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.