Aggregationof aberrant cytoplasmic inclusion such as disaggregated proteins and damagedorganalles are the hallmarks in most of the neurodegenerative diseases,especially in PD.
Consequently, these aggregations within neurons leads to cytotoxiceffects such as overproduction of ROS and oxidative stress mediated cell loss,due to autophagy dysfunctions, but the exact cause of autophagy dysfunctionmediates neurodegeneration still unclear. In this study, MTT assay showed that differentconcentration (0.5-200 nM) of rotenone treatment to SK-N-SH cells significantlyaffects cell viability compared with untreated control cells.
However, we foundthat maximal inhibition (60 %) of cell viability at 100 nM of rotenone doseconcentration, which is corroborating with previous findings (Jayaraj et al., 2013; Kavitha et al., 2013;Tamilselvam et al.
, 2013). Addition of rotenone treatment to SK-N-SHcells directly affects cellular energy and metabolism which consequently leads ROSaccumulation and cell death. However, we found that GE to rotenone treatment inhibitsabove 50% of the cell population at the concentration 60 nM for 24 h asdemonstrated in Figure 1. This is consistent with the finding in other in vitro models with relevance to PD (Jayaraj et al., 2013; Kavitha et al., 2013;Tamilselvam et al., 2013).
Since, oxidative injurywas proposed to be a primary mechanism which involved in mitochondrial toxicitymediated rotenone induced cell death in SK-N-SH cells (Fiskum et al., 2003; Song et al., 2012). The mitochondria are one of the majorprinciple sourcesof ATP biosynthesis through oxidative phosphorylation for thepurpose of providing energy to cellular activities. Oxidative phosphorylationfor ATP synthesis, which is consists of five electron transport chain (ETC; I-V),which together composes different structural proteins. A reduction orinhibition of any one of ETC could lead to disrupt the balance between ATPproduction and consumption, and results reduced ATP stores and causes electronsto accumulate within respiratory chain components(Kavitha et al.
, 2013). Inhibition of ETC’s such as ETC-I,also leads to the shunting of electrons through the ETC-II, which may enhances theformation of ROS upto5-7 times more than normallevel (Selvakumar et al., 2014). This appears to be the primary eventof rotenone induced formation of O2?, which undergoesspontaneous orSOD catalyzed dismutation to form H2O2.Catalase and glutathione peroxidase catalyzes the decomposition of H2O2to H2O and O2(Fiskum et al., 2003).Over accumulationof intracellular ROS (as demonstrated by the DCFDA assay) major culprit for neuronalloss and a key marker of oxidative stress and leads to a rapid consumption anddepletion of endogenous scavenging antioxidants(Moon and Paek, 2015).
In addition, ROS, may quicklyinteracts with nearest cellularcomponents such as proteins, lipids, carbohydrates and nucleic acids, leading to the oxidative damage of these molecules, disaggregated protein accumulationand the subsequent cellular dysfunction (Szewczyk and Wojtczak, 2002). In this study,we found that treatment with rotenone decreased activities of enzymaticantioxidants such as SOD, GSH were probably due to a response towards increasedconcentration of ROS and lipid peroxidation by TBARS as shown in Table 1.Moreover, GSH depletion, the first indicator of oxidative stress during PDprogression, suggests a concomitant increase in ROS accumulation(Sherer et al., 2003; Kavitha et al.
, 2013). It was reported that abnormalproduction of ROS and NO could inhibit the cell growth and induce cell death inSK-N-SH cells(Zhao and Li, 2002; Ezoulin et al., 2008). Our current results also agreementwith previous findings,GE appears to prevented rotenone induced cell death by reducedROS and NO generation, TBARS via enhanced activity of ECT-1 and neutralized theendogenous antioxidant by enhancing the activities of SOD and GSH inexperimental group, which might be due to ROS scavenging property of GE (Tiwari and Kakkar, 2009).
Similarly, the SN of PD brains has areduced level of the antioxidant enzymes suchas catalase, SOD and GPx (Sian et al., 1994a) and antioxidantmoleculessuch as GSH (Sian et al., 1994b), suggesting the presence of asustained burden of oxidative stress that overhelmed the antioxidantcapacity (Liu and Ames, 2005; Liu et al., 2009).
Collectively, rotenone modelrecapitulates mostof the mechanisms thought to be important in PD pathogenesis(Betarbet et al., 2002). Furthermore,rotenone promotes cell death by inducing apoptosis morphological changes suchas chromatin condensation and distended mitochondrial structure.
Cellspre-treated with GE prevent the cell death by reverse of those process. Previouslyit was reported that rotenone induces apoptotic cell death through chromatincondensation and disrupts mitochondrial structure neuronal cells, when cells pretreatedwith baicalein and naringin prevents the apoptotic morphological dependent celldeath(Watabe and Nakaki, 2004; Kim et al., 2009; Song etal., 2012; Shangguan et al., 2017), Our results concurrence withprevious findings.Treatment with rotenonereduces autophagy clearance in the neuronal cells. Chu et al, (Chu et al.
, 2013) reported that rotenone treatmentrupture the mitochondrial transmembrane potential with mitophagy recognitionprotein and reduces intracellular mitophagy in neuronal cells and it is leadsto mitophagy dysfunction(Chu et al., 2013; Hou et al., 2015; Moors et al.,2017). In this study, we found thatrotenone treatment significantly reduces the mitophagy vesicle engulfment andincreased accumulation of oxidatively damaged mitochondrial (called distendedmitochondrial structure)populationsin SK-N SH cells, due to mitochondrialtransmembrane potential depolarization.
Aggregation of ?-Synuclein(14 kDa consists 140 amino acids) pre-synaptic protein localized in various partsin the brain) anearly culprit for dopaminergic cell death in PD(Moors et al., 2017). Treatment of rotenone increases theaggregation of ?-Synuclein protein via oxidative stress in neuronal cells,which is decreased by centella asiaticaextract (McMurray, 2001; Berrocal et al., 2014). In the present study, we found thatGE treatment to rotenone significantly reduces ?-Synuclein expression ascompared with rotenone only treated cells, which concurrence with previousfindings.ER stress arises when protein-foldingcapacity is inadequate due to increased rates of synthesis or accumulation ofmisfolded or abnormal proteins, and it activates the UPR. The UPR has threebranches that govern translational control and chaperone protein expression,PERK, IRE-1?, and ATF6(Velculescu et al., 1995; Ron and Walter, 2007; Walterand Ron, 2011; Back and Kaufman, 2012).
Upon sensing the presence of unfoldedor misfolded proteins, IRE1? undergoes dimerization andtrans-autophosphorylation, activating its endoribonuclease activity; IRE1? thenmediates the excision of a 26-nucleotide intron from X-box binding protein 1(XBP1), resulting in a translational frameshift and formation of a potenttranscriptional activator(Jiang et al., 2016)thereby activating the transcriptionof ER stress target genes.PERK is also a type I ERtransmembrane kinase. Similar to IRE1?, when activated by ER stress, PERKoligomerizes, autophosphorylates and then directly phosphorylates ? subunit ofeukaryotic initiation factor 2 (eIF2?) (Harding et al., 1999). Phosphorylated eIF2? preventsformation of ribosomal initiation complexes leading to global mRNAtranslational attenuation.
This reduction in ER workload protects cells from ERstress-mediated death. Meanwhile some mRNAs require eIF2? phosphorylation fortranslation such as the mRNA encoding ATF4. ATF4 is a b-ZIP transcriptionfactor that regulates several UPR target genes including those involved in ERstress mediated cell death such as CHOP (Harding et al., 2000). A third regulator of ER stresssignaling is the type II ER transmembrane transcription factor, ATF6?.
It wasextensively studied in the context of ER Stress. Upon ER stress conditions,ATF6? translocate to the nucleus to activate UPR genes involved in proteinfolding, processing, and degradation (Yoshida et al., 2000). When activated, the signal transduction pathwaysinitiated by PERK, IRE1? and ATF6? induce a characteristic set of genesencoding ER chaperones and nuclear transcription factors that ultimately leadeither to reduction of ER stress or to death (Mori, 2003). Therefore, ER stress may play a prosurvival orproapoptotic role and severe ER stress triggers neuronal cell death(Vilatoba et al.
, 2005;Wang et al., 2008).Inthe current study, we found that the specifc mechanism of the cell activity ofthe rotenone treated cells, the ER stress (UPR pathway) associated proteins,including PERK, IRE-1? and ATF6? levels were significantly increased inrotenone treated cells, suggesting the presence of increased ER stress in thePD model (Han and Holtzman,2000; Chen et al., 2008; Han et al., 2014a; Han et al., 2014b; Jiang et al.
,2016). And also increased levels of elf 2?, ATF4, CHOP and XBP1(Wu et al., 2013; Chen et al., 2008; Jiang et al., 2016) were observed inrotenone treated SK-N-SH cells. Pretreatment of GE observed protectiveeffect in rotenone treatment by down-regulating the ER stress offeredprotection against rotenone cytotoxicity.
Taken together, the results suggestthat by reducing the ER stress contribute a protection againstrotenone cytotoxicity.Autophagymediates lysosomal degradation of long-lived cytoplasmic proteins, initiatedunder the conditions of differentiation, stress such as oxidative stress, ERstress and protein aggregate accumulation (Yorimitsu et al., 2006; Mizushima, 2007; Sarkar etal., 2007a; Sarkar et al., 2007c).
It was reported that autophagy playa crucial role in a number of neurological diseases especially in PD(Cherra and Chu, 2008; Yue et al., 2008). The strategy of upregulating oraccelerating autophagy to treat neurodegenerative diseases has been tested invarious cell and animal models and shown to decrease protein aggregation,maintain the cytoplasmic homeostasis leads to prevent the cell death (Sarkar et al., 2007c; Pan et al., 2009). Our study focused on the ER stressdependentactivationof autophagy during rotenone induced oxidative stressconditioncan prevent thecell loss in SK-NSH cells by GE treatment.
Autophagyhas revealed that the regulatory molecules that control autophagy are varied, dependson the intracellular homeostasis including AMPKand mTOR (Meijer and Codogno, 2006). Theserine/threonine kinase mTOR, a central controller of cell growth andnegatively regulates autophagy. mTOR kinase regulates autophagy through ATGproteins, resulting in interference with the formation of autophagosomes(Levine and Yuan, 2005). The data fromthe present study encourages the concept that GE induces a pathway of autophagythrough the downregulation of AMPK and mTOR expression in SK-N-SH cells.TheLC3-I and LC3-II proteins are critical markers for autophagy, was produced byautophagy cascade.
LC3-I exists in cytosolic form and LC3-II in membrane boundform. The ratio of conversion from LC3-I to LC3-II is closely correlated withthe extent of autophagosome formation (Kabeya et al., 2000). In addition, the autophagic proteinp62/SQSTM1 is selectively incorporated into autophagosome through directbinding to LC3 and efficiently degraded by autophagy.
The level of p62inversely correlates with autophagic activity (Bjorkoy et al., 2006). Our hypothesisthat autophagy is induced during ER stress is supported by activated theautophagosome formation, which was shown in examinations of LC3-I, LC3-II.Therefore,the AMPK and mTOR dependent or ?independent mechanism may participate inpathway regulation of LC3-associated autophagosome formation in dopaminergicneurons, under normal and pathogenic conditions.