Parkinson’s disease (PD) is a neurodegenerative disorder that leads to the progressive loss of dopaminergic neurons in the substantia nigra. Mitochondrial damage and oxidative stress (OS) due to alterations in iron and redox metabolism, “prion-like protein infection,” defective autophagy, and protein misfolding and aggregation are considered to be associated with PD onset and progression.
Autophagy helps remove protein aggregates and damaged organelles through lysosomal digestion. The autophagy machinery plays a vital role in PD, where several genetic mutations have been identified in the components of the machinery as disease risk factors. Autophagy dysregulation has also been observed to be associated with reactive oxygen species (ROS) in cellular damage as well as signaling. Moreover, all chemicals which induce dopaminergic and ROS damage, also termed Parkinsonian toxins, have been reported to impair or dysregulate autophagy in astrocytes as well as neurons.
Astrocytes help maintain redox balance in the brain through antioxidant detoxification and production, regulation of inflammatory responses in the central nervous system, and formation and maintenance of the blood–brain barrier. Metabolism of dopamine (DA) is reported to be the main ROS source in the brain. Functional goal cells have been observed to neurons against OS through the metabolization of DA and by a battery of antioxidants enzymes in astrocytes. Prolonged astrocyte dysfunction can lead to an increase in the vulnerability of DA neurons and their degeneration during aging, ultimately leading to PD.
6-hydroxydopamine (6OHDA) is extensively used to investigate PD pathogenesis in pre-clinical studies since it causes PD-like symptoms in rodents. 6OHDA is a DA analog whose production takes place through y DA hydroxylation in the presence of H2O2 and Fe + 2 iron. Although 6OHDA is considered to be a purely synthetic toxin, several studies have indicated its origin to be endogenous. 6OHDA toxicity mechanisms are complex and lead to the generation of hydrogen peroxide and hydroxyl radicals through rapid autooxidation. However, it is unclear whether any endogenous toxifying enzymes, such as quinone oxidoreductase 2 (NQO2/QR2), result in this process.
NQO2, which is structurally related to NQO1, is a ubiquitously expressed flavoprotein that carries put two-electron reduction of quinone to hydroquinones. The emergence of NQO2 as a possible target in PD took place more than two decades ago. One previous study indicated that a chronic OS induced by parkinsonian toxin paraquat (PQ) could be inhibited by a specific NQO2 inhibitor S29434/NMDPEF in vivo as well as in vitro in rates after systemic and intranigral injection of PQ. PQ has been observed to inhibit autophagy, while S29434 treatment led to the stimulation of autophagy in astrocytes. Recent studies indicate that NQO2 plays a role in inhibiting autophagy and generation of OS and flavone-induced autophagy by acting as a receptor of pro-autophagic ligands.
A new study under review at the journal Scientific Reports and currently available on the Research Square* preprint server aimed to analyze the toxic effects of 6OHDA along with a related role of NQO2 in murine and human astrocytes.
Study: Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: a potential novel target in Parkinson’s disease. Image Credit: PopTika / Shutterstock
*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
About the study
The study involved the detection of ROS in live astrocytoma U373 cells by two alternative ROS fluorescent indicators, MitoSOX or a dichlorofluorescein (DCF) derivative. Thereafter, measurement of the cellular NQO2 activity took place using U373 cell lysates. This was followed by the preparation of NQO2-overexpressing (N-over) U373 cells and flow cytometry (FACS) analysis of cell viability.
Astroglial and dopaminergic cells were then co-cultured, after which transfection of U373 cells took place along with silencing of NQO2. WT pups of C3H/HeOuJ strain mice were used for primary astrocyte isolation from cerebral cortices. Finally, fluorescence and phase–contrast microscopy was performed along with gene expression omnibus (GEO) datasets analysis.
Study findings
The results indicated an increased reduction of LC3II levels (which indicates the effects of 6OHDA on autophagy in astrocytes) in U373 cells 24 hours post-treatment compared to 6 hours. 100 mM of 6OHDA was observed to have a strong negative effect on the autophagic flux at 24 hours, while 50 mM had at 48 hours. The addition of S29434 restored LC3II levels in 6OHDA-treated U373 cells compared to cells without S29434. A similar induction pattern was also observed for NQO2.
An increase in OS was reported only at high doses of 6OHDA. 6OHDA (100 µM) induced a time-dependent increase in MitoSox fluorescence 15 hours post-treatment, which was much stronger compared to 100 µM PQ. Co-treatment with S29434 resulted in partial attenuation of ROS induction by 6OHDA, while full attenuation was observed in the presence of PQ and S29434. Additionally, U373 cell lysates pre-treated with 6OHDA were observed to have higher NQO2 activity as compared to control cells. However, 6OHDA was reported to not be a substrate of NQO2 but instead induced NQO2 activity in astrocytes in a dose-dependent manner.
6OHDA was reported to induce toxicity leading to cell death in U373 astroglial cells, which could be lowered by S29434 treatment. U373 cells that overexpressed NQO2 were reported to be more sensitive to 6OHDA with a higher mortality rate. Moreover, neuroblastoma cells (SH-SY5Y) were observed to be more sensitive to 6OHDA as compared to U373 cells and were reported to die massively 24 hours after the addition of 10 µM of 6OHDA. S29434 treatment was unable to prevent cell death in SH-SY5Y cells.
Silencing of NQO2 was observed to enhance or restore autophagy in cells that were exposed to lower (25 µM) and higher (50 µM) doses of 6OHDA. 24-hour treatment with 6OHDA was observed to inhibit autophagic flux through a decrease in LC3II levels in a dose-dependent fashion in C3H mice. Moreover, S29434 was observed to increase LC3II levels in mice cells exposed to 6OHDA. Dopaminergic SH-SY5Y cells were observed to be protected by U373 monolayers from 6OHDA-induced cell death, whereas NQO2-overexp U373 were observed to be less protective. Additionally, the protective effect of U373 astrocytes was observed to be increased in both normal and N-over cells by S29434. Finally, higher NQO2 expression was reported in PD patients as compared to mean expression in healthy patients.
Therefore, the current study demonstrated that drugs which target NQO2 are capable of stimulating autophagy as well as protecting against hydroxyquinone and dopamine quinone toxicity. S29434 can delay neuronal damage in PD and other pathological conditions involving OS and autophagy impairment mediated by NQO2. This suggests that NQO2 inhibitors might have a role in treating cerebral injuries involving oxidative neurodegeneration. However, further studies are required to confirm this hypothesis.
*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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