11

1

22

CHAPTER 11

APOPTOSIS AND AGING

33

I. Aging of cell

The sorting of human cells by their recruitment: Human natural life time is about 120 years. But, the cells of that

human body is composed are very different in their life time. We can sort them as 4 types as the follows:

Recruiting tissue: The recruiting tissue needs to be replaced always, for examples, intestine endothelial cells (IEC).

Stable tissue cells: The cells were highly differentiated with special function. Usually, no obvious aging can be found in the stable tissue cells. For examples, liver cells, kidney cells.

Consistent cells: There is no cell replacement in the consistent tissue. For examples, neurons, skeleton muscle cells, heart cell, and others.

Exhausting cells: ovary cells can be exhausted out without any supplement.

44

The signs of cell aging

The changes of forms: Aged cell appears with increased permeability and fragility, cell atrophy, decreased organelle quantity (especially for mitochondria), and accumulation of lipofuscin in cell.

NucleusNucleus Enlarged, Stained darkly, Inclusion contained Enlarged, Stained darkly, Inclusion contained in nucleusin nucleus

ChromatinChromatin Condensed, Agglutinated, Broken, DissolvedCondensed, Agglutinated, Broken, DissolvedPlasma Plasma

membranemembraneIncreased viscosity (mucosity), Decreased Increased viscosity (mucosity), Decreased

mobilitymobility

PlasmaPlasma Accumulation of pigments, Formation of Accumulation of pigments, Formation of vesiclesvesicles

MitochondrionMitochondrionDecreased quantity, Enlarged size, Decreased quantity, Enlarged size, Mutated Mutated and lost and lost mtDNAmtDNA

Golgi BodyGolgi Body BrokenBroken

Nissl BodyNissl Body DisappearedDisappeared

Inclusion Inclusion Decreased glycogen, Accumulated fatDecreased glycogen, Accumulated fatNuclear Nuclear

MembraneMembrane InvaginatedInvaginated

Form changes of an aged cell

55

The changes of molecules:

DNA: The DNA replication and transcription will be inhibited, but some genes will be abnormally activated. Telomere DNA lost. The mtDNA is specifically absented, and the cell DNA is oxygenated, broken, absented, and desmethylated.

RNA: The quantity of mRNA and tRNA is decreased.

Protein: Synthesis is inhibited. The proteins are modified with glycosylation and others resulting in decreased stability, antigenicity, and digestability. Accumulated free radicals break the peptide chains, and peptide chains are conjugated together resulting in denatured proteins.

Enzyme: The activity core is oxygenated. Ca2+, Zn2+, Mg2+, and Fe2+ lost. The secondary structure, solubility, and isoelectric point are changed. So, enzyme molecules are inactivated.

Lipid: Unsaturated fatty acids are oxygenated resulting in membrane mobility decreased.

66

Mechanisms of cell aging

There are many theories about the mechanism of aging. Briefly, error theories and genetic/programmed theories are important.

Error theories: After the cell suffered from damages, repairing system is working not efficiently resulting in the accumulation of errors that cause cell aging. 1. Waste product accumulation: Lipofuscin is a macromolecule conjugated by proteins, DNA, and lipids in lysosomes. Accumulated lipofuscins can inhibit signal transduction and molecules exchanging. For example, Alzheimer’s disease (AD) is caused by the accumulation of β- amyloid protein (β-AP). So, β-AP detection can be used to diagnose AD. 2. Cross linking of large molecules: Excessive crossly linked macromolecules is an important pathogen to aging. For examples, The linkage of DNA and collagen will damage their function. Linkage of collagens is associated with arteriosclerosis and vascular diseases.

77

3 ． Free radical theories: The free radicals in human body is formed by two ways: Some harmful extra-environments cause the level of free radicals raised; Some metabolism reaction in vivo form free radicals. The latter is the major way to raise the free radical level in vivo. Usually, the free radicals with the normal level are beneficial to body to clean the pathogen microorganisms in vivo. But the excessive free radical accumulation is very harmful to cells. Free radical can cause excessive linkage reaction between DNA and proteins, fatty lipid, especially polyunsaturated fatty acids (PUFA), and damage DNA, bio-membrane, structural and functional proteins. Excessive free radicals can inhibit some important bio-reaction in vivo, and cause hard erythrocytes formed that is associated with some important and severe diseases, such as cardiovascular diseases and severe cerebral malaria. The level of free radicals is raised significantly with becoming. The older, the higher level of free radicals, that is why the elders ‘ skins and skin color present special features. There are free radical cleaning systems in body including superoxide dismutase (SOD) and others. But in old people, the expression of the proteins for these system are not enough or deficient, or the receptors for these systems are inhibited or expressed lowly, so, the level of free radicals in old people is higher than young people.

88

Melatonin is the commercial bio-products in America that can clean the excessive free radical accumulation. But, for the elders, melatonin receptor expression is not enough, that is core problem for them.

4. Mitochondrial DNA mutation: Reactive oxygen species (ROS) can be accumulated in mitochondrion because of its features. mtDNA is naked DNA that is easy to be damaged by ROS, and the DNA polymerase for mtDNA replication lacks repairing function, so, mtDNA is very easy to be mutated by excessive ROS. The mutation of mtDNA can block the respiratory chain of cell that can cause further accumulation of free radical. mtDNA mutation and absence is much more obvious in elder than in young. The changes of mtDNA is closely associated with many diseases in elder, for example, AD and other degeneration diseases. Brain, heart is the organs where the oxidative stress is the most high. So, these organs are easy to become old, that is why the cerebral and cardiovascular diseases are major killers for old people. Caloric restriction is beneficial to a long life probably!

99

5 ． Somatic mutation and DNA repair:

Some extra-environmental effects of physics or chemistry can damage DNA, and all of intra-environmental free radicals can damage DNA. Usually, damaged DNA can be repaired by the DNA repairing system immediately. But, in old people, the repairing mechanism is obviously degenerated. So, the mutation and replication mistake will be accumulated to a high level that causes cell old and death. The damage DNA of the cells with active function has priority to be repaired firstly, and the complete repairing happens in DNA replication phase of cell cycle. That is why the stem cells can keep young and powerfully potential proliferation and differentiation ability.

6 ． The inactivation of repeated genes:

The repeated genes in eukaryotic genome can both increase the gene information and protect the gene damage from the gene disappeared. In another hand, the repeated genes can delay the early death of the important cell. If one copy of repeated gene is damaged and blocked, other copies can be activated immediately till to last copy is used. Experimental data shows that the repeated gene copy number for rRNA in liver can be decreased with aging.

1010

Genetic/programmed theories:

1 ． Programmed senescence: This theory believes that all of growth, development, senescence, and death are regulated by some existed genetic programs. The senescence can presents some senescence markers, such as the senescence marker protein-2 in liver. In addition, senescence is associated with the programmed degeneration of nerve-endocrine system and immune system.

2 ． Replicative senescence:

The proliferation of normal cells in vivo or vitro is not unlimited. The number of proliferation is a limit that is called as Hayflick limit, highest division frequency, or passage number. For example, human embryonic fibroblast can be cultured in vitro with a passage number of 60-70. Hayflick limit is associated with the length of telomere DNA. The telomere can be cut off a part at each time of DNA replication. When it become short to the Hayflick limit, the DNA damage checkpoint will be started, p53 will be activated, p21 will be expressed, and the cell will stop the cell cycle to turn to apoptosis. The telomere of human fibroblast becomes short by 14-18bp/year. The length of telomere is closely associated with telomerase activity. Telomerase can synthesize telomere DNA with its RNA as template. In old body, the telomere activity is not so active.

1111

3 ． Longevity genes:

The statistics data shows that the life time of son/daughter is associated with his/her parents. Each species of animal is of consistent average of life time and longest life time. The patients of Werner's syndrome present obvious senescence syndromes at thirty nine years old, and die from this disease at forty seven years old. The kids of Hutchinson-Gilford syndrome present obvious senescence syndromes at one year old, and die from this disease at twelve to eighteen years old. The life time of species is depended on some genes in species’ genome. We call these genes as longevity genes or senescence genes.

1212A patient of Werner's syndrome at 37 years old

(From http://www.nejm.org on)

1313

Normal kid (Left)

Patient of Hutchinson-Gilford syndrome (Right)

1414

When a cell turns to aging way, some senescence associated genes (SAG) are frequently expressed in it, and the level of these expressions are much higher than normal. Investigators have found some SAGs on 1st, 4th chromosomes and X chromosome. At least, there are four mutated genes are associated with AD. The mutation of the gene of amyloid protein precursor (APP) causes its product, β- amyloid protein (β-AP) accumulated in brain tissue and AD developed.

II. Necrosis and apoptosis

The cell death happens frequently in normal tissue. The cell death ways include ① necrosis; ② apoptosis and programmed cell death (PCD).

1515

Necrosis: Necrosis means the cell death caused by chemical, physical, and biological effects. The pathological changes for necrosis are enzyme digestion and protein denaturation. If the enzymes are from the same cell, we call this digestion as autolysis, otherwise, called as heterolysis.

Mitochondria Tumefacient ER Other organelles DisintegratedPrimary stage of necrosis Structural fatty Uncombined and vacuolated Protein granules Increased Nucleus Broken or pyknoted

Basophilic nuclear protein Degenerated Later stage of necrosis Plasma Eosinophilic Dark eosin staining Water rich cell Water vesicle enlarged

Water rich cell Cell structures are disappeared Latest stage of necrosis Membrane and organelles Broken DNA Degenerated Content of cell Flowed out Inflammation

1616

The differences between necrosis and apoptosis

1717

Apoptosis:

Apoptosis was named by Kerr in 1977. The major features of apoptosis are as the follows: ① Chromatin is condensed and moved to nuclear membrane. The nucleus is broken, and cell will form many apoptosomes by germination. ② Apoptosome contains organelles, pyknotic chromatin. Apoptosome can be swallowed and digested by adjacent cells. Because apoptosome is always enveloped by its membrane, it will not release out the content, so, it will not result in inflammation. But necrosis always causes inflammation. ③ Apoptosis cell can synthesize some protein for itself. Necrosis cell can not do so. ④ Endonuclease was activated, so, the chromatin is cut off at nucleosome junction, and form many fragments with a 200bp length difference, so, a ladder electrophoresis result can be obtained for apoptosis cell genome DNA. ⑤ Usually, apoptosis is a physiological procedure, but necrosis is a pathological procedure.

1818

The differences between necrosis and apoptosis

DifferenceDifferencess

ApoptosisApoptosis NecrosisNecrosis

CausesCauses Physiological or pathologicalPhysiological or pathological PathologicalPathological

FieldField Single distributed cellsSingle distributed cells Mass of tissue or cellsMass of tissue or cells

MembraneMembrane No broken and form No broken and form apoptosomeapoptosome BrokenBroken

ChromatinChromatin Condensed under nuclear Condensed under nuclear membranemembrane FlocculentFlocculent

OrganellesOrganelles Almost no changeAlmost no change Tumefacient, disintegrated Tumefacient, disintegrated ERER

Cell sizeCell size Shorten by condensationShorten by condensation EnlargedEnlarged

ApoptosomeApoptosome Yes. Swallowed by adjacent Yes. Swallowed by adjacent macrophagesmacrophages

No. Autolysis. Swallowed No. Autolysis. Swallowed by macrophagesby macrophages

Genome Genome DNADNA

Degenerated with Degenerated with regulation. Ladder DNA regulation. Ladder DNA electrophoresiselectrophoresis

Disintegrated. Smeared Disintegrated. Smeared electrophoresis resultelectrophoresis result

Synthesis of Synthesis of proteinprotein YesYes NoNo

RegulationRegulation By genesBy genes Passively Passively

InflammatioInflammationn

NoNo YesYes

 

1919

Left: Normal thymocyte

Right: Apoptosis thymocyte (Apoptosome)

2020

There are many papers or books describe the apoptosis and programmed cell death (PCD) as same concept. Actually, apoptosis is different from PCD. PCD means that some cells must turn to apoptosis and death at some time points following the spatiotemporal sequence for the individual development, and the death of these cells are designated previously by genetic regulation system. Apoptosis means the cell death regulated by genes, but it is not designated previously by genetic system, and many effects from the cell environment can regulate it. The final result for PCD is apoptosis, but, it is not true that all apoptosises are programmed. Robert Horvitz group of MIT found 14 genes are associated with the apoptosis of C. elegans using a somatic mutation method. Ced-3 and Ced-4 can induce apoptosis, Ced-9 can inhibit Ced-3 and Ced-4 and stop apoptosis. If Ced-9 is deficient, the fetus will die from excessive apoptosis.

2121

On October 7, 2002, an American scientist and two British scientists won the 2002 Nobel prize because of their great contributions to the genetic regulation of organ development and PCD.

2222

III. The molecular mechanism of apoptosis

In an embryo, apoptosis is one of the basic ways to maintain the cell quantity in body for the normal development. In an adult, apoptosis can clean the old and damaged cells for the body health. Like cell proliferation, apoptosis is regulated by gene system exactly. There are two major ways to apoptosis: 1. Activate the apoptotic enzyme, caspase, by extracellular signals; 2. Activate caspase by the caspase activation factor released from mitochondria. Activated caspase can degenerate the important proteins in cell to cause apoptosis.

2323

Apoptosis associated genes or proteins

1. Caspase family: Caspase is a type of protease. These proteases are the key enzymes to cause apoptosis. When they are activated by signals, the important proteins in cell will be degenerated to cause the cell turn to apoptosis irreversibly. The caspases keep the features as the follows: ① The enzyme activity depends on the nuclear affinity of cysteine residue; The substrate is always cut off at the site post aspartate, that is ②why it was named as caspase (cysteine aspartate-specific protease); ③ are the tetramer composed of two large subunits and two small units. Large and small subunits are encoded by same gene. Interleukin-1 β-converting enzyme (ICE) is the homologous gene of the nematode Ced-3 that was earliest found. Because ICE can cleave the precursor of IL-1, it was so named. 11 ICE homologous proteins have been found in human cells. They can be sorted as two types: ICE subgroup and Ced-3 family. The former participates in inflammation. Ced-3 family participates in apoptosis and can be sorted as two types: 1. Executioner or effector, such as caspase-3, 6, and 7, can degenerate the structural and functional proteins in cell to cause apoptosis. But they can not be activated by autocatalytic or self-splicing ways. 2. Initiators, such as caspase-8, and 9 can be activated with signal by self-splicing way, and start caspase cascade reactions. Caspase inhibitor, inhibitors of apoptosis proteins (IAPs), is a big protein family in cell. IAPs, such as XIAP, can bind to caspase by baculovirus IAP repeats domain (BIR domain) to inhibit caspase.

2424

The members of ICE family (A: 3 types of caspases, those in blue mean caspases involved with inflammation, in red mean executioner, in green mean

initiators. B: Structural model of caspase-3. C: Activation of caspase-3)(From Katja C. Zimmermann, et al. 2001)

2525

2 ． Apaf-1: Apaf-1 (apoptotic protease activating factor-1) is important to the apoptosis in mitochondrion. If Apaf-1 is knockout, the mouse brain can not be developed normally. Apaf-1 contains 3 domains: ① CARD (caspase recruitment domain) can enrich caspase-9. ② Ced-4 homologous domain can bind to ATP/dATP. ③ C terminal domain can bind to cytochrome c to activate Apaf-1. Apaf-1/ cytochrome c complex can bind to ATP/dATP, enrich caspase-9 by CARD domain, form apoptosome, activate caspase-3, and finally start caspase cascade reactions.

3 ． Bcl-2 (B-cell lymphoma/Leukemia-2) family: Bcl-2 is the apoptosis suppressor gene and integrin. 19 homologous genes about Bcl-2 have been identified so far. Anti-apoptotic Bcl-2 members: Bcl-2, Bcl-xl, Bcl-w, and Mcl-1. Pro-apoptotic Bcl-2 members: Bax, Bak, Bad, Bid, and Bim. Bcl-2 proteins are mainly located on mitochondrion membrane. Most of pro-apoptotic proteins are located in plasma. When cell receive apoptosis signals, pro-apoptotic proteins will move to mitochondrion membrane to release out the mitochondrion content, such as cytochrome c, to activate caspase, and result in apoptosis. The pro-apoptotic proteins can be activated by dephosphorating, modifying by caspase, releasing from combined protein.

2626Bcl-2 family (From Katja C. Zimmermann, et al. 2001)

2727

4. Fas: Fas is also called as APO-1/CD95, and is the member of TNF receptor family. Fas gene encodes a 45KD transmembrane protein that is distributed on thymocyte, activated T and B cells, macrophages, the cells of liver, spleen, lungs, heart, brain, intestine, testes, ovary, and others. The combination of Fas and Fas ligand can activate caspase to cause apoptosis of target cells.

5 ． p53: p53 is a cancer suppressor gene that can check the DNA replication at G phase. If DNA was damaged, p53 will inhibit cell cycle till to the DNA is repaired. Otherwise, the cell will be introduced to apoptosis.

6. c-myc: c-myc is a proto-oncogene that is excessively expressed in many human cancer tissue. c-myc can enhance cell proliferation and inhibit differentiation. c-myc is excessively expressed in apoptotic cells also. As a transcription regulator, c-myc can activate proliferation genes and apoptotic genes. So, it presents two choices to cells, proliferation or apoptosis. With GF and Bcl-2, c-myc promotes proliferation, otherwise apoptosis.

7. ATM: ATM (ataxia telangiectasia-mutated) gene is a DNA damage detector also. 1% of people are the zygotes with ATM absence. These persons are sensitive to radiation rays and easy to suffer from cancers.

2828

Apoptosis intermediated by Fas:

The apoptosis receptors on cell surface are the TNF receptor (TNFR) including Fas (Apo-1/CD95), TNFR1, DR3/WSL, DR4/TRAIL-R1, and DR5/TRAIL-R2. Fas is transmembrane protein including extracellular part and the intracellular part called death domain (DD). Fas ligand (FasL) combines to Fas and change DD structure to link the DD of Fas associated death domains (FADD), then, the DED (death effector domain) of FADD can bind to caspase-8 to form DISC (death-inducing signaling complex). DISC can activate caspase-8, -10, and start caspase cascade reactions to activate caspase-3, -6, and -7, the important proteins in cell are degenerated, cell turns to apoptosis. Caspase can activate CAD (caspase-activated Dnase). CAD can cut off DNA at the links of nucleosome, and form DNA fragments with 200bp difference in length. Fas/FasL is important immune system. By the Fas/FasL intermediation, the activated T cells can clean the cell clones that take autoimmunity to the cells of self body. This action can protect the body from damage. The abnormal apoptosis of lymphocytes is the major pathogen to the autoimmune diseases. The cell toxic T lymphocytes (CTL) can introduce apoptosis by FasL, but, some cancer cells can introduce lymphocyte apoptosis to escape immunity attack.

2929

Apoptosis intermediated by Fas

(From Avi Ashkenazi and Vishva M. Dixit 1998)

3030

Mitochondria and apoptosis

As the apoptosis inducer, cytochrome c released from mitochondrion can bind to Apaf-1, caspase-9 precursor, and ATP/dATP to form apoptosome, then, activate caspase-3 to start caspases cascade reaction resulting in apoptosis. Mitochondrion keeps always no damaged in apoptotic cell. So, how is the cytochrome c released into plasma? Probably, it is released into plasma through the permeability transition pore (PT pore) or the channel formed by the Bcl-2 family. PT pore is composed of adenine nucleotide translocator (ANT) and voltage dependent anion channel (VDAC). The members of Bcl-2 family regulate the switch of PT pore. Pro-apoptotic members, such as Bax, can promote PT pore opened by binding to ANT or VDAC. Anti-apoptotic members, such as Bcl-2 and Bcl-xL, can bind to ANT or VDAC competently with pro-apoptotic members, or block the pro-apoptotic member to bind to ANT and VDAC directly.

3131

The mutation of Ced-3 and Ced-4 can inhibit the cell death in each develop stage in nematode. Caspase can not be activated in the mouse with Apaf-1 absence, but most of organs can be developed normally excepting excessive nerve cells. The proteins released out with cytochrome c include Smac (second mitochondria-derived activator of caspase), AIF (apoptosis inducing factor) and endonuclease G (Endo G). Smac can bind to the BIR domain of IAP (inhibitor of apoptosis) to stop the inhibition of IAP to caspase. AIF causes nucleus pyknosed and chromatin broken. Endo G cleaves DNA. So, if no caspase involved, apoptosis can be still started by mitochondrion way. In the cells that are responding to Fas, the cells of type I, such as thymocytes, their caspase-8 is powerful to cause apoptosis after activated by Fas. So, excessive expression of Bcl-2 can not inhibit the apoptosis introduced by Fas in type I cells. The cells of type II, such as liver cells, the activation of caspase-8 intermediated by Fas is not enough to cause apoptosis. So, the apoptosis signals must be enhanced by mitochondrion way in this type cells: activated caspase-8 can cleave Bid in plasma to form tBid (truncated Bid), then, tBid enters mitochondrion and causes cytochrome c released out, finally, the apoptotic signal is enhanced.

3232

Apoptosis caused by cytochrome c(From R. Chris Bleackley and Jeffrey A. Heibein 2001)

3333

By the described as above, it is indicated that mitochondrion is both the energy station and the center of apoptosis of regulation. Why is mitochondrion so important to a cell? Each growth factor can promote glycose transported to mitochondrion to enhance energy supply, so, when the GFs were inhibited, the cell will turn to apoptosis. It is easy to understand that the GF inhibition causes apoptosis, but, the detail about it is still kept unknown so far.