W6 Translational - Cell Death

which are the initiator caspases?

C2, 8, 9, 10

which are the executioner caspases?

C3, 6, 7

two key domains of initiator caspases (2, 8, 9, 10)

• Adaptor-binding domain (N-terminal) – binds to adaptor proteins.

• Protease domain (C-terminal) – has the active site, but needs activation.

what causes DNA fragmentation in apoptosis?

1. Caspase 3 cleaves iCAD

2. CAD released and activated

3. active CAD cuts DNA between nucleosomes → produces fragments

Why is apoptosis hard to detect?

1. Specificity: Some features can overlap with necrosis → multiple assays are often needed for accurate detection.

2. Timing → apoptotic cells are cleared quickly → leads to false negatives if sampled too early or too late.

3. Sensitivity: Some assays require a high number of cells (e.g., DNA laddering).

common apoptosis features used for detection

1. DNA Fragmentation (gel electrophoresis/ DNA laddering)

2. Caspase Activity & Substrate Detection (Caspase-3 assay, WB, TUNEL assay)

3. Membrane Alterations (Annexin V binding assay)

4. Mitochondrial Assay (cytochrome C release)

How can gel electrophoresis be used to measure apoptosis?

DNA fragmentation = hallmark feature of apoptosis

• Caspase-3 cleaves iCAD → activates CAD → cuts DNA between nucleosomes → DNA fragmentation

In gel electrophoresis → bands indicate DNA

• as time goes by, increased DNA fragmentation is visualised as increased bands

How does TUNEL assay measure apoptosis?

EARLY STAGE DETECTION

assay endonuclease cleavage products

• TdT enzyme labels DNA strand breaks with fluorescent dUTP

• Detected by light microscopy or flow cytometry.

• Highly sensitive but may give false positives (e.g. from necrosis or active gene transcription).

How does Annexin V measure apoptosis?

EARLY STAGE DETECTION

• Annexin V = high affinity for phosphatidylserine, which flips to the outer membrane early in apoptosis.

• Annexin V = tagged with fluorescent probe

• detected via flow cytometry or fluorescence microscopy.

where and what are the anti-apoptotic Bcl2 proteins? and what do they do?

location: Mitochondrial outer membrane

what: Bcl2, BclX_L

How: inhibit apoptosis by by inhibiting pro-apoptotic proteins → blocking Cytochrome C release

what are the pro-apoptotic Bcl2 proteins? and what do they do?

What: Bax, Bak

Where: Bax = cytosol → mitochondria on signal; Bak = always at mitochondria

How: promote apoptosis by forming transition pore to enhance Cytochrome C release

where and what are the pro-apoptotic BH3-only proteins? and what do they do?

What: Bad, Bim, Puma, Noxa

Where: Cytosol → activated on signal

How: inhibiting anti-apoptotic Bcl2s and/or activating Bax/Bak → promotes apoptosis by enhancing Cytochrome C release

what is the role of IAPs?

Function: Block apoptosis unless it's truly necessary by regulating caspase activation → set an inhibitory threshold that caspases must overcome to trigger apoptosis

Mechanism:

• Contain BIR domains → bind to active caspases and inhibit them.

• Some also polyubiquitylate caspases, marking them for degradation by proteasomes

Why is apoptosis important?

• Balancing Act: crucial for maintaining a healthy balance between cell proliferation (growth) and cell death → This ensures that cells survive only where and when they are needed (prevents cancer growth)

• Development: During development, apoptosis plays a vital role in sculpting tissues and organs. E.g. elimination of excess nerve cells → This ensures that all target cells are properly innervated, and redundant cells are removed.

• Quality Control: Apoptosis removes damaged, infected, or potentially harmful cells (e.g., precancerous cells).

what are the ‘eat me’ signals in apoptosis

Phosphatidylserine → Normally on the inner leaflet of the plasma membrane. During apoptosis, it "flips" to the outer leaflet

Pathological Apoptosis - too little — EFFECT

If cells that should die (e.g., damaged cells, precancerous cells) don't undergo apoptosis, they can survive, proliferate uncontrollably, and lead to tumor formation.

diseases: cancer, autoimmune

Pathological Apoptosis - too little — CAUSE

• Disrupted balance of pro- and anti-apoptotic proteins: E.g., overexpression of anti-apoptotic proteins (like Bcl2) or underexpression of pro-apoptotic proteins.

• Mutations in p53: Mutations in p53 (found in ~50% of cancers) can impair the suppression of tumours, allowing damaged cells to survive.

• Increased expression of IAPs: IAPs inhibit caspases, thus blocking apoptosis.

Pathological Apoptosis - too much — EFFECT

• Neurodegenerative Diseases: Excessive apoptosis of neurons contributes to the progressive loss of brain cells in diseases like Alzheimer's, Parkinson's, and Motor Neuron Disease (MND).

• Ischemic Injury (e.g., Stroke, Heart Attack): While necrosis is primary in the core of an ischemic event, apoptosis can occur in the surrounding "penumbra" region, contributing to tissue damage.

Pathological Apoptosis - too much — CAUSE

• Disrupted balance of pro- and anti-apoptotic proteins: E.g., under-expression of anti-apoptotic proteins (like Bcl2) or over-expression of pro-apoptotic proteins.

• Mutations in p53: Mutations in p53 can increase the suppression of tumours, allowing damaged cells to survive.

• decrease expression of IAPs: IAPs inhibit caspases, thus decreases apoptosis.

Why and How do we target Apoptosis in Cancer Treatment?

• Why: Since decreased apoptosis contributes to many cancers, drugs that restore or stimulate the apoptotic pathway can be effective cancer treatments.

• How: Block anti-apoptotic Bcl2 family proteins (e.g., Bcl2, BclXL). This would unleash the intrinsic apoptotic pathway, leading to increased cancer cell death.

• Example: ABT-737

What is the role of ABT-737 in cancer treatment?

ABT-737 = chemical designed to mimic pro-apoptotic BH3-only proteins

1. binds to the hydrophobic groove of anti-apoptotic Bcl2 proteins (like BclXL).

2. neutralises its anti-apoptotic function

3. promotes apoptosis in cancer cells that depend on BclXL for survival.

How does Flow Cytometry measure apoptosis?

cells suspended in fluid/ solution → laser passed through cells → scattered light and fluorescence are detected → indicates cell parameters

Helps distinguish between early and late apoptosis, and necrotic cells.

• Healthy cells: Annexin V negative, PI negative.

• Early apoptotic cells: Annexin V positive, PI negative (PS externalized, but membrane intact).

• Late apoptotic/Necrotic cells: Annexin V positive, PI positive (PS externalized, and membrane compromised).

importance of apoptosis in CNS

critical for proper development of the brain and spinal cord, regulating cell numbers and forming efficient neuronal networks.

• Mice lacking Apaf1 have enlarged brains due to impaired neuronal apoptosis during development, leading to lethality.

• Deleting the pro-apoptotic gene Bax in mice largely eliminates neuronal cell death in the CNS during development.

Apoptosis in ALS - SOD1 (mutant mice) & Caspase activity (human)

• MND mouse models = SOD1 mutation linked to motor neuron death

• increased levels of caspase-1 (initiator) & caspase-3 (executioner) found in spinal MN of human ALS patients