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Aggregation in PD. Consequently, these aggregations within

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Aggregation
of aberrant cytoplasmic debris such as disaggregated proteins and long lived
and damaged organalles are the hallmarks in most of the neurodegenerative
diseases, especially in PD. Consequently, these aggregations within neurons
leads to cytotoxic effects such as overproduction of ROS and oxidative stress mediated
cell loss, due to autophagy dysfunctions, but the exact cause of autophagy
dysfunction mediates neurodegeneration still unclear. In our study, MTT assay
showed that rotenone treatment to SK-N-SH cells in a dose-dependent manner  (0.5-200 nM). We found that maximal inhibition
(60 %) of cell viability at 100 nM of rotenone dose concentration, 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-SH
cells directly affects cellular energy and metabolism which consequently leads ROS
accumulation and cell death. However, we found that GE to rotenone treatment inhibits
above 50% of the cell population at the concentration 60 nM for 24 h as
demonstrated 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 injury
was proposed to be a primary mechanism which involved in mitochondrial toxicity
mediated rotenone induced cell death in SK-N-SH cells (Fiskum et al., 2003; Song et al., 2012). The mitochondria are one of the major
principle sourcesof ATP biosynthesis through oxidative phosphorylation for the
purpose of providing energy to cellular activities. Oxidative phosphorylation
for ATP synthesis, which is consists of five electron transport chain (ETC; I-V),
which together composes different structural proteins. A reduction or
inhibition of any one of ETC could lead to disrupt the balance between ATP
production and consumption, and results reduced ATP stores and causes electrons
to 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 the
formation of ROS upto5-7 times more than normallevel (Selvakumar et al., 2014). This appears to be the primary event
of rotenone induced formation of O2?, which undergoes
spontaneous orSOD catalyzed dismutation to form H2O2.
Catalase and glutathione peroxidase catalyzes the decomposition of H2O2
to H2O and O2(Fiskum et al., 2003).Over accumulation
of intracellular ROS (as demonstrated by the DCFDA assay) major culprit for neuronal
loss and a key marker of oxidative stress and leads to a rapid consumption and
depletion of endogenous scavenging antioxidants(Moon and Paek, 2015).

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In addition, ROS,  may  quickly
interacts with  nearest cellular
components such as proteins, 
lipids,  carbohydrates  and 
nucleic  acids,  leading to 
the  oxidative  damage 
of  these  molecules, disaggregated protein accumulation
and the subsequent cellular dysfunction  (Szewczyk and Wojtczak, 2002). In this study,
we found that treatment with rotenone decreased activities of enzymatic
antioxidants such as SOD, GSH were probably due to a response towards increased
concentration of ROS and lipid peroxidation by TBARS as shown in Table 1.
Moreover, GSH depletion, the first indicator of oxidative stress during PD
progression, suggests a concomitant increase in ROS accumulation(Sherer et al., 2003; Kavitha et al., 2013). It was reported that abnormal
production of ROS and NO could inhibit the cell growth and induce cell death in
SK-N-SH cells(Zhao and Li, 2002; Ezoulin et al., 2008). Our current results also agreement
with previous findings,GE appears to prevented rotenone induced cell death by reduced
ROS and NO generation, TBARS via enhanced activity of ECT-1 and neutralized the
endogenous antioxidant by enhancing the activities of SOD and GSH in
experimental group, which might be due to ROS scavenging property of GE (Tiwari and Kakkar, 2009).Similarly, the SN of PD brains has a
reduced level of the antioxidant enzymes suchas catalase, SOD and GPx (Sian et al., 1994a) and antioxidant
moleculessuch as GSH (Sian et al., 1994b), suggesting the presence of a
sustained burden of oxidative stress that overhelmed the antioxidantcapacity (Liu and Ames, 2005; Liu et al., 2009). Collectively, rotenone model
recapitulates mostof the mechanisms thought to be important in PD pathogenesis(Betarbet et al., 2002). Furthermore,
rotenone promotes cell death by inducing apoptosis morphological changes such
as chromatin condensation and distended mitochondrial structure.Cells
pre-treated with GE prevent the cell death by reverse of those process. Previously
it was reported that rotenone induces apoptotic cell death through chromatin
condensation and disrupts mitochondrial structure neuronal cells, when cells pretreated
with baicalein and naringin prevents the apoptotic morphological dependent cell
death(Watabe and Nakaki, 2004; Kim et al., 2009; Song et
al., 2012; Shangguan et al., 2017), Our results concurrence with
previous findings.

Treatment with rotenone
reduces autophagy clearance in the neuronal cells. Chu et al, (Chu et al., 2013) reported that rotenone treatment
rupture the mitochondrial transmembrane potential with mitophagy recognition
protein and reduces intracellular mitophagy in neuronal cells and it is leads
to mitophagy dysfunction(Chu et al., 2013; Hou et al., 2015; Moors et al.,
2017). In this study, we found that
rotenone treatment significantly reduces the mitophagy vesicle engulfment and
increased accumulation of oxidatively damaged mitochondrial (called distended
mitochondrial structure)populationsin SK-N SH cells, due to mitochondrial
transmembrane potential depolarization.

Aggregation of ?-Synuclein
(14 kDa consists 140 amino acids) pre-synaptic protein localized in various parts
in the brain) anearly culprit for dopaminergic cell death in PD(Moors et al., 2017). Treatment of rotenone increases the
aggregation of ?-Synuclein protein via oxidative stress in neuronal cells,
which is decreased by centella asiatica
extract (McMurray, 2001; Berrocal et al., 2014). In the present study, we found that
GE treatment to rotenone significantly reduces ?-Synuclein expression as
compared with rotenone only treated cells, which concurrence with previous
findings.

ER stress arises when protein-folding
capacity is inadequate due to increased rates of synthesis or accumulation of
misfolded or abnormal proteins, and it activates the UPR. The UPR has three
branches that govern translational control and chaperone protein expression,
PERK, IRE-1?, and ATF6(Velculescu et al., 1995; Ron and Walter, 2007; Walter
and Ron, 2011; Back and Kaufman, 2012). Upon sensing the presence of unfolded
or misfolded proteins, IRE1? undergoes dimerization and
trans-autophosphorylation, activating its endoribonuclease activity; IRE1? then
mediates the excision of a 26-nucleotide intron from X-box binding protein 1
(XBP1), resulting in a translational frameshift and formation of a potent
transcriptional activator(Jiang et al., 2016)thereby activating the transcription
of ER stress target genes.

PERK is also a type I ER
transmembrane kinase. Similar to IRE1?, when activated by ER stress, PERK
oligomerizes, autophosphorylates and then directly phosphorylates ? subunit of
eukaryotic initiation factor 2 (eIF2?) (Harding et al., 1999). Phosphorylated eIF2? prevents
formation of ribosomal initiation complexes leading to global mRNA
translational attenuation. This reduction in ER workload protects cells from ER
stress-mediated death. Meanwhile some mRNAs require eIF2? phosphorylation for
translation such as the mRNA encoding ATF4. ATF4 is a b-ZIP transcription
factor that regulates several UPR target genes including those involved in ER
stress mediated cell death such as CHOP (Harding et al., 2000). A third regulator of ER stress
signaling is the type II ER transmembrane transcription factor, ATF6?. It was
extensively studied in the context of ER Stress. Upon ER stress conditions,
ATF6? translocate to the nucleus to activate UPR genes involved in protein
folding, processing, and degradation (Yoshida et al., 2000). When activated, the signal transduction pathways
initiated by PERK, IRE1? and ATF6? induce a characteristic set of genes
encoding ER chaperones and nuclear transcription factors that ultimately lead
either to reduction of ER stress or to death (Mori, 2003). Therefore, ER stress may play a prosurvival or
proapoptotic role and severe ER stress triggers neuronal cell death(Vilatoba et al., 2005;
Wang et al., 2008).

In
the current study, we found that the specifc mechanism of the cell activity of
the rotenone treated cells, the ER stress (UPR pathway) associated proteins,
including PERK, IRE-1? and ATF6? levels were significantly increased in
rotenone treated cells, suggesting the presence of increased ER stress in the
PD 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 in
rotenone treated SK-N-SH cells. Pretreatment of GE observed protective
effect in rotenone treatment by down-regulating the ER stress offered
protection against rotenone cytotoxicity. Taken together, the results suggest
that by reducing the ER stress contribute a protection against
rotenone cytotoxicity.

Autophagy
mediates lysosomal degradation of long-lived cytoplasmic proteins, initiated
under the conditions of differentiation, stress such as oxidative stress, ER
stress and protein aggregate accumulation (Yorimitsu et al., 2006; Mizushima, 2007; Sarkar et
al., 2007a; Sarkar et al., 2007c). It was reported that autophagy play
a crucial role in a number of neurological diseases especially in PD(Cherra and Chu, 2008; Yue et al., 2008). The strategy of upregulating or
accelerating autophagy to treat neurodegenerative diseases has been tested in
various 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 stressdependentactivation
of autophagy during rotenone induced oxidative stressconditioncan prevent the
cell loss in SK-NSH cells by GE treatment.

Autophagy
has revealed that the regulatory molecules that control autophagy are varied, depends
on the intracellular homeostasis including AMPKand mTOR (Meijer and Codogno, 2006). The
serine/threonine kinase mTOR, a central controller of cell growth and
negatively regulates autophagy. mTOR kinase regulates autophagy through ATG
proteins, resulting in interference with the formation of autophagosomes(Levine and Yuan, 2005). The data from
the present study encourages the concept that GE induces a pathway of autophagy
through the downregulation of AMPK and mTOR expression in SK-N-SH cells.

The
LC3-I and LC3-II proteins are critical markers for autophagy, was produced by
autophagy cascade. LC3-I exists in cytosolic form and LC3-II in membrane bound
form. The ratio of conversion from LC3-I to LC3-II is closely correlated with
the extent of autophagosome formation (Kabeya et al., 2000). In addition, the autophagic protein
p62/SQSTM1 is selectively incorporated into autophagosome through direct
binding to LC3 and efficiently degraded by autophagy. The level of p62
inversely correlates with autophagic activity (Bjorkoy et al., 2006). Our hypothesis
that autophagy is induced during ER stress is supported by activated the
autophagosome formation, which was shown in examinations of LC3-I, LC3-II.Therefore,
the AMPK and mTOR dependent or ?independent mechanism may participate in
pathway regulation of LC3-associated autophagosome formation in dopaminergic
neurons, under normal and pathogenic conditions.

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