SIRT3-mediated deacetylation of SDHA rescues mitochondrial bioenergetics contributing to neuroprotection in rotenone-induced PD models

doi:10.21203/rs.3.rs-1705902/v1

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SIRT3-mediated deacetylation of SDHA rescues mitochondrial bioenergetics contributing to neuroprotection in rotenone-induced PD models

https://doi.org/10.21203/rs.3.rs-1705902/v1

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published 13 Dec, 2023

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Mitochondrial dysfunction is critically involved in the degeneration of dopamine (DA) neurons in the substantia nigra (SNc), a common pathological feature of Parkinson’s disease (PD). Previous studies have demonstrated that the NAD⁺-dependent acetylase Sirtuin 3 (SIRT3) participates in maintaining mitochondrial function and is downregulated in aging-related neurodegenerative disorders. The exact mechanism of action of SIRT3 on mitochondrial bioenergetics in PD pathogenesis, however, has not been fully described. In this study we investigated the regulatory role of SIRT3-mediatged deacetylation of mitochondrial complex II (succinate dehydrogenase) subunit A (SDHA) and its effect on neuronal cell survival in rotenone (ROT)-induced rat and differentiated MN9D cell models. The results revealed that SIRT3 activity was suppressed in both in vivo and in vitro PD models. Accompanying this downregulation of SIRT3 was the hyperacetylation of SDHA, impaired activity of mitochondrial complex and decreased ATP production. It was found that the inhibition of SIRT3 activity was attributed to a reduction in the NAD⁺/NADH ratio caused by ROT-induced inhibition of mitochondrial complex I. Activation of SIRT3 by icariin and honokiol inhibited SDHA hyperacetylation and increased complex II activity, leading to increased ATP production and protection against ROT-induced neuronal damage. Furthermore, overexpression of SDHA also exerted potent protective benefits in cells treated with ROT. In addition, treatment of MN9D cells with the NAD⁺ precursor nicotinamide mononucleotide increased SIRT3 activity and complex II activity and promoted the survival of cells exposed to ROT. These findings unravel a regulatory SIRT3-SDHA axis, which may be closely related to PD pathology. Bioenergetic rescue through SIRT3 activation-dependent improvement of mitochondrial complex II activity may provide an effective strategy for protection from neurodegeneration.

SIRT3

mitochondrial complex II

succinate dehydrogenase subunit A (SDHA)

neurodegeneration

rotenone

deacetylation

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DArgic) neurons in the substantia nigra (SN) [1]. A large number of studies have shown that damaged mitochondrial respiratory chain (MRC) and dysfunctional mitochondria often exist in the SN of PD patients and various animal models [2, 3]. Many PD associated proteins or molecular signaling pathways that may be involved in PD development have been found to be closely related or interact with mitochondrial function [4]. However, given the multifaceted functions of intracellular mitochondria and the pathogenic complexity of PD, the important role of mitochondrial dysfunction in PD neurodegeneration is still far from fully understood.

Epidemiological studies have shown that mitochondrial complex I inhibitor rotenone (ROT) increases the risk of PD, and animal models have demonstrated that ROT can recapitulate PD-like symptoms [5]. With regards to the hypothesis of mitochondrial complex I inhibition in PD pathology, one of the consequences associated with complex I interference is the collapse of ATP production, resulting in neuronal cell damage and loss of function [6]. Although insufficient energy supply will adversely affect neural function, the decisive role of ATP decline in PD pathological neurodegeneration remains to be fully elucidated [7]. In addition, damage to other mitochondrial complexes, including complex II, has also been frequently reported in animal models and the brain of human PD patients. Complex II, succinate dehydrogenase (SDH), plays a central role in mitochondrial metabolism, linking the tricarboxylic acid cycle (TAC) and mitochondrial electron transport system [8–11]. It has been reported that two SDH subunits SDHA and SDHB are preferentially lost in the striatum of patients with Huntington’s disease, accompanied by mitochondrial dysfunction [12]. Research on PD has demonstrated that post-mortem brain tissues harbored a dramatic decrease in complex II activity in cortical regions, which was not owing to the gene expression, because no corresponding downregulation of complex II subunits was observed [10]. In addition, varying degrees of both complex I and complex II deficiency have been found in individual SN neurons from PD patients [13]. These results suggest that mitochondrial complex II may be another important player in the loss of DA neurons. However, the mechanism underlying the impairment of mitochondrial complex II and its role in PD pathology are poorly studied.

Sirtuin3 (SIRT3) is a NAD⁺-dependent protein deacetylase that regulates mitochondrial proteins including components of oxidative phosphorylation (OXPHOS) [8, 14]. SIRT3 directly deacetylates SDHA, and studies have shown that SDH activity is reduced in SIRT3 knockout cells and mice [15]. In our previous studies we have demonstrated that icariin (ICA), a flavonoid glucoside extracted from Epimedium, can upregulate SIRT3 and protect ROT-induced neuronal cell death in PD model [8, 16]. Whether activation of SIRT3 can regulate complex II activity through modulation of its subunit SDHA in the context of PD pathogenesis thus has yet to be defined.

In the present study we explored the role of activation of SIRT3 in the regulation of mitochondrial complex II activity and neuroprotection in ROT-induced PD models. SIRT3 was activated by ICA and honokiol (HNK) [16, 17], and NAD⁺ precursor nicotinamide mononucleotide (NMN). We investigated the molecular mechanism by which SIRT3 activation inhibits SDHA acetylation and elucidated SIRT3-dependent rescue of mitochondrial complex II activity and bioenergy supply in ROT-induced PD models. Neuroprotection was also observed in SDHA-overexpressing cells. These findings suggest a regulatory SIRT3-SDHA axis that may be a key player in the pathogenesis of neurodegenerative diseases and highlight the potential therapeutic value of SIRT3 activation in PD treatment.

2.1 Chemicals and reagents

ROT and TZ were purchased from MedChemExpress (New jersey, USA), and NaBu was purchased from Sigma-Aldrich (St Louis, MO, USA). HNK was obtained from Dalian meilunbio Biological Technology Co., Ltd (Da Lian, China), ICA was purchased from Nanjing Zelang Biological Technology Co., Ltd (Nanjing, China), and the purity of HNK and ICA used in the experiment was more than 98%. Roswell Park Memorial Institute (RPMI) 1640 medium was supplied by Gibco (USA), fetal bovine serum was from Medical Research Council (UK), and 0.25% trypsin was purchased from Hyclone (Logan, UT). GAPDH and TH antibodies were obtained from Proteintech (Wuhan, China). SDHA and Acetylated-Lysine antibody were purchased from Cell Signaling Technology (Abcam, Danvers, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were supplied by Solarbio Science & Technology Co. Ltd (China).

2.2 Animals and experimental design

Healthy male SPF Sprague-Dawley rats (190 to 220 g) were from Changsha Tianqin Biotechnology Co., Ltd. The animals were maintained under a 12 h light/dark cycle in temperature (23 ± 1°C) and humidity (relative, 60%)-controlled rooms and allowed free access to food and water. SD rats were randomly divided into 6 groups, including Control, HNK (5 mg/kg/day), ICA (15 mg/kg/day), ROT (1.5 mg/kg/day), ROT (1.5 mg/kg/day) + HNK (5 mg/kg/day), and ROT (1.5 mg/kg/day) + ICA (15 mg/kg/day) group, 15 animals for each group. Two weeks after HNK i.p. injection and ICA oral gavage, SD rats were administrated subcutaneously with 1.5 mg/kg/day ROT, and HNK and ICA were given daily at the specified dose of each group, for 42 consecutive days. The control group was given the same amount of solvent. HNK, ICA, and ROT concentrations were chosen according to previous studies [18–20]. ICA was dissolved in ddH₂O, and HNK and ROT were dissolved in DMSO and then diluted in physiological saline to make a final injection solution (saline containing 1% DMSO). The control group was subject to an equivalent volume of vehicles (ddH₂O and solvent were administrated by oral gavage and subcutaneous injection). Twenty-four hours after the last administration of HNK, ICA and ROT, the rotarod test was conducted to detect the motor coordination of rats [21]. The animals were executed 12 h after the behavioral test. All the animal procedures conformed to the National Institute of Health Guideline for the Animal Care and Use of Laboratory Animal, and the protocols were approved by the institutional Animal Care and Use Committee at Zunyi Medical University (Zunyi, China).

2.3 Rotarod test

The rotarod test was conducted to evaluate motor coordination of rats. The procedure for the rotarod test was described previously [16]. Twenty-four hours after the last injection of ROT and administration of ICA and HNK, animals were extracted freely from each group, and the motor coordination was evaluated using the rotarod test. Prior to the test start, the rats were trained to adapt to the rotating axis (20 rpm, constant speed) of a rotarod. When the rats stayed on the rotating axis for at least 30 s without falling down, the training stopped. At the test, all rats were replaced on the rotating axis and the latency to fall was measured with a cutoff at 600 s. Each animal was repeatedly evaluated 3 times within 24 h, and the performance was averaged.

2.4 Immunostaining of DArgic neuron

DA neurons were identified by an antibody that detects TH through immunohistochemical staining using an antibody for TH. After assessment of the motor performance, rats were anesthetized and then were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were removed and transferred into 30% sucrose to dehydrate for 48 h. The brains were embedded in paraffin and sectioned. For immunostaining TH, a series of 5-µm thick sections of SN were performed. Brains SN sections were dewaxed and removed of endogenous peroxidase. The brain tissue sections were treated with 0.3% Triton for 15 min, 3% H₂O₂ for 15 min, blocked in goat serum for 30 min, and then incubated with a primary antibody to TH (1: 200, Abcam) for 24 h at 4°C. After washing, the sections were incubated with biotinylated goat anti-rabbit IgG secondary for 30 min and biotinylated for 1 h at 37°C. The sections were visualized using a DAB Color Development Kit (cat. no. PV-9000; ZSGB-BIO, Beijing, China). Images of TH-positive neurons in SN compacts of the brain were obtained by an Olympus microscope (Olympus, Tokyo, Japan). For the quantification of TH-positive neurons, four representative areas per well were counted. Three slices were applied for cell enumerating on each rat.

2.5 Cell culture

The mouse DArgic neuron cell line MN9D commonly used as a DA neuron model to study PD was obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). MN9D cells were cultured in RPMI 1640 media replenished with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.

2.6 Differentiation of MN9D cells and drug treatment

To simulate accurately the characteristics of neurons, sodium butyrate was used to promote the differentiation and maturation of MN9D cells to DArgic neuronal cells. MN9D cells were seeded in dishes, cultured in medium for 1–2 days until approximately 75–80% confluence and were then administered with 1 mM NaBu for 6 days. After 6 days of NaBu incubation, the morphology of neurons was captured by Olympus microscope. ROT, HNK, and ICA were diluted in DMSO and prepared freshly before corresponding experiments. Upon the completion of differentiation, cells were divided into different treatment groups for subsequent exposure to the tested chemicals. The cells were pretreated with HNK and ICA for 2 h, then exposed to ROT for 24 h before being subject to the subsequent experiments and assays.

2.7 Cell transfection

The empty lentiviral vector and lentiviral vector encoding mouse SDHA were synthesized by Genomeditech (Shanghai, China). MN9D cells were infected with lentivirus medium at a multiplicity of infection (MOI = 100). The green fluorescence signal was observed with a fluorescence microscope (Olympus, Tokyo, Japan) and the expression of SDHA was determined by western blot after infection for 72 h.

2.8 Evaluation of cell viability

Viability of MN9D cells was evaluated by the MTT assay. To observe the ROT toxicity and the effects of HNK, ICA, NMN and TZ on ROT toxicity, cells were pretreated with these chemicals at indicated concentrations for 2 h before exposure to 100 nM ROT for 24 h. Upon the completion of treatment of differentiated MN9D cells with the tested drugs, cells were exposed to MTT (0.1 mg/mL) for 4 h at 37°C. Following the incubation, supernatant was discarded, the cells were solubilized with 120 µL of DMSO and the solution was shaken for 10 min. The concentration of the resultant blue formazan solution was spectrophotometrically determined. The viability of cells was expressed as a percentage of the absorbance measured in relative to the control cells.

2.9 Extraction of mitochondria

Freshly harvested cell pellets or brain tissues were washed with ice-cold PBS. To the cell and tissue samples, pre-cooled mitochondrial separation solution (sucrose 85.6 g, Tris-HCl 1.57 g, EDTA 0.29 g, and PMSF 0.17 g, pH 7.4) was added, and samples were hand-homogenized in ice-cold isolation buffer by Dounce homogenizer (homogenized on ice for 5 min). The homogenized samples were centrifuged at 1,000 g at 4°C for 10 min to collect supernatants which were then centrifuged at 10,000 g at 4°C for 10 min. The resultant sample pellets were collected and stored at -80°C for subsequent experiments.

2.10 NAD⁺/NADH quantitative test

Tissue samples and MN9D cells were lysed in 200 µL NAD⁺/NADH extraction buffer by three freeze/thaw cycles. The lysate was centrifuged at 12,000 rpm, and supernatant was collected for measurements. Samples were split into 2 fractions to separately measure total NAD (containing total NAD⁺ and NADH) in one fraction and in the other fraction in which samples were heated to 60°C for 30 min in a water bath and all NAD was decomposed into the NADH. The levels of NAD⁺ were estimated by subtracting NADH from total NAD. Absorbance was measured at 450 nm using Multiskan Microplate Reader (Thermo Fisher Scientific, USA) in a clear-bottom 96-well plate. The NAD⁺/NADH ratio can be calculated as: NAD⁺/NADH = (total NAD-NADH)/NADH.

2.11 Activity of SIRT3

The activity of SIRT3 was measured by means of a fluorometric method using the FLUOR DE LYS® SIRT3 Fluorometric Drug Discovery Assay Kit (Enzo life science, Switzerland). The assay is based on NAD⁺-dependent deacetylation of the substrate, which comprises the p53 sequence Gln-Pro-Lys-Lys (Ac)-AMC, by SIRT3. Upon deacetylation the substrate becomes Gln-Pro-Lys-Lys-AMC and is sensitized to a Developer to generate a fluorophore. Cells and tissue samples were lysed and treated with 0.5% triton for 30 min and then centrifuged at 2500 rpm for 5 min at 4°C. Supernatant was collected and quantified using BCA Protein Assay Kit (Shanghai Generay Biotech, China). SIRT3 assay buffer, 20 µg protein lysates and solvent were added into 96 well plates successively and incubated on a shaker for 45 min at 37°C. The developing solution was then added to each well and incubated for 30 min at room temperature. The fluorophore was excited with 355 nm light and the emitted light (460 nm) was detected on Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, USA). The activity of SIRT3 was calculated and expressed as a percentage of the control.

2.12 Measurement of mitochondrial complex II activity

Mitochondrial complex II activity was determined using with ETC complex II Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). The assay is based on the succinate dehydrogenase (SDH) catalytic reduction of dichlorophenol-indophenol (DCPIP) to reduced dichlorophenolindophenol (DCPIPH2) with the oxidation of succinate and decreases in the absorbance of DCPIP that was colorimetrically determined at OD600. Protein concentrations of MN9D cell lysates and brain tissue isolated mitochondria were determined using BCA Protein Assay Kit. Catalytic reaction of samples was started by adding the reactant DCPIP at 30°C. The absorbance was measured at OD600 and recorded for 3 min to calculate the complex II activity.

2.13 Total cellular ATP content

ATP measurement was measured by the chemiluminescence method using the Luminescent ATP Detection Assay Kit (Abcam, ab113849) as per the manufacturer’s instruction. In a black 96-well plate, 100 µL MN9D cells suspension (5 × 10⁴ cells/well) were added. To lyse the cells and stabilize ATP, 50 µL detergent was added to each well of the plate and incubated for 5 min in an orbital shaker at 600–700 rpm. Fifty µL substrate solution was then added to each well and incubated in the orbital shaker at 600–700 rpm for 5 min and covered for 10 min. The measurement of luminescence was then conducted by using Varioskan LUX Multimode Microplate Reader [22]. The plate was protected against exposure to light in the process of detection. A standard curve was plotted and ATP amount in sample well was calculated from standard curve (nmol).

2.14 Western blot analysis

SN tissues in brains and MN9D cells were collected and processed in ice-cold RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) mixed with the protease inhibitor PMSF (1 mM). Samples were incubated on ice for 30 min, and the protein lysates were centrifuged at 12,000 rpm for 15 min at 4°C. Protein was quantified using BCA Protein Assay Kit. Then samples were boiled in loading buffer at 100°C for 10 min. Samples (30 µg) were resolved by 10% SDS-polyacrylamide gels and transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membrane. Skim milk (5%) was used to block nonspecific antigens for 2 h at room temperature, the membranes were then incubated for 18 h at 4°C with the appropriate primary antibodies: anti-p-GAPDH (1: 1,000), anti-TH (1: 1,000), anti-Acetylted-Lysine (1: 1,000), and anti-SDHA (1: 1,000). The membranes were washed with TBST for three times and were subjected to horseradish peroxidase-conjugated secondary antibody (1: 2000) for 2 h. The membrane-bound secondary antibodies were detected with an ECL Western Blot Detection Kit (Thermo scientific). The densities of immunoreactive bands were quantified by using Image Lab software, version v6.0 (Bio-Rad Laboratories, Inc. USA).

2.15 Immunoprecipitation

Brain tissues and MN9D cells were lysed by RIPA lysis buffer, and protein concentration was measured using BCA Protein Assay Kit. Protein (500 µg) was equilibrated with 0.1% SDS and Np-40 lysis buffer. Lysate was incubated with 20 µL protein A/G PLUS and 1µg anti-SDHA antibody for 18 h at 4°C. Samples were spun down, washed four times with PBS, added 10 µL loading buffer to separate the protein from protein A/G PLUS and boiled for 10 min at 100 ℃. Protein samples were resolved by electrophoresis on 7.5% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. BSA (5%) was used to block nonspecific antigens for 2 h at room temperature, the membranes were then incubated for 18 h at 4°C with the appropriate primary antibodies: anti-Acetylted-Lysine (1: 1,000), and anti-SDHA (1: 1,000). The membranes were washed with TBST for three times and were subjected to horseradish peroxidase-conjugated secondary antibody (1: 2,000) for 2 h. The blots were detected with ECL Western Blot Detection Kit (Thermo scientific).

2.16 Statistical analysis

Statistical analysis was performed with the GraphPad Prism software, version v.7.0.0.159 (GraphPad Software, Inc. La Jolla California, USA) and SPSS version 18.0 software (SPSS Incorporated, USA). The statistical significance was analyzed using a two-tailed Student’s t-test (for two groups) and ANOVA and Tukey post hoc comparison were applied in multi-group/factorial design. Fisher’s exact test was used to examine whether two sets were significantly overlapped/enriched. p value < 0.05 was considered as statistical significance. Data were expressed as mean ± SE of three or more independent experiments.

3.1 Neurotoxicity of ROT in differentiated DA MN9D cells

To study the cellular mechanisms of ROT-induced DA neuronal degeneration, we developed a cellular model derived from immortalized midbrain MN9D cells, a DA neuronal cell line that has been extensively characterized as a cell model of DA neurons [23]. We differentiated MN9D cells with 1 mM sodium butyrate (NaBu). NaBu induces cell cycle arrest and morphological neural differentiation by inhibiting histone deacetylase [23]. After treatment with NaBu for 6 days, MN9D cells stopped dividing and adopted a mature neuronal morphology, characterized by the formation of long neurites (Fig. 1B). We first characterized the cytotoxicity of ROT on MN9D cells and identified a suitable dosage for subsequent experiments (Fig. 1C). The viability of MN9D cells was significantly reduced after being exposed to ROT, and the inhibitory effect showed a dose-dependent response (Fig. 1D-E). Our previous study and other research have demonstrated that HNK and ICA may confer beneficial effects in the pathogenesis of neurodegenerative disorders via activating SIRT3 activity [16, 24]. Therefore, we questioned whether HNK and ICA could attenuate the damage to differentiated MN9D cells induced by ROT. The cells pretreated with 20 µM HNK and 10 µM ICA displayed significant improvements in cell survival (p < 0.05, compared with ROT alone) (Fig. 1D-E). The differentiated MN9D cells had regular and mature neuronal morphology, while cells treated with ROT for 24 h exhibited roundish somata with occasional damaged neurites, and at the same time cells were reduced in number. MN9D cells pretreated with HNK and ICA for 2 h exhibited fewer damaged cell synapses than those treated with ROT alone (Fig. 1F-G). These results demonstrated that HNK and ICA attenuated ROT-induced neurotoxicity in MN9D cells.

3.2 HNK and ICA decrease acetylation of SDHA through activation of SIRT3 in ROT-induced PD model

Previous reports have shown that SIRT3 functions as a major deacetylase of mitochondria. To identify the effect of ROT on SIRT3 activity and its deacetylase activity, we analyzed SIRT3 activity by fluorometric assay kits. As illustrated in Fig. 2A-B, our results demonstrated that treatment with ROT resulted in profound inhibition of SIRT3 activity in differentiated MN9D cells. Previous research suggests that the anti-hypertrophic effects of HNK is dependent on activation of the deacetylase SIRT3 [17]. We recently reported that ICA protects neurodegeneration from oxidative stress through elevation of SIRT3 activity [16]. Thus, we analyzed the effects of the two SIRT3 activators HNK and ICA on the acetylation status of mitochondrial proteins and SIRT3 activity in the ROT-exposed differentiated MN9D. Different doses of HNK and ICA were co-incubated with ROT in differentiated MN9D cells for 24 h, where the SIRT3 activity was then detected. The results showed that the ROT-elicited suppression on the activity of SIRT3 was markedly abolished after administration of HNK and ICA (Fig. 2A-B). Since ROT treatment inhibited SIRT3 activity, we next sought to determine whether NAD⁺-dependent SIRT3 is involved in the modulation of acetylation of mitochondrial proteins. We performed immunoblot analysis of mitochondrial proteins acetylation in MN9D cells and found that cells exposed to ROT for 24 h exhibited a profound elevation of acetylation levels in mitochondrial proteins, whereas addition of HNK and ICA significantly decreased the acetylated levels of mitochondrial proteins (Fig. 2C-D). Given that SDHA is a subunit of mitochondrial complex II and a direct target of SIRT3 deacetylase activity [15], we examined whether the major mitochondrial deacetylase SIRT3 could modulate the acetylation status of SDHA and thus affect its catalytic function. We determined acetylation levels of SDHA using immunoprecipitation. Consistent with the effect on SIRT3 activity, we observed increased acetylation of SDHA in differentiated MN9D cells treated with ROT, whereas the acetylation level of SDHA protein was significantly reduced in MN9D cells exposed to a combination of ROT with HNK or ICA (Fig. 2E-F). As a NAD⁺ dependent deacetylase, SIRT3 functions depending on the maintenance of the NAD⁺/NADH ratio [8, 25]. ROT inhibits complex I activity which may result in significant decline in mitochondrial NAD⁺/NADH ratio. We therefore determined whether ROT can directly inhibit NADH conversion to NAD⁺ and thus reduce the NAD⁺/NADH ratio. The results showed that treatment with ROT caused a significant decrease in the NAD⁺/NADH ratio, suggesting that ROT-induced inhibition of SIRT3 enzymatic activity may be regulated through the decline of NAD⁺/NADH ratio exerted by ROT (Fig. 2G-H). However, although HNK and ICA significantly reduced SDHA acetylation caused by ROT, they did not reverse the decreased NAD⁺/NADH ratio (Fig. 2G-H).

3.3 Activation of SIRT3 by HNK and ICA improves complex II activity and rescues ATP production

It has been suggested that SIRT3 may be an important physiological regulator of SDH activity [15]. Previous studies demonstrated that there were no changes in protein expression of the complex II subunits in the brain tissues of PD, but a decrease in the complex II activity was observed in the occipital lobe using frozen post-mortem brain tissues [26]. Also, the diminished complex II activity was found in liver mitochondria of SIRT3 knockout mice [15], suggesting a critical role of SIRT3 in the regulation of complex II. Given that SDHA is a catalytic subunit of the mitochondrial complex II succinate dehydrogenase, we proposed that the treatment of ROT may affect the complex II activity through SIRT3-mediated modulation on the acetylation status of SDHA. To test this hypothesis, we determined the protein expression of SDHA and the activity of complex II. Consistent with previous findings [26], we did not observe changes in the protein expression of SDHA, however, a significant decrease in SDH activity was found in the cells treated with ROT. Furthermore, pretreatment with HNK and ICA markedly negated the suppression of complex II activity caused by ROT (Fig. 3A-D). Collectively, these results suggest that there may exist a regulatory NAD⁺/NADH-SIRT3-SDHA axis that regulates complex II activity, which may be a new mechanism involved in ROT-induced neurodegeneration. As the complex II is a member of the MRC that transfers electrons directly to the ubiquinone pool, a decrease in the activity of complex II thus impairs the electron transport, leading to the decline of ATP production. We assessed ATP levels in the cells following 24 h exposure to 100 nM ROT with or without HNK and ICA using a luminescence assay kit [27]. ATP content was 0.44 µM/10⁵ cells in the control cells and decreased to 0.32 µM /10⁵ cells in the ROT-treated MN9D cells, whereas the presence of HNK and ICA significantly reversed the decline in the ATP production induced by ROT (Fig. 3E-F). Collectively, our data indicate that ROT treatment results in decreased ATP production due to inhibition of complex I and resultant suppression of complex II activity, whereas mitochondrial ATP production can be enhanced by HNK and ICA through activation of the complex II activity in the differentiated MN9D cells. These results clearly demonstrate that the activation of SIRT3 by HNK and ICA increases SDH activity via inhibiting hyperacetylation of SDHA, and this in turn can rescue bioenergetic deficit attenuating ROT-induced damage to neuronal cells.

3.4 Activation of SIRT3 by supplementation of NAD improves the survival of ROT-treated cells

To further determine whether the rise of SIRT3 activity is a key to the change of acetylation, the decrease of SDH activity and the corresponding ATP collapse exerted by ROT, we treated cells with NMN, a precursor of NAD⁺. We questioned whether NMN treatment can protect cells from ROT-induced damage. We evaluated the viability of differentiated MN9D cells after incubation with ROT and NMN. The results showed that NMN could reduce the neurotoxicity caused by ROT (Fig. 4A). The analysis of SDH activity showed that NMN treatment significantly increased SDH activity (Fig. 4B). Then we interrogated the potential involvement of SDH function in ATP synthesis and detected ATP content in MN9D cells. Interestingly, the decrease of ATP level induced by ROT was significantly weakened in MN9D cells treated with NMN (Fig. 4C). These results strongly support that the damage of SDH activity is related to ROT-induced neurotoxicity, and the protection of SDH activity improves cellular bioenergetic status. Since the increase of ATP exerts a protective effect on the neurotoxicity induced by ROT in MN9D cell model, we further evaluated whether terazosin (TZ), a clinical drug that can increase the level of ATP and is related to the reduction of PD diagnosis frequency in individuals taking TZ [28], can eliminate the cytotoxicity induced by ROT in differentiated MN9D cells. Cells were cultured with ROT and TZ for 24 h, and the cell viability was detected. As shown in Fig. 4D-E, although ROT treatment alone resulted in decrease in cell viability and reduction of ATP content, the addition of TZ significantly increased the ATP level in differentiated MN9D cells and rescued cell survival. These results clearly showed that the increase of ATP content exerts a protective effect on the neurotoxicity caused by ROT. To further evaluate the role of upregulation of SDH in protection against ROT-induced neuronal damage, we constructed SDHA overexpressed MN9D cells by lentivirus transfection. The overexpression of SDHA was confirmed by fluorescent imaging and western blot (Fig. 4F-H). Compared with normal control (vector transfection), SDHA overexpression could significantly reduce the loss of cell viability caused by ROT (p < 0.05, compared with ROT group) (Fig. 4I).

3.5 Protective effect of HNK and ICA on ROT-induced neurodegeneration

Rats were injected subcutaneously with ROT (1.5 mg/kg/day) for 6 weeks to induce DA neurodegeneration and establish PD model. To determine the effects of HNK and ICA on DA neurons, rats were administered with HNK (5 mg/kg/day) intraperitoneally and ICA (5 mg/kg/day) by gavage for 6 weeks. The motor behavior of rats was assessed by rotarod test. The results showed that the latency time of rats in the ROT group was significantly shortened. Compared with the control group, the latency on the rod decreased by about 39% (p < 0.05), while the latency time of rats co-administered with HNK or ICA increased significantly to 93.6% and 101.7% of the control level, respectively (Fig. 5B). To evaluate the ROT-induced damage of DA cells, the expression of tyrosine hydroxylase (TH), a catalyzing enzyme involved in DA production in DA cells, was detected by immunohistochemical staining. As shown in Fig. 5C-D, it was found that the number of DA neurons decreased significantly in SN of rats administered with ROT, while rats co-treated with HNK and ICA had a significant protective effect on the damage of DA neurons induced by ROT. The TH expression was detected by western blotting to further evaluate ROT-induced DA neuron degeneration and the neuroprotective effects mediated by HNK and ICA (Fig. 5E-F). These results suggest that HNK and ICA have protective effects on DA neuron loss in rats treated with ROT.

3.6 Activation of SIRT3 by HNK and ICA regulate SDH activity and improve ROT-induced bioenergetic decline in PD rat model

To further prove the role of SIRT3-SDHA axis in ROT-induced neurodegeneration, the protein expression of SDHA, the activities of SIRT3 and SDH, and the content of ATP were detected in PD rat model. Based on previous evidence that HNK and ICA treatment induced SIRT3 activity and ROT treatment significantly reduced SIRT3 activity, we asked whether ROT also affected SIRT3 activity and global acetylation of total proteins in the SN of rat brain. We analyzed the effects of HNK and ICA on SIRT3 activity and mitochondrial protein acetylation. Consistent with the decrease of SIRT3 activity in MN9D cells, we observed that ROT inhibited SIRT3 activity in rats and increased the acetylation level of mitochondrial total proteins. However, when rats were co-treated with HNK or ICA, the decrease of SIRT3 activity induced by ROT was rescued and the increase of total protein acetylation was significantly reversed (Fig. 6A-B). Next, we tested whether the increase of SIRT3 activity was related to the acetylation of SDHA and mitochondrial SDH activity, we analyzed the acetylation status of SDHA by immunoprecipitation and western blotting. We found that immunopurified SDHA was highly acetylated in rat PD model induced by ROT. Similarly, hyperacetylation of SDHA was rescued in the brain tissue of PD rats administered in combination with HNK or ICA (Fig. 6C). To determine whether the protein expression of SDHA played a role in SDH activity, SDHA protein expression and SDH activity were examined in brain tissues of PD rats. Interestingly, ROT, HNK and ICA had no effect on the protein expression level of SDHA in rats (Fig. 6D), which suggests that ROT inhibited SDH activity through the deacetylation modification mechanism of SIRT3. Then, we determined the activity of complex II in purified brain mitochondria. Our results demonstrated that ROT significantly inhibited SDH activity, while the SDH activity of HNK and ICA treated rats was returned to the control level (Fig. 6E). Based on previous research results, we propose that the change of SDH activity may affect the ATP synthesis of OXPHOS, which plays a key role in the pathogenesis of PD [29]. Therefore, we detected ATP content, and the results revealed that ATP level decreased significantly reduced in rats exposed to ROT but remained at the control levels in rats co-treated with HNK or ICA. Together, these results suggest that SDHA is the target of SIRT3 deacetylase activity, and ROT may inhibit SDH activity by increasing the acetylation of SDHA, which is due to the downregulation of SIRT3 caused by ROT. In addition, our results demonstrate that pharmacological activation of SIRT3 can rescue bioenergetic failure and thus prevent neuronal degeneration in rat PD model induced by ROT.

Mitochondrial dysfunction plays a central role in the pathogenesis of PD [31]. In the present study, we found that SIRT3 activity was significantly reduced and the activity of mitochondrial complex II was impaired in both cellular and rat models of PD. The decrease in mitochondrial complex II activity was attributed to downregulation of SIRT3, resulting in increased SDHA acetylation. We demonstrated that activation of SIRT3 by HNK and ICA, as well as NAD precursors, reduces SDHA acetylation and confers a neuroprotective effect over ROT-induced neuronal injury. Furthermore, overexpression of SDHA was protective against ROT-induced MN9D cells. These findings strongly suggest that acetylation-dependent inhibition of mitochondrial complex II activity is involved in the mode of action of DA neuron degeneration, thereby contributing to the pathogenesis of PD. More importantly, direct activation of SIRT3 by HNK and ICA can have direct effects on cell survival and neuroprotection. Our data reveal a novel mechanism by which SIRT3-mediated improvement in mitochondrial complex II activity rescues bioenergetic decline to ameliorate PD pathology.

Increasing studies have shown that mitochondrial complex activities of MRC are often regulated by post-translational regulation of reversible lysine acetylation [10, 15, 26, 30]. In previous study, we found that SIRT3 plays a protective role in ROT-induced PD cell model by improving mitochondrial function and oxidative status [16]. SIRT3 is a major NAD⁺-dependent mitochondrial protein deacetylase, and its activity is regulated by mitochondrial NAD⁺/NADH ratio. Maintaining cellular and mitochondrial NAD⁺ level and the optimal NAD⁺/NADH ratio are essential for SIRT3 activity and mitochondrial function [8, 31]. NAD⁺ deficit and reduced NAD⁺/NADH ratio are often found in ROT-induced PD models [32, 33]. However, it has yet to fully characterize the role of SIRT3-mediated deacetylation in PD pathologies.

Although studies have shown that many factors may affect the NAD⁺/NADH ratio, the function of complex I (NADH/coenzyme Q reductase) is very important to maintain the mitochondrial NAD⁺/NADH ratio, because NADH molecules are oxidized to NAD⁺ in complex I, coupled with OXPHOS and ATP production. Therefore, persistent inhibition of complex I activity results in the decrease of NAD⁺/NADH ratio and ATP production [34, 35]. Remarkably, our study not only found that ROT reduced ATP levels, but also found that ROT inhibited SIRT3 activity, which was the result of complex I inhibition caused by ROT and led to the suppression of mitochondrial complex II activity, thus aggravating the bioenergetic collapse. To further explore the mechanism of impaired complex II and its involvement in neurodegeneration, we employed pharmacological approaches to activate SIRT3 in ROT-induced rat and cellular PD models. Our work revealed that HNK and ICA confer protective effects by activating SIRT3 in ROT-induced cells and animal models. Our results suggest that the decrease of SIRT3 activity was attributed to the decrease of NAD⁺/NADH ratio, which is a consequence of the inhibition of complex I elicited by ROT. However, HNK and ICA treatment did not prevent the decline of NAD⁺/NADH ratio caused by ROT. We hypothesized that in the case of HNK, the activation of SIRT3 may be that the incubation of SIRT3 with HNK enhanced its affinity for NAD [17]. How ICA activates SIRT3 has yet to be further studied. It has been well demonstrated that NAD⁺ is an important cofactor of mitochondrial and cellular metabolism, and the decrease of NAD⁺/NADH ratio is closely related to the pathogenesis of a variety of age-related and metabolic disorders [25, 36]. Therefore, we further explored the role of regulation of NAD⁺/DADH in the ROT-induced neuronal cell damage in our PD models. It has been reported that NMN acts as a precursor of NAD⁺ and restores SIRT3 function [37–40]. Our work in the present study showed that NMN administration exhibited potent protection against DArgic neurons damage and rescued bioenergetic deficits, accompanied by increased SDH activity in the ROT-induced PD model.

Previous studies demonstrated that the physical interaction of SIRT3 with HNK may enhance SIRT3’s deacetylase activity, which can affect complex II activity directly [15, 30]. Here we revealed that SDHA was hyperacetylated and SDH activity was decreased in both cellular and rat models. Importantly, we demonstrated that pharmacological activation of SIRT3 by HNK and ICA reduced acetylation levels of SDHA in the brain tissues of rats administered with ROT and in ROT-treated MN9D cells, resulting in the improvement of SDH activity. It is worth emphasizing that aging is an important factor implicated in the pathogenesis of PD, and the deacetylation capacity of SIRT3 significantly decreases as one ages [41]. Therefore, a decrease in SIRT3 activity may lead to a decrease in SDH activity, and impaired SIRT3 function may be one of the key culprits entangled in the net of aging-mediated pathogenesis of PD.

The complex II is a key enzyme in the mitochondrial citric acid cycle and ETC and it plays a unique role because it represents a branching point of two essential mitochondrial pathways (mitochondrial OXPHOS and TAC) [42]. It is well known that one of the consequences of inhibiting mitochondrial respiration is a loss of ATP production and a decrease in bioenergy [43]. The inhibition of complex II not only affects the production of ATP by interfering with OXPHOS, but also leads to the accumulation of fumarate in the matrix, thus directly inhibiting the production of ATP [44]. Despite the important role of complex II in energy metabolism, less research has been done on the role of SDH-related energy deficits in neurodegeneration. In fact, loss of SDH activities has been well documented in human PD and animal models [8–11]. Therefore, fully exploring the regulation of ATP levels by complex II is crucial for understanding the pathological progression of neuronal cell death in PD and identifying new therapeutic targets for PD [30]. Our work in this study demonstrated that inhibition of complex II is involved in the deficit of ATP production and plays a key role in pathological progression of PD, revealing a novel mechanism by which mitochondrial damage contributes to PD pathogenesis. Importantly, our results showed that activation of SIRT3 by HNK and ICA significantly improved ATP generation and reduced ROT-induced cell death. We believe that neuronal cell death is closely linked to ATP level and bioenergetic improvement confers beneficial effects on neuronal survival. To further test this hypothesis, TZ was used to treat differentiated MN9D cells. TZ is an approved clinical drug for treatment of patients with benign prostatic hyperplasia, and it was recently discovered to enhance glycolysis and reduce human PD progression[28, 45]. Our results demonstrated that TZ improved neuronal cell survival in a ROT-induced cell model, which was closely related to rescue of ATP synthesis. These results further confirmed that intracellular ATP levels are critical for neuronal survival and that ATP supplementation can delay PD pathogenesis.

Our data suggest that the SIRT3 enzymatic activity is decreased due to the drop in the NAD⁺/NADH ratio as the result of the inhibition of complex I caused by ROT. The reduction of SIRT3 activity resulted in a decrease in complex II activity because of increased acetylation of SDHA, which could be reversed via activation of SIRT3 by HNK and ICA. To sum up our results, we propose a model illustrating the mechanism by which SIRT3 regulates the complex II activity and ATP production in PD pathology (Fig. 7). ROT directly inhibits mitochondrial complex I and results in inhibition of NADH to NAD⁺ conversion [46]. Our results demonstrated that SIRT3 activity was reduced due to the decrease in the NAD⁺/NADH ratio caused by ROT. Furthermore, activation of SIRT3 enhanced complex II activity and improved cellular ATP biosynthesis, thereby preventing bioenergetic exhaustion and conferring neuroprotective benefits in DArgic neurons (Fig. 7). These findings open new opportunities to develop a drug targeting the modulation of SIRT3-SDH axis to intervene and treat PD.

In conclusion, in the present study, we demonstrate that mitochondrial complex II is regulated by acetylation of SIRT3, which is inhibited as a result of the ROT-induced decrease in the NAD⁺/NADH ratio. Our findings suggest that there may be an NAD⁺/NADH-SIRT3-SDHA axis that regulates complex II activity and affects ATP synthesis, and that SIRT3 activation may provide a potential strategy for intervening in PD pathology.

DArgic: dopaminergic

DCPIP: dichlorophenol-indophenol

DCPIPH2: dichlorophenolindophenol

ETC: electron transport chain

HNK: honokiol

ICA: icariin

MRC: mitochondrial respiratory chain

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NaBu: sodium butyrate

NAD⁺: nicotinamide adenine dinucleotide

NMN: nicotinamide mononucleotide

PD: Parkinson’s disease

ROT: rotenone

SDH: succinate dehydrogenase

SDHA: succinate dehydrogenase subunit A

SIRT3: sirtuin3, SN: substantia nigra

TAC: tricarboxylic acid cycle

TZ: terazosin.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation (NSF) of China (Grant No. 81460548), Tutorial Studio Foundation of Education Department of Guizhou Province (Grant No. 99050), and Special Project for 2019 Academic New Seedling Cultivation and Innovation Exploration, Zunyi Medical University (Grant No. CK-1187-042)

Author Contribution

All authors contributed to the study conception and design. Erin McCarthy carried out the cell culture work. Erin McCarthy and Gerard O’Keeffe carried out the bioinformatics analysis. Martina Mazzocchi generated the stable cell lines. Aaron Barron carried out the Seahorse experiments. Noelia Morales-Prieto, Cathal McCarthy, Louise Collins, Aideen Sullivan and Gerard O’Keeffe supervised the work. All authors edited and co-wrote the manuscript.

Funding

Data Availability The data that support the fndings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

Ethics Approval The bioinformatics work was conducted using open-source transcriptome data. MN9D cells are commercially available and no ethics approval was required.

Consent to Participate Not applicable to this study.

Consent for Publication Not applicable to this study.

Competing Interests

The authors declare no competing interests.

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SIRT3-mediated deacetylation of SDHA rescues mitochondrial bioenergetics contributing to neuroprotection in rotenone-induced PD models

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Chemicals and reagents

2.2 Animals and experimental design

2.3 Rotarod test

2.4 Immunostaining of DArgic neuron

2.5 Cell culture

2.6 Differentiation of MN9D cells and drug treatment

2.7 Cell transfection

2.8 Evaluation of cell viability

2.9 Extraction of mitochondria

2.10 NAD⁺/NADH quantitative test

2.11 Activity of SIRT3

2.12 Measurement of mitochondrial complex II activity

2.13 Total cellular ATP content

2.14 Western blot analysis

2.15 Immunoprecipitation

2.16 Statistical analysis

3. Results

3.1 Neurotoxicity of ROT in differentiated DA MN9D cells

3.2 HNK and ICA decrease acetylation of SDHA through activation of SIRT3 in ROT-induced PD model

3.3 Activation of SIRT3 by HNK and ICA improves complex II activity and rescues ATP production

3.4 Activation of SIRT3 by supplementation of NAD improves the survival of ROT-treated cells

3.5 Protective effect of HNK and ICA on ROT-induced neurodegeneration

3.6 Activation of SIRT3 by HNK and ICA regulate SDH activity and improve ROT-induced bioenergetic decline in PD rat model

4. Discussion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1

SIRT3-mediated deacetylation of SDHA rescues mitochondrial bioenergetics contributing to neuroprotection in rotenone-induced PD models

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Chemicals and reagents

2.2 Animals and experimental design

2.3 Rotarod test

2.4 Immunostaining of DArgic neuron

2.5 Cell culture

2.6 Differentiation of MN9D cells and drug treatment

2.7 Cell transfection

2.8 Evaluation of cell viability

2.9 Extraction of mitochondria

2.10 NAD+/NADH quantitative test

2.11 Activity of SIRT3

2.12 Measurement of mitochondrial complex II activity

2.13 Total cellular ATP content

2.14 Western blot analysis

2.15 Immunoprecipitation

2.16 Statistical analysis

3. Results

3.1 Neurotoxicity of ROT in differentiated DA MN9D cells

3.2 HNK and ICA decrease acetylation of SDHA through activation of SIRT3 in ROT-induced PD model

3.3 Activation of SIRT3 by HNK and ICA improves complex II activity and rescues ATP production

3.4 Activation of SIRT3 by supplementation of NAD improves the survival of ROT-treated cells

3.5 Protective effect of HNK and ICA on ROT-induced neurodegeneration

3.6 Activation of SIRT3 by HNK and ICA regulate SDH activity and improve ROT-induced bioenergetic decline in PD rat model

4. Discussion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1

2.10 NAD⁺/NADH quantitative test