《Cell》细胞老化：决定未来的路

Cell179, October 31, 2019 a 2019 

Cellular Senescence: Defining a Path Forward

Definition and Characteristics of Cellular Senescence 

Cellular senescence is a cell state 

triggered by ：stressful insults and certain physiological processes,

characterized by ：aprolongedand generally irreversible cell-cycle arrest withsecretory features, macromolecular damage, andaltered metabolism 

These features can be inter-dependent 

Cell-Cycle Arrest 

mammalian cells 

retinoblastoma(RB) familyandp53 proteinsare important for establishing senescent cell-cycle arrest 

RB1 and its family members p107 (RBL1) and p130 (RBL2) are phosphorylated by specific cyclin-dependent kinases (CDKs; CDK4, CDK6, CDK2). 

This phosphorylation reduces the ability of the RB family members torepressE2F family transcription factor activity, which is required for cell-cycle progression 

CDK2 inhibitor p21WAF1/Cip1 (CDKN1A) and CDK4/6 inhibitor p16INK4A (CDKN2A) accumulate. 

Secretion 

SASP的功能

the SASP reinforces and spreads senescence in autocrineandparacrinefashions 

activates immune responses that eliminate senescent cells 

SASP factors mediate developmental senescence 

tissue plasticity 

 contribute to persistent chronic inflammation (known as inflammaging) 

the SASP can recruit immature immune-suppressive myeloid cells to prostate and liver tumors （肝脏肿瘤！！！看一下Distinct Functions of Senescence-Associated Immune Responses in Liver Tumor Surveillance and Tumor Progression 

）

控制SASP的相关通路

enhancer remodeling 

activation of transcription factors, such as NF-kB, C/EBPb, GATA4 

mammalian target of rapamycin(mTOR) 

p38MAPK signaling pathways 

上游因子控制SASP的方式不同并且和老化诱导的途径有关

type 1 interferon response：DNA damage, cytoplasmic chromatin fragments (CCFs) 

inflammasome ：damage-associated molecular patterns (DAMPs) 

SASP的成分和强度不同决定于：duration of senescence, origin of the prosenescence stimulus, and cell type 

SASP可以和外界的微环境通过：juxtacrine NOTCH/JAG1 signaling；release of ROS；cytoplasmic bridges；extracellular vesicles （egexosomes）

）

Macromolecular Damage 

DNA Damage ：The first molecular feature associated with senescence wastelomere shortening, a result of the DNA end-replication problem, during serial passages 

端粒导致DDR的过程

Type1

Telomere shortening during proliferation 

telomeric DNA loop destabilization &telomere uncapping 

generating telomere dysfunction-induced foci (TIFs) 

ctivate the DDR 

cell-cycle arrest 

（This response can also be elicited by inhibiting or altering genes involved in telomere maintenance ）

Type2

oxidative DNA damage at telomeric G-reach repeats 

telomere-associated foci (TAFs), can exist at telomeres （irrespective of telomere length or shelterin loss ）

DNA损伤种类

half of the persistent DNA damage foci in senescent cells localize to telomeres

other stressful subcytotoxic insults can trigger senescence by inducing irreparable DNA damage 

genotoxic agents： radiation (ionizing and UV),

                                 pharmacological agents (e.g., certain chemotherapeutics),

                                 oxidative stress 

activated oncogenes can induce senescence (known as OIS) as a tumor-suppressive response, restricting the uncontrolled proliferation of potentially oncogenic cells. 

——OIS is often mediated by the tumor suppressorsp16INK4A andARF, both encoded by theCDKN2A locus, imposing a cell-cycle arrest 

DDR也可以产生OIS的激活

oncogene-driven hyperproliferation→damage signal originates at collapsed replication forks 

DNA-SCARSs (DNA segments with chromatin alter- ations reinforcing senescence) ：Senescent cells harbor persistent nuclear DNA damage foci 

特点：associate with promyelocytic leukemian (PML) nuclear bodies （？？？）

           lack theDNA repair proteinsRPAandRAD51as well as single- stranded DNA (ssDNA) 

           contain activated forms of the DDR mediatorsCHK2andp53 

DNA-SCARSs 可以调节细胞生长停滞和SASP，但是并不是一个老化细胞的特征

Protein Damage 

Proteotoxicity is a hallmark of aging and cellular senescence 

damaged proteins help identify senescent cells 

蛋白质损伤的途径

ROS 

↓

oxidize both methionine and cysteine residues 

↓

alter protein folding and function 

eg

（蛋氨酸和半胱氨酸，很多络氨酸磷酸酶都含有cys，因此他们所去磷酸化的部位就会被inactivate）PTPs：protein tyrosine phosphatases 

↓

hyperactivating ERK signaling （similar to the effect of activated oncogenes ）

↓

trigger senescence 

在肿瘤前期的病变处可以发现高磷酸化的ERK

Most protein oxidative damage：degradation by the ubiquitin proteasome system(UPS) orautophagy因此这两个特点可以作为检测老化情况的指标

promyelocytic leukemian (PML) 小体可以作为ROS和氧化损伤的sensor；同样也可以作为细胞老化的一个非特异性指标

Lipid Damage 

老化细胞的清除可以减少老年小鼠肝脏和大脑中脂肪的沉积

（特定的脂代谢和老化相关知之甚少）

虽然很多方法可以检测脂肪在组织和细胞之前的变化，但是作为老化的biomarker还是未知的，因为不同的老化相关的脂肪十分不同。

比如在原癌基因激活的老化和复制性老化中脂肪代谢非常不同

Deregulated Metabolic Profile 

Mitochondria 

Mitochondria in senescent cells are less functional, showingdecreased membrane potential,increased proton leak, reduced fusion and fission rates, increased mass, andabundance of tricarboxylic acid (TCA) cycle metabolites 

Altered AMP:ATP and ADP:ATP ratios during senescence 

↓

activating AMPK (AMP-activated protein kinase), a main sensor of energy deprivation 

↓

contribute to cell-cycle withdrawal 

线粒体的功能失调和SASP的调节也相关

Mitophagy (mitochondrial clearance) in senescent cells appears tosuppressthe SASP 

genetic or pharmacological inhibition of the ETC（electron transport chain ） can induce senescence even though cells lack expression of key pro-inflammatory SASP factors

 NAD+/NADH ratios are reduced n senescent cells →alter the activity of poly-ADP ribose polymerase (PARP) and sirtuins （乙酰化酶）——both involved in activation of the SASP regulatorNF-kB 

lysosomes

 溶酶体的代谢是有细胞的代谢或者降解需要决定的

当AA在溶酶体的腔内含量比较高的时候，mTOR1 is recruited and activated 

并且溶酶体可以和线粒相互关联

老化的细胞中溶酶体的数量和大小都有所增加，但是活性并没有增加

↓

lysosome-mitochondrial axis degrades 

↓

decreased mitochondrial 

↓

ncreases ROS production 

↓

ROS targets cellular structures, including lysosomes, which forms a vicious feedback loop that induces more damage

溶酶体的数量和SA-b-gal 的活性有关

从治疗角度来讲，溶酶体增大的三部分可以为捕捉药物增加空间，比如CDK4/6 in- hibitors palbociclib哌柏西利, ribociclib, and abemaciclib 

Senescence-Associated (Epi-)genetic and Gene Expression Changes

上述的表型变化大多是由于基因的表达所导致，这里我们解释下相关基因的情况

老化相关基因查询网址(http://Senequest.net) 

Chromatin Landscape 

表关遗传的变化大多和环境相关。replicative senescence has been correlated with global loss of DNA methylation at CpG sites ；Cells undergoing OIS fail to show such alterations in DNA methylation 

老化的细胞呈现普遍的increase in chromatin accessibility, but the genome-wide profilevariesdepending on the stimulus 

大多是组蛋白的变化individual histone modifications and variants

H4K16ac is often enriched at active pro- moters in senescent, but not proliferating, cells 

N terminus proteolytic cleavage of H3.3 correlates with gene repression in a different subset of genes during senescence 

Certain histone modifications are crucial for senescence, such as elevatedH4K20me3andH3K9me3,which contribute to theproliferation arrest ；elevatedH3K27acat gene enhancers promotes aSASP 

Senescence is also associated with chromatinmorphological changes. 

Senescence-associated heterochromatin foci (SAHFs), visualized as DAPI-dense foci, are enriched in heterochromatin protein (HP) 1. 

 SAHFs derive from chromatin factors—

 including RB,

  histone variant macroH2A, 

 high mobility group A proteins, 

 the HIRA/UBN1/CABIN1, a

 ASF1a chaperones—and increased nuclear pore density 

SAHF主要组成复制后基因的poor eterochromatic 部分，因此和细胞老化的相关性很小

细胞的老化同时也和大面积的H1丢失有关

enescence-associated distension of satellites (SADSs) ——先于SAHF，并且和细胞老化相关

Retrotransposable elements

repressed LINE-1 (L1) retrotransposons are activated, stimulating the cGAS-STING pathway that elicits a type 1 interferon response ，从而和SASP相关

Downregulation of lamin B1（a major component of the nuclear lamina,）——也是老化细胞的一个主要特点

Lamin B1 和表观基因、enescence-associated chromatin structures (SAHFs and SADSs) 相关

主要发生在H3K9me3-rich regions：liberate H3K9me3 from the nuclear lamina promoting spatial rearrangement of H3K9me3 heterochromatin to form SAHFs 尤其是在OIS中发挥作用，然而复制性老化并没有什么相互作用

lamin B1 loss and reduced nuclear integrity 也可以促进SASP的形成

Transcriptional Signatures 

genes linked to thecell-cycle arrestand SASPare frequently interrogated in combination with other biomarkers to validate the senescence phenotype or type of senescence 

eg：CDKN1A (p21WAF1/Cip1), CDKN2A (p16INK4A), and CDK2B (p15INK4B), and a subset of SASP genes, along with decreased expression of cyclinsCCNA2andCCNE2andLMNB1 should be determined.

为了更好研究转录情况——Whole-transcriptome studies，可以预测药物靶向；

但是现在的转录因子数据还是不足，因此相关转录因子的发现还是很重要的

 miRNAs and Non-coding RNAs （）

尤其是miRNA和细胞老化的关联更大；可以直接或者间接的调节老化关键因子，

「p53, p21WAF1/Cip1, and SIRT1 」

negative：miR-504 targets the p53 30UTR, reducing p53 abundance and activity 

                  Gld2-mediated stabilization of miR-122 enables its binding to the CBEP 30 UTR, resulting in decreased p53 mRNA polyadenylation and translation 

                  multiple miRNAs downregu- late p21WAF1/Cip1, including 28 miRNAs that block OIS 

                  miR-24 suppresses p16INK4a 

positive：miR-605 targets MDM2, triggering p53-mediated senescence 

miRNA feedback loops can modulate senescence programs. 

——For example, ap53/ miRNA/CCNA2 pathwaydrives senescence independently of the p53/p21WAF1/Cip1 axis ；p53- dependent upregulation ofmiR-34a/b/cdownregulates cell proliferation and survival factors 

「regulate the SASP 」

MiR-146a/b ：dampens a proinflammatory arm of the SAS 

miRNAs also downregulate repressors of senescence, including polycomb group(PcG)members CBX7, EED, EZH2, and SUZ12 (miR-26b, 181a, 210, and 424), leading top16INK4a derepressionand senescence initiation 

「the role of miRNAs in senes- cence extends beyond their classical functions」

Argonaute 2 (AGO2) binds let-7f in the nucleus, forming a complex with RB1(pRB), resulting inrepressivechromatin atCDC2andCDCA8promoters . Silencing theseE2Ftarget genes is required for senescence initiation. 

Long non-coding RNAs (lncRNAs) (>200 nt) canbind RNA, DNA, or proteinsto regulate senescence. 

ANRIL, a 30-40kb antisense transcript encoded by the CDKN2A locus, binds CBX7 torepress INK4b/ARF/INK4a expression 

lncRNA PANDA recruits PcG com- plexes, suppressing senescence-promoting genes 

silencing ofGUARDIN, a p53-responsive lncRNA, causes senescence or apoptosis 

following OIS induced by RAF, the lncRNA VADpreserves senescence by decreasing repressive H2A.Z deposition at INK promoters 

lncRNA UCA1 disrupts association of the RNA-binding protein hnRNP A1 with p16INK4A

Immune-Regulation and Anti-apoptotic Proteins 

虽然细胞老化的诱导环境可以激活一些炎症反应因子，尤其是一些细胞表面的marker，可以作为研究从单个细胞到组织的方式，但是DCR2 and NKG2D ligands are not conserved among species, making mouse-to-human comparisons not possible. 

Notch1 in OIS and DPP4 in replicative and OIS：upregulated cell surface markers, 并且对于调节SASP有着一定的作用

increased expression of anti-apoptotic BCL-2 family members 也可以，因为老化的细胞是有凋亡抵抗的。

in Vivo Models to Study Cellular Senescence 

Senescence Reporter Mice

通过报告基因estimatep16Ink4a expression 

Murine Models of Accelerated Senescence and Aging 

（各种早衰小鼠模型的建立方法）

Identification of Cellular Senescence In Vivo

A Simplified Algorithm for Detecting Senescent Cells In Situ

实验室免疫染色/WB+用显微镜观察下

不同方法联合检测

Challenges to Detect Senescent Cells in Humans 

将细胞的老化和人类的疾病联系起来的

neurodegenerative disorders, glaucoma青光眼, cataract白内障， atherosclerosis and cardiovascular disease, diabetes, osteoarthritis, pulmonary, and renal and liver fibrosis 

组织的检测可以：fresh samplesby SA-b-gal staining 

 or indirect markers in formalin-fixed tissues 

histochemical dye SBB interacts with lipofuscin, another hallmark of senescent cells 

Lipofuscin is preserved in fixed material 

reagent (GL13) is amenable to immunohistochemistry （identified senescent Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin lymphomas (cHL) where they predicted poor prognosis ）

Another method for identi- fying and quantifying senescent cellsin vivo is SA-b-gal staining combined with ImageStream X analysis 

We recommendcombining cytoplasmic (e.g., SA-b-gal, lipofuscin), nuclear (e.g., p16INK4A, p21WAF1/Cip1, Ki67) and SASP, context and/or cell-type-specific markers 

老化相关仍然存在的问题

不可逆的细胞周期停滞并不一定存在所有的老化细胞中！

遗传学和表观遗传学在细胞老化中的关联？

在不同的老化诱导条件下细胞的修复方式又是什么？

抗衰老治疗的应用？

（我个人觉得mirNA&Non-coding RNAs是不是也挺热门的）