Cell death

Question 1

Autophagy

Answer 1

Autophagy is a cell survival mechanism that aims to derive energy by eliminating defective organelles, very rarely will autophagy result in cell death.

Question 2

Give some different mechanisms of cell injury

Answer 2

Loss of cell membrane integrity: breakdown of concentration gradients of ions and metabolites

Decreased ATP concentration: rapid shutdown of homeostatic pathways

Protein unfolding and protein aggregation

Generation of ROS: activates multiple signalling pathways, kinases, phosphatases, NF-kB, phospholipases

Protein trafficking defects

Cation imbalance (Na+, Ca2+, H+)

Loss of integrity of the genome

Mitochondrial dysfunction: increase in membrane permeability, leads to H+ leakage which dissipates the proton-motive force and acidification of the cytosol

Question 3

How are organelles recycled in autophagy?

Answer 3

Autophagosomes are generated and fuse with a lysosome containing cellular material. This is a autophagolysosome, the products can be recycled for ATP production and protein synthesis.

Question 4

What is apoptosis?

Answer 4

Apoptosis is ‘programmed cell death’ directed by expression of specific genes. It does not contribute to inflammation.

Question 5

Signs of apoptosis

Answer 5

Membrane blebbing

Cell shrinkage

Nuclear fragmentation

Chromatin condensation

Chromosomal DNA fragmentation

Global mRNA decay

Question 6

Intrinsic apoptosis mechanism

Answer 6

Activated by intracellular signals generated when cells are stressed and depends on the release of proteins from the intermembrane space of mitochondria. Or can be initiated by developmental programming / lack of specific growth factor.

Question 7

Extrinsic apoptosis mechanism

Answer 7

The extrinsic pathway is activated by extracellular ligands binding to cell-surface death receptors, which leads to the formation of the death-inducing signaling complex (DISC).

Caspase activation

Question 8

Two pathways of extrinsic apoptosis

Answer 8

The binding of TNF-alpha to TNFR1 has been shown to initiate the pathway that leads to caspase activation

The Fas-Fas ligand-mediated model The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10.

Caspase 8 cleaves caspase 3 which cleaves I-CAD the inhibitor of CAD which is released into the nucleus to cleave DNA

Question 9

Describe how caspases cause apoptosis

Answer 9

Caspases cleave key enzymes involved in DNA repair and replication as well as key structural proteins that maintain nuclear architecture.

PARP: The ability of the enzyme PARP (poly (ADP-ribose) polymerase) to repair DNA damage is prevented following cleavage of PARP by caspase-3.

DNA topoisomerase II is a nuclear enzyme essential for DNA replication and repair. Caspases can inactivate this enzyme leading to DNA damage.

Lamins are intra-nuclear proteins that maintain the shape of the nucleus and mediate interactions between chromatin and the nuclear membrane. Degradation of lamins by caspase 6 results in chromatin condensation and nuclear fragmentation.

Question 10

Why may some tissues express FAS-L

Answer 10

Confers immune privilege to the organ - immune cells killed that are nearby

Question 11

What caspase degrades lamins?

Answer 11

Caspase 6

Question 12

What caspase cleaves PARP?

Answer 12

Caspase 3

Question 13

What caspase cleaves I-CAD

Answer 13

Caspase 3 (cleaved by caspase 8)

Question 14

What is necrosis?

Answer 14

Necrosis is a sequence of morphological changes that follow cell death following irreversible injury in living tissue.

Question 15

What does necrosis promote?

Answer 15

Inflammation

Question 16

What is pyknosis?

Answer 16

Shrunken basophilic nucleus (less visible chromatin)

Question 17

What is Karyorrhexis?

Answer 17

More basophilic chromatin, fragmented nucleus (appears almost granular)

Question 18

What is Karyolysis?

Answer 18

Loss of basophilic chromatin - appears to have no nucleus

Question 19

What happens when macrophages interact with apoptotic cells?

Answer 19

When macrophages interact with apoptotic cells (bind with CD-14/ICAM-3 and PS interactions). Macrophage expression is altered, production of anti-inflammatory cytokines (TGF-B1) and down-regulation of inflammatory cytokines.

Question 20

What happens when macrophages interact with necrotic cells?

Answer 20

When macrophages interact with necrotic cells via opsonin complement receptor interactions there is upregulation of pro-inflammatory cytokines (IL-1B, IL-8)

Question 21

Why do apoptotic cells not promote inflammation but necrotic ones do?

Answer 21

Blebbing leads to the hiding of endogenous DAMPs, apoptotic cells are phagocytosed.

Necrotic membrane lysis leads to the display and/or release of these intracellular molecules. Exposure of certain intracellular molecules (DAMPs) can trigger inflammation via innate immune cells.

Question 22

What is pyroptosis?

Answer 22

Pyroptosis is carried out in a deliberate and highly self-programmed process that generally
involves innate immune cells and activation of inflammasomes.

Question 23

How is the inflammasome activated?

Answer 23

TLR signalling leads to NFKB stimulations, leads to transcription of a large number of genes in response to inflammatory stimuli

These mRNA are translated into protein for pro-IL-1B and caspases.

Proteins could be secreted, but must be first cleaved by active cysteine protease (caspase).

Inactive NLRP3 inflammasome activated - leads to cleavage of caspases - and cleavages and release of IL-1b and IL-18.

Leads to pyroptosis

Question 24

What pathogens is the inflammasome specifically important for?

Answer 24

Important for intracellular pathogens

Question 25

What caspases does the inflammasome activate?

Answer 25

1,4 and 5

Question 26

What is produced by the inflammasome that leads to pyroptosis?

Answer 26

Pore forming gasdermins

Question 27

What is cellular senescence?

Answer 27

‘permanent’ arrest of the cell division cycle.

It is associated with hyperactivated secretion of pro-inflammatory factors that affect a range of patho-physiological processes such as impaired wound healing, cancer and aging.