Transcript Slide 1

APOPTOSIS, NECROPTOSIS
and AUTOPHAGY
Associate Professor
Dr. Alexey Podcheko
Spring 2015
OBJECTIVES
1. Define apoptosis
2. Compare and contrast the morphologic
changes seen in apoptosis versus
necrosis
3.Outline the 4 phases of apoptosis
4. Describe the intrinsic and extrinsic
pathways of apoptosis
OBJECTIVES
5. List 4 physiologic examples of apoptosis
6. List one role of apoptosis in the Pathology of
the immune system
7. Describe the role of p53 in carcinogenesis
8. Describe the role of apoptosis in
neurodegenerative diseases
9. Define Autophagy
10. List 2 diseases where Autophagy play role
in pathogenesis
Stages of the cellular response to stress
and injurious stimuli
APOPTOSIS
DEFINITION:
ENERGY DEPENDENT CONTOLLED
PROCESS OF PROGRAMMED CELL
DEATH.
ENERGY DEPENDENT CONTOLLED PROCESS
OF PROGRAMMED CELL DEATH
Apoptosis
Why should I care?
(Chronic
myelogenous
leukemia)
(maintains normal size of the organ)
Complexity of Apoptotic pathways
Comparative morphologies of
Apoptosis and Necrosis
1
2
3
4
The two major routes to Apoptosis:
Intrinsic and Extrinsic
The extrinsic (death receptor–initiated)
pathway of apoptosis
The extrinsic (death receptor–initiated)
pathway of apoptosis
Click here to see video
The intrinsic or mitochondrial
pathway
1. Involves Bcl family of
proteins
2. Both pro and anti
apoptotic members
3. Best known is antiapoptotic Bcl2
4. Production of Bcl2
can be induced by
Growth Factors (IGF1)
deprivation or IR
The mitochondrial pathway of Apoptosis
BCL2
antagonistsBim, Bid, and
Bad
Smac/DIABLO
(blocks IAPs)
The equilibrium between proapoptotic and antiapoptotic members of the Bcl-2
family of proteins reflects binding of antiapoptotic members to themselves
(homodimers) or to the proapoptotic proteins Bak and Bax
Opening of the mitochondrial
permeability transition pore
leads to Apaf-1 activation and
triggering the apoptotic
cascade.
CytC- cytochrome c
PTP= permeability transition
pores
ROS = reactive oxygen
species
An experiment introduces a “knockout” gene
mutation into a cell line. The frequency of
shrunken cells with chromatin clumping and
cytoplasmic blebbing is increased
compared with a cell line without the
mutation. Overall survival of the mutant cell
line is reduced. Which of the following genes
is most likely to be affected by this mutation?
(A) BAX
(B) p53
(C) C-MYC
(D) FAS
(E) BCL-2
How to detect or measure
apoptotic events in the tissues ?
Inducer of
Apoptosis
-
+
-
+
Poly (ADP-ribose) polymerase (PARP) is a protein involved in a
number of cellular processes- mainly DNA repair and programmed
cell death
Phosphatidylserine exposure during
apoptosis
“Eat-me” signal for professional phagocytes
Annexin-V-FITC
Phosphatidylserine exposure during apoptosis
Propidium Iodide stains
in red only cells with
damaged membranes
DNA condensation and fragmentation of 50-300.000 base pair fragments is
one of the earliest stages of apoptosis! Process of fragmentation will
continue until fragment size will reach app. 200 base pairs. Why?
DNA Fragmentation
Components of a nucleosome
DNA Fragmentation
Nucleosomal Banding Pattern
Accumulation of cells in sub-G1 phase
of cell cycle during apoptosis
G2/M
S
G1
How apoptotic cells removed from
tissues?
• Phagocytosis is the main mechanism
• Signals for phagocytosis are:
1. Flip of phosphatidylserine from inner to outer
leaflet of cell membrane (ligand for
macrophages receptors)
2. Secretion of soluble factors that recruit
phagocytes
3. Production of thrombospondin on cell surface
of apoptotic cells
4. Coating of apoptotic cells with C1q
complement
Apoptosis
Why should I care?
Role of Apoptosis in the Hand Plate
A 40 y/o male brought to ED post-MVA. Patient has a
history of fever, headache, chills, and pain in RUQ 5
days ago. Histologic exam shows liver section
w/disruption of normal hepatic lobule. Small
shrunken hepatocytes w/intense eosinophilic
cytoplasm, fragmented nuclear chromatin, &
cytoplasmic bleb formations are noted. Which
process is most likely occurring in the hepatocytes
described?
A) apoptosis
B) Atrophy
C) Caseous necrosis
D) Coagulation necrosis
E) Dysplasia
F) Fatty change
G) Hetrophagy
H)Liquefaction necrosis
I) metaplasia
CLINICO-PATHOLOGIC CORRELATIONS:
APOPTOSIS AND DISEASE
1. Reduced Apoptosis – Cancer, Autoimmune
Diseases (SLE, Rheumatoid Arthritis, etc)
2. Increased Apoptosis – Neurodegenerative
Diseases, Myocardial Infarction, Stroke, Viral
diseases (Hepatitis, AIDS)
• A 55-year-old woman with colon cancer and a
healthy 55-year-old woman are participating a
study of colon cancer. Malignant cells are
obtained from the tumor in the affected patient,
and normal colonic epithelial cells are procured
from the healthy subject. After both cell types
are treated with transforming growth factor-beta,
the number of normal cells decreases, whereas
the number of tumor cells remains unchanged.
The tumor cells most likely express a mutation
that inhibits which of the following physiologic
processes?
A) Apoptosis
B) Cell cycle progression
C) DNA repair
D) Migration
E) Necrosis
Mechanisms of increased apoptosis:
• Growth Factor Deprivation: Lymphocytes that are not
stimulated by antigens and cytokines, neurons
deprived of nerve growth factor, endometrial glands in
post menopause
• DNA Damage: Radiation or chemotherapeutic agents
induces DNA damage
Role of P53 Tumor suppressor protein in
apoptosis and cancerogenesis
•P53 is a 53kDa protein present mostly in the nuclei of all
cells, rapidly accumulates after injury and become
phosporylated
•Phosphorylation allows to bind to DNA and activate
transcription of cell cycle inhibitors or proapoptotic proteins
•P53 block transition of cells from G1 to S phase if DNA
damage is not repaired
P53 mediated apoptosis
Role of P53 Tumor suppressor protein in
apoptosis and cancerogenesis
If repair is unsuccessful then P53 will
induce expression of BAX. BAX acts
against bcl2 and causes apoptosis
Hormonally related cancers seem to
particularly involve loss of p53 related
apoptosis
Chemo/rad work best when p53 is intact
as also act through apoptosis
Neurodegenerative Diseases and
Apoptosis
-
Withdrawal of growth factors favors
actions of pro-apoptotic members of Bcl
family
- For neurons dependent on nerve growth
factors the loss of this stimulation can lead
to neural apoptosis and degenerative
neurologic diseases:
(Alzheimer's disease, Amyotrophic lateral sclerosis,
Friedreich's ataxia, Huntington's disease, Lewy body
disease, Parkinson's disease, Spinal muscular atrophy)
Example: SOD-1 mutations activate cell death pathways in
familial amyotrophic lateral sclerosis
SOD-1 mutations can activate caspase-1 and caspase-3, and might increase freeradical generation, leading to motor neuron apoptosis. The activation of caspase-1
leads to interleukin-1 production, which can induce a local microglial inflammatory
response and increase the number of neurons affected.
Mechanisms of IR
induced cell death
Cell injury caused by virus infection
Direct injury:
-Depletion of cellular resources
-Activation of Apoptosis
Immunologically mediated injury:
-Antibodies+complement
-T-lymphocytes/Granzyme
Protein Misfolding and unfolded protein
response (aka ER-stress)
• Proteins which called “Chaperones” play crucial
role in the proper folding of newly synthesized
proteins
• Normally misfolded polypeptides are
ubiquitinated and targeted for proteolysis in
proteasomes.
• Accumulation of misfolded proteins in the ER
trigger a number of cellular responses,
collectively called the unfolded protein response
• If cells are unable to cope with the accumulation
of misfolded proteins, the cell activates
caspases and induces apoptosis.
• Examples: Alzheimer, Huntington, and
Parkinson diseases, type 2 diabetes
Mechanisms of protein folding and the unfolded protein
response
Autophagy
•Definition: Autophagy is the process in which a cell
sequesters and recycles damaged organelles and
macromolecules ( eats its own contents)
What can activate Autophagy:
1. Nutrient Deprivation( low level
of a.acids)
2. Accumulation of long-living
proteins
What regulate Autophagy?
1. MTOR signaling pathway
2. Atgs genes/proteins
What Mechanism?
1. Atgs proteins create autophagic
vacuoles
2. Autophagic vacuoles fuse with
lysosomes and content is
digested
Autophagy and Disease
1. Cancer: This is an area of active investigation, autophagy
can both promote cancer growth and act as a defense
against cancers.
2. Neurodegenerative disorders: In Alzheimer disease,
formation of autophagosomes is accelerated. In Huntington
disease, mutant protein huntingtin impairs autophagy.
3. Infectious diseases: Many pathogens are degraded by
autophagy; these include mycobacteria, Shigella spp., and
HSV-1. This is one way by which microbial proteins are
digested and delivered to antigen presentation pathways.
Macrophage-specific deletion of Atg5 increases
susceptibility to tuberculosis
4. Inflammatory bowel diseases: Studies have linked both
Crohn disease and ulcerative colitis to autophagy related
genes.
Necroptosis
• Necroptosis was first recognized as a caspaseindependent form of cell death that can be triggered
by treatment with TNF only in the presence of a pancaspase inhibitors
• Main executing molecule of the process is called
RIPK3 - receptor-interacting protein kinase 3 (RIPK3)
• Necroptosis requires that the function of caspase 8 be
inhibited or disrupted.
• Unlike apoptosis, in which several of the highly
immunogenic intracellular proteins are sequestered in
the dead cell, necroptosis is a strong trigger of
innate and adaptive immune responses
Formation of the
Necrosome is key
point in the
induction of
Necroptosis
Molecular mechanism
of
TNF-mediated necroptosis:
Cross-linking
of TNFR1
by TNF
causes
recruitment
of
RIP1
and
RIP3 along with
caspase
8.
Inhibition
of
caspase
8,
as may occur in
some viral
infections,
allows RIP1 and
RIP3
to initiate signals that affect
mitochondrial generation
of ATP and ROS. This
is followed by events
typical of necrosis.
Key Concepts of Necroptosis
• Necroptosis resembles necrosis morphologically and
apoptosis mechanistically as a form of programmed cell
death.
• Necroptosis is triggered by ligation of TNFR1, and viral
proteins of RNA and DNA viruses.
• Necroptosis is caspase-independent but dependent on
signaling by the RIP1 and RIP3 complex.
• RIP1-RIP3 signaling reduces mitochondrial ATP
generation, causes production of ROS, and
permeabilizes lysosomal membranes, thereby causing
cellular swelling and membrane damage as occurs in
necrosis.
• Release of cellular contents evokes an inflammatory
reaction as in necrosis.
Clinical implications of the
necroptosis
• Activation of Necroptosis will be beneficial
to induce strong anti-viral immune
response, e.g. for vaccination or for
treatment of viral infections
• Inhibition of Necroptosis will be beneficial
for treatment of ischemia-reperfusion
injury (MI, strokes, transplantation of
organs)
• More details:
Mediation of Programmed Cell Death by
Apoptosis or Regulated Necrosis
mitochondrial
permeability
transition
Necrostatin1, Necrosulfonamide and Cyclosporine are
effective in the prevention of the Ischemia-reperfusion injury
Pyroptosis
• Another form of programmed cell death
• Accompanied by the release of fever-inducing
cytokine IL-1 from ells
• Has some biochemical similarities with apoptosis
• Pyroptosis occurs in cells infected by microbes
(microbes in the cytoplasm of cells)!!!
• Involves activation of caspase-1, generation by
cell IL-1
• IL-1 recruit leukocytes to the site of infection
From : Cell death in the host response to infection Cell Death and Differentiation (2008) 15, 1339–1349;
doi:10.1038/cdd.2008.91; published online 20 June 2008 , K Labbé1 and M Saleh1,2
Pathogen-induced host cell death
The type of death the cell undergoes depends on the nature of the pathogen, pathogen
load and site of infection.
Pyroptotic, apoptotic, autophagic or oncotic cells display a distinct set of morphological
and biochemical characteristics
Apoptosis and autophagy do not induce inflammation
Apoptosis, pyroptosis and autophagy are generally beneficial to the host, oncosis favors
pathogen dissemination
Please, take your pen and answer
on the following questions
You have 80 seconds to answer
each question
Q1: A 43-year-old man presents with a scaly,
erythematous lesion on the dorsal surface of his
left hand. Skin biopsy reveals atypical
Keratinocytes and inflammatory cells, as well as
numerous scattered apoptotic bodies. Which of
the following proteins plays the most important
role in mediating programmed cell death in these
Keratinocytes?
A. Catalase
B. Cytochrome c
C. Cytokeratins
D. Myeloperoxidase
E. Superoxide dismutase
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Q2: You observe a slide containing numerous
epithelial cells. A colleague looks at the unstained
cells under the light microscope and says that most
of the cells are undergoing apoptosis. You look at
the slide in the microscope and agree. What
alterations in the cellular structure did you observe
in the light microscope that led to the conclusion
that the cells were undergoing apoptosis?
A Plasma membrane was intact
B Mitochondria were absent
C Plasma membrane showed blebbing
D Nucleus was absent
E Polysomes were associated with the
endoplasmic reticulum
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Q3: On day 28 of her menstrual cycle, a 23-year-old
woman experiences onset of menstrual bleeding
that lasts for 6 days. She has had regular cycles for
many years. Which of the following processes is
most likely occurring in the endometrium
just before the onset of bleeding?
(A) Apoptosis
(B) Caseous necrosis
(C) Heterophagocytosis
(D) Atrophy
(E) Liquefactive necrosis
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Q4: A cell pathologist performs experiments to
examine the biochemical and physiological changes
that occur in cells undergoing apoptosis. Which of
the following characteristics is associated with the
first stage of apoptosis of cells?
A DNA ladder formation
B Nuclear condensation
C Blebbing or "boiling" of the cytoplasmic
membrane
D Chromatin condensation
E Fragmentation of DNA into 50-300 kilobase
fragments
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A male newborn is found to have syndactyly of three fingers of his right hand.
As the attending resident you are called upon to explain to the child's parents
as to why this happened to their child. Which of the following cellular
processes is most likely to have failed during development in utero and
caused the malformation?
V A Apoptosis
B Differentiation
C Fusion
D Fission
E Migration
F Proliferation