Unit I: Apoptosis

Question 1

Necrosis- 4 major differences from apoptosis

1. ​
2. 3. 4.

Answer 1

Necrosis

1. Cell dissolution with extrusion of contents
2. acute inflammatory response
3. tissue architecture is lost, cellular loss is obvious because lots of cells die at once
4. cell swells and blebs before dying

Question 2

Apoptosis- 4 main different features

Answer 2

1. cell fragments, but contents do not get out
2. no inflammation
3. tissue changes aren’t apparent (one cell dies at a time)–architecture preserved
4. condensation of cell, break up of cell

Question 3

apoptotic bodies

Answer 3

cell fragments due to apoptosis. No extrusion of intracellular contents. remember cell SHRINKS before fragmenting.

Question 4

Mechanisms of apoptosis (x5)

Answer 4

1. loss of growth factor/stimulation
2. Fas-Fas killing of infected cells
3. P53 killing of damaged cells
4. mis-folded protein accumulation
5. maintenance of cell populations (embryogenesis, epithelial layers, immune)

Question 5

Apoptotic cells microscopic features

Answer 5

EOSINOPHILIC

1. cell shrinks (condensation of nucleus and cell)
2. chromatin is at periphery of nucleus under nuclear membrane
3. membrane blebbing

Question 6

Why isn’t cell loss apparent in __(apoptosis or necrosis)___ ?

(x3)

Answer 6

• Apoptosis- cell loss is not apparent due to:
  
  • cells die singly or in small clusters
  • space is filled in by movement of surrounding cells
  • no inflammation

Question 7

In viral hepatitis, cells undergo

Answer 7

BOTH apoptosis and necrosis

Question 8

What happens to cell fragments in

1. Apoptosis
2. Necrosis

Answer 8

1. Apoptosis- cell fragments are phagocytosed by surrounding cells (magrophages and parynchyma)
2. Necrosis- phagocytosed by macrophages, initiate acute inflammatory response?

Question 9

Caspase

1. what is it?
2. activation?

Answer 9

1. cysteine proteases that cleave aspartic acid= Caspase
2. zymogens that require activation by initators or autolytic activation.

Question 10

Caspases- function/effect (x2 major ones)

Answer 10

• cleave nuclear and cytoskeletal scaffold (LAMINS)==> protein cross-linking and cytosolic condensation
• triggers endonuclease activity

Question 11

Endonucleases

1. activation?
2. function?
3. mechanism?

Answer 11

1. activated by caspases, require Mg or Ca
2. DNA breakdown
3. Calcium and Mg dependent endonucleases break down DNA into 50-300kB pairs==> 180-200 bp. Internucleosomal DNA cleavage.

Question 12

How are apoptotic cells phagocytosed (mechanism)? X2 “markers”

Answer 12

• Phosphatidyl serine on plasma membrane is flipped and faces outwards, marking a cell as an apoptotic body.
• external expression of thrombospondin

Phagocytosed by MACROPHAGES= minimal inflammation

Question 13

Physiological vs Pathological processes of apoptosis

Question 14

Initiation of apoptosis: Major pathways

Answer 14

1. Bcl Pathway
2. Fas-Fas pathway

Question 15

Bcl-2 proteins

1. Sensors- what are they, how are they activated, what is their function?
2. Effectors- what are they, function, regulation?
3. Regulators - what are they, function, regulation?

Answer 15

• Bcl-2 family of proteins in mitochondria- pro and anti-apoptotic members
  
  • cytoplasmic signaling influences balance of pro/anti

1. Sensors: BH3
   
   • Activated by decreased survival signals, DNA damage, ER stress (protein misfolding)
   • activate Bax/Bak==>form pore
   • inhibit Bcl-xl, Bcl-2
2. Effectors: pro-apoptotic= Bax/Bak- form pore
   
   • ​​activated by BH3 proteins
   • inhibited by Bcl-xl, Bcl-2
3. Regulators: anti-apoptotic: Bcl-xl, Bcl-2- inhibit Bax/Bak and other pro-apoptotic proteins
   
   • increased by growth factors and other survival signals
   • inhibited by BH3 (= sensors)

Question 16

Apoptosis Figure

(shows intrinsic and extrinsic pathways)

Answer 16

Question 17

What happens after Bax/Bak are activated?

Answer 17

• Bax/Bak= pore formed in mitochondria==> cytochrome C leaks out of outer membrane
• Cytochrome C binds Apaf-1 and activates caspase 9

Question 18

Follicular lymphomas- oncogene?

Answer 18

have an oncogene that activates bcl-2==> inhibition of apoptosis in neoplastic cells

Question 19

Death receptor pathway- what is it?

2 initiators

Answer 19

Extrinsic pathway of apoptosis

1. Fas (CD95) binds FasL (virally infected t-cells)
2. TNF/TNF-R crosslinking

Question 20

Fas-Fas ligand

1. mechanism
2. purpose
3. viral adaptation

Answer 20

1. activation of FADD (FAS associated death domain)==> pro caspase 8 activation ==> autolytic activation of other caspases
2. killing of autoreactive t-cells
3. FLIP (made by viruses) blocks activation of caspase 8

Question 21

How doe live cells protect themselves from phagocytosis? (how are they differentiated from apoptotic bodies?)

Answer 21

Cd31 expression

Question 22

TNF family of receptors

Answer 22

• includes TNFR1 and Fas
• Activate FADD or TRADD==> caspase activation

Question 23

Spinal muscle atrophy- what’s the mutaiton?

Answer 23

mutation in regulatory proteins that inhibit activation of TNF induced apoptosis.

Question 24

CTL-induced cell death- 2 ways

Answer 24

1. CTL’s express FasL and can activate extrinsic pathway via Fas receptor ligation
2. CTL recognizes foreign antigen==> expression of perforins create transmembrane pores==>exocytosis of granzyme B (cleave proteins at aspartate residues) ==> activated cellular caspases
   
   • no death receptor activation, no mitochondrial engagement– direct activation of effector phase= caspases.

Question 25

Decrease in growth factor

Answer 25

**Intrinsic pathway-**neurons and hormonal signaling decreased Bcl-2, increased BH3 sensors (bim)

Question 26

ER stress- what causes it? mechanism? disease examples?

Answer 26

from accumulation of misfolded proteins * misfolded proteins are ubiquitinized and destroyed by proteosome, but too many overwhelm the process * ER stress response activates caspases==\> apoptosis (intrinsic) * degenerative CNS disease= alzheimers, parkinsons, maybe DMII

Question 27

DNA damage apoptosis 1. what causes DNA damage 2. Cell response? 3. Disease example

Answer 27

1. heat, radiation, chemo, 2. p53 accumulates in cell==\> arrest cell cycle at G1==\> [if the damge can't be repaired] trigger apoptosis via sensors (activate Bax/Bak; inhibit Bcl-xl/2) 3. p53 mutations allow damaged cells to divide= carcinogenesis

Question 28

Disease with inhibited apoptosis (x2) Diseases with increased apoptosis (x3)

Answer 28

1. **Inhibited apoptosis** * ​​p53 mutation * autoimmune disorder (cant remove autoreactive t-cells) 2. **Increased apoptosis** * CNS degenerative disease * ischemic injury * virus induced lymphocyte depletion

Question 29

Autophagy

Answer 29

* **_autophagic vacuole_** formed from ribosome free regions of ER digest cell components * activated by multi-protein units that sens nutrient deprivation * involved in clearance of misfolded proteins in neurons and hepatocytes * may trigger apoptosis