Discussion
The protective effects of SA against neurodegenerative diseases and other diseases such as obesity and osteoarthritis have been studied, but there are no mechanistic studies regarding the effect of SA on circadian rhythms and mitochondrial function (Kim et al., 2010; Li et al., 2019; Yang et al., 2019; Zare, Eidi, Roghani & Rohani, 2015). The present study demonstrates that SA ameliorates the neurotoxicity induced by MPTP and its metabolite MPP+ through REV-ERB α-modulated mitochondrial fission.
Mitochondrial dysfunction is closely linked to the pathogenesis of PD, and maintenance of mitochondrial function is considered a potential cure for the disease (Celardo, Martins & Gandhi, 2014). MPTP is one of most commonly used chemical reagents for the PD model, and its treatment causes dopaminergic neuronal degeneration in the SNpc and behavioural abnormalities, which are based on mitochondrial damage. In the present study, MPTP or its metabolite MPP+-treated mice or cells increased dopaminergic neuronal damage by decreasing TH protein expression and mitochondrial damage by decreasing ATP levels and altering GDH activity. However, SA treatment restored TH protein expression and mitochondrial function. Additionally, SA also improved behavioural function in both tests using the MPTP-treated mouse model. Zare et al. reported that SA has neuroprotective potential against 6-OHDA in a PD animal model by regulating oxidative stress and lowering nigral iron levels (Zare, Eidi, Roghani & Rohani, 2015). Our results confirmed that SA protects against dopaminergic neuronal cell damage and mitochondrial function.
Damaged mitochondria are regulated by several maintenance mechanisms, such as mitophagy, mitochondrial biogenesis, and mitochondrial fission and fusion, all working in coordination. Mitochondrial fission is considered the first step in the maintenance of mitochondrial homeostasis. However, excessive mitochondrial fission induces cell injury through ATP depletion, ROS generation and apoptosis activation, which are common causes of the development of neurodegenerative diseases (Roe & Qi, 2018; Suen, Norris & Youle, 2008). Drp1 is considered the most important protein in the fission process, and its activity is controlled by phosphorylation of serine 616 and serine 637. An impaired balance between these two types of phosphorylation results in excessive mitochondrial fragmentation and neuronal cell death (Cho, Choi, Cho, Kim & Sun, 2013). Recently, accumulating evidence has demonstrated that inhibition of Drp1 and/or phospho-Drp1 Ser616 ameliorates neurotoxicity and deficits in dopamine release in PD animal models. Simone et al. showed that treatment with mdivi-1, a putative inhibitor of Drp1, decreased mitochondrial dysfunction and oxidative stress in α-synuclein-overexpressing rats (Bido, Soria, Fan, Bezard & Tieu, 2017; Mishra, Singh, Tiwari, Bano & Shukla, 2019; Park et al., 2019). Our findings demonstrate that MPTP-induced mitochondrial fission was significantly weaker after SA treatment through decreased protein expression of phospho-Drp1 Ser616 and is correlated with recovery of mitochondrial function. Therefore, our data suggested that SA attenuated excessive mitochondrial fission, especially the reduction of Drp1 phosphorylation at serine 616.
Circadian rhythms, which control the repeated approximately 24-hour sleep-wake cycle, manage various physiological functions, such as antioxidant and inflammatory responses, by changing gene expression (Zheng, Yuan, Wu, Lv & Zhu, 2017). Thus, disruption of the circadian cycle is considered to be a key factor in several diseases, including cancer, metabolic disease, and neurodegenerative disease (Xie et al., 2019). The most well-known clinical characteristic of the abnormal circadian cycle is sleep disorder. For this reason, disruption of the circadian cycle is widely accepted as a novel risk factor for the development of PD because sleep and circadian disorders are easily discovered in PD patients (Videnovic & Golombek, 2013). Several studies using animal PD models showed dopaminergic neuronal death and deregulated circadian symptoms, including behavioural and physiological outputs (Hayashi et al., 2013; Wang et al., 2018). Furthermore, MPTP treatment with environmental circadian disruption induced by long-term exposure to a 20:4 light/dark cycle exacerbated motor and cognitive deficits through excessive neuronal cell loss and the neuroinflammatory response (Lauretti, Di Meco, Merali & Pratico, 2017). In addition, MPTP intoxication led to sleep disorders, including sleep episodes in the daytime and sleep fragmentation at night, whereas melatonin and L-dopa significantly improved these sleep symptoms in MPTP-treated monkeys (Belaid, Adrien, Karachi, Hirsch & Francois, 2015). Collectively, these studies suggest that abnormal circadian and PD are closely related. REV-ERB α is responsible for the stability of the circadian rhythm by regulating circadian-related protein expression and is considered to be involved in the development of PD. Deletion of REV-ERB αcontributed to exacerbated motor deficits and dopaminergic neuronal loss in a 6-OHDA-treated PD mouse model (Kim et al., 2018). In this study, SA treatment restored the protein level of REV-ERB α in MPTP-treated mice. Our data suggested that SA treatment has an effect on the recovery of circadian cycle disruption by regulating REV-ERB α.
Although disruption of the circadian cycle and its contribution of onset to the development of PD were found in previous studies, whether disruption of the circadian cycle contributes to the progression of PD remains controversial. To adapt to the constant changes in the environment caused by daily changes during the day and night, a continuous supply of energy is essential to maintain and improve cell function (de Goede, Wefers, Brombacher, Schrauwen & Kalsbeek, 2018). Thus, the regulation of mitochondrial function is considered a possible candidate for this argument. Interestingly, several studies have suggested that mitochondrial functions and morphologic changes are dependent on a viable circadian clock. Bmal1 knockout mice showed mitochondrial defects accompanying morphological changes and functional abnormalities, which led to severe, progressive, age-dependent heart failure (Kohsaka et al., 2014). Altered mitochondrial gene expression and increased mitochondrial oxidative stress were discovered in aged mice with mutations in the clock gene (Gong et al., 2015). Moreover, Woldt et al. demonstrated that genetic deletion ofrev-erb α in mice resulted in impaired mitochondrial content and oxidative function; however, the amount of mitochondria and respiratory capacity were improved after treatment with the REV-ERB α-expressing vector (Woldt et al., 2013a). To investigate whether the protective effect of SA is mediated by mitochondrial fission through the regulation of REV-ERB α, we tested an antagonist and agonist of REV-ERB α with SA in an in vitro and in vivo PD model. The protective effect of SA disappeared after co-treatment with SA and REV-ERB α antagonists, as shown by decreased mitochondrial function and TH protein expression, while SA and agonist co-treatment did not change in the in vitromodel. These in vivo results using an antagonist with SA indicated that the recovery effect of SA was diminished, as shown by decreased behavioural function and TH protein expression. In addition, mitochondrial fission boosted after co-treatment with SA and antagonist. Our data also showed that the level of Drp1 protein expression driven by the plasmid was decreased after treatment with REV-ERB α recombinant protein (Figure S1). Together, these data support a prominent role for SA in the regulation of mitochondrial fission via the REV-ERB α protein.
In this study, we identified the pharmacological activation of SA in a neurotoxin-induced PD model through the regulation of REV-ERB α protein expression and a novel role of REV-ERB α in managing mitochondrial homeostasis by maintaining the balance of two kinds of Drp1 phosphorylation. Comprehensively, SA could be a promising therapeutic option for the prevention and treatment of PD.