SEMINAR ON CELLULAR AGING AND CELL DEATH
Department of Pharmacology Sree Siddaganga college of Pharmacy Tumkur-572 102 Karnataka
Contents: o
Cell aging
• Introduction • Factors affecting aging • Theories of aging • Programmed theories • Error theories • Cellular mechanisms in aging o
Cell death
• Introduction • Types o
Apoptosis
• In physiological condition • In pathological condition
Contents: •
Morphological changes
•
Biochemical changes
•
Apoptosis inducers
•
Mechanisms of apoptosis – Intrinsic pathway – Extrinsic pathway
•
Apoptosis inducing factor
•
Apoptosis and diseases
o
Necrosis
•
Types
•
Mechanism
o
Autophagy
•
Mechanism
•
References
CELL AGING
• Aging is defined as the normal process accompanied by the progressive alteration in the body’s / cell’s c ell’s homeostatic adaptive responses. • Progressive time related loss of structural and functional capacity of cells leading to death.
Senescence • Cell aging is also called as Cellular senescence. • Senescence is a stage in which the cells are no longer capable of dividing but these cells will remain metabolically active.
Life expectancy vs. Lifespan • Life Expectancy: Average number of years a body is expected to live. • Maximum Lifespan: Greatest age reached by any member of the species.
STAGES OF LIFE SPAN
Factors affecting Ageing: • Genetic – Clock genes, • Diet – malnutrition, obesity etc. • Social conditions Diseases – Atherosclerosis, diabetes etc. • Werner’s syndrome.
Senescense inducers
The Two Main Aging Theory Categories • Programmed Theories Aging has a biological timetable or internal biological clock. • Error Theories Aging is a result of internal or external assaults that damage cells or organs so they can no longer function properly. Many theories are a combination of programmed and error theories.
Programmed vs. Error Theories Error Theories
Programmed Theories • Programmed Senescence Theory • Endocrine Theory • Immunology Theory
• • • • •
Wear and Tear Theory Rate-of-Living Theory Cross-linking Theory Free Radical Theory Error Catastrophe Theory • Somatic Mutation Theory
Programmed Senescence Theory • The result of sequential switching “off” or “on” of specific genes. • Example – “Hayflick’s Limits” – Functional changes within cells are responsible for aging. – Cumulative effect of improper functioning of cells and eventual loss of cells in organs and tissues.
Telomeric Theory • Telomere: – Telomere is molecular cap made up of DNA which compose the linear end of the chromosome. – It is made up of thousands of building building blocks with a sequence TTAGG that repeats over and over – It ensures complete replication of the chromosomal end. – Protects the chromosomal terminal from fusion and degradation.
T E L O M E R A S E I N A G I N G
Telomerase in aging
Endocrine Theory • Biological clocks act through hormones to control the pace of aging. Hormones H ormones effects growth, metabolism, temperature, inflammation and stress. • Examples- Menopause – Decreased level of estrogen & progesterone pr ogesterone – Hot flashes, insomnia
Immunologic Theory • A programmed decline in the immune system leads to an increased vulnerability to disease, aging and death • Example- Decreased T cells (helper cells) in adults – Increased diseases in older adults – Increased autoimmune diseases in adults
Wear and Tear Theory • Years of damage to cells, tissues and organs eventually wears them out, killing both them and the body • Example- Wearing out of the skeletal system such as in osteoarthritis • Wear and tear can be viewed as a result of aging and not the cause of it.
Rate of Living Theory Metabolic Rate Theory – The higher the basal metabolic rate (the rate, at which the body at rest, uses energy), the shorter the life span.
Cross-Linking Theory • The accumulation of cross-linked proteins damages cells and tissue, slowing down bodily processes. • Example Non-enzymatic glycosylation reactions occur when glucose molecules attach to proteins causing a chain of chemical reactions resulting in a structural change to the proteins. – Loss of flexibility of connective tissue – Microvascular changes in arteries
Free Radical Theory • A free radical is a molecule with an unpaired, highly reactive electron. One type of very reactive free radical is the oxygen free radical, which may be produced during metabolism or as a result of environmental pollution.
Super oxide anions H2O2 Hydroxyl radical e.g.
What does free radicals do in a living system? Free radicals
Mutation Membrane damaged
DNA damaged
Free Radical Theory The free radical “grabs” a electron from any molecule it its vicinity. It does this because electrons like to exist in pairs. When it “grabs” an electron from another molecule, it damages the other molecule.
Free Radical Theory • During aging, damage produced by free radicals cause cells and organs to stop functioning.
• Some of the molecules that may be damaged by free radicals are fats, proteins, and DNA (both in the nucleus and in mitochondria). • If membrane fats are attacked, then causes breakdown of the cell membrane. If it is a red blood cell membrane, it leads to hemolysis. • If DNA is attacked, leads to mutation that may cause aging or cancer.
Free Radical Theory • Antioxidants: – Natural antioxidants in the body, such as bilirubin. – Enzymes such as superoxide dismutase (SOD), catalase, & glutathione peroxidase. – Dietary antioxidants such as beta carotene, and the vitamins C and E.
Free Radical Theory • Not all free radicals cause damage. • Free radicals are part of immunological response system. – Macrophages engulf bacteria – Free radical reactions produced inside the macrophage oxidize and kill bacteria.
Error Catastrophe Theory – DNA errors promote senescence, programmed cell death (apoptosis). – Aging results from gene interference with the ability of the cells to reproduce.
Somatic Mutation Theory • Genetic mutations occur and accumulate acc umulate with age in the somatic cell causing the cell to: – Deteriorate – Malfunction
• Accumulation of mutations result in : – Damage to the DNA The theory states that aging is an imbalance between DNA’s ability to repair itself and accumulating DNA damage. – When the damage exceeds the repair, the t he cell malfunctions and this can lead to senesence.
Werner’s Syndrome • Werner’s Syndrome is a genetic disease that accelerates aging and as a result people at a young chronological age develop characteristics seen in old people.
CELLULAR MECHANISMS OF AGING • Cross linking proteins & DNA. • Accumulation of toxic by-products. • Ageing genes. • Loss of repair mechanism. • Free radicle injury • Telomerase shortening.
Ageing –changes: • Gradual atrophy of tissues and organs. • Dementia • Loss of skin elasticity • Greying and Loss of hair • BV damage – atherosclerosis. • Loss of Lens elasticity opacity affects vision.
CELL DEATH
CELL DEATH • Death - Permanent cessation of all vital functions, including those of heart, lungs, and brain. • Extinction of life or End of life.
TYPES OF CELL DEATH • Apoptosis • Autophagy • Necrosis
APOPTOSIS • Apoptosis is a pathway of cell death that is induced by a tightly regulated intracellular program in which cells destined to die activate enzymes that degrade the cells’ own nuclear DNA and nuclear and cytoplasmic proteins. • The word “apoptosis” is derived from Greek, meaning ‘falling off’ or ‘dropping off’
• It is also also defined as a form of cell death designed to eliminate unwanted host cells from the body through activation of a coordinated, internally programmed series of events which is significant in a variety of physiologic and pathologic conditions.
APOPTOSIS IN BIOLOGIC PROCESSES
Physiologic conditions.
Pathological conditions.
IN PHYSIOLOGICAL CONDITIONS The
death of the cell is necessary for the organism to reach its final form during embryonic development. By the fourth week of human embryonic development, development, limb buds appear as outpockets from the body wall. wall. By six-weeks, six-weeks, the terminal (end) portion portion of the limb buds flatten to form handplates and footplates. Forty-eight days after fertilization, select cells within the interdigital spaces of the hand and footplates undergo apoptosis apoptosis allowing allowing the digits digits to separate. separate. Digit separation is complete by fifty-six days after fertilization.
5 weeks
8 weeks
• If the cells in the interdigital space fail to undergo apoptosis, the fetus will be born with webbed hands and/or webbed feet. The medical term for this condition is syndactyly.
Hormone dependent involution in adult, such as • Endometrial cell breakdown during the menstrual cycle, ovarian follicular atresia in the menopause. • The regression of the lactating brest after weaning, and
The
cell has obtained serious damage that cannot be repaired or requires too much energy to be repaired.
Cell
deletion in proliferating cell populations, such as intestinal crypt epithelia, in order to maintain a constant number.
The cell has outlived its usefulness.
• During the immune response, Helper T-cells stimulate the proliferation of B-cells and T-cells allowing for the production of antibodies and cytotoxic T-cells. • The cytotoxic T-cells destroy infected body cells. • When all traces of the pathogen have been removed from the body, suppressor T-cells shut the cellmediated and humoral responses off.
• However, millions of cytotoxic T-cells are still circulating looking for infected body cells. • The body removes these cells by programmed apoptosis and subsequent by ingestion by macrophages thus leaving only memory B and T-cells behind from the encounter.
Elimination
of potentially harmful self-reactive lymphocytes, either before or after they have completed their maturation.
IN PATHOLOGICAL CONDTIONS • Cell death in tumors exposed to chemotherapeutic agents. • Cell death by cytotoxic T cells in immune mechanisms such as in graft versus host disease and rejection reaction. • Cell injury in certain viral diseases. e.g. viral hepatitis, in which loss of infected cells is largely because of apoptotic death. • Pathologic atrophy of organs and tissues on withdrawal of stimuli. e.g. prostatic atrophy after orchiectomy.
• Progressive depletion of CD 4 + T cells in the pathogenesis of AIDS. • Even in situations in which cell death is mainly by necrosis, the pathway of apoptosis may contribute. e.g. injurious stimuli that cause increased mitochondrial permeability trigger apoptosis.
MORPHOLOGICAL CHANGES •
Cell rounds up, communication with neighbors stops
•
Membrane blebs
•
Phosphatidylserine flips over (inner membrane to outer membrane).
•
Proteins crosslink
•
β-tubulin, actin polymerize
•
Cell shrinks
•
Chromatin condenses
•
DNA cleaved stepwise (DNA ladder)
•
50,000 - 300,000 bp then 185 bp (internucleosomal)
•
Macrophages phagocytize apoptotic bodies, using lectin receptors to recognize altered carbohydrates
•
No inflammatory response
Normal Cell Cell shrinkage away from neighbouring cells Plasma membrane blebbing Cytoplasmic and nuclear condensation
Margination of condensed chromatin
Nuclear and cellular fragmentation
Apoptotic Bodies Phagocytosis
BIOCHEMICAL CHANGES PROTEIN CLEAVAGE: • Proteins are hydrolysed by a specific family of proteases – caspases. • Active caspases cleave many vital cellular proteins, such as lamins, and thus break up nuclear scaffold and cytoskeleton. • Caspases also activate DNAses, which degrade nuclear DNA.
DNA Breakdown: • DNA is broken down into 50 to 300 kilobase pieces. • Subsequently, there is internucleosomal cleavage of DNA into oligonucleosomes, in multiples of 180 to 200 base bas e pairs, by Ca2+ and Mg2+ dependent endonucleases.
Phagocytic recognition: • Apop poptoti totic c cel cells expr expre ess Phosphatidylserine Phosphatidylserine in the outer layers of their plasma membranes. • In some some typ types of ap apoptos ptosiis, thrombospondin, an adhesive glycoprotein, is also expressed on the surfaces of apoptotic bodies. • Thes These e alte altera rati tion ons s perm permit it the the early recognition of dead cells by macrophages, resulting in phagocytosis without the release of pro-inflammatory cellular components.
Inducers of Apoptosis Physiological Activators TNF family Fas Ligand TNF
TGFβ Neurotransmitters glutamate dopamine N-methyl-D-aspartate
Growth Factor withdrawal Loss of matrix attachment Calcium Glucocorticoids
Damage-Related Activators Heat Shock Viral Infection Bacterial Toxins Oncogenes myc,rel, E1A tumour suppressors p53 Cytolytic T cells Oxidants Free Radicals Nutrient Deprivation anti-metabolites
Therapy-associated Agents Chemotherapeutic drugs cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate, vincristine Gamma-radiation UV-radiation
MECHANISMS OF APOPTOSIS • One generated by signals arising within the cell; [Intrinsic (Mitochondrial) Pathway]. Pathway]. • Another triggered by death activators binding to receptors at the cell surface: – TNF-α – Lymphotoxin – Fas ligand (FasL) [Extrinsic (Death Receptor - Initiated) Pathway]. Pathway ]. • A third that may be triggered by dangerous reactive oxygen species.
INTRINSIC PATHWAY • In a healthy cell, the outer membranes of its mitochondria express the protein Bcl-2 on their surface. • Bcl-2 is bound to the molecule of the protein Apaf-1 (apoptotic protease activating factor-1) • Internal damage to the cell (e.g . from reactive oxygen species) causes • Bcl-2 to release apaf-1 • a related protein, Bax, to penetrate mitochondrial membranes, causing • cytochrome C to leak out.
• The release cytochrome C and Apaf-1 bind to molecules of caspase 9. • The resulting complex of o o o o
Cytochrome c Apaf-1 caspase 9 (and ATP)
is called the apoptosome apoptosome..
• These aggregate in the cytosol.
Bind to and neutralize
ROS
A I R D N O H C O T I M
Other pro-apoptotic proteins (AIF)
Cyt c
Inhibitors of apoptosis (IAP)
o r P
s a p s a c
Apaf - 1
Bcl-2
9 e
Caspase activation
e vi t c A
e s a p s a c
9
CYTOSOL Executioner caspases
INTRINSIC
Bind to and neutralize
ROS
A I R D N O H C O T I M
Other pro-apoptotic proteins (AIF)
Cyt c
Bcl-2
Inhibitors of apoptosis (IAP)
o r P
s a p s a c
9 e
Caspase activation
Apaf - 1 e vi t c A
e s a p s a c
9
CYTOSOL Executioner caspases
INTRINSIC
• Caspase 9 is one of the family of over a dozen caspases caspases.. They are all proteases. They get their name because they cleave proteins – mostly each other – at aspartic acid (asp residues). • Caspase 9 cleaves and, in so doing, activates other caspases.
• The sequential activation of one caspase by another creates an expanding cascade of proteolytic activity leads to digestion of structural proteins in the cytoplasm, and degradation of chromosomal DNA, and • Phagocytosis of the cell.
EXTRINSIC PATHWAY • Fas and the TNF receptor are receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell • binding of the complementary death activator (FasL and TNF respectively) transmits a signal to the cytoplasm that leads to • activation of caspase of caspase 8 • caspase 8 (like caspase 9) initiates a cascade of caspase activation leading to • phagocytosis of the cell.
Death receptors and adaptors • Death receptors: ◘ Fas/CD95, DR4/DR5, DR3 ◘ TNFR (Tumor Necrosis Factor Receptor). • Adaptors: FADD (Fas-associated death domain protein) and TRADD (TNFRassociated death domain protein).
EXTRINSIC PATHWAY
APOPTOSIS - INDUCING FACTOR (AIF) • Neurons, and perhaps other cells, have another way to self-destruct that — unlike the two paths described above — does not use caspases. • Apoptosis-inducing Apoptosis-inducing factor (AIF) AIF) is a protein that is normally located in the intermembrane space of mitochondria. When the cell receives a signal telling it that it is time to die, AIF is released from the mitochondria (like the release of cytochrome c in the first pathway); • migrates into the nucleus; • binds to DNA, which • triggers the destruction of the DNA and cell death.
WHAT MAKES A CELL TO DECIDE TO COMMIT SUICIDE? 1. The The with withdr draw awal al of of posi positi tive ve sign signal als s i.e. signals needed for continued survival. 2. The The recei receipt pt of of nega negati tive ve sig signal nals. s.
WITHDRAWAL OF POSITIVE SIGNALS • The continued life of most cells requires that they receive continuous signals from the cells and for may continued adhesion to the surface on which they are growing. Some examples of positive signals •Growth factors for neurons. •Interleukin – 2 (IL-2) an essentila factor for the mitosis of lymphocytes.
RECEIPT OF NEGATIVE SIGNALS • Increased level of oxidants with in the cell. • Damage to DNA b these Oxidants or other agents like UV – light X – Rays Chemotherapeutic drugs • Molecules that bind to specific receptors on the cell surface and signal the cell to design the apoptosis program.
• These death activators include: • Tumor necrosis factor apha (TNFα) that binds to the TNF receptor. • Lymphoma (also known as TNFα) that also binds to the TNF receptors. • FAS ligand (FasL) a molecule that binds to a cell surface receptor named Fas (also called CD95)
Diseases Associated with Deregulated Apoptosis Increased Apoptosis AIDS Neurodegernative disorders Alzeheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis Retinitis pigmentosa Myelodysplastic syndromes Aplastic anaemia Ischaemic Injury Myocardial infarction, Stroke, Reperfusion injury Toxin-Induced liver disease Alcohol
Inhibition of Apoptosis Cancer Follicular lymphomas carinomas with p53 mutations hormone dependent tumours: breast cancer, prostate cancer, ovarian cancer Autoimmune Disorders Systemic lupus erythematosus Immune-mediated glomerulonephritus Viral Infections Herpesvirus, poxvirus, adenovirus
Neurodegenerative Neurodegen erative Disease and Apoptosis • Developmental apoptosis in brain triggered by GF withdrawal • Toxicity of abnormal protein aggregates may be initiator of apoptosis in Alzeheimer’s , Parkinson’s, Huntington’s Disease and amyotrophic lateral sclerosis amyloid-β
IC Ca2+ Oxidative stress
Neuronal Receptors
Apoptosis
Huntington Aggregates
FADD/ Caspase 8
Apoptosis
APOPTOSIS AND VIRUS • Some viruses associated with cancers use tricks to prevent apoptosis of the cells they have infected. Several human papilloma viruses (HPV) HPV) have been implicated in causing cervical cancer. One of them produces a protein (B6) that binds and inactivates the apoptosis promoter p53 promoter p53.. • Epstein-Barr Epstein-Barr Virus (EBV), EBV), the cause of mononucleosis and associated with some lymphomas – produces a protein similar to Bcl-2 – produces another protein that causes the cell to increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).
Apoptosis and Cancer • Some B-cell leukemias and lymphomas express high levels of Bcl-2 of Bcl-2,, thus blocking apoptotic signals they may receive. • Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1. Apaf-1.
[Breast cancer cells dividing]
Contd….
• Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cell. • Other cancer cells express high levels of FasL of FasL,, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).
APOPTOSIS AND AIDS • The hallmark of AIDS (acquired ( acquired immunodeficiency syndrome) syndrome) is the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (µl) of blood). CD4 + T cells are responsible, directly or indirectly, for all immune responses. When their number declines below about 200 per µl, the patient is no longer able to mount effective immune responses and begins to suffer a series of dangerous infections.
ROLE IN HIV • HIV (human immunodeficiency virus) invades CD4+ T cells, and one might assume that it this infection by HIV that causes the great dying-off of these cells. However, that appears not to the main culprit. Fewer than 1 in 100,000 CD4 + T cells in the blood of AIDS patients are actually infected with the virus.
WHAT KILLS THE UNINFECTED CD4 CELLS? The mechanism is not clear, but several possibilities, • All T cells, both infected and uninfected, uninfected, express Fas. Fas. • Expression of a HIV gene (called Nef ) in a HIV-infected cell causes – the cell to express high levels of FasL of FasL at its surface – while preventing an interaction with its own Fas from causing it to self-destruct. • However, when the infected T cell encounters an uninfected one (e.g. in a lymph node), the interaction of FasL with Fas on the uninfected cell kills it by apoptosis.
Detection of apoptotic changes in DNA • Nucleic acid staining – nuclear morphology. • Detection of nuclear DNA fragmentation. • TUNEL staining (terminal deoxynucleotidyl transferase– mediated dUTP nick end-labeling). • Single-cell electrophoresis (Comet assay).
NECROSIS Necrosis
refers to a spectrum of morphologic changes that follow cell death in living tissue, largely resulting from the progressive degradative action of enzymes on the lethally injured cells.
• It’s a premature death of a cells and living tissues • Necrosis is caused by external factors, such as infection, toxins, or trauma.
TYPES There are seven distinctive morphologic patterns of necrosis: • Coagulative Coagulative necrosis is typically seen in hypoxic environments, such as an infarction. Cell outlines remain after cell death and can be observed by light microscopy. • Liquefactive necrosis is usually associated with cellular destruction and pus formation (e.g. pneumonia). pneumonia). This is typical of bacterial or, sometimes, fungal infections because of their ability to stimulate an inflammatory reaction.
Contd….
• Gummatous necrosis is restricted to necrosis involving spirochaetal infections (e.g. syphilis). • Haemorrhagic necrosis is due to blockage of the venous drainage of an organ or tissue. • Caseous necrosis is a specific s pecific form of coagulation coagulation necrosis typically caused by mycobacteria (e.g. tuberculosis), fungi, and some foreign substances. It can be considered a combination of coagulative and liquefactive necrosis.
Contd …..
• Fatty necrosis results from the action of lipases on fatty tissues (e.g. acute pancreatitis, breast tissue necrosis). • Fibrinoid necrosis is caused by immunemediated vascular damage. It is marked by deposition of fibrin-like proteinaceous material in arterial walls.
Sequence of Events in Necrosis • Membrane permeabilizes • Ca++ enters or is released from ER stores • Cell swells • Ca activates calpain, ruptures lysosomes • Lysosomal enzymes are released and activated by Ca ++ • DNA degraded (random smear on gel) • Inflammatory response
NECROSIS MECHANISM
AUTOPHAGY • Autophagy, Autophagy, or autophagocytosis or autophagocytosis,, is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. • It is a tightly-regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products
MECHANISM • The formation of a membrane around a targeted region of the cell, separating the contents from the rest of the cytoplasm. • The resultant vesicle then fuses with a lysosome and subsequently degrades the contents.
References • Molecular Biology of The Cell – Bruce Alberts. • Pathologic Pathologic Basis of Disease – Kumar. • www.caspases.org/ • www.ccs.k12.in.us/chsBS/kons/kons/apoptosis.htm • www.mainsgate.com/spacebio/mo www.mainsgate.com/spacebio/modules/gs_teare.htm dules/gs_teare.htmll • www.sciencemuseum.org.uk/online www.sciencemuseum.org.uk/online/lifecycle/172.a /lifecycle/172.asp sp • www.sciencedaily.com/releases/20 www.sciencedaily.com/releases/2007/01/0701101 07/01/070110124142.h 24142.h tm • Nucleic Acids Research, 2007,Vol. 35, No. 22 7417-7428 • www.daviddarling.info/ www.daviddarling.info/.../A/apoptosis.html .../A/apoptosis.html
THAN K YOU
