Mitochondrial dysfunction and apoptosis are attenuated through activation of AMPK/ GSK-3β/PP2A pathway in Parkinson’s disease

Jianhua Su, Junhua Zhang, Rui Bao, Changbo Xia, Yu Zhang, Zhujun Zhu, Qi Lv, Yingjie Qi, Jianqin Xue

PII: S0014-2999(21)00355-1
DOI: Reference: EJP 174202

To appear in: European Journal of Pharmacology

Received Date: 22 February 2021
Revised Date: 19 May 2021
Accepted Date: 21 May 2021

Please cite this article as: Su, J., Zhang, J., Bao, R., Xia, C., Zhang, Y., Zhu, Z., Lv, Q., Qi, Y., Xue, J., Mitochondrial dysfunction and apoptosis are attenuated through activation of AMPK/GSK-3β/ PP2A pathway in Parkinson’s disease, European Journal of Pharmacology, j.ejphar.2021.174202.

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CRediT authorship contribution statement

Jianhua Su: Conceptualization, Methodology, Writing-Original draft. Junhua Zhang: Methodology, Data collection. Rui Bao and Changbo Xia: Data collection and preparation. Yu Zhang and Zhujun Zhu: Visualization, Investigation. Qi Lv and Yingjie Qi: Supervision, Software. Jianqin Xue: Validation, Writing-Reviewing.

1 Mitochondrial dysfunction and apoptosis are attenuated through
2 activation of AMPK/GSK-3β/PP2A pathway in Parkinson’s
3 disease

4 Jianhua Su 1, Junhua Zhang 1, Rui Bao 2, Changbo Xia 3, Yu Zhang 4, Zhujun Zhu 4,

5 Qi Lv 4, Yingjie Qi 1, Jianqin Xue 4, *

7 1 Neurology Department, Jintan Hospital affiliated to Jiangsu University, Changzhou

8 213200, Jiangsu, China.

9 2 School of pharmacy, Jiangsu University, Zhenjiang 212013, Jiangsu, China.

10 3 Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese

11 Medicine, Nanjing 210023, Jiangsu, China.

12 4 Department of Rehabilitation Medicine, Jintan Hospital affiliated to Jiangsu

13 University, Changzhou 213200, Jiangsu, China.


15 * Corresponding author: Jianqin Xue. Address: No.16, Nanmen Street, Jintan District,

16 Department of Rehabilitation Medicine, Jintan Hospital affiliated to Jiangsu

17 University, Changzhou 213200, Jiangsu, China. Tel.: +86-15190532868. E-mail

18 address: [email protected]


20 Abstract

21 Parkinson’s disease (PD) is a common neurological disorder worldwide, characterized

22 by loss of dopaminergic neurons and decrease of dopamine content. Mitochondria

23 plays an important role in the development of PD. Adenosine

24 5‘-monophosphate-activated protein kinase (AMPK), glycogen synthase kinase 3

25 (GSK-3β) and protein phosphatase 2A (PP2A) are all key proteins that regulate

26 mitochondrial metabolism and apoptosis, and they are involved in a variety of

27 neurodegenerative diseases. Here, we aimed to explore the involvement of

28 mitochondrial dysfunction and apoptosis in 1-methyl-4-phenyl-1, 2, 3,

29 6-tetrahydropyridine hydrochloride (MPTP)-induced PD mice and MPP+

30 iodide-induced PC12 cells. MPTP-induced mice were subjected to behavioral testing

31 to assess PD-like behaviors. Various molecular biological techniques including

32 ELISA, Western blot, TUNEL assay, flow cytometry, and the important instruments

33 Seahorse XF24 Extracellular and high performance liquid chromatography (HPLC),

34 were used to identify the underlying molecular events of mitochondria. Treatment

35 with the AMPK activator GSK621 dramatically ameliorated PD by increasing the

36 levels of dopamine and rescuing the loss of dopaminergic neurons, which is

37 dependent on the mitochondrial pathway. Moreover, regulation of

38 AMPK/GSK-3β/PP2A pathway-related proteins by GSK621 was partially inhibited

39 the development of PD, suggesting a negative feedback loop exists between AMPK

40 action and mitochondrial dysfunction-mediated apoptosis. Our data preliminarily

41 indicated that mitochondrial dysfunction and apoptosis in the pathogenesis of PD

42 might be mediated by AMPK/GSK-3β/PP2A pathway action, which might be a

43 promising new option for future therapy of PD.


45 Keywords: Parkinson’s disease; Mitochondrial dysfunction; Apoptosis;

46 AMPK/GSK-3β/PP2A pathway; MPTP/MPP+

48 1. Introduction

49 Parkinson’s disease (PD) is a common neurological disorder worldwide (Lau and

50 Breteler, 2006). The main pathological changes of PD are progressive loss of

51 dopaminergic neurons in the substantia nigra pars compacta (SNpc) and decrease of

52 dopamine content in striatum (Choi et al., 2017). However, to date, the pathogenesis

53 of PD has not been clarified. There are many functional analyses of familial PD

54 causative genes that suggest the loss of dopaminergic neurons is caused by multiple

55 pathological mechanisms, such as mitochondrial dysfunction, oxidative stress,

56 α-synuclein accumulation, protein hydrolysis damage, autophagy and the ubiquitin

57 proteasome pathway (Kalia and Lang, 2015).

58 Recent studies have indicated that mitochondrial dysfunction is a key factor in

59 the pathophysiology of PD (Yamaguchi et al., 2020). The whole process of central

60 nervous system injury is accompanied by massive neuronal apoptosis, and

61 mitochondria also attach importance to the development of apoptosis (Thornton and

62 Hagberg, 2015). The reduction in mitochondrial membrane potential leads to the

63 opening of permeability transition pore of mitochondrial membrane following the

64 release of cytochrome C, which is implicated as a critical effector in apoptotic GT1-7

65 neural cells (Rego et al., 2001). Therefore, study of the mechanism of

66 mitochondrial-mediated apoptosis in central nervous system diseases is particularly

67 important, which will help to reveal key regulatory targets of mitochondrial

68 dysfunction and provide new ideas for the prevention and treatment of PD.

69 Many mechanisms have been reported to mediate mitochondrial apoptosis in

70 neurologic diseases (Thornton and Hagberg, 2015). 5‘-monophosphate-activated

71 protein kinase (AMPK) is a crucial cellular energy sensor that promotes the level of

72 ATP by increasing the activity or expression of proteins involved in catabolism and

73 conserving ATP through switching off some biosynthetic pathways (Hardie et al.,

74 2012). AMPK is also able to regulate metabolic energy balance throughout the body.

75 The activation of AMPK shifts the energy balance from a negative state to neutral

76 state and plays a role in regulating mitochondrial biogenesis and reducing reactive

77 oxygen species production (ROS) (Bayliss and Andrews, 2013). AMPK kinase was

78 activated by metformin to confer neuroprotection to PD mice, possibly due to

79 enhancing autophagy and mitochondrial ROS clearance (Lu et al., 2016).

80 Mitochondrial dysfunction and apoptosis were attenuated by κ-opioid receptor

81 activation via AMPK/Glycogen synthase kinase 3 (GSK-3β) pathway following

82 myocardial ischemia and reperfusion (Tian et al., 2019). GSK-3β dysregulation plays

83 an important role in the pathogenesis of central nervous system disorders,

84 encompassing both neuroinflammation and neurodegenerative diseases (Golpich et al.,

85 2015). Protein phosphatase 2A (PP2A), as a downstream factor of GSK-3β, is mainly

86 involved in mitochondrial apoptosis (Lin et al., 2007). The direct action of PP2A

87 contributed to the pathogenesis of Alzheimer’s disease (Wei et al., 2020). The

88 assembly and activity of PP2A were regulated by reversible methylation of the C

89 subunit α-Synuclein, which aggregated in Lewy bodies of PD and dementia (Park et

90 al., 2016). Based on the above evidence, we speculated that activation of

91 AMPK/GSK-3β/PP2A signaling pathway could delay the development of PD.

92 In the present study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

93 (MPTP)-induced PD mice model was established to explore the protective effects on

94 mitochondrial dysfunction and apoptosis through activation of AMPK/GSK-3β/PP2A

95 pathway in PD. And an in vitro PD model was constructed using retinoic acid-induced

96 PC12 cells to further investigate this mechanism.

97 2. Materials and methods

98 2.1. Animals

99 Thirty male C57/BL6 mice (10-week-old, 25~30 g) were obtained from

100 Changzhou Cavens experimental animal Co., Ltd (Jiangsu, China). Before the start of

101 the experiment, mice were allowed to acclimatize for one week at a suitable condition

102 (22 ± 2℃, 60% humidity, and 12/12 h light/dark cycle), with food and water ad

103 libitum.

104 2.2. Animal experimental design

105 All mice were randomly divided into 3 groups (10 mice per group), including (1)

106 Control group: the mice were given equivalent volume of normal saline for three

107 weeks; (2) MPTP group: the mice were intraperitoneally injected with 30 mg/kg

108 MPTP twice a week for three weeks; (3) MPTP+GSK621 group: the mice were first

109 intraperitoneally injected with 30 mg/kg MPTP twice a week, and one week later,

110 mice were given with 30 mg/kg GSK621 once a day, accompanied with 30 mg/kg

111 MPTP twice a week until the end of experiment.

112 In the fourth week, mice were anesthetized with secobarbital (50 mg/kg) and

113 killed by cervical dislocation. Next, the brains of mice were immediately stripped and

114 cut sagittally into two identical hemispheres. One hemisphere was fixed in 4%

115 formaldehyde, and the section of SNpc was used for further histopathology studies.

116 SNpc and striatum were quickly separated from another hemisphere and stored at -80 ℃

117 for further use as previously reported (Lin et al., 2019). The study guideline of animal

118 experiment was confirmed by the Institutional Animal Care and Use Committee of

119 Jiangsu University (Authorization number: UJS-IACUC-2020032102, 21 March

120 2020).

121 2.3. Behavioral analyses

122 Behavior analyses such as the pole test, traction test and rotarod test were carried

123 out once a week to detect the coordination of body movement. For the pole test, mice

124 were placed on the top of a vertical wooden rough-surfaced pole (height 50 cm,

125 diameter 1 cm) to evaluate motor balance and coordination of mice as previously

126 described (Yoon and Lee, 2014).

127 For traction test, all mice were suspended on the horizontal wire with both

128 forelimbs. The score was evaluated according to the following criteria: if the mice

129 grasped the wire with both hind limbs, the score was 3 points; if the mice seized the

130 wire just with the only one hind limb, the score was 2 points; if two hind limb of mice

131 both could not grasp the wire, the score was 1 point; Mice were scored 0 point for

132 falling.

133 For the rotarod test, mice were accustomed to the rotarod (5 min with a fixed

134 speed of 12 rpm) for 5 days before the initiation of MPTP treatment. After the mice

135 were placed on the rod at 20 rpm for 5 min, the latency time (each mouse could stay

136 on the rod at test speed) were recorded. The tester was blinded to all treatment groups

137 for each behavioral testing.

138 2.4. Immunohistochemistry

139 Immunohistochemistry method was described in the previous publication

140 (Hasegawa et al., 2016). In short, brain tissues were fixed with 4% paraformaldehyde

141 in phosphate buffer (pH 7.4) overnight and cryoprotected by soaking in 20% sucrose

142 overnight. Then, the frozen tissues were cut into 30 μm thick sections. The sections

143 were incubated overnight at 4°C with appropriate anti-TH polyclonal antibody

144 (1:1000; No. SAB4200697, Sigma). Then a goat anti-rabbit secondary antibody and

145 diaminobenzidine (ZLI-9017, ZSGB-BIO, China) were used to assess

146 immunoreactivity. The brain slices were observed and stereoscopic counting with

147 Stereo-Investigator software (Lu et al., 2012; Tang et al., 2020). The total number of

148 tyrosine hydroxylase (TH) positive neurons in entire extent of SNc from 3 mice in

149 each group was measured as follows: 21 consecutive slices were collected and

150 numbered (1, 2, 3, … 21), and then the first, seventh, thirteenth and nineteenth slices

151 (at intervals of six) was counted. Images were observed and photos were taken under

152 a microscope (at ×10 magnification, ×20 magnification, respectively).

153 2.5. High performance liquid chromatography (HPLC) analysis

154 Determination of dopamine (DA) and 5-hydroxytryptamine (5-HT) levels were

155 performed by a HPLC system coupled with a Waters 2465 electrochemical detector

156 (Waters, Milford, USA). Dissected striatal tissues were homogenized with 0.1M

157 HClO4 containing 0.01% ethylene diamine tetraacetic acid (EDTA) buffer and

158 centrifuged at 15,000 g for 25 min to precipitate proteins. Then, the supernatant was

159 filtered through 0.22 μm filter membrane. 10 μL of sample was injected into

160 auto-sampler at 4℃ and eluted through C18 column (2.2 μm, 2.1×100 mm, Waters,

161 Milford, USA). The mobile phase consisted 90 mM NaH2PO4, 50 mM citrate, 1.7 mM

162 1-octanesulfonic acid. Concentrations of DA and 5-HT were expressed as ng/mg

163 tissue weight.

164 2.6. Cell culture and treatments

165 The rat pheochromocytoma cell line PC12 cells were obtained from American

166 Type Culture Collection and maintained as previously described (Cantelmo et al.,

167 2020). Briefly, PC12 cells were cultured in Dulbecco’s modified Eagle medium

168 (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 5% foetal bovine

169 serum (FBS, GIBCO, Grand Island, NY, USA), 10% horse serum, and 1% penicillin

170 and 1% streptomycin, which was replaced every 48 h. To harvest PC12 cells, after

171 cultures were washed with PBS buffer, PC12 cells were detached with EDTA solution

172 containing 0.25% trypsin and reseeded to 5×105 cells/ml required for each assay.
173 2.7. Establishment of MPP+ -induced PC12 cells and grouping

174 MPP+ is the active metabolite of MPTP, which is recognized as an environmental

175 cause of PD injury, and its compounds are widely used in cell culture and rodent

176 models of PD (Zhu et al., 2012). After cell differentiation and maturation, PC12 cells

177 were incubated with different doses of MPP+ iodide (0, 50, 100, 200, 400, 800 µM)

178 for 24 h, and then the cell viability was detected by MTT assay to select the

179 appropriate dosage for cell modelling. PC12 cells were also treated with different

180 dosages of GSK621 (0, 10, 20, 30, 40, 50 μM) in the absence of MPP+ iodide to

181 confirm appropriate usage as a positive control. Next, PC12 cells were divided into 3

182 groups, namely, NC group, MPP+ group, and MPP++GSK621 group. PC12 cells in

183 NC group were treated without MPP+ iodide; PC12 cells in MPP+ group were treated

184 with 200 µM MPP+ iodide; PC12 cells in MPP++GSK621 were incubated with 200

185 µM MPP+ iodide for 24 h, and then GSK621 was added into cell culture for 24 h.

186 After administration, PC12 cells in various groups were collected for next

187 examination.

188 2.8. Detection of ATP and pyruvate

189 The contents of ATP and pyruvate were detected by commercial kits (ATP, No.

190 BC0305, Solarbio; pyruvate, No. BC2205, Solarbio) according to their instructions,

191 respectively. In brief, 0.02 g tissue was added to 0.2 ml of extraction solution,

192 homogenised and centrifuged at 8000 g for 10 min at 4 ℃. The supernatant was added

193 to 100 μL of chloroform and shaken well, centrifuged at 10000g for 3min at 4°C. The

194 sample was mixed with the working solution and the absorbance value A1 was

195 measured at 340 nm. After the reaction in a water bath at 37°C for 3 min, the

196 absorbance value A2 was measured and the ATP content of the sample was calculated

197 197

from the ATP standard solution.

ATP content (

μmol g

) = 0.625 ∗

△ A sample
△ A standard

198 Note: ΔA sample = A2 sample – A1 sample, ΔA standard = A2 standard – A1 standard,

199 W: sample mass

200 2.9. TUNEL assay

201 Apoptosis of SNpc or PC12 cells was evaluated by TUNEL assay according to

202 the instruction of an in-situ cell death detection kit (Roche Molecular Biochemicals,

203 Mannheim, Germany) using an inverted fluorescence microscopy (U-RX-T/IX73,

204 OLYMPUS, Japan). The apoptotic index was expressed as the number of positively

205 stained apoptotic cells/the total number of cells.

206 2.10. Flow cytometry

207 The apoptosis of PC12 cells was also detected by flow cytometry. After drug

208 administration, PC12 cells were harvested and washed with PBS. Next, fluorescein

209 isothiocyanate-annexin V and propidium iodide were added to PC12 cells and

210 incubated in the dark condition for 15 min at room temperature. The fluorescence

211 signals were measured by a flow cytometer (lexcitation = 488 nm; lemission = 530

212 nm, Guava Easycyte 8, Millipore).

213 2.11. Measurement of mitochondrial membrane potential

214 5,5,6,6-tetrachloro-1,1,3,3- tetraethylbenzimidazolylcarbocyanine iodide (JC-1),

215 a fluorescence probe is used to monitored the mitochondrial membrane potential by

216 flow cytometry analysis. PC12 cells were incubated with JC-1 dye (Enzo Life

217 Sciences, USA) for 30 min, and then washed twice in PBS. Relative fluorescence

218 intensity was monitored by flow cytometry (Guava Easycyte 8, Millipore) with

219 excitations at 530 nm and emissions at 585 nm.

220 2.12. Oxygen consumption rate (OCR) assay

221 Cultured PC12 cells were rinsed once with DMEM containing 0.04 mM phenol

222 red (pH=7.4) and pre-incubated in DMEM containing 0.04 mM phenol red and 2.5

223 mM D-glucose (pH=7.4). Subsequently, PC12 cell cultures were placed in the

224 Seahorse XFe96 Flux Analyzer and the following operation process was: a calibration

225 period (12 min), 3 cycles of mixing (1 min) and measuring (3 min) followed by

226 consecutive injections of 250 μM L-glutamate or Seahorse medium, 0.5 μM carbonyl

227 cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and a mixture of 0.5 μM

228 antimycin A and 0.5 μM rotenone. A sequence of mixing (1 min), waiting (1 min) and

229 measuring (3 min) was performed three times between each injection. The OCR of

230 PC12 cells was monitored in real time using the Seahorse extracellular flux analysis

231 system (Agilent Technologies, USA).

232 2.13. Western blot analysis

233 Tissues of SNpc or PC12 cells were homogenized with RIPA lysis buffer

234 containing 1mM PMSF. Lysates were centrifuged at 4 ℃, 12000 g for 10 min.

235 Subsequently, the supernatant was collected to measure the concentration of total

236 proteins using a Nanodrop 2000 instrument (Thermo Fisher Scientific, America).

237 Proteins were separated with 8-12% of sodium dodecyl sulfate-polyacrylamide

238 (SDS-PAGE) and then transferred onto polyvinylidene fluoride membrane. The bands

239 were visualized by the first antibodies as follows: AMPK antibody (1:1000, No.

240 AF6423, Affinity), phosphor-AMP-actived protein kinase antibody (p-AMPK, 1:1000,

241 No. AF3423, Affinity), GSK-3β antibody (1:1000, No. 9832, CST), p-GSK-3β

242 antibody (1:1000, No. 9336, CST), PP2A antibody (1:1000, No. DF8594, Affinity),

243 Cleaved Caspase 3 antibody (1:1000, No. 9661, CST), Caspase 3 antibody (1:1000,

244 No. 9662, CST). The protein intensities were normalized to the internal standard

245 signal intensity (β-actin) using the ECL Plus Assay Kit (Affinity, K002). All

246 experiments were repeated three times.

247 2.14. Statistical analysis

248 Data are expressed as mean ± standard error of the mean (S.E.M.). Statistical

249 differences were determined by one-way analysis of variance (ANOVA) followed by

250 Dunnett test or two-way ANOVA followed by Tukey multiple comparisons test, where

251 P<0.05 was considered statistically significant. Data were analyzed using Graphpad

252 Prism 7.0 (Graphpad Software, Inc., San Diego, CA).

253 3. Results

254 3.1. GSK621 effectively improved motor impairment and neuronal damage of

255 MPTP-induced PD mice

256 To investigate whether AMPK activation improved the neuroprotective effects on

257 PD, we determined the effect of AMPK activator GSK621 on MPTP-induced motor

258 deficits by testing performance in the behavior analysis, including the pole test,

259 traction test, and rotarod test (Fig. 1A-1C). Mice were evaluated every week after

260 MPTP injection by measuring motor time. Traction test was utilized to measure

261 muscle strength and equilibrium. Traction scores were significantly lower in

262 MPTP-treated mice, while GSK621 increased traction scores. GSK621 also

263 significantly reduced the behavioral deficits elicited by MPTP treatment as measured

264 by accelerating latency time and lessening T-turn and T-total time. In other words,

265 GSK621 administration relieved MPTP-induced motor impairment, which also

266 suggested that AMPK activation played a vital role in the improvement of PD.

267 To investigate whether lower activity of AMPK might be responsible for the

268 more severe pathology in brains of MTPT-induced mice, we explored changes of

269 dopaminergic neurons in the absence or presence of GSK621 after MPTP injection.

270 Firstly, the levels of DA and 5-HT were detected by HPLC analysis (Fig. 1D and 1E).

271 Compared with Control group, MTPT induction obviously decreased the content of

272 DA and 5-HT, while GSK621 reversed these changes after MPTP treatment. In

273 addition, after treatment with MPTP, there were remarkably greater losses of

274 TH-positive cells in the SNpc than that in Control group (Fig.1F). However, due to

275 the action of AMPK by GSK621, the number of TH-positive cells in MPTP +

276 GSK621 group was higher than that in MPTP group. These results showed that

277 GSK621 highly increased AMPK activity, which effectively attenuated

278 MPTP-induced neuronal damage.

279 3.2. GSK621 attenuated mitochondrial damage and apoptosis in MPTP-induced PD

280 mice

281 As shown in Fig. 2A and 2B, in MPTP-treated mice, the level of ATP was

282 significantly decreased and pyruvate production was obviously increased due to the

283 disorder of mitochondrial function, compared with Control group. In contrast to

284 MPTP group, GSK621 apparently enhanced the level of ATP and reduced pyruvate

285 production.

286 After MPTP treatment, TUNEL assay was used to observe apoptosis of SNpc

287 under a fluorescence microscope (Fig. 2C). The green apoptotic signal in MPTP group

288 was much higher than that in Control group, while less apoptotic cells were evident in

289 MPTP+GSK621 group, that is, GSK621 weakened cell apoptosis of SNpc.

290 3.3. Effects of AMPK/GSK-3β/PP2A pathway on MPTP-induced PD mice

291 Considering that GSK621 alleviated the pathological features of PD, we

292 speculated that AMPK activation might regulate mitochondrial disorder and apoptosis

293 in PD. Therefore, we examined how GSK621 affected mitochondrial dysfunction and

294 apoptosis after AMPK activation by western blot analysis. The expressions of

295 AMPK/GSK-3β/PP2A pathway-related proteins were as shown in Fig. 3. Compared

296 with Control group, the expressions of AMPK, p-AMPK, GSK-3β, p-GSK-3β were

297 obviously decreased, and PP2A level was significantly increased by MPTP treatment,

298 which further significantly enhanced the expression of apoptotic factor Caspase 3.

299 However, the trends were reversed by GSK621. These results showed that

300 mitochondrial dysfunction and apoptosis might be attenuated through the activation of

301 AMPK/GSK-3β/PP2A pathway.

302 3.4. GSK621 protected mitochondrial function in MPP+-induced PC12 cells

303 In order to better clarify the protective effects of GSK621 on mitochondrial

304 disorder and apoptosis in vitro, we constructed MPP+ -induced PC12 cell model.

305 Based on the study of the effects of MPP+ iodide at different dosages on cell viability

306 (Fig. 4A), we found that cell viability was significantly reduced at the dosage of 100

307 µM MPP+ iodide, while cell viability of other groups (≤50 µM MPP+ iodide) had no

308 statistical difference. In particular, the cell viability significantly decreased by 40% at

309 the dosage of 200 µM MPP+ iodide, which was used for modeling. Then, we studied

310 the effects of GSK621 at different dosages (0, 10, 20, 30, 40, 50 µM) on cell viability,

311 respectively (Fig. 4B). The results show that the cell viability of PC12 cells was

312 decreased with 40 µM GSK621, so we selected 30 µM GSK621 as an appropriate

313 dosage used in further study. After PC12 cell were treated with MPP+, we found

314 GSK621 administration improved PC12 cell viability (Fig. 4C).

315 As shown in Fig. 4D and 4E, the level of ATP was much lower and pyruvate

316 production was much higher in MPP+ group than those in NC group, respectively.

317 Nevertheless, GSK621 treated PC12 cells showed an apparently opposite trend to

318 MPP+ group.

319 The mitochondrial membrane potential of PC12 cells was observed by JC-1

320 staining (Fig. 4F). Compared with NC group, the ratio of relative red/green

321 fluorescence in MPP+ group was remarkably decreased. However, the ratio of relative

322 red/green fluorescence in MPP++GSK621 group was much higher than that in MPP+

323 group, which showed that GSK621 effectively increased mitochondrial membrane

324 potential.

325 In order to further test the effect of AMPK activation on mitochondrial energy

326 metabolism, Seahorse instrument was used to carry out extracellular flux analysis of

327 OCR profiles in MPP+ -induced PC12 cells (Fig. 5). MPP+ -induced PC cells

328 displayed a reduced OCR, while GSK621 enhanced it on the whole (Fig. 5B).

329 Additional signs of mitochondrial dysfunction in PC12 cells were detected following

330 MPP+ treatment (Fig. 5C-5G). The performance of the electron transport chain was

331 reduced in MPP+ group, which displayed an impaired spare respiratory capacity,

332 suggesting that mitochondria could not cope with an increased energy demand.

333 However, GSK621 significantly recovered impaired spare respiratory capacity. There

334 were only slight changes of ATP production, basal respiration and proton leak

335 between MPP+ group and MPP++GSK621 group. In contrast, in order to provide a

336 summary profile of the combination of the spare respiratory capacity, ATP-coupled,

337 proton leak and non-mitochondrial OCR in a single parameter, we found GSK621

338 restored the mitochondrial energy metabolism in MPP+ -induced PC12 cells.

339 3.5. GSK621 inhibited cell apoptosis in MPP+ -induced PC12 cells

340 To verify the effect of AMPK activation on PC12 cell apoptosis after MPP+

341 iodide treatment, TUNEL staining was used to observe the percentage of apoptotic

342 cells. More apoptotic cells (fluorescing green signal) were evident in MPP+ group

343 than that in MPP++GSK621 group under a fluorescence microscope (Fig. 6A).

344 Secondary assessment of apoptosis was performed using Annexin V/PI

345 double-staining to distinguish viable, necrotic, and apoptotic cells using flow

346 cytometry (Fig. 6B). Compared to NC group, PC12 cells showed obvious apoptosis in

347 MPP+ group. There were significantly fewer (early and late) apoptotic cells in

348 MPP++GSK621 group than that in MPP+ group. Thus, GSK621 inhibited PC12 cell

349 apoptosis caused by MPP+ intervention.

350 3.6. Effects of AMPK/GSK-3β/PP2A pathway on MPP+ -induced PC12 cells

351 To further explore AMPK-related regulation mechanism, we measured the

352 protein expressions of AMPK/GSK-3β/PP2A pathway in vitro (Fig. 7). Compared

353 with NC group, due to MPP+ iodide treatment, AMPK expression and

354 phosphorylation were significantly decreased in PC12 cell model, which was further

355 used to elucidate the induction of energy metabolism and apoptosis. Then, GSK-3β

356 expression and phosphorylation in MPP+ group were significantly lower than those in

357 NC group, which promoted PP2A mediated apoptosis protein expression, such as

358 Caspase 3 and Cleaved Caspase 3. However, compared to MPP+ group, GSK621 also

359 reversed the expression levels of these protein, which was almost similar with the fact

360 of animal experiments, further suggesting GSK621 activated AMPK/GSK-3β/PP2A

361 pathway to regulate mitochondrial dysfunction and apoptosis in PD.

362 4. Discussion

363 PD is a neurodegenerative disorder caused by the progressive loss of

364 dopaminergic neurons in the midbrain, and mitochondrial dysfunction and apoptosis

365 are involved in the pathogenesis of PD (Yamaguchi et al., 2020). However, the

366 mechanisms that impair homeostatic responses to mitochondrial dysfunction and lead

367 to apoptosis remain unclear. AMPK exerts potential therapeutic effects in

368 degenerative diseases, including cerebral ischemic/reperfusion injury and Alzheimer’s

369 disease (Du et al., 2015; Shen et al., 2017). There are accumulating evidences that

370 AMPK plays a protective role in the progression of PD (Choi et al., 2010; Chen et al.,

371 2020). Thus, our study explored the potential mechanism of inhibiting mitochondrial

372 disorder and apoptosis in PD, possibly through the activation of AMPK-mediated

373 signaling pathway, which was further verified by AMPK agonist GSK621.

374 MPTP-induced PD model exhibited locomotion deficits reproducing the

375 pathological features of PD, due to dopaminergic neuron loss and subsequent

376 dopamine depletion (Yao et al., 2012). In our study, the behavior tests between normal

377 and MPTP-treated mice showed significant differences on several parameters of gait

378 and posture. These parameters included the reduced the support base and swinging

379 speed, and increased total ambulation time, which consistently pointed to the

380 emergence of movement obstacles in MPTP-treated mice, while GSK621 treatment

381 restored motor dysfunction. At the same time, GSK621 rescued the number of TH

382 positive neurons. Overall, GSK621 attenuated neuron loss in the SNpc and partially

383 restored behavioral impairments caused by MPTP intoxication, accompanied by the

384 activation of AMPK.

385 Mitochondria play an important role in not only energy metabolism but also

386 regulation of cell apoptosis in PD (Mounsey and Teismann, 2010; Ruberg et al., 2010).

387 Therefore, mitochondria have emerged as a potential target for treating PD (Bagnoli et

388 al., 2020; Thomas et al., 2020). AMPK activation mitigated dopaminergic dysfunction

389 and mitochondrial abnormalities in PD (Ng et al., 2012), which was consistent with

390 the results that GSK621 restored the reduction of DA and 5-HT levels.

391 Then, the effect of GSK621 on mitochondrial function was evaluated both in

392 vivo and in vitro. Treatment with GSK621 effectively alleviated mitochondrial

393 disorder and cell apoptosis in MPTP-induced PD mice and MPP+ -induced PC12 cells.

394 The energy in brain mainly comes from the oxidative phosphorylation of

395 mitochondria, so neurons are more sensitive to ATP synthesis damage. The inhibition

396 of mitochondrial complex I and II activities could lead to mitochondrial ATP damage

397 and abnormal function of intracellular ATP dependent enzymes, resulting in abnormal

398 energy metabolism and cell death (Zhu et al., 2019). Measurement of mitochondrial

399 dysfunction was confirmed that GSK621 stabilized energy metabolism, such as the

400 increase of ATP production and decrease of pyruvate production in vivo. In particular,

401 in vitro experiments, after AMPK activation, MPP+ -induced PC12 cells displayed a

402 significant increase of mitochondrial membrane potential, as well as the increase of

403 ATP and the decrease of pyruvate level when compared with those in MPP+ group.

404 We also measured OCR in living PC12 cells utilizing the Seahorse XF24 Extracellular

405 as previously described, which made it possible to quantify mitochondrial respiration

406 and glycolysis directly (Bagnoli et al., 2020). We found that the OCR in

407 MPP++GSK621 group was effectively enhanced compared with MPP+ group,

408 suggesting that mitochondrial disorder and respiration were improved by AMPK

409 activation.

410 We confirmed that the activation of AMPK could effectively alleviate

411 mitochondrial disorder and apoptosis in PD, but its downstream pathway is still

412 unclear. It has been reported that AMPK/GSK-3β pathway highly attenuated

413 mitochondrial dysfunction and apoptosis (Tian et al., 2019). Furthermore, targeting

414 phosphatase is considered as the next generation of a modification therapy for PD,

415 such as PP2A (Braithwaite et al., 2012; Clarka and Ohlmeyerb, 2019). Emerging

416 evidences have suggested that PPA2 is regarded as a mitochondrial metabolic gene

417 regulating mitochondrial functions and cellular longevity (Muid et al., 2019). In

418 addition, the mechanism of PP2A activation contributed to JS-K induced

419 caspase-dependent apoptosis in human hepatocellular carcinoma cells (Ling et al.,

420 2018). Therefore, we speculated that AMPK/GSK-3β/PP2A pathway might attach

421 importance to regulate mitochondrial disorder and apoptosis in PD. In order to verify

422 this hypothesis, we further investigated the expression levels and activities of AMPK,

423 GSK3β and PP2A both in vivo and in vitro. We found that after MPTP/MPP+

424 administration, the expression of AMPK and GSK-3β and their phosphorylation were

425 significantly inhibited, respectively, and PP2A expression was further promoted.

426 However, GSK621 not only enhanced the activity of AMPK, but also increased its

427 expression level, especially the phosphorylation of AMPK (p-AMPK), and

428 subsequently regulated AMPK/GSK-3β/PP2A pathway. Previous study has shown

429 that mitochondrial damage in hippocampal neurons of rats was closely related with

430 Caspase 3 protein expression, which further led to cell death (Feng et al., 2018).

431 Treatment with GSK621 reduced the apoptosis rate and the expression of active

432 Caspase 3 both in MPTP-induced PD mice and MPP+ -induced PC12 cells.

433 However, there are still some limitations in our study. Even though MPTP is a

434 common model of PD which causes death of dopaminergic neuron, it seems to differ

435 from PD. Therefore, we just offered a possibility that the inhibition of mitochondria

436 and apoptosis was able to provide neuroprotection in PD, which still need further

437 research. Besides, AMPK also plays other important roles in the progression of PD.

438 For example, modulating the catalytic activity of AMPK had neuroprotective effects

439 against α-synuclein toxicity (Bobela et al., 2017). Metformin prevented dopaminergic

440 neuron death in MPTP/P-induced PD mice via enhancement of AMPK-mediated

441 autophagy and mitochondrial ROS clearance (Lu et al., 2016). The above evidences

442 are something that our research has not covered.

443 5. Conclusion

444 In conclusion, GSK621 alleviated the reduction of mitochondrial membrane

445 potential and OCR, as well as ATP production, suggesting that the mitochondrial

446 mechanism is vital for treating PD. At the same time, after activating AMPK-mediated

447 pathway, GSK621 significantly reduced the apoptosis rate and the expression of

448 apoptosis related proteins. Based on these above studies, we demonstrated for the first

449 time a link between the regulation of mitochondria and apoptosis mediated

450 neuroprotection in PD, possibly through AMPK/GSK-3β/PP2A pathway.

451 Acknowledgements

452 Research project was funded by Changzhou Science and Technology Bureau,

453 Jiangsu Province (No. WZ201043) and the 18th batch of science and technology

454 projects in Changzhou (No. CE20185003).

455 Conflict of interest

456 The authors have no conflicts of interest to declare.

457 CRediT authorship contribution statement

458 Jianhua Su: Conceptualization, Methodology, Writing-Original draft. Junhua

459 Zhang: Methodology, Data collection. Rui Bao and Changbo Xia: Data collection and

460 preparation. Yu Zhang and Zhujun Zhu: Visualization, Investigation. Qi Lv and

461 Yingjie Qi: Supervision, Software. Jianqin Xue: Validation, Writing-Reviewing.

462 Data availability

463 All data included in this study are available upon request by contact with the

464 464

465 465

corresponding author.

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593 Figure legends

594 Fig. 1. GSK621 improved motor behavior and neuronal damage in MPTP-induced PD mice.

595 (A) Latency time on rod was measured by rotarod test (n=8). (B) Traction score was analyzed by

596 traction test (n=8). (C) Latency time on rod was measured by rotarod test (n=8). (D) The

597 quantification of DA in striatum was analyzed by HPLC (n=6). (E) The quantification of 5-HT in

598 striatum was analyzed by HPLC (n=6). (F) Immunohistochemical staining and stereological

599 counts of TH positive neurons in the SNpc (at ×200 magnification and ×100 magnification, n=3).

600 Values are expressed as mean ± SEM. Statistical differences were determined by one-way analysis

601 of variance followed by Dunnett multiple comparisons test. *P<0.05 and **P<0.01 versus Control

602 602

603 603

group; &P<0.05 and &&P<0.01 versus MPTP group.

604 Fig. 2. GSK621 attenuated mitochondrial damage and apoptosis in MPTP-induced PD mice.

605 (A) ATP production in the SNpc of MPTP-induced PD mice (n=5). (B) Pyruvate production in

606 the SNpc of MPTP-induced PD mice (n=5). (C) TUNEL assay was used to observe apoptosis of

607 SNpc under a fluorescence microscope (n=3, Bar: 50μm). Values are expressed as mean ± SEM.

608 Values are expressed as mean ± SEM. Statistical differences were determined by one-way analysis

609 of variance followed by Dunnett multiple comparisons test. *P<0.05 and **P<0.01 versus Control

610 610

611 611

group; &P<0.05 and &&P<0.01 versus MPTP group.

612 Fig. 3. Effect of GSK621 on AMPK/GSK-3β/PP2A pathway in MPTP-induced mice was

613 analyzed by Western blot (n=6). (A) Relative expressions of AMPK, p-AMPK, GSK-3β,

614 p-GSK-3β, PP2A Cleaved Caspase 3, and Caspase 3. (B) Density quantitative results of these

615 above proteins. Values are expressed as mean ± SEM. Statistical differences were determined by

616 one-way analysis of variance followed by Dunnett multiple comparisons test. *P<0.05 and

617 617

618 618

**P<0.01 versus Control group; &P<0.05 and &&P<0.01 versus MPTP group.

619 Fig. 4. GSK621 protected the mitochondrial function in MPP+ -induced PC12 cells. (A) MTT

620 method was used to analyze PC12 cell viability at different modeling dosages of MPP+ iodide

621 (n=6). The cell viability significantly decreased by 40% at the dosage of 200 µM MPP+ iodide,

622 which was used for modeling. (B) The effects of GSK621 at different dosages (0, 10, 20, 40, 50

623 µM) on PC12 cell viability (n=6). PC12 cell viability was decreased with 40 µM GSK621, so 30

624 µM GSK621 was considered as an appropriate dosage used in further study. (C) The protective

625 efficacy of GSK621 on MPP+ -induced PC12 cells was assessed by MTT assay (n=6). (D) ATP

626 production in MPP+ -induced PC12 cells (n=6). (E) Pyruvate production in MPP+ -induced PC12

627 cells (n=6). (F) The mitochondrial membrane potential of MPP+ -induced PC12 cells was

628 observed by JC-1 probe and flow cytometry (n=3). JC-1 Red Fluorescence represents normal

629 mitochondrial membrane potential, whereas JC-1 Green Fluorescence indicates damaged

630 mitochondrial membrane potential. Values are expressed as mean ± SEM. Statistical differences

631 were determined by one-way analysis of variance followed by Dunnett multiple comparisons test.

632 632

633 633

*P<0.05 and **P<0.01 versus NC group; &P<0.05 and &&P<0.01 versus MPP+ group.

634 Fig. 5. Extracellular flux analysis of energy metabolism of PC12 cells was measured by

635 Seahorse instruments. The OCR in living PC12 cells was measured utilizing the Seahorse XF24

636 Extracellular, which made it possible to quantify mitochondrial respiration and glycolysis directly.

637 (A) Typical OCR readout profile and its components were shown in this assay (n=6). (B) The

638 figure of OCR in MPP+ iodide-induced PC12 cells (n=6). Parameters of mitochondrial function

639 derived from OCR profiles, including (C) ATP production (n=6); (D) Basal respiration (n=6); (E)

640 Maximal respiration (n=6); (F) Proton leak (n=6); (G) Spare respiratory capacity (n=6). Values are

641 expressed as mean ± SEM. Statistical differences were determined by one-way analysis of

642 variance followed by Dunnett multiple comparisons test. *P<0.05 and **P<0.01 versus NC group;

643 643

644 644

&P<0.05 and &&P<0.01 versus MPP+ group.

645 Fig. 6. GSK621 restored cellular apoptosis of MPP+ -induced PC12 cells. (A) Representative

646 images of DAPI staining and TUNEL (n=3, Bar: 50 μm). Apoptotic cells (Green fluorescence)

647 increased after MPP+ iodide treatment, but this was attenuated when treatment was combined with

648 GSK621. (B) Secondary assessment of apoptosis was analyzed by flow cytometry (n=3).

649 Secondary assessment of apoptosis was performed using an Annexin V-FITC binding assay, with

650 results plotted as a histogram. Model+GSK621 group exhibited significantly fewer apoptotic cells

651 than that in Model group. Values are expressed as mean ± SEM. Statistical differences were

652 determined by one-way analysis of variance followed by Dunnett multiple comparisons test for

653 TUNEL assay, and one-way analysis of variance followed by Tukey multiple comparisons test for

654 flow cytometry analysis. *P<0.05 and **P<0.01 versus NC group; &P<0.05 and &&P<0.01 versus

655 655

656 656

MPP+ group.

657 Fig. 7. Effect of GSK621 on MPP+ -induced PC12 cells was analyzed by western blot (n=6).

658 (A) Relative expressions of AMPK, p-AMPK, GSK-3β, p-GSK-3β, PP2A, Cleaved Caspase 3,

659 and Caspase 3. (B) Density quantitative results of these above proteins. Values are expressed as

660 mean ± SEM. Statistical differences were determined by one-way analysis of variance followed by

661 Dunnett multiple comparisons test. *P<0.05 and **P<0.01 versus NC group; &P<0.05 and

662 662

663 663

&&P<0.01 versus MPP+ group.

Graphic abstract

Mitochondrial damage

Abnormal mitochondrial membrane potential
Mitochondrial energy metabolism disorder

Author agreement

We declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere.

We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.

We understand that the Corresponding Author is the sole contact for the Editorial process. He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs.