Apoptosis

• Definition: Programmed and controlled cell death of individual cells.

• cell shrinks

• doesn't release contents

• no inflammation

• doesn't require transcriptional activation

• every cell is sitting primed and ready for signal

• obvious sign ( hallmark ) = DNA fragmentation = DNA ladder

Necrosis

• Definition: Uncontrolled cell death due to acute cellular injury

• cytoplasm swells

• bursts and releases inflammatory cytokines

Caspases

• tri-peptide recognition sequence

• ... - X - X - X - D || X - X - ...

• Cuts after aspartic acid ( D )

• leaves exposed aspartic acid

Apoptosis via Caspases

1. Caspase Activation

   • 1.1 = the inhibitory domain gets cleaved

   • 1.2 = initiator caspases start signaling cascade

   • 1.3 = signaling cascade activates effector caspases

2. Phosphatidylserine Externalization

   • 2.1 = activated caspases trigger scrablase activation

   • 2.2 = scrablase cases PS to flip from inner to outer leaflet

     • PS can now be used as "eat-me" signal for phagocytes

     • PS can also now be used as docking site for Annexin V ( labeling marker )

3. Substrate Cleavage

   • 3.1 = activated effector caspases start degrading all kinds of intracellular components

     • Structural Proteins: Leading to cytoskeletal breakdown and cell shrinkage.

     • Signaling Molecules: Disruption of survival pathways.

     • Transcription Factors: Preventing new protein synthesis.

     • DNA Repair Enzymes: Ensuring irreversible DNA damage.

4. DNA Fragmentation

   • 4.1 = High Molecular Weight Endonucleases degrade DNA from 700 kbp ➡️ 300 kbp ➡️ 50 kbp

   • 4.2 = caspase-3 cuts off the inhibitory domain on Caspase-Activated DNase ( CAD ) , thereby activating it

   • 4.3 = some oligosomal endonuclease , like CAD DNAase , can act on the remaining 50 kbp

     • it cuts between histones ( spaced 180 base pairs apart ) , producing uniform DNA "ladder" fragments

     • the ladder pattern is used to confirm / track apoptosis

Phosphatidylserine ( PS )

• binding site for annexin 5

• regulated by :

  • floppase = uni-directional

  • flippase = moves from outer to inner leaflet

  • scrablase = moves from inner to outer leaflet

    • activated via caspase

Apoptotic Substrates

• asdf

Phagocytosis

• in necrotic tissue , it leads to inflammation

• in apoptotic cells , it causes the release of anti-inflammatory cytokines

• macrophages use the PS on the outer leaflet as a targeting / recognition sequence

Pro-Survival ( Anti-Apoptotic ) Signals

• have conserved BH domain

  • Bcl-2

  • Bcl-x2

• you also have IAPs

  • inhibitors of apoptosis

  • they bind to and inhibit caspases

    • usually the upstream , inhibitory caspases

• viruses evolved anti-apoptotic homologous

Pro-Apoptotic ( Apoptotic ) Signals

• BH ( Bcl-2 )

  • Bax , Bak , Bid

    • Promote MOMP and cytochrome C release

• BH3 domain is essential for inducing apoptosis

  • dimerization domain

• p53 = classic

  • responds to DNA damage by inducing cell cycle arrest or apoptosis

Apoptosis via Molecular Pathway

• this is the primary pathway

• it uses cytochrome c from the mitochondria ( ETC )

• it also uses TNF𝛼 , FAS , and FADD

  • caspase 8 , 10

1. apoptosis is initiated via some disturbance in the balance of pro vs anti apoptotic signals

2. cytochrome c is released from mitochrondria into cytosol

3. cytochrome c's then stabilize a caspase dimer , forming apoptosome

4. apoptosome $\underset{\text{"hand wave"}}{\overset{\text{causes}}{\to }}$$\ce{->[\text{causes}][\text{"hand wave"}]}$ apoptosis

Extrinsic vs Intrinsic Apoptosis

• Both pathways lead to the production of apoptotic substrates

• Extrinsic ( Death Receptor ) Pathway :

  1. TNF-α and Fas biding activates FADD

  2. FADD activates caspases

  3. Caspases $\underset{\mathrm{Bid}}{\overset{}{\to }}$$\ce{->[][Bid]}$ T-Bid

• Intrinsic ( Mitochondrial ) Pathway :

  1. Stress

  2. Mitochondria release cytochrome c

     • has Bak , and Bax on outside

• in the extrinsic pathway , caspases not only lead to CAD DNA slicing , but also activate T-Bid

  • T-Bid also activates caspase 9 , which then potentiates mitochondrial export of cytochrome c ?

Apoptosis is Normal

• its used for structural remodeling

  • cuts out webbing of fingers , toes

  • but if something goes wrong , it leads to syn or polydactyl problems

  • it can also present as cleft palette

    • failure of ridge cells to die

• it is used in neuronal selection to prune 50% of neurons

• it is also used in clonal selection / deletion of lymphocytes to retain the top 5% of adaptive candidates

Huntingtin's

• the huntingtin protein is a caspase substrate

• if it gets cleaved , it becomes activated

  • leads to apoptosis in neuronal cells

Familial Alzheimer's Disease ( FAD )

• hereditary and early onset ( > 35 )

• 50% of the mutations are in presenillin 1

  • its a membrane protein that regulates calcium levels

• mutations ➡️ alpha beta plaque build up ➡️ apoptosis

Spinal Muscular Atrophy ( SMA )

• mutation in NAIP protein

• makes it impossible to inhibit caspase activation

Parkinsons

• apoptosis of dopamanergic substantia nigra neurons

• treatment = give L-DOPA

• alpha-synuclein normally help regulate pre-synaptic mutations vesicles

• mutations in alpha-synuclein lead to apoptosis

Stroke

• normally caused by necrosis , leading to clot formation

• but if you could prevent apoptosis in generally ,

  • especially around the surrounding tissue ,

    • it would slow down and reduce necrosis

Lymphoproliferative Diseases

• mutated caspase 10

  • allows uncontrolled lymphocyte growth

• some DNA translocations can lead to over expression of anti-apoptotic factors

AIDS

1. virus infection

2. loss of immune system

3. decrease in CD4+ T-Cells

4. increase in apoptosis

Cancer

• caused by both :

  • over-expression of anti-apoptitc factors

  • the in-activation of apoptotic inducers

Imaging Use Cases

1. transplant rejection monitoring

2. early identification of ischemia or brain injury

3. real time chemo drug monitoring

4. real time bone remodeling monitoring

   • osteoporosis = apoptosis of osteoclasts