Wnt2 overexpression protects against PINK1 mutant‑induced mitochondrial dysfunction and oxidative stress
- Authors:
- Published online on: April 8, 2020 https://doi.org/10.3892/mmr.2020.11066
- Pages: 2633-2641
Abstract
Introduction
PTEN-induced kinase 1 (PINK1) is located in the mitochondrial membrane, and helps to regulate mitochondrial morphology and autophagy (1). Mutations in PINK1 have been recognized to be the second most common cause of autosomal recessive adolescent Parkinson's disease (PD) (2). Loss of Drosophila PINK1 leads to mitochondrial morphological disorders and mitochondrial complex function damage (3). Furthermore, neurons are dependent on mitochondria, which act as the major energy producers (4). Therefore, when PINK1 is mutated, neurotoxicity is increased and this may be associated with mitochondrial defects, thus contributing to the loss of dopaminergic (DA) neurons (5). Oxidative stress is a prominent and common feature of all forms of PD, and may have a toxic effect that causes neuronal cell death (6). Moreover, oxidative stress in PD is closely associated with a series of pathogenic factors, including mitochondrial dysfunction, DA metabolism and metal ion dysregulation (7).
The human Wnt gene family, known as the wingless-type MMTV integration site family, consists of 19 members, and the interaction between Wnt1 and Wnt5a promotes the development of DA neurons in the midbrain (8). Moreover, the Wnt2 gene is one of the 19 Wnt family members that is highly expressed in the human thalamus, and plays an important role in the late development of human genes and the brain (9,10). A previous functional study has shown that Wnt2 promotes the migration of primitive neurons and increases the number of DA neurons (11), thus enhancing DA function in the midbrain, and affecting mRNA and protein expression levels. The Wnt signaling pathway can be cross-linked with a number of signaling pathways (12). The interaction of β-catenin with the forkhead box sub-group O (FOXO) signaling pathway can inhibit Huntington protein toxicity (13). Furthermore, the Wnt signaling pathway also regulates mitochondrial energy, metabolism and oxidative stress (14,15).
The present study selected Drosophila as the experiment model, as Drosophila genes have been fully sequenced and annotated, and are highly homologous to human genes (16,17). Compared with other models, the Drosophila life cycle is very short; therefore, it is easier to observe the whole process of disease development (18). Mature genetic systems, abundant strain resources and powerful genome editing techniques also make Drosophila one of the primary choices for genetic research in neurodegenerative diseases (19).
The present study identified that overexpression of Wnt2 gene had a significant effect on PINK1B9 transgenic Drosophila. While the Wnt pathway may have neuroprotective effects, the role of Wnt2 in neurodegenerative diseases remains unknown. Therefore, the aims of the present study were to investigate the function of Wnt2 in PINK1 mutant transgenic Drosophila, and to identify its association with the Wnt/β-catenin and PPARG coactivator 1α (PGC-1α)/FOXO/manganese superoxide dismutase (MnSOD) signaling pathways.
Materials and methods
Drosophila stocks
A total of five fly stocks were used: two stocks of Drosophila melanogaster (UAS-Wnt2OE and UAS-Wnt2RNAi) were purchased from the Bloomington Drosophila Stock Center. In total, three stocks (UAS-PINK1B9/FM7; MHC-Gal4, W1118 and MHC-GAL4) were provided by Institute of Life Sciences of Fuzhou University. W1118 is a wild-type genotype. UAS-Wnt2OE is a stock that overexpresses the Wnt2 gene. UAS-Wnt2RNAi is a Drosophila that has lost the function of the Wnt2 gene by using RNA interference technology. MHC-GAL4 is a Drosophila with an indirect flying muscle promoter. UAS-PINK1B9/FM7; MHC-Gal4 is a PD Drosophila model, in which the PINK1 mutation gene can be specifically expressed in indirect flying muscles. The classic GAL4/UAS system is divided into two parts: GAL4 and UAS. The GAL4 stock and UAS stock are two independent stocks (20). The fusion of GAL4 and tissue-specific promoter can regulate the expression of GAL4 protein in different tissues of Drosophila (21). Furthermore, UAS and target genes are fused to construct a transgenic line with a UAS-target gene (22). Only when the two hybridize, can GAL4 recognize the UAS promoter and induce expression of UAS downstream genes in specific tissues (23). Drosophila were placed in Drosophila culture tubes containing corn medium and cultured at a constant temperature of 25°C and 60% relative humidity.
Drosophila construction
The flies were driven via the muscular driver MHC-GAL4. MHC-GAL4 virgin flies were crossed with W1118 male flies, and the F1 generation genes were W1118/+; MHC-GAL4/+, which served as the control group. In the PINK1B9 disease group, UAS-PINK1B9/FM7; MHC-GAL4 virgin flies were crossed with W1118 male flies, which produced the F1 generation with a genotype of UAS-PINK1B9/+; MHC-GAL4/+. In the overexpression (OE) intervention group, UAS-PINK1B9/FM7; MHC-GAL4 virgin flies were hybridized with male flies of UAS-Wnt2OE, and the F1 generation genotype was UAS-PINK1B9/y; MHC-GAL4/Wnt2 OE, UAS-PINK1B9/y. In the RNA interference (RNAi) intervention group, UAS-PINK1B9/ FM7; MHC-GAL4 virgin flies were crossed with males of UAS-Wnt2 RNAi Drosophila, and the F1 generation obtained had the genotype UAS-PINK1B9/y; MHC-GAL4/Wnt2RNAi.
Morphological observation of Drosophila (24)
Flies carrying the MHC-GAL4/UAS systems were grouped as follows: Normal control group (W1118), disease control group (PINK1B9), Wnt2OE intervention group (PINK1B9; Wnt2OE) and Wnt2 knockdown intervention group (PINK1B9; Wnt2 RNAi). On day 5, ~100 male flies were selected from each group. After being anesthetized by CO2, the 100 flies were divided into transparent glass tubes with five flies per tube. After the flies completely woke up (after ~1 h), the shape of their wings and whether they could fly were observed. The number and the ratio of abnormal wings and flying were calculated. All assays were performed in triplicate and independently repeated three times.
Drosophila mRNA expression detection
The experimental groups were the same as aforementioned. On day 5, the head and abdomen were removed, and the chest was kept from 30 male flies from each group. TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract the total RNA from the chest, according to the manufacturer's protocol. The primers were synthesized by Sangon Biotech Co., Ltd. and are presented in Table I. Subsequently, RNA samples were reverse transcribed into cDNA using PrimeScript™ reverse transcription (RT) reagent kit with gDNA Eraser (Takara Bio, Inc.). Following the manufacturer's instructions, this reaction was a two-step process. The first step was to remove the genomic DNA, adding 1 µl gDNA eraser and 2 µl gDNA eraser buffer to the RNA sample, which was then incubated at 42°C for 2 min. The second step was to synthesize cDNA by adding the 1 µl enzyme, 1 µl primer, 4 µl buffer and 4 µl RNase-free H2O to the 10 µl gDNA eraser-treated sample. The reaction temperature was 37°C for 15 min and 85°C for 5 sec. Quantitative PCR (qPCR) was conducted using an Applied Biosystems ABI 7500 system (Thermo Fisher Scientific, Inc.) using Power SYBR® Green PCR Master mix (Thermo Fisher Scientific, Inc.). The RT PCR conditions were as follows: Initial denaturation at 50°C for 20 sec and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec; annealing at 60°C for 1 min and extension at 72°C for 1 min. Moreover, 18S served as an endogenous control for data normalization. The 2−ΔΔCq method (25) was used to analyze the relative expression.
Western blotting for detection of protein expression
Drosophila grouping was the same as aforementioned. The the chest tissue of 20 Drosophila was cut on ice. Fresh tissues were lysed with 200 µl ice-cold RIPA buffer (Solarbio Science & Technology Co., Ltd.) containing 1 mM phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, tissues were ground to a homogenate, followed by centrifugation at 14,000 × g for 15 min at 4°C. Then, 120 µl supernatant liquid was collected and added to 40 µl 4Xloading buffer (Beijing Solarbio Science & Technology Co., Ltd.), mixed and boiled at 100°C for 10 min. The samples were stored at −20°C prior to further experiments.
For analytical SDS-PAGE, 10 µl protein was loaded per lane to 5% concentrated gel (30% acrylamide, 10% SDS; 10% APS; 1 M Tris-HCl pH 6.8; TEMED) and dissociated in 10% separation gel (30% acrylamide; 10% SDS; 10% APS; 1.5 M Tris-HCl pH 8.8; TEMED). The proteins were then transferred to 0.2-µm PVDF membranes (Beijing Solarbio Science and Technology Co., Ltd.), blocked in 5% milk for 2 h at room temperature and washed in TBS-T (TBS, 3 M NaCl, 200M Tris; pH 7.5, containing 0.1% Tween-20). Subsequently, overnight incubation at 4°C was performed with the following primary antibodies: Rabbit anti-MnSOD (1:1,000; cat. no. ab13534; Abcam; polyclonal), rabbit anti-α tubulin (1:1,000; cat. no. ab52866; Abcam; monoclonal) and mouse anti-β-catenin (1:1,000; cat. no. AB_528089; Developmental Studies Hybridoma Bank; monoclonal). All the primary antibodies were Drosophila-specific. Following washing with TBS-T, the membrane was incubated with the corresponding secondary antibody, horseradish peroxidase-AffiniPure Goat Anti-Rabbit/Mouse IgG (H+L; 1:5,000; cat. nos. EM35111–01 and EM35110-01; Emarbio Science and Technology Co., Ltd.), for 1 h at room temperature. All the resulting immune complexes were visualized with chemiluminescence reagent (Thermo Fisher Scientific, Inc.,), followed by imaging using Image Lab 5.1 (National Institutes of Health). The target protein bands were quantified by scanning densitometry using ImageJ software (v.1.49v; National Institutes of Health).
ATP, malondialdehyde (MDA) and reactive oxygen species (ROS)
In total, ten Drosophila thoraxes were cut on ice from each group, and 1,000 µl lysate was added to homogenize the chest tissue on ice. Samples were then heated at 100°C for 2 min and centrifuged at 12,000 × g for 5 min at 4°C. The thoracic ATP level was measured using a luciferase-based bioluminescence assay (cat. no. S0027; Beyotime Institute of Biotechnology). Then, 40 fly chests from each group were ground by adding 500 µl 1X PBS and centrifuge at 1,425 × g for 10 min at 4°C. The supernatant was collected and the MDA content was measured by the thiobarbituric acid method (26). Analyses were performed according to the instructions of the reagent kit for MDA (cat. no. A003-1-2; Nanjing Jiancheng Bioengineering Institute). ROS were measured using the CellROX Orange reagent (cat. no. BB-470512; Shanghai Bio-Tech Co., Ltd.). The thoraxes of 30 Drosophila were obtained, homogenized, centrifuged at 1,000 × g for 10 min at 4°C and the supernatant was collected. The supernatant and 20 µM CellROX Orange Reagent were mixed and incubated at 37°C in the dark for 30 min, and the fluorescence intensity at 510 and 610 nm (maximum excitation light and maximum emission wavelength) was measured using a multi-function microplate reader.
Transmission electron microscopy analysis
Drosophila was grouped as aforementioned, and 10 male flies from each group F1 generation were randomly selected on day 5. Flies were anesthetized by CO2, and the chest was cut carefully so as not to damage the muscle tissue. Thoraxes were fixed overnight at 4°C in 2.5% glutaraldehyde, washed several times with 0.1 mol/l phosphate buffer and post-fixed in 1% osmiumtetroxide in distilled water for 2 h at room temperature. The samples were dehydrated in a 50, 70 and 90% graded ethanol series, and embedded in Epoxy resin for 48 h at room temperature. The polymerization conditions in the polymerization tank were 36°C for 24 h, 45°C for 12 h and 65°C for 48 h.
The embedded polymer samples were cut into 1 µm sections using a Leica UC7 ultrathin slicer, stained with 1% toluidine blue for 30 sec at room temperature and observed under an optical microscope (magnification, ×100). The samples were then cut into ultra-thin sheets (70 nm), stained with 3% uranyl acetate for 15 min at room temperature and 3% lead citrate for 15 min at room temperature. Sections were observed with a Hitachi H-7650 transmission electron microscope (magnification, ×30,000).
Statistical analysis
Statistical analysis of data was performed using SPSS 16.0 (IBM Corp.). Normally distribution continuous variable data were compared by one-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.
Results
Wnt2 overexpression rescues the abnormal phenotype caused by PINK1B9 mutation
The Wnt2 gene was specifically expressed in Drosophila flight muscles using the MHC-GAL4 promoter. The phenotypic changes of Drosophila were demonstrated by changes in wing morphology and flight ability. In the normal control group, most of the wings completely overlapped and were parallel to the body (Fig. 1A). However, in the transgenic disease model of PINK1B9 the wings had bifurcations, erections and sagged. Furthermore, compared with the low rate of abnormal wings in the control group, the disease group had a significantly higher rate of abnormal wings (Fig. 1B) and a significant decrease in flight ability (Fig. 1C). In the Wnt2OE intervention group, the incidence of wing anomalies was significantly reduced and the flight capabilities were improved, compared with the disease group. Moreover, there were no significant differences between the Wnt2 RNAi intervention group and the PD disease model group. Therefore, the present results suggested that Wnt2OE may have a protective effect on the phenotype of PINK1B9 transgenic Drosophila.
Wnt2 gene overexpression enhances mitochondrial function in PINK1B9 transgenic Drosophila
RT-qPCR results demonstrated that in PINK1B9 disease model group, the mRNA expression levels of the mitochondrial complex subunit-related genes, Complex I [NADH-ubiquinone oxidoreductase chain 1 (ND1), ND42 and ND75], Complex II (succinate dehydrogenase complex subunits B), Complex III (Cytochrome b) and Complex IV (Cyclooxygenase 1), decreased significantly. While Wnt2OE intervention in PINK1B9 transgenic Drosophila increased the mRNA expression levels of these related genes (P<0.05), in the Wnt2 RNAi intervention group there was no significant difference compared with the disease model group (Fig. 2A). The results of the ATP assay demonstrated that ATP production in the PD model group was decreased. Furthermore, the amount of ATP produced by mitochondria in the Wnt2OE intervention group was ~1.5 times higher compared with the disease model group (Fig. 2B). Ultrastructural transmission electron microscopy analysis identified that mitochondria were disrupted in PINK1B9 transgenic Drosophila, and mitochondrial morphology was not recognizable. Moreover, Wnt2OE could rescue mitochondrial defects in PINK1B9 flies (Fig. 2C). Therefore, the present results suggested that overexpression of Wnt2 can improve the mitochondrial function of PINK1B9 transgenic Drosophila.
Wnt2OE reduces oxidative stress damage in PINK1B9 transgenic Drosophila
ROS production in the PINK1B9 disease model group was significantly higher compared with the normal control group (P<0.05; Fig. 3A). Furthermore, following Wnt2OE intervention in PINKB9 transgenic Drosophila, ROS production was significantly reduced (P<0.05) and almost returned to normal levels. MDA, a commonly used indicator of lipid peroxidation injury (27), was significantly increased in the PINK1B9 disease model group compared with the normal control (P<0.05; Fig. 3B). Moreover, after Wnt2OE intervention, MDA production was reduced (P<0.05). It was demonstrated that the content of ROS and MDA were not significantly different between the Wnt2RNAi intervention groups and the disease model group (Fig. 3A and B).
Western blot analysis results revealed that the protein expression of MnSOD in the PINK1B9 disease model group was significantly lower compared with the normal control group (P<0.05; Fig. 3C). However, the expression of MnSOD was significantly increased (P<0.05) following Wnt2OE intervention in the PINK1B9 disease model. Collectively, the present results indicated that Wnt2 overexpression reduced oxidative damage in PINK1B9 transgenic Drosophila.
Possible mechanism of Wnt2 overexpression-mediated protection
Western blot analysis results revealed that there was no significant difference in the protein expression of β-catenin, a key molecule of the classic Wnt/β-catenin signaling pathway (28), between each group (Fig. 4A). Moreover, RT-qPCR showed that the mRNA expression levels of FOXO and PGC-1α were decreased in the PINK1B9 disease model group, and were increased following Wnt2OE intervention in the PINK1B9 disease model (Fig. 4B). Therefore, it can be speculated that Wnt2 may activate the FOXO/PGC-1α signaling pathway to regulate mitochondrial function and inhibit anti-oxidative stress-induced damage.
Discussion
In Drosophila melanogaster, mutations in PINK1 causes phenotypic abnormalities and decrease exercise capacity, resulting in dysfunction of mitochondria and decreased ATP levels, which leads to selective degeneration of DA neurons and sensitivity to stress (16,17). In the present study, the disease model demonstrated a pathological phenotype that was consistent with previous experimental results (24,29), including a high abnormal wing rate, low flight rate, lower mRNA expression of mitochondrial complex subunits and lower ATP level, which suggested that PINK1 mutations could cause serious damage in mitochondria. Furthermore, overexpression of Wnt2 gene rescued the phenotype of PINK1B9 transgenic Drosophila, increased ATP production level and enhanced the expression of the mitochondrial biosynthesis-related gene PGC-1α. PGC-1α is a transcriptional cofactor for numerous mitochondrial proteins, which can regulate mitochondrial function to meet cellular needs and protect from oxidative damage (30). Therefore, it is speculated that Wnt2OE inhibited mitochondrial dysfunction caused by the PINK1 mutation and eventually protects PINK1B9 transgenic fruit flies.
When mitochondria are damaged, particularly damage to the mitochondrial respiratory chain complex, electron chain leakage may occur and cause the production of superoxide and hydrogen peroxide (31). These products, not only participate in the damage of DNA, proteins and lipids, but also pose a serious threat to cells and tissues (31). Mitochondrial dysfunction and oxidative stress play key roles in the development of PD, as both lead to excessive ROS production (32). This in turn leads to damage and death of DA neurons (33). A small level of ROS is essential for normal physiological functions; however, the accumulation of large amounts of ROS further destroys mitochondria and exacerbates oxidative stress (34). It has been shown that maintenance of ATP and inhibition of ROS production protects DA neurons (35). Reactive oxygen damages polyunsaturated lipids and forms MDA, which is a marker of lipid damage in oxidative stress (36). Furthermore, the present results indicated that the PINK1 mutation increased ROS and MDA levels, but reduced MnSOD protein expression.
MnSOD, which is mainly distributed in the mitochondrial matrix, is an important scavenger for the superoxide anion that is produced during mitochondrial oxidative phosphorylation (37). Previous findings have shown that overexpression of MnSOD serves an important role in the protection of PINK1-mutant PD Drosophila (38). In the PINK1 mutants, the present study identified increased ROS and MDA production, and a reduced MnSOD, thus suggesting that the PINK1 mutation leads to oxidative stress damage. Moreover, overexpression of Wnt2 improved mitochondrial function, which also partially reduced ROS production. However, Wnt2 overexpression also appeared to directly participate in the regulation of oxidative stress levels in PINK1B9 transgenic Drosophila, as it not only reduced ROS and MDA production levels, but also increased the protein expression levels of MnSOD.
The Drosophila gene Wnt2, homologous to the human gene Wnt7a, is involved in the Wnt/β-catenin signaling pathway (39). However, it remains unknown whether the protective effect of Wnt2 overexpression on PINK1B9 transgenic Drosophila is related to the Wnt/β-catenin signaling pathway. The present results indicated that Wnt2 overexpression did not increase the protein expression of β-catenin, a key protein of the Wnt/β-catenin signaling pathway (28), which suggested that it did not protect PINK1B9 transgenic flies via the Wnt/β-catenin signaling pathway. Furthermore, a previous study found that Wnt2 does not act via the Wnt/β-catenin pathway, but activates the non-canonical pathway (40). Thus, this raises the question of how Wnt2 improves mitochondrial function, reduces oxidative damage and enhances antioxidant capacity. The present study evaluated the expression of FOXO, a gene associated with mitochondrial oxidative stress, which regulates the expression levels of PGC-1α and MnSOD (38,41). Under conditions of mitochondrial dysfunction and oxidative stress caused by PINK1 mutation, the present study hypothesized that the overexpression of Wnt2 directly regulates the expression of PGC-1α/FOXO/MnSOD, improves mitochondrial function and improves antioxidant capacity to rescue PINK1B9 transgenic fruit flies.
The FOXO family members are key regulators of neuronal processes, such as dendritic structural function and memory consolidation (42,43). FOXO is also an important regulator of cellular stress response, which enhances cellular antioxidant defenses (44). A previous study revealed that the expression of genes, such as MnSOD, could be controlled by the forkhead transcription factor FOXO3a (44). Moreover, PGC-1α has been shown to regulate FOXO activity in different systems. For example, it has been reported that PGC-1α is a positive regulator of fasting-induced hepatic gluconeogenesis, which is mediated by its interaction with FOXO1a (45). Similarly, overexpression of PGC-1α enhances the stimulatory effect of FOXO1a on selenoprotein P promoter activity and insulin attenuation (46). PGC-1α has also been shown to interact with FOXO3a, which regulates antioxidant gene expression in endothelial cells and skeletal muscle (47). In addition, upregulation of PGC-1α and FOXO3a protects against oxidative stress injury induced by a high-fat diet and inhibits adipocyte apoptosis (47,48). The Wnt signaling pathway is also involved in the regulation of mitochondrial energy metabolism and oxidative stress (49). When ROS levels exceed the body's ability to scavenge, Wnt and β-catenin interact with FOXO under stimuli of oxidative stress (13,50). Furthermore, FOXO1 interacts with PGC-1α in different systems (45,46). PGC-1α is a mitochondrial energy-metabolizing enzyme. When ROS are in excess, the human body can enhance the detoxification ability of mitochondrial ROS by increasing the expression levels of FOXO1 and PGC-1α to promote the expression of downstream antioxidant systems, such as MnSOD (49).
To the best of our knowledge, the condition of mitochondrial dysfunction caused by PINK1 mutation and oxidative stress remains to be determined. Although the Wnt signaling pathway is linked to the FOXO signaling pathway via β-catenin (50), the present results suggested that Wnt2 did not change the β-catenin protein expression level, but did affect the mRNA expression levels of PGC-1α and FOXO, and the expression levels of their target protein MnSOD. Based on these results, it can be speculated that Wnt2 may be directly involved in the PGC-1α/FOXO/MnSOD signaling pathway; however, the specific mechanism remains to be investigated. Moreover, the regulation of signaling pathways is complex and there will be cross-effects between the pathways (47–48), with both upregulation and inhibition of the expression of related factors leading to cascade reactions. Therefore, it will be beneficial to examine how the Wnt2 pathway interacts with or influences the PGC-1α/FOXO/MnSOD pathway in future experiments.
The present study demonstrated that Wnt2 overexpression protected PINK1B9 transgenic flies by improving flight muscle and mitochondrial morphology, and enhancing mitochondrial complex I and II function. Furthermore, it was found that Wnt2 overexpression exhibited a protective effect on early mitochondria and oxidative stress-related PD by improving mitochondrial function and reducing oxidative stress damage. However, the present study does have some limitations. First, the model is monotonous and limited to fruit flies, and thus requires further examination in higher animal models such as mice. Secondly, further research into the underlying pathways and mechanisms is required, such as whether it is the experimental effects that are related to the non-classical Wnt signaling pathway. Furthermore, how Wnt2 cross-links with the PGC-1α/FOXO/MnSOD signaling pathway is not fully understood. In the PINK1 mutant PD Drosophila model of mutation constructed by Park et al (24), mitochondrial dysfunction and oxidative stress injury are primarily exhibited in the early stage, while DA neuronal loss predominantly occurs in the middle and late stages of the Drosophila, which is after 25 days (24,50). This is consistent with the progressive DA neuronal loss observed in clinical patients with PD. Thus, this may indicate that damage to DA neurons in PD is the result of further neurological damage caused by these pathogenic factors.
To the best of our knowledge, there are currently no treatments that mitigate disease progression or prevent neurodegeneration in all neurodegenerative disease. Therefore, it is necessary to develop interventions that are effective prior to severe damage of the neuronal in the early stages of PD. The present results indicated that Wnt2 overexpression had a protective effect on early mitochondrial damage and oxidative stress in PD, improving mitochondrial function and reducing oxidative stress damage. Due to the limitations of the present study, the specific mechanism of action of Wnt2 is not fully understood. However, the improvement of mitochondrial function and the reduction of oxidative stress damage can support the experimental results of the Drosophila model, providing a strong basis for future experiments.
In conclusion, the present results suggested that the Wnt2 gene may have a protective effect on PINK1B9 transgenic Drosophila. Therefore, it can be hypothesized that the reduction of oxidative stress and the restoration of mitochondrial function via Wnt2 gene overexpression in the PINK1 mutant transgenic Drosophila may be related to the PGC-1α/FOXO/MnSOD signaling pathway.
Acknowledgements
The authors would like to thank Dr Yu-Feng Yang, from Institute of life Sciences of Fuzhou University, for donating the PINK1B9 Drosophila model; Dr Ru-Jia Liao, Dr Jing-Xin Mo and Dr Qing-Tuan Meng, from Guangxi Clinical Research Center for Neurological Disease of Affliated Hospital of Guilin Medical University, for sharing their expertise; Miss Liang-Xian Li, intermediate research assistant, from Guangxi Key Laboratory of Brain and Cognitive Neuroscience of Guilin Medical University; Miss Ying Cui and Miss Xiao-Jun Diao, PhD candidate, Department of Neurology from Xiangya School of Medicine of Central South University; Miss Wen-Jing Wang, postgraduate student, Department of Neurology from Guilin Medical University; Miss Fang Shi (intermediate research assistant), Miss Ning Tian (inter- mediate research assistant) and Miss Mei-Rong Chen (intermediate research assistant), from Guangxi Clinical Research Center for Neurological Disease of Affliated Hospital of Guilin Medical University, for their help.
Funding
The present study was supported by the National Natural Science Foundation of China (grant nos. 81460180 and 31460256), 2017 Youth and Middle School Teachers' Basic Ability Improvement Project of the Education Department of Guangxi Zhuang Autonomous Region (grant no. 30606017018) and the Innovation Project of Guangxi Graduate Education (grant no. YCSW2018206).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
SRX was responsible for construction of the Drosophila models, western blotting, MDA and ROS level determination, and data analysis. XYW was responsible for construction of the Drosophila models, morphological observation of Drosophila, O2K detection and data statistics. SRX and XYW wrote the first draft of this article. XLF was responsible for electron microscopy analysis. XRC performed the ATP determination experiment. ZWW was responsible for mRNA expression level detection. QHL and LS undertook project funding, project design, manuscript revision and quality control. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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