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J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (6): 750-757    DOI: 10.3785/j.issn.1008-9292.2020.12.10
    
Sirt3 gene knockout protects mice from Alzheimer's disease through activating autophagy
SHU Min1(),ZHANG Wenzhe1,JIN Xiangbo1,ZENG Linghui1,*(),XIANG Yingchun2,*()
1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Pharmacy, Zhejiang Hospital, Hangzhou 310012, China
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Abstract  

Objective: To investigate the protective effect of Sirt3 gene knockout on Alzheimer's disease (AD) in mice. Methods: The animal model of AD was established by intraperitoneal injection of D-galactose and brain-localized injection of amyloid β-protein (Aβ)1-40 in wild type C57BL/6 mice and Sirt3 gene knockout mice. Morris water maze, Y maze and tail suspension test were used to assess the cognitive function and anxiety-like behaviors in mice. Aβ deposition in the hippocampus was detected by immunofluorescent staining. Western blotting analysis was conducted to detect the expression of related proteins in the brain. Mouse cortical primary neurons were cultured and AD cell model was established. MTT assay was used to detect cell viability after modeling. Results: Behavioral results showed that cognitive deficits were found in wide type mice after induction of AD as its prolonged escape latency (P < 0.05) and decreased crossing number of platform and target zone duration (all P < 0.05); while the knockout of Sirt3 alleviated cognitive deficit induced by AD (all P < 0.05). Aβ immunofluorescence staining showed that the deposition of Aβ in the hippocampal region and expression of cleaved caspase 3 in the brain in Sirt3 knockout mice was reduced compared with that of wild type mice (all P < 0.05). The expression of LC3-Ⅱ and P62 increased after AD was induced in wild type mice, while the autophagy in Sirt3 knockout mice was activated as the increase expression of LC3-Ⅱ and decrease expression of P62 (all P < 0.05). In the AD cell model, the results of MTT assay were consistent with the animal experiments, and the protective effect of Sirt3 knockdown was eliminated after the treatment of the autophagy inhibitor chloroquine (all P < 0.05). Conclusion: The knockdown of Sirt3 shows a protective effect on AD induced by D-galactose and Aβ1-40 in mice, which may be related to its function of activating autophagy.



Key wordsAlzheimer's disease      Sirt3      Autophagy      Amyloid β-protein      Learning      Memory      Mice     
Received: 20 September 2020      Published: 14 January 2021
CLC:  R741.02  
Corresponding Authors: ZENG Linghui,XIANG Yingchun     E-mail: shumin123@zju.edu.cn;zenglh@zucc.edu.cn;xych999@126.com
Cite this article:

SHU Min,ZHANG Wenzhe,JIN Xiangbo,ZENG Linghui,XIANG Yingchun. Sirt3 gene knockout protects mice from Alzheimer's disease through activating autophagy. J Zhejiang Univ (Med Sci), 2020, 49(6): 750-757.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2020.12.10     OR     http://www.zjujournals.com/med/Y2020/V49/I6/750


敲除Sirt3后激活自噬对阿尔茨海默病有保护作用

目的: 探究Sirt3在阿尔茨海默病(AD)发生发展中的作用及可能机制。方法: 体内实验以C57BL/6野生型小鼠和Sirt3基因敲除小鼠为对象,采用腹腔注射D-半乳糖联合脑定位注射β-淀粉样蛋白(Aβ)1-40建立AD小鼠模型。莫里斯(Morris)水迷宫实验、自发活动开场实验和悬尾实验观察造模前后小鼠学习记忆和焦虑抑郁状态的改变,免疫荧光染色观察脑内海马区域Aβ沉积情况,蛋白质印迹法检测小鼠脑组织中自噬和凋亡相关蛋白表达。体外实验选小鼠皮层原代细胞作为对象,给予Aβ1-40建立AD体外模型,MTT法检测细胞活性。结果: 体内实验结果显示,野生型小鼠诱发AD后平台潜伏期延长(P < 0.05),穿越平台次数减少、目标象限停留时间缩短(均P < 0.05),提示造模后小鼠的学习记忆能力下降;而Sirt3敲除能够缓解AD所致的学习记忆障碍(均P < 0.05)。相较于野生型小鼠,Sirt3基因敲除小鼠脑内海马区域的Aβ沉积减少(P < 0.05),凋亡蛋白cleaved caspase 3表达减少(P < 0.05)。另外,野生型小鼠诱发AD后LC3-Ⅱ和P62蛋白表达增加(均P < 0.05),提示自噬流受阻;而Sirt3基因敲除小鼠诱发AD后LC3-Ⅱ蛋白表达增加,P62蛋白表达减少(均P < 0.05),提示自噬被激活。小鼠皮层原代细胞AD模型中,Sirt3基因敲除的细胞较野生型细胞死亡减少(P < 0.05);加用自噬抑制剂氯喹后,Sirt3基因敲除的保护作用消失(P < 0.05)。结论: Sirt3敲除对于D-半乳糖联合Aβ1-40诱发的AD有保护作用,这种保护作用可能与自噬流活化有关。


关键词: 阿尔茨海默病,  Sirt3,  自噬,  β-淀粉样蛋白,  学习,  记忆,  小鼠 
组别 n 平台潜伏期(s) 穿越平台次数 目标象限停留时间(s) 平均游泳速度(cm/s)
与野生型对照组比较,*P<0.05;与野生型模型组比较,#P<0.05.
野生型对照组 7 36.6±11.9 4.7±1.3 40.3±4.1 18.6±1.2
基因敲除对照组 7 29.8±12.4 4.6±1.3 40.8±5.6 15.8±2.5
野生型模型组 9 67.2±8.7* 1.0±0.4* 21.0±5.7* 14.6±2.4
基因敲除模型组 7 32.8±8.0# 3.0±0.9# 34.3±5.1 19.1±1.6
Tab 1 Results of Morris water maze test in wild type mice and Sirt3 knockout mice before and after Alzheimer's disease induction ($\bar x \pm s$)
组别 n 跨格次数 站立次数 静止时间所占比例(%)
与野生型对照组比较,*P<0.05;与野生型模型组比较,#P<0.05.
野生型对照组 7 48±11 1.14±0.49 22±5
基因敲除对照组 7 41±9 0.80±0.34 21±3
野生型模型组 9 22±3* 0.30±0.11* 30±5
基因敲除模型组 7 37±6# 0.42±0.17 25±5
Tab 2 Results of open field and tail suspension tests in wild type mice and Sirt3 knockout mice before and after Alzheimer's disease induction ($\bar x \pm s$)
Fig 1 Amyloid β-protein deposition in wild type mice and Sirt3 knockout mice after Alzheimer's disease induction
Fig 2 Expression of apoptosis- and autophagy-related proteins in wild type mice and Sirt3 knockout mice by Western blotting
组别 n cleaved caspase 3 LC3-Ⅱ/LC3-Ⅰ P62
与野生型对照组比较,*P<0.05;与基因敲除对照组比较,#P<0.05;与野生型模型组比较,ΔP<0.05.
野生型对照组 3 1.00 1.00 1.00
基因敲除对照组 3 1.05±0.07 1.04±0.23 1.13±0.09
野生型模型组 3 1.63±0.10* 1.67±0.21* 1.29±0.18*
基因敲除模型组 3 1.16±0.15Δ 2.03±0.35# 0.80±0.10
Tab 3 Expression of cleaved caspase 3, LC3 and P62 in wild type mice and Sirt3 knockout mice before and after Alzheimer's disease induction ($\bar x \pm s$)
Fig 3 Cell survivability of mouse cortical neurons under different treatment (n=3)
[1]   COSTANZO M , ZURZOLO C . The cell biology of prion-like spread of protein aggregates: mechanisms and implication in neurodegeneration[J]. Biochem J, 2013, 452 (1): 1- 17
doi: 10.1042/BJ20121898
[2]   KRSTIC D , KNUESEL I . Deciphering the mechanism underlying late-onset Alzheimer disease[J]. Nat Rev Neurol, 2013, 9 (1): 25- 34
doi: 10.1038/nrneurol.2012.236
[3]   KAUSHIK S , CUERVO A M . Chaperone-mediated autophagy: a unique way to enter the lysosome world[J]. Trends Cell Biol, 2012, 22 (8): 407- 417
doi: 10.1016/j.tcb.2012.05.006
[4]   TAKáTS S , JUHáSZ G . A genetic model with specifically impaired autophagosome-lysosome fusion[J]. Autophagy, 2013, 9 (8): 1251- 1252
doi: 10.4161/auto.25470
[5]   HAIGIS M C , SINCLAIR D A . Mammalian sirtuins: biological insights and disease relevance[J]. Annu Rev Pathol, 2010, 5:253- 295
doi: 10.1146/annurev.pathol.4.110807.092250
[6]   BAUSE A S , HAIGIS M C . SIRT3 regulation of mitochondrial oxidative stress[J]. Exp Gerontol, 2013, 48 (7): 634- 639
doi: 10.1016/j.exger.2012.08.007
[7]   DONMEZ G , OUTEIRO T F . SIRT1 and SIRT2: emerging targets in neurodegeneration[J]. EMBO Mol Med, 2013, 5 (3): 344- 352
doi: 10.1002/emmm.201302451
[8]   RAMADORI G , LEE C E , BOOKOUT A L et al. Brain SIRT1: anatomical distribution and regulation by energy availability[J]. J Neurosci, 2008, 28 (40): 9989- 9996
doi: 10.1523/JNEUROSCI.3257-08.2008
[9]   NORTH B J , VERDIN E . Sirtuins: Sir2-related NAD-dependent protein deacetylases[J]. Genome Biol, 2004, 5 (5): 224
doi: 10.1186/gb-2004-5-5-224
[10]   LI S , DOU X , NING H et al. Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity[J]. Hepatology, 2017, 66 (3): 936- 952
doi: 10.1002/hep.29229
[11]   ZHANG M , DENG Y N , ZHANG J Y et al. SIRT3 protects rotenone-induced injury in SH-SY5Y cells by promoting autophagy through the LKB1-AMPK-mTOR pathway[J]. Aging Dis, 2018, 9 (2): 273- 286
doi: 10.14336/AD.2017.0517
[12]   秦川, 吴善球, 陈保生 et al. 灵芝制剂治疗APP/PS-1阿尔茨海默病转基因小鼠模型的病理学改变[J]. 中国医学科学院学报, 2017, 39 (4): 552- 561
QIN Chuan , WU Shanqiu , CHEN Baosheng et al. Pathological changes in APP/PS-1 transgenic mouse models of Alzheimer's disease treated with ganoderma lucidum preparation[J]. Acta Academiae Medicinae Sinicae, 2017, 39 (4): 552- 561
doi: 10.3881/j.issn.1000-503X.2017.04.015
[13]   SUN C , OU X , FARLEY J M et al. Allopregnanolone increases the number of dopaminergic neurons in substantia nigra of a triple transgenic mouse model of Alzheimer's disease[J]. Curr Alzheimer Res, 2012, 9 (4): 473- 480
doi: 10.2174/156720512800492567
[14]   YANG W , SHI L , CHEN L et al. Protective effects of perindopril on d-galactose and aluminum trichloride induced neurotoxicity via the apoptosis of mitochondria-mediated intrinsic pathway in the hippocampus of mice[J]. Brain Res Bull, 2014, 109:46- 53
doi: 10.1016/j.brainresbull.2014.09.010
[15]   HUANG Y , SHEN W , SU J et al. Modulating the balance of synaptic and extrasynaptic nmda receptors shows positive effects against amyloid-β-induced neurotoxicity[J]. J Alzheimers Dis, 2017, 57 (3): 885- 897
doi: 10.3233/JAD-161186
[16]   HAN P C , YIN J , TANG Z et al. Pituitary adenylate cyclise-activating polypeptide (PACAP) protects against beta-amyloid toxicity and potentiates mitochondrial function[J]. Alzhe & Demen, 2013, 9 (4): P355
doi: 10.1016/j.jalz.2013.05.672
[17]   WEIR H J , MURRAY T K , KEHOE P G et al. CNS SIRT3 expression is altered by reactive oxygen species and in Alzheimer's disease[J]. PLoS One, 2012, 7 (11): e48225
doi: 10.1371/journal.pone.0048225
[18]   YIN J X , TURNER G H , LIN H J et al. Deficits in spatial learning and memory is associated with hippocampal volume loss in aged apolipoprotein E4 mice[J]. J Alzheimers Dis, 2011, 27 (1): 89- 98
doi: 10.3233/JAD-2011-110479
[19]   CHEN H K , JI Z S , DODSON S E et al. Apolipoprotein E4 domain interaction mediates detrimental effects on mitochondria and is a potential therapeutic target for Alzheimer disease[J]. J Biol Chem, 2011, 286 (7): 5215- 5221
doi: 10.1074/jbc.M110.151084
[20]   YIN J X , NIELSEN M , CARCIONET et al. Apolipoprotein E regulates mitochondrial function through the PGC-1α-sirtuin 3 pathway[J]. Aging (Albany NY), 2019, 11 (23): 11148- 11156
doi: 10.18632/aging.102516
[21]   YIN J , NIELSEN M , LI S et al. Ketones improves apolipoprotein E4-related memory deficiency via sirtuin 3[J]. Aging (Albany NY), 2019, 11 (13): 4579- 4586
doi: 10.18632/aging.102070
[22]   KROEMER G . Autophagy: a druggable process that is deregulated in aging and human disease[J]. J Clin Invest, 2015, 125 (1): 1- 4
doi: 10.1172/JCI78652
[23]   LEVINE B , KROEMER G . Autophagy in the pathogenesis of disease[J]. Cell, 2008, 132 (1): 27- 42
doi: 10.1016/j.cell.2007.12.018
[24]   MENZIES F M , FLEMING A , RUBINSZTEIN D C . Compromised autophagy and neurodegenerative diseases[J]. Nat Rev Neurosci, 2015, 16 (6): 345- 357
doi: 10.1038/nrn3961
[25]   SU H , WANG X . P62 stages an interplay between the ubiquitin-proteasome system and autophagy in the heart of defense against proteotoxic stress[J]. Trends Cardiovasc Med, 2011, 21 (8): 224- 228
doi: 10.1016/j.tcm.2012.05.015
[26]   ZAFFAGNINI G , SAVOVA A , DANIELI A et al. P62 filaments capture and present ubiquitinated cargos for autophagy[J]. EMBO J, 2018, 37 (5): e98308
doi: 10.15252/embj.201798308
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