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Tai HR, Wang Z, Gong H, Han XJ, Zhou J, Wang XB, Wei XW, Ding Y, Huang N,
Qin JQ, Zhang J, Wang S, Gao F, Chrzanowska-Lightowlers ZM, Xiang R, Xiao
HY.
Autophagy impairment with lysosomal and mitochondrial dysfunction is an
important characteristic of oxidative stress-induced senescence.
Autophagy 2017, 13(1), 99-113.
Copyright:
This is an Accepted Manuscript of an article published by Taylor & Francis in Autophagy on 28/10/2016,
available online: http://www.tandfonline.com/doi/full/10.1080/15548627.2016.1247143.
DOI link to article:
https://doi.org/10.1080/15548627.2016.1247143
Date deposited:
18/07/2017
Embargo release date:
28 October 2017
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Autophagy impairment with lysosomal and mitochondrial
1
dysfunction is an important characteristic of oxidative
2
stress-induced senescence
3
4
Haoran Tai
1*
, Zhe Wang
1*
, Hui Gong
1*
, Xiaojuan Han
1
, Xiawei Wei
1
, Jiao Zhou
1
, Xiaobo Wang
1
,
5
Yi Ding
1
, Ning Huang
1
, Jianqiong Qin
1
, Jie Zhang
1
, Shuang Wang
1
, Fei Gao
2
, Zofia M.
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Chrzanowska-Lightowlers
2
, Rong Xiang
3
, Henyi Xiao
1#
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8
Author affiliation:
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1
Lab for Aging Research, Center of Gerontology and Geriatrics, State Key Laboratory of
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Biotherapy & Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan
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University, Chengdu 610041, China
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2
Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle
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University, Newcastle upon Tyne NE2 4HH, UK
14
3
Department of Clinical medicine, Medical School of Nankai University, Tianjin 300071, China
15
*
These authors contributed equally to this work.
16
#
To whom correspondence should be addressed.
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2
Abstract
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Autophagy has profound implications with aging. However, the true features of autophagy in
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aging development remain to be clarified. In the present study, we explored the status of
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autophagic flux during the development of cell senescence induced by oxidative stress. In this
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system, with increased autophagic structures though, the degradation of SQSTM1/p62 protein,
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the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic
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enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal
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dysfunction. The influence of autophagy activity on senescence development is confirmed by
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both positive and negative autophagy modulators and mTOR dependent autophagy activators,
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rapamycin and PP242, efficiently suppress cellular senescence through a mechanism relevant
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to restoring autophagic flux. Moreover, by time phased treatment of cells with antioxidant
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N-acetylcysteine (NAC), mitochondria uncoupler carbonyl cyanide m-chlorophenylhydrazone
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(CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation,
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we found that mitochondrial dysfunction plays a initiative role, while lysosomal dysfunction is
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more directly responsible for autophagy impairment as well as senescence. Interestingly, the
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effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both
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lysosomal and mitochondrial functions. Together, this study demonstrates that autophagy
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impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy
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activity should be a promising way for senescence retardation.
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Key words: autophagy, senescence, oxidative stress, mitochondria, lysosomes, rapamycin
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Abbreviations
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3
PBS = Phosphate buffered saline
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SA-β-gal = Senescence associated β-galactosidase
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SAHF = Senescence-associated heterochromatin foci
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qRT-PCR = Real-Time Quantitative Reverse Transcription PCR
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MTT = Methyl thiazolyl tetrazolium
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ROS = Reactive oxygen species
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DAPI = 4',6-diamidino-2-phenylindole
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DMEM = Dulbecco's modified eagle medium
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H
2
O
2
= Hydrogen peroxide
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SIPS = Stress induced premature senescence
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GFP = Green fluorescent protein
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mRFP = Monomeric red fluorescence protein
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LC3 = Microtubule-associated protein 1 light chain 3
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mTOR = Mammalian target of rapamycin
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TFEB = Transcription factor EB
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HCQ = Hydroxychloroquine
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3-MA = 3-Methyladenine
57
VPA = Valproic acid
58
COX = Cytochrome c oxidase
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CHX = Cycloheximide
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NAC = N-acetylcysteine
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MMP = Mitochondrial membrane potential
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4
PtdIns3K = Phosphoinositide 3-kinase
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UCP = Uncoupling protein
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IL = Interleukin
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CCCP = Carbonyl cyanide m-chlorophenylhydrazine
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EBSS = Earle’s balanced salt solution
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ULK = Unc-51-like kinase
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UPR
mt
= Mitochondrial unfold protein response
69
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Introduction
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Aging is a complicated process with its mechanism remains to be explored. Cellular
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senescence happens during aging development and is widely used as
in vitro
model for aging
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research. Stress-induced premature senescence (SIPS) develops faster than replicative
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senescence and has been established for studying the influence of extracellular or intracellular
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stress on aging process.
1
Known features of senescent cells include flat and enlarged cellular
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morphology, increased senescence associated β-galactosidase (SA-β-gal) activity, the
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appearance of senescence-associated heterochromatin foci (SAHF), cell growth arrest and
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senescence-associated secretory phenotype (SASP) such as IL-6 and IL-8 secretion.
2-6
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Autophagy plays cytoprotective roles by turnover long lived proteins and scavenging damaged
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cellular components. Among different types of autophagy, macroautophagy is the most
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studied one and is composed of two stages: the early stage acting for the formation of
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autophagosome with capturing damaged cellular molecules and organelles; the late stage
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responsible for the digestion of entrapped components within autolysosome formed by the
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