Apoptosis - PowerPoint Presentation

Slide1

Apoptosis

Jalal GhasemzadehAndrology lab

Yazd Reproductive Sciences Institute Slide2

بسم الله الرحمن الرحیمSlide3

Introduction into cell death

In humans, the rate of cell growth and cell death is balanced to maintain the weight of the body. Slide4

Life cannot exist without cellular deathSlide5

Types of cell

death Cell death can occur via several processes (about 11 type) : 1. Apoptosis 2. Necrosis 3. Autophagy 4. Entosis

5

.

Oncosis

6

.

Pyroptosis

.

.

.

11.Slide6

Two main mechanism

of cell deathApoptosis = “normal” or “programmed” cell deathNecrosis = “accidental

”

or “ordinary” cell deathSlide7

Introduction of apoptosis

The word ‘‘apoptosis’’ comes from the ancient Greek, meaning the: ‘‘falling of petals from a flower’’ or ‘‘of leaves from a tree in autumn’’

The

term

apoptosis

(

a-

po

-toe-sis

)

was

first used in

a

now-classic paper by

Kerr et al 1972 to describe a morphologically distinct form of cell death.Slide8

Apoptosis definition

Apoptosis or programmed cell death (PCD) is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise (“cellular suicide”).It is important for the development of multicellular organism (embryonic development) and homeostasis of their tissues (adult).Slide9

Importance of

apoptosisApoptosis is a beneficial and important phenomenon: In embryo1. During embryonic development, help to digit formation.Lack of apoptosis in humans can lead to webbed fingers called “ syndactyly ”. Slide10

Importance of

apoptosis2. Normal event in development of the nervous systemSlide11

Importance of

apoptosisApoptosis is a beneficial and important phenomenon: In adultNormal cell turn over Tissue homeostasisInduction and maintenance of immune toleranceDevelopment of the nervous system Endocrine-dependent tissue atrophyElimination of activated, damaged and abnormal cellsSlide12

Importance of

apoptosisApoptosis is a beneficial and important phenomenon: In animals

Embryonic Chicken Foot

Embryonic Mouse Paw

Tail absorption of the tadpoleSlide13

A

poptosis versus NecrosisApoptosis is the physiological cell death which unwanted or useless cells are eliminated during development and other normal biological processes. Necrosis is the pathological

cell death

which occurs when cells are exposed to

a serious physical or chemical

insult

(hypoxia, hyperthermia, ischemia).Slide14

Differences

between apoptosis and necrosisThere are many observable morphological and biochemical differences between necrosis and apoptosis: Morphological features Necrotic cells Apoptotic cells  Volume enlargement  Volume reduction  Swelling of cytoplasm 

S

hrinking of

cytoplasm

& mitochondria



No loss of membrane

integrity



Loss of membrane integrity



No vesicle formation



Formation of

apoptotic bodies  Condensation of chromatin & DNA fragmentation Slide15
Slide16

Differences

between apoptosis and necrosis Biochemical features Necrotic cells Loss of regulation of ion homeostasis No energy requirement (passive process, also occurs at 4 °C) Apoptotic cells



Tightly regulated

process



Energy

(

ATP

)

- dependent

(

active process

,

doesn’t

occur at 4 °C) Release of various factors into cytosol by mitochondria



Activation

of

caspase

cascadeSlide17

Differences between

apoptosis and necrosis Physiological significance Necrosis Affects groups of contiguous cells Evoked by non-physiological disturbances (lytic viruses, hypoxia) Phagocytosis by macrophages Significant inflammatory response

Apoptosis



Affects

single cells

or

small clusters of cells



Induced

by physiological

stimuli(lack

of growth

factors, DNA damage)

 Rapidly phagocytized by adjacent epithelial cells or macrophages  No inflammatory responseSlide18

Inflammatory reaction

 There is essentially no inflammatory reaction associated with the process of apoptosis nor with the removal of apoptotic cells because: Apoptotic cells do not release their cellular constituents into the surrounding interstitial tissue. They are quickly phagocytized by surrounding cells thus likely preventing secondary

necrosis.



The

engulfing cells

do

not produce

anti-inflammatory

cytokines.Slide19
Slide20

Apoptotic cells -Eat me signalsSlide21



Takes about 30 - 60 min. In the tissue only about 5% cells is affected by the PCD. Physiological changes and phagocytosis

is

very fast.

Apoptosis timingSlide22

APOPTOSIS

Apoptosis pathwaysSlide23

Apoptosis pathways

1. Extrinsic pathway (death receptor- mediated events)2. Intrinsic pathway

(mitochondria- mediated events)Slide24

Extrinsic Pathway

Components: Death Receptors Death Ligands Adaptor Proteins



CaspasesesSlide25

CaspasesCaspases= Cysteinyl aspartate specific proteasesA family of intracellular cysteine proteases that play a pivotal role in the initiation and execution of apoptosis.At least

14

different

members of

caspases

in mammalian cells have

been identified

All are synthesized as

i

nactive proenzymes

(

zymogen

)

with 32-56 kDaSlide26

Caspase structureSlide27

Caspase subgroups

To date, ten major caspases have been identified and broadly categorized into:  Signaling or Initiator caspases

( 2, 8, 9, 10)



Effector

or

Executioner

caspases

(

3, 6, 7

)



Inflammatory

caspases

( 1, 4, 5)

The other

caspases

that have been identified include:

Caspases

11

, 12, 13,

14

Central role

in cascade of apoptotic events is played by

caspase

3

(CPP32)Slide28

Extrinsic

Pathway “Death receptors” that are members of the tumor necrosis factor (TNF) receptor superfamily.Death receptors have a cytoplasmic domain of about 80 amino acids called the “death domain”.This death domain plays a critical role

in

transmitting

the death signal

from the

cell surface to the intracellular signaling

pathways.Slide29

Death Receptor

s & their ligands The best characterized receptors & ligands corresponding death include: FasR (CD95/APO1) FasL DR3 Apo3L

DR4 (TRAIL-R1

)

Apo2L

DR5

(

TRAIL-R2

)

Apo2L

TNFR1

TNF-α TNFR2 TNF-ß

Ligands

ReceptorsSlide30

Death Ligands

and Death ReceptorsSlide31

Extrinsic Pathway

Binding of trimeric FasL to Fas Trimerization and clustering of Fas Recruitment of Fas-associated death domain (FADD) to Fas

Recruitment of caspase-8 to FADD

Formation of Death-Inducing Signaling

Complex

(DISC )

Activation of caspase-8 (autoactivation

)

Activation of effector

caspases

ApoptosisSlide32

Intrinsic Pathway

Components: Bcl-2 family proteins Cytochrom c  Adaptor proteins



CaspasesesSlide33

Intrinsic Pathway

The stimuli that initiate the intrinsic pathway produce intracellular signals such as radiation (DNA damage), absence of certain growth factors, hormones and cytokines. All of these stimuli cause changes in the mitochondrial

outer membrane

permeabilization

(MOMP

)

Release

of

pro-apoptotic proteins

such as

cytochrome

c,

Smac

/DIABLO, AIF, endonuclease G and CAD

from the

inter-membrane space into the cytosol.Cytochrome c binds and activates Apaf-1 as well as procaspase-9, forming an “apoptosome”.

Caspase-9

activation, subsequent caspase-3 activation and

cell

death.Slide34

Intrinsic

PathwaySlide35

Intrinsic

Pathway & Bcl2 familyThe control & regulation of apoptotic mitochondrial events occurs through members of the Bcl-2 family of proteins  Anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-w  Pro-apoptotic

proteins

include

Bax

,

Bak

, Bid, Bad,

Bim

,

Bik

T

he

main mechanism of action of the Bcl-2 family of

proteins

is the regulation of cytochrome c release from the mitochondria via alteration of mitochondrial membrane permeability.Slide36

Sperm apoptosisSlide37

Spermatogenesis & Apoptosis

 Apoptosis plays two primary roles in spermatogenesis: It is essential in limiting the population of germ cells to a number that can be supported by Sertoli cells.Selective depletion of abnormal germ cells, i.e. cells with abnormal morphology, altered biochemical function or DNA damage.Slide38

Fas System in sperm apoptosis

The main factor postulated to be implicated in sperm apoptosis is the cell surface protein, Fas (CD95 or APO1)Fas is a type I membrane protein that belongs to the tumor necrosis factor receptor family.Binding of Fas ligand (FasL) or agonistic anti-Fas antibody to Fas, kills cells by apoptosis.Slide39

Fas mediated Apoptosis in sperm

DISC(3,7)Slide40

A

bortive apoptosisIt has been shown that any defects in the remodeling of cytoplasm during spermatogenesis and presence of cytoplasmic retentions with increased expression of apoptotic markers like Fas, caspase1, P53 and P21 may lead “abortive apoptosis”. A phenomenon in which defective sperm cells escape PCD and are present in the ejaculate.Slide41

Abortive apoptosis

Sakkas et al (1999) have proposed that presence of Fas-positive sperm in ejaculate, is an indicative of sperm cells which are labeled for apoptosis. they escape apoptosis because: 1) There are too many abnormal spermatozoa for the available Fas-L (Fas ligand produced by sertoli cell) 2) May signaling through Fas system is not functional. Slide42

A

bortive apoptosis According to this model, these spermatozoa that escape the process, may indicate the presence of combination of abnormalities including histone-protamine substitution, DNA remodeling and also cytoplasmic and membrane anomalies.Abortive apoptosis can explain why a high percentage of spermatozoa with DNA damage and abnormal forms spermatozoa which show markers of apoptosis have been seen in patients with abnormal semen parameters. Slide43

Abortive apoptosis

Regarding to this evidence, there is not complete clearance of abnormal spermatozoa through apoptosis. Abortive apoptosis in sperm associated with: - abnormal morphologies - aberrant biochemical functions - nuclear DNA damageSlide44

Abortive

apoptosis in spermSlide45

Apoptosis markers

The markers of apoptosis expressed by a varying proportions of ejaculated sperm and are including:Externalization of Phosphatidyl serine (PS) to the sperm outer membrane leafletActivated caspasesLoss of the integrity of the mitochondrial membrane potential (MMP)

DNA

fragmentationSlide46

Studies …

Production of ejaculated spermatozoa that possess apoptotic markers (such as Fas positivity and DNA damage) indicate that in some men with abnormal sperm parameters, an ‘abortive apoptosis’ has taken place.In men with normal sperm parameters, the percentage of Fas-positive spermatozoa is low.Samples with low sperm concentration are more likely to have a high proportion of Fas-positive spermatozoa.

The apoptotic mechanism of spermatozoa is already triggered

before ejaculation

. After ejaculation, spermatozoa do not become apoptotic spontaneouslySlide47
Slide48

Apoptosis and male fertility

Male infertility appears to be positively correlated with increased levels of sperm with apoptotic markers.In the natural selection process, spermatozoa with damaged DNA would have a low chance of achieving conception with an oocyte.All data indicate that increased levels of sperm apoptosis during in vitro fertilization (IVF) correlate to impaired embryo morphology at early cleavage stages, failure to progress to the blastocyst stage in culture and decreased pregnancy rates.Slide49
Slide50

Assay for apoptosisSlide51

Apoptosis assay methods

 Apoptosis assays, based on methodology, can be classified into six major groups:1. Cytomorphological alterations2. DNA fragmentation3. Detection of caspases, cleaved substrates, regulators

and inhibitors

4.

Membrane alterations

5.

Detection of apoptosis in whole

mounts

6.

Mitochondrial assaysSlide52

Cytomorphological alterations

The evaluation of hematoxylin and eosin-stained tissue sections with light microscopy does allow the visualization of apoptotic cells.This method detects the later events of apoptosis. TEM is considered the gold standard to confirm apoptosis: (1) electron-dense nucleus (2) nuclear fragmentation (3) intact cell membrane (4) disorganized cytoplasmic organelles (5) large clear Vacuoles (6) blebs at the cell surfaceSlide53

DNA fragmentation

DNA fragmentation occurs in the later phase of apoptosis.TUNEL (Terminal dUTP Nick End-Labeling) assay quantifies the incorporation of deoxyuridine triphosphate (dUTP) at single and double stranded DNA breaks in a reaction catalyzed by the template-independent enzyme, terminal deoxynucleotidyl transferase (TdT).Incorporated dUTP is labeled such that breaks can be quantified either by flowcytometry, fluorescent microscopy, or light microscopy.Slide54

Tunel

, light microscopy

TUNEL positive cell

TUNEL

negative

cellSlide55

Tunel

, light microscopy

TUNEL positive cell

TUNEL positive cellSlide56

Tunel

, flourscent microscopy

TUNEL positive cell

TUNEL

negative

cellSlide57

Tunel

, flourscent microscopySlide58

Relationship between tunel & caspasesSlide59

Membrane alterations

Fluorescein isothyocyanate (FITC) conjugated Annexin

V

During

the process of apoptosis,

one of the

earliest

events

is

Externalization of

Phosphatidyl

serine

(PS)

from the inner

to the outer plasma

membrane

of apoptotic cells.

These cells can be demonstrated by

bound with

FITC-labeled

Annexin

V

and detected with fluorescent

microscopy

.

Positive cell in the

Annexin

V assay beginning of the apoptotic processSlide60

Membrane alterations

Fluorescein isothyocyanate (FITC) conjugated

Annexin

V + PI

The membranes of necrotic cells are labeled with

annexin

V. Since

loss of membrane integrity

is a pathognomonic feature of

necrotic cell

death, necrotic cells will stain with specific membrane-

impermeant

nucleic acid dyes such as

propidium

iodide

and

trypan

blue.

The membrane integrity of apoptotic cells can be demonstrated by the

exclusion of these dyes

.Slide61

Fluorescein isothyocyanate (FITC)-conjugated Annexin V + PI

The vital dye propidium iodide (PI) should be used in combination with annexin V. By using an additional labeling with the vital dye propidium iodide (PI), it is possible to distinguish viable, apoptotic and necrotic sperm populations at the same timeSlide62

FITC

conjugated Annexin V + PISlide63

Detection of Apoptosis in Whole Mounts

Apoptosis can also be visualized in whole mounts of embryos or tissues using dyes such as acridine orange (AO), Nile blue sulfate (NBS), and neutral red (NR).Since these dyes are acidophilic, they are concentrated in areas of high lysosomal and phagocytotic activity.Slide64

Mitochondrial Assays

Mitochondrial assays and cytochrome c release allow the detection of changes in the early phase of the intrinsic pathway.The mitochondrial outer membrane (MOM) collapses during apoptosis, allowing detection with afluorescent cationic dye. Cytochrome c release from the mitochondria can also be assayed using fluorescence and electron microscopy in living or fixed cells. Apoptotic or anti-apoptotic regulator proteins such as Bax, Bid, and Bcl-2 can also be detected using fluorescence and confocal microscopySlide65

Importance

of apoptosis in human diseasesAberrant cell death can lead to many human diseases: Decreased apoptosis Cancer, Autoimmune disorders

Excessive

apoptosis

Neurodegenerative

and

immunodeficiency

(AIDS)

disorders ,

Ischemia

NOTE:

Properties

of carcinogenic agents (chemical agents as well

as radiations) are the growth-inhibition power and the ability to induce cell death. These properties are widely used in anticancer chemo- and radiotherapiesSlide66

(Tissue atrophy)

(Hyperplasia)Slide67

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