example, one recent study reported that aerobic exercise can decrease Drp1 expression and reduce excessive mitochondrial fission, thereby maintaining mitochondrial functional and structural stability ⁵⁶. Another study suggested that long-term treadmill running alleviated excessive mitochondrial fission, apoptosis, and neuronal damage, leading to the improvement of neurodegenerative conditions ⁴⁶. While these findings significantly confirm the positive impact of exercise on mitochondrial fission and neuronal apoptosis in PD, the regulatory mechanism still requires further investigation.

Recent research has shown that skeletal muscle produces myokines during exercise, facilitating communication between muscle and other organs, including the brain, fat, bone, and heart ^22,57–59. Among these myokines, irisin is a promising target for the beneficial effects of exercise.

Concurrent administration of recombinant irisin and bone marrow stromal cells (BMSC) to PD rats protected DA neurons from apoptosis and degeneration ⁶⁰. This beneficial effect involved irisin triggering the migration of BMSCs to impaired brain regions, boosting the count of TH-positive neurons in the substantia nigra and striatum, and enhancing symptoms ⁶⁰. Additionally, irisin positively affected mitochondrial function by promoting mitochondrial biogenesis and alleviating oxidative stress in a PD model ³¹. These studies highlight the potential of irisin in PD treatment. Our results revealed that irisin levels were significantly decreased in the brain tissues of PD mice. Interestingly, ten weeks of treadmill exercise reversed this reduction, which may be related to the neuroprotective effects of exercise. This hypothesis is supported by our finding that blocking irisin receptor αV/β5 signaling with Cyclo RGDyk significantly inhibited the beneficial effects of exercise including the improvement of mitochondrial function and cell apoptosis. A recent study also supported our findings, showing that levels of FNDC5 in skeletal muscle, peripheral blood, and brain were significantly higher in the PD group receiving eight weeks of treadmill exercise compared to the sedentary PD group, which alleviated MPTP-induced motor dysfunction and degeneration of DA neurons, thereby ameliorating the pathological process of PD ⁶¹. In conclusion, our findings, along with these studies, suggest that irisin may modulate the protective effects of exercise on PD by improving mitochondrial function.

Emerging evidence has suggested that myokines, including irisin, AMPK, and Sirt1, frequently work together to regulate the impacts of exercise ^{22,57–59,62}. For instance, a study reported that treadmill exercise increased the expression levels of P-AMPK, Sirt1, and BDNF in the striatum of PD mice, significantly improving mitochondrial and motor dysfunction ⁶³. Additionally, eight weeks of treadmill exercise reduced α-synuclein levels, promoted autophagy, and further alleviated the loss of dopaminergic neurons in an MPTP-induced rodent PD model ⁶⁴. This reduction was achieved by enhancing mitochondrial function and activating the Sirt1 signaling pathway, downstream of AMPK ⁶⁴. However, it is not yet clear whether irisin can upregulate the AMPK/Sirt1 pathway during exercise in MPTP-induced PD models. Our findings revealed that phosphorylated P-AMPK and Sirt1 expression were elevated in the substantia nigra of the PD exercise group. However, blocking irisin signaling reversed this phenomenon. Moreover, blocking irisin signaling also impaired the enhanced mitochondrial function and reduced apoptosis observed in PD mice undergoing exercise intervention. In conclusion, the interplay between Irisin and the AMPK/Sirt1 signaling pathway may collaboratively govern the neuroprotective effects of exercise on PD pathology.

In conclusion, our research suggests that exercise can mitigate excessive neuronal apoptosis, improve mitochondrial fission abnormalities, lessen the loss of dopaminergic neurons, and enhance motor function in an MPTP-induced PD model. Furthermore, our findings indicate the involvement of the Irisin/AMPK/Sirt1 signaling pathway in these processes. Nevertheless, our study has some limitations. We did not evaluate the activity of mitochondrial complex I in PD. Additionally, we did not conduct in vitro experiments or employ transgenic models to further clarify the precise mechanisms related to irisin. In future investigations, we will address these issues by exploring different models and experimental approaches to comprehensively examine the specific mechanisms underlying the protective effects of exercise in PD.

Acknowledgements

Sincere thanks to Dr. Li for the guidance given on the experimental techniques.

Funding

This work was funded by Grants from the Natural Science Foundation of Jiangsu Province (BK20201435).

Availability of data and materials

The corresponding author will provide the necessary data supporting the findings of this study upon a reasonable request. Authors are accountable for ensuring the continued availability of the data.

Ethics approval statement

All animal experiments in the study were approved by the Animal Ethics Committee of Yangzhou University (NO. 202303133).

Declaration of interests

The authors assert the absence of any conflicting interests.

Author Contributions

Nan Li and Renqing Zhao designed and wrote the manuscript. Experiment material preparation was undertaken by Bin Wang, Yuanxin Wang, Xin Tian, Junjie Lin, Xun Sun, Yu Sun, Xin Zhang, Haocheng Xu, Mingzhi Li, Fanxi Zeng. The experiment and subsequent analysis were carried out by Nan Li. The final manuscript was read and approved by all authors.

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Exercise Mitigates MPTP-Induced Mitochondrial Fragmentation through the Irisin/AMPK/SIRT1 Pathway

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Highlights

1 Introduction

2 Materials and methods

2.1 Experimental animals and groups

2.2 Drug injection program

2.3 Treadmill exercise program

2.4 Rotarod test

2.5 Immunohistochemistry and immunofluorescence staining

2.6 Enzyme-linked immunosorbent assay (ELISA)

2.7 Western blot analysis

2.8 Statistical analysis

3 Results

3.1 Exercise intervention alleviates MPTP-induced Motor Deficits and restores dopaminergic neuron loss

3.2 Exercise reduces excessive apoptosis in the nigrostriatal neurons of mice with PD

3.4 Exercise enhances AMPK/SIRT1 signaling, however, this effect is inhibited by an irisin receptor blocker

4 Discussion

Declarations

References

Additional Declarations

Supplementary Files

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