Apoptotic Signaling

Question 1

Forms of cell death

Answer 1

1. Necrosis (passive play in cell murder)
2. Apoptosis
3. Autophagic Cell Death
4. Cornficatoin (epidermis)

• all modes of cell death exhibit distinct morphologic features

Question 2

Apoptosis

Answer 2

tightly regulated cell suicide process; cell numbers controlled at level of cell growth and by managing rate of cell death destroy cells that are no longer needed or are a threat to the organism
2 types: extrinsic and intrinsic apoptosis

Question 3

Apoptosis main concepts

Answer 3

1. fundamental cellular process essential in normal development and physiology
2. highly regulated by specialized signaling pathways initiated form within or outside cells
3. Excessive apoptosis or too little apoptosis contributes to range dx pathologies
4. Novel clinical treatments in development for selected dx based on controlling apoptosis

Question 4

Why do cells undergo apoptosis

Answer 4

cells die throughout the life cycle of animal in order to:

1. Allow for proper development
   - formation of fingers and toes
   - proper nerve connects
   - resorption tadpole tail at time of metamorphosis
2. Allow for tissue growth and regression in adult tissue
   - remodels lactating gland after weaning via apoptosis milk secreting alveolar cells
   - phenobarbital -> liver division and enlargement apoptosis increases when treatment stops apoptosis remains increased until liver returns to normal size
3. Remove cells that threaten organism survival
   - cells with DNA damage
   - cells infected with viruses (cytotoxic T cells kill infected cells by apoptosis)
   - cells of immune system (regulate number of immune cells to prevent them from eliciting immune response against normal body constituents)

Question 5

Morphologic changes of cells associated with apoptosis

Answer 5

Early events:

• cell shrinkage
• aggregation of chromatin at nuclear membrane
• bubbling and blebbing from cell surface but no loss cell integrity

Later events:

• Break up of nuclear envelope
• Separation of cell into smaller membrane bound bodies
• engulfment by resident phagocytes
• ** no inflammatory response during apoptosis, cells are eaten and digested by macrophage or neighboring cell; usually few dead cells in apoptotic tissue

Question 6

Biochemical changes of cells associated with apoptosis

Answer 6

1. Microscopy:
2. Gel electrophoresis based assay
3. Enzymatic assay

Question 7

biochemical changes of cells associated with apoptosis Microscopy

Answer 7

1. Detect relocalization of cytochrome C from mitochondria to cytoplasm (mirochondria becomes leaky during apoptosis bc pores formed in mitochondria membrane)
2. Detect externalization fo phosphatidylserine; normally exclusively located in inner leaflet of PM in apoptotic cells PS flips to outer leaflets serving as “eat-me” signal -> rapid engulfment by macrophage (rapid clearance apoptotic cells prevent inflammatory response) (measure externalization with antibody annexin V)

Question 8

biochemical changes of cells associated with apoptosis gel electrophoresis based assay

Answer 8

detect DNA ladder of 180 bp intervals (characteric of apoptosis) caused by internucleosomal DNA cleavage

Question 9

biochemical changes of cells associated with apoptosis enzymatic assays

Answer 9

Measure caspase protease activity (detect proteolytic cleavage products of caspase activity)

Question 10

Signals for apoptosis

Answer 10

can be intracellular or extracellular

Question 11

Extracellular signals apoptosis

Answer 11

Changes in levels of extracelluar molecules

• increase in bone morphogenic proteins leads to cell death of cells btwn developing digits
• Binding of ligands called “death activators” to cell surface receptors including TNF-alpha and FAS ligand (FASL); T cell clearance stimulated by presence of death ligands
• withdraw of survival factors initiates apoptosis in nerve cells (produced in excess in development and compete for limited survival factors, insufficient amounts -> apoptosis)

Question 12

Intracellular signals apoptosis

Answer 12

usually DNA damge

• increase levels of oxidants within cell
• increase in P53 levels resulting from DNA damage b/c UV light or chemo drugs

Question 13

Two main apoptotic pathways

Answer 13

Extrinsic and intrinsic pathways

• have diff triggers and regulators
• both result in activation of caspase (protease cascade)
• is some “cross talk” cascade (synergy to amplify signal)

Question 14

Extrinsic pathway

Answer 14

aka death receptor pathway (responds to extracellular signals)
2 main players= death receptors (link btwn signal and stress) and caspases (aka effectors execute final stages of apoptosis)
- signal
- death receptor activation
-caspase activaiton
- cell death

Question 15

Death receptors

Answer 15

transmembrane proteins present on cell-surface; contain extra-celluar ligand-binding domain and intracellular death domain; receptors belong to tumor necrosis receptor family (TNF)
bind death ligands (pro-apoptotic signals) (ligand examples TNF and FasL, form trimers recruit FADD protein links to initiator caspase forming disc

Question 16

DISC

Answer 16

death inducing signal complex; includes Death receptor, FADD protein (= adaptor, form glue that binds complex), initiator caspace

Question 17

Caspase

Answer 17

family or proteases have a cystine active site and cleave their targets at aspartic acid; synthesized in cell as inactive precursors (procaspases or zymogens) activated upon cleavage that removes prodomain

Question 18

How does caspase work

Answer 18

Synthesized as inactive precurosr -> cleavage that removes pro domain -> activation -> cleave and activate other procaspases -> amplifying proteolytic caspase cascade within cells (at critical point cascade becomes irreversible)
* caspases form cascade of multiple caspases, initially activated caspases = initiator caspases later executioner caspases

Question 19

initiator caspases

Answer 19

initially activated caspases are most closely linked to apoptotic signal upon activation they cleave and activate executioner capspaces

Question 20

executioner caspases

Answer 20

activated by initiator caspases; cleave target proteins, targets include cytoskeleton and nuclear proteins and cell-cell adhesion proteins their cleavage -> loss cell structure -> cell destruction and dissolution

Question 21

central effectors of apoptosis

Answer 21

caspases

Question 22

Extrinsic (death receptor) pathway

Answer 22

1. Process extracellular signals
2. initiated by binding of extracellular ligands (“death ligands”) to cell surface death- receptors -> receptor homo-trimerizatoin
3. Activated death receptors recruit adaptor proteins which in turn recruit initiator procaspase-8 forming DISC
4. Procaspase cleaved to form active caspase
5. Caspase-8 begins caspase cascade and activates executioner caspases which cleave cellular proteins essential for cell structure
6. cellular protein cleavage leads to cell destruction (cleave cytoskeletal protein activate DNAses, ect.)

Question 23

Intrinsic pathway

Answer 23

Main players: Bcl-2 proteins and caspases
Bcl-2 Proteins- Process and initiate signaling pathways in response to stress or stimuli
Caspases (effectors, execute final stages of apoptosis)

Question 24

BCL-2 protein family

Answer 24

Group of proteins that share regions protein homology called BH (Bcl-2 Homology) domains
- mitochondria proteins that control release of proteins from mitochondria into cytosol by controlling pore formation in mitochondrial membrane (regulate cytochrome c release from mitochondria in response to intracellular stress signal)
2 main forms
Anti-apoptotic
Pro-apoptotic

Question 25

Anti-apoptotic Bcl-2 proteins

Answer 25

prevent protein release from mitochondria Bcl-2 and Bcl-Xl (cell Lives, these usually have L in them)

Question 26

Pro-apoptotic Bcl-2 Proteins

Answer 26

``` enhance protein release from mitochondria (lead to pores opening and cytochrome C release) ex. Bax, Bak (Apoptosis Inducers tend to have A and I in them) Bax Bak Bad Puma Bid Bim Noxa ```

Question 27

Regulation of the intrinsic pathway

Answer 27

Ratio fo active pro and anti apoptotic Bcl-2 proteins regulates intrinsic pathway; Bcl-2 protein family controls release of proteins from mitochondria into cytosol

Question 28

Absence of stress

Answer 28

anti-apoptotic members prevent proapoptotic proteins Bax and Bak from associating

Question 29

Apoptotic signal

Answer 29

Bcl-2 inhibition released allows pro-apoptotic proteins to self associate and form multimers; Bak and Max protein complex forms allows for release proteins from inner membrane space to cytoplasm (known as mitochondrial outer membrane permeabilization) -> cytochrome C release -> binds Apafl -> apoptosome -> cleavage pro-caspase 9 by apoptosome -> caspase cascade and ultimately cell death

Question 30

Cytochrome C

Answer 30

component electron transport chain in mitochondria; when released to cytosol binds to procaspase activating protein (Apaf1) which oligomerizes into heptameter called apoptosome which cleaves initiator pro-caspase 9 and begins caspase casade leading to cell death

Question 31

Intrinsic (mitochondrial) pathway

Answer 31

1. Triggered from inside the cell 2. Activated Bcl-2 proteins release mitochondria proteins into cytoplasm (cytochrome C) 3. Cytochrome C (with Apafl) forms complex called apoptosome 4. Apoptosome causes cleavage of initiator caspase 9 beginning caspase cascade 5. Executioner caspase cleaves cellular proteins 6. Cellular protein cleavage ultimately leads cellular destruction

Question 32

shared portion of 2 apoptotic pathways

Answer 32

activation of initiator caspase (8 and 10 extrinsic 9 intrinsic) -> caspase cascade cell protein cleavage and cell death

Question 33

Summary of both pathways (intrinsic and extrinsic apoptotic pathways)

Answer 33

3 overall stages Induction: insult, stress, or ligand Integration: death-receptors and Bcl-2 family Execution: caspases alter cells

Question 34

Cross talk between intrinsic and extrinsic apoptotic pathways

Answer 34

Initiator caspase 8 from extrinsic pathway cleaves and activates proapoptotic Bcl-2 family member Bid; cross-talk serves to amplify signal from extrinsic stimuli

Question 35

Apoptosis and pathology

Answer 35

too much and too little cell death is detrimental to organism and contributes to range of sieases

Question 36

excessive cell death and degenerative dx

Answer 36

- neurodegenerative conditions like alzheibers and cognitive dysfunction dx (dog) exhibit substantial loss of function and death of neurons in part b/c of apoptosis (characteristic beta-amyloid accumulation; brain pathology includes cortical atrophy, accumulation of degraded proteins, some apoptotic cell death of neurons)

Question 37

treatments for neurodegeneration

Answer 37

selegeline hydrochloride (L-deprenyl or anipryl) use for Parkinson's and alzheimers in ppl and canine cognitive dysfunction syndrome in canines its monoamine oxidase type B inhibitor, has protective effects against neuronal apoptosis

Question 38

Alzheimers brain

Answer 38

active caspase 3 detected in dying neurons of alzheimers brain

Question 39

progressive retinal atrophy

Answer 39

- conglomerate of many autosomal recessive diseases of the eye - degeneration of photoreceptor cells in part bc of apoptosis - mutations in progressive rod cone dysplasia gene in retina account for one form PRA in canines

Question 40

nonalcoholic fatty liver disease (NAFLD)

Answer 40

chronic liver disease; increased synthesis of fatty acids in hepatocytes, retention of lipids due to impaired hepatocyte, apo-lipoportein secretion or beta-oxidation; increased cell death in liver and peripheral tissues

Question 41

emricasan

Answer 41

caspasce inhibitor surpasses apoptosis and inflamation= for fatty liver dx +/- use in Zika and prevent killing of fetal brain cells; in phase II trials for non-alchoholic steatohepatitis; treatment of liver fibrosis or cirrhosis post liver transplant

Question 42

Excessive cell survival and proliferative diseases

Answer 42

inability to initiate apoptosis can lead to uncontrolled cell proliferation associated with cancer and autoimmune disorders

Question 43

Autoimmune lyphoproliferative syndrom (ALPS)

Answer 43

genetic dx in humans typified by splenic enlargement and lymph node enlargement; body can't properly regulate number of immune cells (lymphcytes); characterized by production of abnormally large number lymphocytes; ppl with ALPS have increased risk of lymphatic cancer; majority of cases bc mutation in Fas (death ligand) -> defective apoptosis lymphocytes

Question 44

cancer and cell surival

Answer 44

mutations P53 gene which usually initiation apoptosis in response to DNA damage overexpression of Bcl-2 observed in numerous cancers -> antiapoptotic condition Cancer cells are living when they should die

Question 45

Chemoteraputic agents

Answer 45

aim to induce apoptosis in tumor cells - Navitoclax - Venclexta - Gleevec

Question 46

ALPS

Answer 46

Known mutations: - FAS gene - Cas 8 - Cas 10 Mutations detrimental b/c normally immune response stimulated leading to lymphocyte production, lymphocytes destroyed by apoptosis when no longer required but deffective apoptosis leads to excess lymphocytes and cells multiplying out of control -> cancer

Question 47

Navitoclax

Answer 47

mimics proteins that bind to Bcl-2 and Bcl-Xl inhibiting their function allowing apoptosis kill tumor cells, this is in phase II trials but can -> thrombocytopenia bc Bcl-Xl reduces platelet lifespan which limits dose

Question 48

Venclexta

Answer 48

inhibitor of Bcl-2 (1st FDA approved Bcl-2 inhibitor); approve for treatment of chromic lymphocytic leukemia; can have v serious side effects, being tested for treating cancer

Question 49

Gleevec

Answer 49

tyrosin kinsae inhibitor; kills chornic myeloid leukemia by up regulating pro-apoptotic Bcl2 proteins (BIM and BAD)

Question 50

Masitinib

Answer 50

tyrosine kinase inhibitor promotes apoptosis used to treat canine mast cell tumors