SC79, a novel Akt activator, protects dopaminergic neuronal cells from MPP+ and rotenone
Jian‑liang Zhu1 · Yu‑ying Wu2 · Di Wu3 · Wei‑Feng Luo2 · Zhi‑qing Zhang2,3 · Chun‑feng Liu2
Received: 15 May 2019 / Accepted: 15 July 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
In pathogenesis of Parkinson’s disease (PD), mitochondrial dysfunction causes substantial reactive oxygen species (ROS) production and oxidative stress, leading to dopaminergic (DA) neuronal cell death. Mitochondrial toxins, including MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone, induce oxidative injury in cultured DA neuronal cells. The current study tested the potential effect of SC79, a first-in-class small-molecule Akt activator, against the process. In SH-SY5Y cells and primary murine DA neurons, SC79 significantly attenuated MPP+- and rotenone-induced viability reduction, cell death, and apoptosis. SC79 activated Akt signaling in DA neuronal cells. Akt inhibition (by LY294002 and MK-2206) or CRISPR- Cas9-mediated Akt1 knockout completely abolished SC79-induced DA neuroprotection against MPP+. Further studies demonstrated that SC79 attenuated MPP+- and rotenone-induced ROS production, mitochondrial depolarization, and lipid peroxidation in SH-SY5Y cells and primary DA neurons. Moreover, upregulation of Nrf2-dependent genes (HO1 and NQO1) and Nrf2 protein stabilization were detected in SC79-treated SH-SY5Y cells and primary DA neurons. Together we show that SC79 protects DA neuronal cells from mitochondrial toxins possibly via activation of Akt-Nrf2 signaling.
Keywords Dopaminergic neuronal cells SC79 · Akt · Parkinson’s disease (PD) · Nrf2
Introduction
Parkinson’s disease (PD) is a common neurodegenerative disorder. It is characterized by impaired motor functions, caused by the progressive depletion of dopaminergic (DA) neurons in the substantia nigra (SN), striatum, and puta- men [1, 2]. The typical PD symptoms include distal rest- ing tremor, rigidity, and disturbance in balance [1, 2]. The
pathological mechanisms of PD remain unclear [2–4]. Mito- chondrial complex I dysfunction and subsequent oxidative stress play a crucial role in DA neuronal cell injury [1, 2].
Mitochondrial toxins are added to cultured DA neuronal cells to mimic PD-associated oxidative injures [5, 6]. Rote- none can rapidly cross the blood–brain barrier and accu- mulate in the brain, damaging mitochondrial functions to ultimately induce DA neuronal cell death. MPP+ (1-methyl-
4-phenylpyridinium ion), the neurotoxic byproduct of MPTP
Jian-liang Zhu, Yu-ying Wu, and Di Wu have contributed equally to this work.
Zhi-qing Zhang [email protected]
Chun-feng Liu [email protected]
1 Department of Emergency and Intensive Care Unit, The Second Affiliated Hospital of Soochow University, Suzhou, China
2 Department of Neurology, The Second Affiliated Hospital, Institute of Neuroscience, Soochow University, 1055 Sanxiang Road, Suzhou 215004, China
3 Institute of Neuroscience, Soochow University, Suzhou, China
(1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) metabolic oxidation, is selectively taken up by DA neurons, inhibiting complex I of mitochondrial electron transport [5, 6].
Recent studies have characterized a first-in-class small- molecule Akt activator, namely SC79 [7]. It is a novel, effi- cient, and cell-permeable Akt activator [7]. SC79 physically binds to Akt PH domain, thereby inducing Akt conforma- tional change [7]. SC79 uniquely suppresses Akt membrane translocation, while promoting its activation in the cytosol [7]. SC79 is shown to phosphorylate Akt at both Ser-473 and Thr-308, causing significant Akt activation [8–11].
Researchers have proposed a cytoprotective function of SC79. For instance, Zheng et al. reported that SC79 acti- vated Akt signaling to protect myocardiocytes from oxygen
and glucose deprivation/re-oxygenation [8]. Activation of Akt by SC79 inhibited dexamethasone-induced injury in osteoblasts [9]. Furthermore, the Akt activator attenuated Ultra-violet (UV)-induced retinal pigment epithelium cell apoptosis [10]. The results of the current study show that SC79 protects DA neuronal cells from mitochondrial toxins, rotenone, and MPP+.
Materials and methods
Reagents and antibodies
SC79, MK-2206, and LY294002 as well as rotenone and MPP+ were obtained from Sigma-Aldrich (St. Louis, Mo). The antibodies were all provided by Cell Signaling Tech (Danvers, MA). Cell culture reagents were obtained from Gibco Co. (Suzhou, China).
SH‑SY5Y cells
SH-SY5Y DA neuronal cells were purchased from the Cell Bank of Biological Institute of CAS (Shanghai, China). SH- SY5Y cells were cultured and differentiated as previously described [12].
Primary murine DA neurons
Isolation and primary culture of murine DA neurons were described in detail previously [11, 13]. Briefly, murine ventral mesencephalon tissues containing DA neurons were dissected from murine embryo (C57/B6) on day-14 of gestation. Single cells were cultured in mixed hormone MEM (MHM) supplemented with 5% FBS. Neurons were plated in poly-L-lysine-coated six-well plates at a density of 1 × 106 neurons per mL. The medium was renewed every 2–3 days. DA neuron cultures were grown for 8–10 days before further experiments. The protocols were approved by the Ethics Board of Soochow University. The animal proto- col was approved by the Soochow University Institutional Animal Care and Use Committee (IACUC).
Lactate dehydrogenase (LDH) assay
Cells were cultured in six-well tissue plates. After treatment, LDH levels in the conditional medium and cell lysates were measured by the two-step LDH detection kit (Promega). Medium LDH was normalized to the total LDH (medium LDH plus lysate LDH), reflecting cell death percentage.
Cell viability assay
Cells were cultured onto 96-well tissue culture plates. After treatment, cell viability was tested by CCK-8 assay. CCK-8 optical density (OD) values at 550 nm were recorded.
Western blotting
Total cell lysates were separated by SDS-PAGE gels [14], which were transferred to PVDF blots (Millipore). The blots were blocked (in 10% milk-PBST), followed by incu- bation with designated primary and secondary antibodies. The protein band was detected via enhanced chemilumi- nescence (ECL) reagents (Pierce, Shanghai, China) by X-ray films. ImageJ software (NIH) was always utilized to quantify the total gray of each band.
Caspase activity assay
After treatment, the caspase-3 and caspase-9 activities were measured via using the fluorometric caspase assay kits (Beyotime Biotechnology, Suzhou, China) [15]. The caspase-3 and caspase-9 p-nitroaniline (pNA) absorbance was detected at 405 nm.
TUNEL assay
Cells were seeded onto six-well tissue culture plates (2 × 105 cells per well). TUNEL [terminal deoxynucleoti- dyl transferase (TdT)-mediated dUTP nick end labeling] In Situ Cell Death Detection Kit (Roche, Shanghai, China) was applied to quantify TUNEL-positive nuclei in apop- totic cells. TUNEL percentage (% vs. Hoechst staining) of 200 cells per treatment in five random views (1: 100 magnification) was recorded.
ROS assay
Cells were seeded onto 12-well plates at 3 × 104 cells per well. Following treatment, ROS production was measured via the cell-permeable CM-H2DCF-DA reagent (Life Technologies, Grand Island, NY) according to the proto- col. Cells were incubated with 10 μM of CM-H2DCF-DA for 30 min. The DCF-DA fluorescence absorbance was detected by a fluorescent photometer (BD Biosciences, Shanghai, China) with the excitation and emission wave- lengths at 485 nm and 538 nm, respectively.
Mitochondrial depolarization assay
After treatment, mitochondrial depolarization (“∆Ψ”) was examined by using the mito-dye JC-1. JC-1 will aggregate to form the green monomers following mito- chondrial depolarization, and JC-1 fluorescence absorb- ance was recorded at 550 nm, indicating mitochondrial depolarization.
Lipid peroxidation assay
Following an early protocol [16], cellular lipid peroxidation levels were measured by the TBAR (thiobarbituric acid reac- tive substances) activities. In brief, following the treatment, 50 μg total cell lysates (TCL) per treatment were mixed with 20% of acetic acid and thiobarbituric acid solution. After heating, the mixture was centrifuged, with the red pigment dye in the supernatant tested by a microplate reader.
CRISPR/Cas9‑mediated Akt1 knockout
The small guide RNA (sgRNA) targeting human Akt1 with the targeted DNA sequence, 5′-TCACGTTGGTCCACA TCCTG-3′ (sgRNA-1, PAM, CGG) or 5′-GAGCGACGT
GGCTATTGTGA-3′ (sgRNA-2, PAM, AGG), was inserted into the lenti-CRISPR-GFP plasmid (Addgene). Cells were cultured in six-well plates. Each of the two CRISPR/Cas9 Akt1-knockout construct was transfected to SH-SY5Y cells. Stable cells were selected via FACS sorting of GFP-positive SH-SY5Y cells. Akt1 knockout (KO) in the resulting stable cells was verified by Western blotting assay.
The quantitative real‑time reverse transcriptase polymerase chain reaction (qPCR) assay
Following treatment, total RNA was extracted by the TRI- zol reagents (Sigma). Reverse transcription and qPCR were performed by the TOYOBO ReverTra Ace qPCR kit (Tokyo, Japan), under the ABI Prism 7600H fast Real-Time PCR system (Foster City, CA). The mRNA primers for nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase1 (HO1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and GAPDH were provided by Dr. Di [17]. The melt curve analy- sis was performed to calculate the product melting tempera- ture. The 2−∆∆Ct method was utilized for mRNA quantifica- tion, using GAPDH as the reference gene.
Nrf2 KO
The lentiCRISPR-GFP-Nrf2-puro KO construct was pro- vided by Dr. Di [17], transfected to SH-SY5Y neuronal cells.
GFP-positive stable cells were sorted by FACS. Single cells were cultured onto 96-well plate to generate the monoclonal cells. Nrf2 KO was confirmed by Western blotting.
Statistics
Data were presented as mean ± standard deviation (SD). Statistical differences were analyzed by one-way analysis of variance (ANOVA) followed by multiple comparisons performed with post hoc Bonferroni test (SPSS). Values of P < 0.05 were considered statistically significant.
Results
SC79 protects dopaminergic neuronal cells from MPP+ and rotenone
To establish the cytotoxic effect of MPP+ in DA neuronal cells, differentiated SH-SY5Y cells were treated with differ- ent concentrations of MPP+, from 0.3–10 mM. CCK-8 assay was performed to test cell viability, and results show that MPP+ (at 3–10 mM, treatment for 48 h) significantly inhib- ited survival (CCK-8 OD) of SH-SY5Y cells (Fig. 1a). Fur- ther experimental results showed that MPP+ dose-depend- ently induced LDH release to the medium, indicating cell death (Fig. 1b). Significantly, co-treatment of the Akt acti- vator SC79 (10 μM) potently attenuated MPP+ (3–10 mM)- induced viability reduction and LDH release (Fig. 1a and b). These results suggest that SC79 can protect SH-SY5Y cells from MPP+.
SC79′s dose response was studied next. As shown, SC79 dose-dependently inhibited MPP+ (3 mM, 48 h)-induced viability reduction (Fig. 1c) and LDH release (Fig. 1d) in SH-SY5Y cells. The Akt activator was effective at 1–10 μM, but not at 0.3 μM (Fig. 1c and d). Among the tested concen- trations, SC79 at 10 μM demonstrated highest efficiency in protecting SH-SY5Y cells (Fig. 1c–d). Importantly, rote- none (300 nM, 48 h)-induced SH-SY5Y cell viability reduc- tion (Fig. 1e) and death (Fig. 1f) were attenuated by SC79 (10 μM) co-treatment as well (Fig. 1e and f). SC79 single treatment, at the tested concentrations, did not affect SH- SY5Y cell functions (Fig. 1a–d).
The activity of SC79 was also tested in the primary cul- tured murine DA neurons. As shown, SC79 (10 μM) largely inhibited MPP+-induced viability reduction (Fig. 1g) and LDH release (Fig. 1h) in DA neurons. Furthermore, rote- none-induced neuronal death was significantly alleviated by SC79 co-treatment (Fig. 1i). SC79 alone was ineffective on neuronal functions (Fig. 1g and h). Collectively, these results show that SC79 protects DA neuronal cells from MPP+ and rotenone.
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Fig. 1 SC79 protects dopaminergic neuronal cells from MPP+ and rotenone. SH-SY5Y cells (a–f) or the primary murine dopaminergic neurons (“DA neurons,” g–i) were treated with MPP+ (0.3–10 mM), rotenone (300 nM), or together with applied concentration of SC79, cells were further cultured in the medium for 48 h, and cell survival and cell death were tested by CCK-8 assay and LDH release assay,
respectively. “Ctrl” stands for untreated control group (same for all figures). “PBS” stands for SC79’s vehicle PBS. Bars stand for mean ± standard deviation (SD, n = 5). *P < 0.05 vs. “Ctrl” cells. #P < 0.05 vs. MPP+ or rotenone single treatment. Experiments in this figure were repeated five times, with the similar results obtained
SC79 inhibits MPP+‑ and rotenone‑induced apoptosis in dopaminergic neuronal cells
MPP+ disrupts mitochondrial functions, leading to sub- stantial ROS production and oxidative stress, which shall induce the release of pro-apoptosis proteins, causing cell apoptosis [18]. Since SC79 significantly inhibited MPP+-induced DA neuronal cell death, we next tested its effect on cell apoptosis. As shown, treatment with MPP+ (3 mM, 16 h) increased the activities of caspase-3 (Fig. 2a) and caspase-9 (Fig. 2b) in SH-SY5Y cells. Co-treatment with SC79 (10 μM) potently inhibited caspase-3/-9 acti- vation by MPP+ (Fig. 2a and b). Western blotting analy- ses in SH-SY5Y cells demonstrated that SC79 signifi- cantly attenuated MPP+-induced cleavages of caspase-3, caspase-9 and poly (ADP-ribose) polymerase (PARP) (Fig. 2c). MPP+-induced activation of caspases induces DNA damages, causing accumulation of Histone-bound single-strand DNA (Fig. 2d), again inhibited by SC79 (Fig. 2d). To further evaluate the anti-apoptosis activity of SC79, TUNEL staining assay was performed. Results show that SC79 co-treatment significantly attenuated MPP+-induced increases of TUNEL-positive SH-SY5Y
cells (Fig. 2e). SC79 single treatment had no effect on caspase-PARP activation nor cell apoptosis (Fig. 2a–e).
In the primary DA neurons, SC79 inhibited MPP+-induced Histone-bound DNA accumulation (Fig. 2f) and TUNEL-nuclei ratio increase (Fig. 2g). Rotenone treat- ment similarly induced Histone-bound DNA accumula- tion in SH-SY5Y cells (Fig. 2h) and primary DA neurons (Fig. 2i), again attenuated by SC79 (Fig. 2h and i). These results show that SC79 potently inhibited MPP+- and rote- none-induced DA neuronal cell apoptosis.
Akt inhibition abolishes SC79‑mediated neuroprotection against MPP+
Next, we tested the potential effect of SC79 on Akt activa- tion. In SH-SY5Y cells, SC79 dose-dependently induced phosphorylation of Akt at Ser-473 and Thr-308 (Fig. 3a). Total Akt1 level was unchanged (Fig. 3a). SC79-induced SH-SY5Y cell protection against MPP+ was completely nullified by the Akt inhibitors (Fig. 3b). LY294002 and MK-2206 also enhanced MPP+-induced viability reduction (Fig. 3b), indicating that basal Akt activation is cytoprotec- tive in MPP+-treated SH-SY5Y cells.
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Fig. 2 SC79 inhibits MPP+- and rotenone-induced apoptosis in dopaminergic neuronal cells. SH-SY5Y cells (a–e and h) or the pri- mary murine dopaminergic neurons (“DA neurons,” f–g and i) were treated with MPP+ (3 mM), rotenone (300 nM), or together with SC79 (10 μM), cells were further cultured for indicated time periods, and cell apoptosis was evaluated by the assays mentioned in the text.
Expression levels of the listed proteins were quantified, normalized to the loading control (Tubulin) c. Bars stand for mean ± standard devia- tion (SD, n = 5). *P < 0.05 versus “Ctrl” cells. #P < 0.05 versus MPP+ or rotenone single treatment. Experiments in this figure were repeated four times, with the similar results obtained
To rule out the possible off-target toxicities of the Akt inhibitors, CRISPR/Cas9 gene-editing method was employed to knockout Akt1 in SH-SY5Y cells. Each of the two applied lenti-CRISPR Akt1 knockout constructs, containing non-overlapping sgRNA sequences (see Meth- ods), led to complete Akt1 knockout (KO) in SH-SY5Y cells (Fig. 3c). SC79-induced Akt activation, or Ser-473 and Thr-308 phosphorylation, was blocked in Akt1-KO cells (“L1/L2,” Fig. 3c). Importantly, in Akt1-KO SH- SY5Y cells, SC79 was unable to inhibit MPP+-induced viability reduction (Fig. 3d) and cell death (Fig. 3e). This genetic evidence further supported that Akt activation is required for SC79-mediated DA neuroprotection against MPP+. This is further supported by the fact that Akt1-KO cells were more vulnerable to MPP+ (Fig. 3d and e).
In the primary murine DA neurons, SC79 induced potent Akt activation or increased phosphorylation of Akt at Ser-473 and Thr-308 (Fig. 3f). Significantly, LY294002 and MK-2206 abolished SC79-mediated neuroprotection in MPP+-treated primary neurons (Fig. 3g). Akt inhibitors also intensified MPP+-induced viability reduction in DA neurons (Fig. 3g). These results suggest that Akt activa- tion mediates SC79-induced DA neuroprotection against MPP+.
SC79 inhibits MPP+‑ and rotenone‑induced oxidative injury in dopaminergic neuronal cells
Rotenone and MPP+ are known to disrupt mitochondrial respiratory chain, leading to ROS production and oxidative stress, eventually causing DA neuronal cell death. In SH- SY5Y cells, MPP+-induced ROS production, mitochondrial depolarization (∆Ψ), and lipid peroxidation, which were evi- denced by increases of DCF-DA intensity (Fig. 4a), JC-1 intensity (Fig. 4b), and TBAR activity (Fig. 4c), respec- tively. Significantly, co-treatment of SC79 potently inhib- ited MPP+-induced oxidative injury in SH-SY5Y cells (Fig. 4a–c). Similarly, rotenone-induced ROS production was attenuated by SC79 (Fig. 4d).
Nrf2 signaling is a key cellular defensive mechanism against oxidative stress, which could be activated follow- ing Akt activation [9, 19, 20]. qPCR assay results show that SC79 dose-dependently increased mRNA and protein expression of Nrf2-dependent genes, including HO1 and NQO1 (Fig. 4e and f). Furthermore, Nrf2 protein was sta- bilized in SC79-treated SH-SY5Y cells (Fig. 4f), with Nrf2 mRNA levels unchanged (Fig. 4e). Importantly, CRISPR- Cas69-mediated KO of Nrf2 blocked SC79-induced expression of HO1 and NQO1 in SH-SY5Y cells (Fig. 4g).
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Fig. 3 Akt inhibition abolishes SC79-mediated neuroprotection against MPP+. SH-SY5Y cells (a) or the primary murine dopamin- ergic neurons (“DA neurons,” f) were treated with SC79 for 1 h, and the listed proteins in total cell lysates (TCL, 30 μg per treatment) were analyzed by Western blotting assays. SH-SY5Y cells (b) or the primary murine dopaminergic neurons (g) were treated with SC79 (10 μM) or plus MK-2206 (“MK,” 5 μM, 30 min pre-treatment)/ LY294002 (“LY,” 2 μM, 30 min pre-treatment), followed with MPP+ (3 mM) treatment for applied time, and cell viability was tested by
CCK-8 assay. SH-SY5Y cells with the lenti-CRISPR-Cas9-Akt1 KO construct (two different sgRNAs, “L1/L2”) or lenti-CRISPR-Cas9 control (“Cas-9-C”) were treated with MPP+ (3 mM) or plus SC79 (10 μM), and Akt signaling proteins were analyzed by Western blot- ting assay (c); cell viability and death were tested by the CCK-8 assay
(d) and LDH release (e), respectively. Bars stand for mean ± standard deviation (SD, n = 5). *P < 0.05 versus “Ctrl” cells. #P < 0.05. Experi- ments in this figure were repeated three times, with the similar results obtained
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Fig. 4 SC79 inhibits MPP+- and rotenone-induced oxidative injury in dopaminergic neuronal cells. SH-SY5Y cells (a–d) or the pri- mary murine dopaminergic neurons (“DA neurons,” i) were treated with MPP+ (3 mM), rotenone (300 nM), or together with SC79 (10 μM), cells were further cultured for applied time periods, and ROS production (a, d, and i), mitochondrial depolarization (b),
and lipid peroxidation (c) were tested by the assays mentioned in the text. SH-SY5Y cells (e and f) or the primary murine DA neu- rons (j and k) were treated with SC79 (at applied concentration) for indicated time period, and expression levels of Nrf2 pathway genes
were analyzed by qPCR and Western blotting assays. Stable SH- SY5Y cells with lentiCRISPR-GFP-Nrf2-puro KO construct (“Nrf2- KO”) or empty vector (“Vec”) were treated with MPP+ (3 mM) or plus SC79 (10 μM), and listed proteins were shown (g); cell viability was tested (h). Expression of the listed proteins were quantified, nor- malized to the loading control (Tubulin) (f, g and k). Bars stand for mean ± standard deviation (SD, n = 5). *P < 0.05 versus “Ctrl” cells. #P < 0.05 versus MPP+/rotenone single treatment. Experiments in this figure were repeated three times, with the similar results obtained
Moreover, SC79-induced SH-SY5Y cell protection against MPP+ was alleviated in the Nrf2-KO cells (Fig. 4h).
In the primary cultured murine dopaminergic neurons, co-treatment of SC79 also significantly inhibited MPP+- and rotenone-induced ROS production (DCF-DA intensity increase, Fig. 4i). Nrf2 protein stabilization and increased
expression of Nrf2-dependent genes (HO1 and NQO1) were detected in SC79-treated primary DA neurons (Fig. 4j and k). These results suggest that SC79 activates Nrf2 signaling, inhibiting MPP+- and rotenone-induced oxidative stress in DA neuronal cells.
Discussion
PD pathogenesis is multi-factorial, including genetic and environmental factors [1, 2]. DA neuronal dysfunction and death in PD progression involve several biochemical factors, including free radicals, mitochondrial dysfunc- tion, abnormal protein aggregation, excitotoxicity, and inflammation [1, 2]. Of particular relevance, mitochon- drial dysfunction and substantial ROS production are the main causes of DA neuronal cell death [1, 2]. Akt is a key pro-survival signaling. Akt phosphorylates its substrates, including mTOR, BAD, CREB, and the Forkhead tran- scription factors, to promote DA neuronal cell survival [21–23] and to protect neuronal cells from oxidative stress [24–26].
In the current study, we show that SC79, a first-in-class small-molecule Akt activator, significantly inhibited MPP+- and rotenone-induced viability reduction, cell death, and apoptosis in SH-SY5Y cells and the primary murine DA neurons. SC79 activated Akt in DA neuronal cells, which is required for DA neuroprotection against mitochondrial tox- ins. Akt inhibitors or CRISPR-Cas9-mediated Akt1 knock- out abolished SC79-induced DA neuroprotection against MPP+. Therefore, activation of Akt by SC79 protects DA neuronal cells from mitochondrial toxins.
Activation of Nrf2 signaling is an established strat- egy to protect DA neuronal cells from oxidative injury (i.e., by mitochondrial toxins) [27–29]. Recent studies have implied that activated Akt could lead to Nrf2 signal- ing activation in different cells. For example, Lee et al. showed that sulforaphane-induced Nrf2 activation is the downstream PI3 K-Akt activation [30]. Xu et al. demon- strated that PI3 K-Akt activation is required for pyocya- nin-induced Nrf2 signaling activation [31]. Furthermore, Zhang et al. showed that Salvianolic acid A (Sal A) acti- vated Akt-dependent Nrf2 signaling in human retinal pig- mentation epithelial (RPE) cells [32].
In the current study, SC79 induced Nrf2 protein stabili- zation and increased expression of Nrf2-dependent genes (HO1 and NQO1) in SH-SY5Y cells and primary DA neu- rons, confirming Nrf2 cascade activation. SC79 potently attenuated MPP+- and rotenone-induced ROS production, mitochondrial depolarization, and lipid peroxidation in DA neuronal cells. Therefore, we implied that SC79 activated Akt-Nrf2 signaling to inhibit MPP+- and rotenone-induced oxidative injury in DA neuronal cells. This is further sup- ported by the fact that Nrf2 KO alleviated SC79-induced SH-SY5Y cell protection against MPP+. The underlying mechanisms of Nrf2 signaling activation by SC79 in DA neuronal cells and how SC79-activated Nrf2 signaling inhibits oxidative stress by mitochondrial toxins warrant further studies.
Conclusion
Together we show that SC79 protects DA neuronal cells from mitochondrial toxins possibly via activation of Akt- Nrf2 signaling.
Author contributions All listed authors designed the study, performed the experiments and the statistical analysis, and wrote the manuscript. All authors have read the manuscript and approved the final version. Author D.W. performed the experiments in the primary neurons.
Funding This work was supported by funds from a special project “diagnostic and therapeutic technology of key clinical diseases” of Suzhou (LCZX201404) and a grant from Suzhou Key Medicine Project Fund of China (Szxk201504) and a grant from National Natural Sci- ence Foundation of China (81502162).
Compliance with ethical standards
Conflict of interest None of the authors have any competing interests.
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