APOPTOSIS IN HEALTH & DISEASES

MODERATOR : PROF. PARAMESH

PRESENTER : DR. MEENU . E.V.

INTRODUCTION

Apoptosis - Definition

• A pathway of cell death induced by a tightly

regulated suicidal program, in which the

cells destined to die activate enzymes that

degrade cells own nuclear DNA and nuclear,

cytoplasmic proteins.

sclero dinesh

Kerr Wyllie and Currie paper, British Journal of Cancer, 1972 Aug;26(4):239-57

"We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word "apoptosis" (ἁπόπτωσισ) is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees”.

mev

Historical aspects

• German scientist Carl Vogt - Principle of apoptosis (1842).

Mev

• Walther Flemming – Process of programmed cell death (1845).

mev

• John Foxton Ross Kerr – Distinguish apoptosis from traumatic cell death (1962).

mev

Nobel prize in 2002 – Sydney Brenner , Horvitz, John

Buston.

‘identified gene that control apoptosis’ Study done in “Caenorhabditis Elegans”

Significance of apoptosis• During development many cells are produced in excess which

eventually undergo programmed cell death and thereby contribute to

sculpturing many organs and tissues [Meier, 2000]

• In human body about one lakh cells are produced every second by

mitosis and a similar number die by apoptosis (Vaux and Korsmayer

,1999, cell)

• Between 50 and 70 billion cells die each day due to apoptosis in the

average human adult. For an average child between the ages of 8 and

14, approximately 20 billion to 30 billion cells die a day. ( Karam, Jose A.

(2009). Apoptosis in Carcinogenesis and Chemotherapy. Netherlands: Springer.

ISBN 978-1-4020-9597-9)

• Without apoptosis, human gut can grow up to 12 miles in length

• Whole epithelial lining in our body changes every 23 days

sclero dinesh

APOPTOSIS NECROSIS

NATURAL YES NO

EFFECTS BENEFICIAL DETRIMENTAL

Physiological or

pathological

Always pathological

Single cells Sheets of cells

Energy dependent Energy independent

Cell shrinkage Cell swelling

Membrane integrity

maintained

Membrane integrity lost

sclero dinesh

APOPTOSIS NECROSIS

Role for mitochondria and cytochrome C No role for mitochondria

No leak of lysosomal enzymes Leak of lysosomal enzymes

Characteristic nuclear changes Nuclei lost

Apoptotic bodies form Do not form

DNA cleavage No DNA cleavage

Activation of specific proteases No activation

Regulatable process Not regulated

Evolutionarily conserved Not conserved

Dead cells ingested by neighboring cells Dead cells ingested by neutrophils and

macrophages

WHY APOPTOSIS?To eliminate cells that :

• are potentially harmful

• have outlived their usefulness / aged

• are damaged beyond repair

• Since it is genetically regulated , apoptosis is

sometimes referred to as programmed cell death.

• Certain forms of necrosis , called “ necroptosis”

are also genetically programmed, but by a distinct

cell of genes.

MECHANISM OF APOPTOSIS

2 phases of apoptosis

1. Initiation phase - activate the caspases

2 pathways:

Intrinsic / mitochondrial pathway – caspase 9

Extrinsic / receptor mediated pathway - 8

2. Execution phase – caspases causes degradation

INTRINSIC PATHWAY

( MITOCHONDRIAL PATHWAY )

INTRINSIC PATHWAY

[ MITOCHONDRIAL PATHWAY ]

PLAYERS

1) Sensors – BAD , BIM , BID

2) Proapoptotic- BAX , BAK

3) Cytochrome C

4) Apaf-1 [ apoptosis activating factor – 1 ]

5) Caspases

DEFENDERS

1) Antiapoptotic -BCL 2,

BCL XL,

2) IAPS: [ inhibitors of apoptosis

1.Caspases:

• ‘c’ - cysteine protease(an enzyme with cysteine in its active site)

‘aspase’- ability of these enzyme to cleave after aspartic acid residues

• More than 10 members

• Central executioners of cell death

• Depending on order in which they are activated during apoptosis, they are divided into

• Initiator caspase - 8, 9

• Executioner caspase -3, 6,7 Presence of cleaved,active caspases is a marker for cells

undergoing apoptosis

2.Bcl-2 proteins:Family of more than 20 members - categorised in 3 groups

[ based on proapoptotic and antiapoptotic properties , and presence of BCL-2 homology (BH) domains ]

1) Antiapoptotic - BCL 2, BCL XL

• present on outer mito.membrane, cytosol and ER

Functn : make outer mitochondrial membrane impermeable and prevent leakage of cytochromec.

Stimulated by : growth factors , survival signalsBCL XL - BCL2 related protein, long isoform

2) Proapoptotic – BAX , BAK

Funtn : promote outer mito. Membrane permeability by forming a channel and promote leakage of cytochrome c

3) Sensors – BAD , BIM , BID

• Act as sensors of cellular stress and damage

• Regulate other two groupsBAX – BCL2 associated X protein,

BAK- BCL2 antagonist killer 1, BAD- BCL2 antagonist of cell death,

BID – BH3 interacting domain death agonist

BIM – BCL2 interacting membrane

3. Cytochrome C – Mitochondrial protein present at inter

mitochondrial space

– Can activate caspase cascade

4. Apaf-1 [ apoptosis activating factor – 1 ]

– Present in cytosol

– Forms apoptosome after combining with cytochrome c

5 . IAPS: [ inhibitors of apoptosis ]

– Newly discovered group of anti-apoptotic proteins present in cytosol

– 7 members identified-NAIP,cIAP-1,cIAP-2 and survivin

– Bind to and inactivate caspases

– Survivin is involved in spindle cell formation

INTRINSIC PATHWAY [MITOCHONDRIAL PATHWAY]Withdrawal of Radiation , Toxins, Free radicals

survival signals

DNA damage / misfolded proteins

Loss of antiapoptotic(BCL2, BCLXL )

function ER STRESS

activation of sensors of Bcl 2 family

activation of proapoptotics- BAX, BAK

oligomerization and increase in outer mitochondrial membrane permeability

Leakage of cytochrome c leakage of SMAC which bind into cytosol to neutralise IAP’s in cytosol

Contd……

Cytochrome c in cytosol + APAF 1

Form Hexamer ( Apoptosome )

Binds to caspase- 9 ( critical initiator caspase)

Enzyme cleaves the adjacent caspase 9 molecules

i.e . Autoamplification process

Execution phase

EXTRINSIC PATHWAY

Death ReceptorsMembers of TNF receptor family which contain a

cytoplasmic domain involved in protein-protein interactions .i.e called Death Domain .

2 types of death receptors are there : • TNFr (tumour necrosis factor receptor )• FasR (fatty acid synthetase receptor )

The ligand for Fas is called FasLThe ligand for TNFr is TNFα

Adaptor Proteins also contain death domain

a. FADD [ Fas associated death domain ]b. TRADD [ TNF receptor associated death domain]

FAS ligand TNF

Deathdomains

Adaptor proteins

Pro-caspase 8 (inactive) Caspase 8 (active)

Pro-execution caspase(inactive)

Execution caspase (active)

• Fas and the TNF receptor

are integral membrane

proteins with their receptor

domains exposed at the surface of

the cell

• Binding of the complementary

deathactivator (FasL and TNF resp

ectively) transmits a signal to the

cytoplasm

• FADD /TRADD attach to death receptors

• Binds an inactive form of caspase-8

• M/L procaspase 8 molecules are brought

• They cleave and generate active caspase 8

• This pathway can be inhibited by a protein FLIP which binds to pro –caspase 8 and cannot cleave it

• Some viruses and normal cells produces FLIP and use this inhibitor to protect themselves from Fas mediated apoptosis

FLIP – FLICE inhibitory protein

• Extrinsic and intrinsic pathway involve fundamentally different molecules for their initiation, but their may be an interconnection between them.

Eg : in hepatocytes and pancreatic β cells, caspase 8 produced by Fas signaling cleaves and activate sensors of bcl family i.e BID which then stimulate mitochondrial pathway

EXECUTION PHASE

• Final phase of apoptosis

• Mediated by proteolytic cascade

• After initiator caspases ( 2, 8, 9 and 10) are cleaved to generate its active form, the enzymatic death program is set in motion by rapid sequential activation of the executioner caspases – 3,6

• Caspase -3 activates DNase which causes degradation of chromosomal DNA within the nuclei and causes chromatin condensation.

• Caspase -3 induces cytoskeletal reorganisation and disintegration of cell into apoptotic bodies.

REMOVAL OF DEAD CELLS

Factors by which apoptotic cells attracts phagocytes towards them :

• Apoptotic bodies- “bite size”-edible for phagocytes

• Phosphatidyl serine “flips” out from inner to outer layer –recognized by macrophages receptors

• Some apoptotic bodies express thrombospondinrecognized by phagocytes

• They are coated by Ab and C1q

32

MORPHOLOGY OF CELLS IN APOPTOSIS

• Cell Shrinkage and dense cytoplasm• Chromatin condensation to periphery and later

fragmentation.• Plasma membrane is intact• Formation of cytoplasmic blebs and apoptotic

bodies.• Structurally altered so that the apoptotic cell

becomes “tasty” for phagocytosis• Phagocytosis of apoptotic bodies by macrophages• Dead cell is rapidly cleared before contents are

leaked out• No inflammatory reaction

MORPHOLOGY OF APOPTOSIS

Progressive cell shrinkage

Chromatin condensation

Plasma membrane blebbing

Apoptotic bodies

Phagocytosis - no inflammation

MORPHOLOGY OF CELLS IN APOPTOSIS

Various methods available for detection of apoptotic cells

1) Light microscopy

• In H&E the apoptotic cell appears as round or oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin

MICROSCOPY

Apoptosis in neutrophils

Councilman bodies- liver tissue

2) Electron microscopy

Defines subcellular changes like

• Chromatin condensation

• Plasma membrane blebbing

3) Gel electrophoresis

DNA ladder pattern Single cell gel electrophoresis comet assay

showing apoptotic cell

4) TUNEL [ terminal deoxy transferasemediated dUTP nick end labelling ]

• Identify DNA breaks in apoptosis

Apoptotic cells detected by TUNEL and fluoresce green;while necrotic cells are stained with red-fluorescent

propidium iodide

5) Immunohistochemistry

Immunohistochemicaldetection of apoptotic cells using antibodies against a wide range of substrates most importantly :

• Caspase 3

• P53

• Annexin V

APOPTOSIS IN PHYSIOLOGIC CONDITIONS

1) Programmed destruction of cells during embryogenesis including implantation, organogenesis , developmental involution and metamorphosis.

Eg : separation of webbed fingers and toes in embryo

Apoptosis in bud formation during which many interdigital cells die. They are stained black by a TUNEL method

Incomplete differentiation in two toes due to lack of apoptosis

sclero dinesh

2) Involution of hormone-dependent tissues upon hormone withdrawal…

Eg :

• Endometrial cell breakdown during menstrual cycle

• Ovarian follicular atresia in menopause

• Regression of the lactating breast after weaning

• Prostatic atrophy after castration

3) Cell loss in proliferating cell populations

Eg :

• Immature lymphocytes in bone marrow and thymus that fail to express useful antigen receptors

• B lymphocytes in germinal centers

• Epithelial cells in intestinal crypts

So as to maintain a constant number…

4) Elimination of potentially harmful self-reactive lymphocytes

5) Death of host cells that have served their useful purpose

Eg :

- Neutrophils in an a/c inflammatory response

- Lymphocytes at the end of an immune response

In these situations , cells undergo apoptosis because they are deprived of necessary survival signals such as growth factors.

Apoptosis: Role in Disease

TOO MUCH: Tissue atrophy

TOO LITTLE: Hyperplasia

NeurodegenerationThin skin

etc

CancerAthersclerosis

etc

APOPTOSIS IN PATHOLOGIC CONDITIONS

Pathological condition arise as a result of dyregulation in apoptosis……………….

i.e defective apoptosis with increase cell survival

OR

Increased apoptosis with increased cell death

DYSREGULATED APOPTOSIS

DISORDERS WITH DEFECTIVE APOPTOSIS AND INCREASE CELL SURVIVAL

Eg :

• Mutations of p53 -------------CANCERS

• AUTOIMMUNE DISEASES

DISORDERS WITH INCREASED APOPTOSIS AND EXCESS CELL DEATH

Eg :

• NEURODENEGERATIVE D/S

• Ischemic injury

• Death of virus infected cells. Eg-AIDS

Apoptosis: Role in DiseaseAging

Aging --> both too much and too little apoptosis(evidence for both)

Too much (accumulated oxidative damage?)---> tissue degeneration

Too little (defective sensors, signals?---> dysfunctional cells accumulatehyperplasia (precancerous lesions)

sclero dinesh

PREGNANCY ASSOCIATED DISEASE AND APOPTOSIS

• During pregnancy trophoblast cells from placenta invade the uterine environment inorder to remodel the maternal blood vessels and help establishing and maintain sucessful pregnancy.

• Strict control over the cell proliferation and apoptosis is required to achieve this.

• In some cases the process can be compromised and excessive apoptosis of trophoblast cells failure of fully remodel the maternal environment complication of pregnancy

Eg : preeclampsia of pregnancy

AUTOIMMUNE D/S AND APOPTOSIS

IN HEALTHY BODY• T and B – lymphocytes are cells of immune system that

are responsible for destroying infected or damaged cell in the body.

• They mature in thymus, but before they can enter the blood stream they are tested to ensure that they are effective against foreign antigen and are also not reactive against normal healthy cells.

• Any ineffective or self reactive T-cells are removed through induction of apoptosis.

AUTOIMMUNE D/S AND APOPTOSIS ….contd

IN DISEASE :• Poor regulation of apoptosis in T- lymphocyte results

Autoimmune D/s of cytotoxic T lymphocytes. Eg : Behcet’s D/S, Ankylosing Spondylitis, SLE

• Poor regulation of apoptosis in B –cellEg : SLE , Scleroderma , Multiple sclerosis

• RHEUMATOID ARTHRITIS : excessive proliferation of synovial cells is thought to be due in part to the resistence of these cells to apoptosis.

• AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME [ALPS] : mutation in FAS gene

Neurodegenerative D/S and Apoptosis

IN HEALTHY BODY :

• During development of central and peripheral nervous system, many neurons undergo apoptosis that coincides with synaptogenesis.

• Signals that determine whether or not developing neurons live or die may include competition for a limited supply of target derived neurotrophic factors and activation of receptors for excitatory neurotransmittor Glutamate.

• Initial overproduction of neurons followed by death of some is an adaptive process that provide enough neurons to form nerve cell circuits.

Neurodenerative D/S and Apoptosis…contd

Metabolic stress [ stroke, aging ]

Oxidative stress and free radicals DNA damage

Inherited mutations misfolded proteins

ER stress

APOPTOSIS increase Ca influx

Neurodenerative D/S and Apoptosis…contd

IN DISEASE :• apoptosis of hippocampal neuron Alzheimer’s D/s

Over expression of Bcl 2• apoptosis of midbrain neurons that uses NT’s

dopamine Parkinson’s Disease• apoptosis of neurons in striatum which control body

movements Huntington’s Disease• apoptosis of lower motor neurons Amyotrophic

Lateral Sclerosis • In stroke activation of glutamate receptors which

act as a trigger to stimulate apoptosis

Apoptosis: Role in DiseaseCancer

• Apoptosis eliminates damaged cells (damage => mutations => cancer)

• Apoptosis is regulated by two major genes p53 & Bcl-2.• Tumor suppressor p53 controls senescence and apoptosis

responses to damage.

• Mutations or overexpression of these genes will result in Cancer.Most cancer cells are defective in apoptotic response (damaged, mutant cells survive)

• High levels of anti-apoptotic proteinsor

Low levels of pro-apoptotic proteins ===> CANCER

Apoptosis: Role in Disease

Cancer

Virus associated cancer

•Several human papilloma viruses (HPV) have been implicated

in causing cervical cancer. One of them produces a protein (E6)

that binds and inactivates the apoptosis promoter p53.

•Epstein-Barr Virus (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).

• Some B-cell leukemia and lymphomas express high

levels 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.

Apoptosis: Role in DiseaseCancer

•Other cancer cells express high levels 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).

•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 cells

Apoptosis: Role in DiseaseCancer

– Cancer cells

• Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells.

sclero dinesh

Fig. 1: induced apoptosis in stomach carcinoma cellsLeft: Before inductionMiddle: 24h after induction Right: 48h after induction

• Apoptosis and AIDS

Human Immunodeficiency Virus infects CD4+

T cells and HIV Tat protein increases the

expression of Fas receptor, resulting in

excessive apoptosis of T cells.

Hallmark- the decline in the number of the

patient's CD4+ T cells (normally about 1000

per microliter (µl) of blood).

sclero dinesh

ANTI-APOPTOTIC THERAPY IN DISEASES

• Stimulation of IAP - for the treatment of stroke, spinalcord injuries , multiple sclerosis

• Synthetic nonspecific caspase inhibitors – trials going on treatment of myocardial reperfusion injury, RA

• Aim of the treatment in neurodegenerative d/s is to block apoptotic triggers and activation of anti -apoptotic pathway ( by neurotrophic factors )

Eg : block amyloid β production t/t Alzheimer’s d/sblock glutamate receptor activation strokeusing neurotrophic factors like

vitamin E t/t alzheimer’sInsulin like Growth factors t/t ALS

CONCLUSIONS• Apoptosis is regarded as a carefully regulated energy dependent

process, characterized by specific morphological and biochemical features in which caspase activation plays a central role.

• The importance of understanding the mechanistic machinery of apoptosis is vital because programmed cell death is a component of both health and disease, being initiated by various physiologic and pathologic stimuli.

• Moreover, the widespread involvement of apoptosis in the pathophysiology of disease lends itself to therapeutic intervention at many different checkpoints.

• Understanding the mechanisms of apoptosis, and other variants of programmed cell death, at the molecular level provides deeper insight into various disease processes and may thus influence therapeutic strategy.

References

• Robbins and Cotran,pathologic basis of disease, Kumar et al, 9th

edi.,2014

• Henry’s clinical diagnosis and management,21st edi,2007,546

• Review article on apoptosis, Indian Journal of Cancer ,vol 35, No.4, sep- 2007 , 495-516

• Review article on various method available for detection of apoptotic cell, Indian Journal of Cancer, july-sep 2013 , vol 50, issue 3.

• www.ncbi.nlm.nih.gov• www.reading.ac.uk

THANK YOU