Essay: Aggregation of aberrant proteins and inclusion bodies

Aggregation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of ROS and oxidative stress leads to cell loss. Autophagy is a significant intracellular mechanism that removes damaged organelles and misfolded proteins in order to maintain cell homeostasis [41]. In recent years, growing evidence obtained from in vitro and in vivo PD models and PD patients has demonstrated that autophagy plays a pivotal role in PD pathogenesis. Emerging data support the view that regulation of autophagy might play a critical role in the progression of PD [41, 42]. Therefore, we need to better understand the role of autophagy in the pathologic process of PD prior to any clinical application of autophagy based medications in PD subjects. Hence, we suggest that GE offers neuroprotection against rotenone caused cell death in SK-N-SH cells via P13/AKT-mTOR dependent autophagic pathway activation and diminished overload of pathogenic stress. Recent evidences concerning that SK-N-SH neuroblastoma cells shows neuronal phenotypic expression of numerous neurochemical markers, and they quickly respond to various neurotoxin insults and this model is highly reproducible and may provide an excellent tool to test new neurotoxic and neuroprotective strategies [43, 26].
In our study, MTT assay showed that rotenone destruct SK-N-SH cells in a dose-dependent manner and approximately half maximal inhibition of cell viability was obtained at 100 nM of rotenone concentration. 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 [26, 27 43].
Since, oxidative injury was proposed to be a primary mechanism of mitochondrial toxicity in the rotenone induced cell death in SK-N-SH cells [45, 46]. Mitochondria are one of the major principle sources of ATP biosynthesis via oxidative phosphorylation for the purpose of providing energy to power cellular activities. 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 [27]. Progressive inhibition of ETC-I, also leads to the shunting of electrons through the ETC-II, which may generate the production of ROS 5-7 times more than normal [12, 13]. This appears to be the primary mechanism of rotenone induced formation of O2‾, which undergoes spontaneous or SOD catalyzed dismutation to form H2O2. Catalase and glutathione peroxidase catalyzes the decomposition of H2O2 to H2O and O2 [46]. Over accumulation of intracellular ROS (as demonstrated by the DCFDA assay) during neuronal loss is a key marker of oxidative stress and leads to a rapid consumption and depletion of endogenous scavenging antioxidants [46, 47].
In addition, ROS, may interact with nearest proteins, lipids, carbohydrates and nucleic acids, leading to the oxidative damage of these molecules and the subsequent cellular dysfunction [48]. Furthermore, we found that treatment with rotenone decreased activities of enzymatic antioxidants such as SOD, catalase and GPx were probably due to a response towards increased concentration of ROS and lipid peroxidation. Moreover, GSH depletion, the first indicator of oxidative stress during PD progression, suggests a concomitant increase in ROS accumulation [27, 49, 47]. It was reported that abnormal production of ROS and NO could inhibit cell growth and induce cell death in SK-N-SH cells [50, 51]. Our current results also agreement with previous findings, GE appears to prevented rotenone induced cell death by reduced ROS and NO generation via enhanced activity of ECT-1 and neutralized the endogenous antioxidant by enhancing the activities of SOD, GPx and catalase in experimental group, which might be due to ROS scavenging property of GE [23]. Similarly, the SN of PD brains has a reduced level of the antioxidant enzymes such as catalase, SOD and GPx [52] and antioxidant molecules such as GSH [53], suggesting the presence of a sustained burden of oxidative stress that over whelmed the antioxidant capacity. Collectively, rotenone model recapitulates most of the mechanisms thought to be important in PD pathogenesis [54].
Normal balance between the formation and degradation of cellular proteins is required for cell survival. Recently, there has been a growing interest in identifying the role of the autophagy in neurodegeneration [55]. Autophagy mediates lysosomal degradation of long-lived cytoplasmic proteins, initiated under the conditions of differentiation, stress such as oxidative stress, endoplasmic reticulum stress, protein disaggregation and accumulation [56, 57, 58]. Alterations in this pathway have been linked to neurodegenerative diseases [59], cancer [60] and cardiomyopathy [61]. It was reported that autophagy play a crucial role in the process and has been implicated in a number of neurological diseases especially in PD [62, 63]. Increased number of autophagic vacuoles and related structures of autophagy have been found in PD patients [64], animal models of PD [65] and in other disorders, including Huntington’s disease [66] and Alzheimer’s disease [67, 68]. This increase in autophagic markers raises the argument whether autophagy is a cause or a protective factor of neuron death. It has been suggested that the increased number of autophagic vacuoles is responsible for the neuronal cell death, but an alternative view is emerging that autophagy is induced to protect neurons by enhancing degradation of abnormal proteins that might trigger injury or apoptosis in the early stages of cell death [68, 69]. In our study the autophagic signaling molecules such as P13k/AKT-mTOR mediated LC3-I, LC3-II and Atg proteins highly increased in rotenone treated cells. This evidence indicates that once the ubiquitin proteasome system (UPS) is inhibited, autophagy is upregulated and the remaining aggregated proteins are degraded [70, 71], our present study also agreement with this previous reports. Furthermore, paraquat induces the accumulation of autophagic vacuoles and increases the degradation of long-lived proteins in the cytoplasm of human neuroblastoma SH-SY-5Y cells [72] indicate that enhanced oxidative stress possibly actives autophagy during the early stage of mitochondrial dysfunction and helps to resist the oxidative stress [72]. It seems that autophagy is induced in the early stage and impaired in the later period of the neurodegerative process [73, 69]. Thus, this is considered to be a default pathway when an aggregate-prone substrate cannot efficiently be cleared by the proteasome [74, 75]. The auto-regulative mechanism that accelerates the degradation of misfolded proteins as a defence or protection may be one of the explanations of the increased number of autophagic vacuoles in the brains of PD patients [64], possibly in response to dysfunction of the UPS. However, with pathogenic deterioration, this compensatory auto-regulative mechanism is ultimately unable to maintain the cellular balance and eventually results in neuronal death [76]. This auto-regulative concept to explain the increased the number of autophagic signaling molecules and markers is also supported by the finding that autophagic structures occur in the early stage of the rotenone treated SK-N-SH cells of PD. This line clearly explain the over action of P13K/AKT-mTORmediated autophagic markers such as LC3-I and LC3-II and Atg proteins. Recently, Pandey et al. [77] reported that histone deacetylase 6 (HDAC6) is an essential mechanistic link in this compensatory induction of autophagy when the UPS is impaired in Drosophila melanogaster [77].
O¨ztap and Topal, [65] reported that neurotoxin based progressive PD models with lysosomal malfunction accompanied by intracellular protein disaggregation of α-synuclein aggregation with UPS malfunctions [78]. The strategy of regulating autophagy to treat neurodegenerative diseases has been tested in various cell and animal models and shown to decrease protein aggregation and cell death [79, 58]. Our study focused on inhibition of PI3/Akt-mTOR pathway during rotenone induced oxidative stress situation can prevent the cell loss in SK-NSH cells by GE. Molecular mechanism study of autophagy has revealed that the regulatory molecules that control autophagy are varied, including ERK1/2, AMP kinase, class I and class III PI3K, Akt, mTOR and so on [80]. Phosphorylated class I PI3K enzymes and protein kinase B (Akt/PKB) can activate mTOR. The serine/threonine kinase mTOR, a central controller of cell growth, negatively regulates autophagy. mTOR kinase regulates autophagy through two general mechanisms. First, mTOR with the help of various downstream effector proteins controls transcription and translation [81]. Secondly, it affects the Atg proteins, resulting in interference with the formation of autophagosomes [82]. The data from the present study encourages the concept that GE regulates over action of pathway of autophagy through the downregulation of mTOR.