L15 4BBY1030: Apoptosis, necrosis and excitotoxicity

Learning Objectives

• To appreciate that there are different forms of cell death.

• To understand the differences between them.

• To emphasize that a specific type of cell death (apoptosis) is required for the development of normal tissues and organs.

• To understand that a disturbance in glutamate homeostasis can cause a form of cell death that is specific to neural tissue (excitotoxicity).

• To appreciate that apoptosis has been conserved across much of the eukaryotic lineage in terms of morphology and the proteins that drive it.

Types of Cell Death

Recognition of Different Forms of Cell Death

• Necrosis:

  • Traumatic cell death from acute injury.

  • Involves activation of a death programme.

  • strokes or catastrophic injury 

  • excessive traumatic injury towards cell or deprivation of oxygen 

  • can’t maintain normal metabolic processes 

• Apoptosis:

  • Proposed in the 1970s, but general acceptance took another 20 years.

  • deliberately die 

  • excessive neurotramsitters —-

• Excitotoxicity:

  • Specific to neural tissue only.

  • A specialized form of cell death confined to one type of tissue.

Comparison of Necrosis and Apoptosis

I. Causes

• Necrosis:

  • Results from injury/insult.

    • Causes include:

    • Ischemia.

    • Hypoxia.

• Apoptosis:

  • Causes include:

  • Withdrawal of growth factors.

  • Chemotherapy.

  • Contact with cytotoxic T cells.

  • Following a developmental programme.

II. Characteristics

• Necrosis:

  • Membrane damage.

  • contents of the brain spill out —- 

  • Chromatin flocculation. random process 

  • Energy levels rapidly depleted. all regulatory processes no longer happen in the cell, atp levels drop to 0 

  • Leakage of cellular contents. 

  • Elicits an inflammatory response. 

• Apoptosis:

  • Intact membrane (with blebbing).

  • make protrusions (blebs), deformation of the surface, membrane buldges out 

  • Chromatin condensation. aggravating into —-

  • Energy levels maintained (or depleted slowly). atp decllining slowly, need cellular process to carry out the first steps of apoptis —- 

  • No leakage of material to the outside

  • No inflammatory response; apoptotic cells are rapidly engulfed by phagocytes (before they lyse, will be released inside the phagocyte). remove cells at a particular time and place and leave the rest of the organism intact

Apoptosis Process

• Apoptosis follows a pre-determined path involving:

  • Chromatin condensation.

  • Membrane blebbing.

  • cell fragmentation, no flammetory response

  • apoptotic bodies recognised by phagocytic cells 

  • Engulfment by phagocytic cells.

  • lysosomes fills up with digestive enzymes which break down the fragments 

• Importance: Prevents release of intracellular molecules, especially in the nervous system, wherein dying cells releasing excitotoxic mediators (e.g., glutamate) may injure adjacent neurons.

Reasons for Cell Apoptosis

1. During Metamorphosis

• Lokshin and Williams (1964) described regulated cell death during insect metamorphosis.

• Vogt (1842) noted physiological cell death during the resorption of the notochord during vertebral development.

• cells of the tail are degraded and those part are used to create gills and lungs turning frogs from a water breather into a air breather 

2. why do cells commit apoptosis 

• Example: Interdigital tissue in mouse paws, which is webbed in the embryo but removed during development.

• Cancer cells. 

• Cells Bearing Excessive DNA Damage

• cells infected by viruses 

• to promote self-tolerance. Autoreactive lymphocytes undergo apoptosis before full development.

• Common Theme: Removal of unwanted cells is prevalent.

Biochemical Characteristics of Apoptosis

1. DNA Cleavage

• DNA is cleaved by an endonuclease, leading to a ladder pattern when analyzed via electrophoresis 

• Example: DNA from mouse thymus lymphocytes after apoptosis induction shows distinct fragment sizes due to cleavage in linker regions.

• TUNEL assay 

2. Changes in Phosphatidylserine Location

• Phosphatidylserine is initially located exclusively on the inner leaflet of the plasma membrane lipid bilayer.

• Apoptosis leads to the release of scramblases (flippases) activated = loss of asymmetry 

• In apoptotic cells, phosphatidylserine flips to the outer leaflet, detectable by labeled annexin V.

• This externalization acts as an 'eat me' signal for phagocytes.

3. Loss of Electrochemical Potential

• Apoptotic cells lose the electrochemical potential across the inner mitochondrial membrane.

• The change can be measured using positively charged fluorescent dyes.

Evolutionary Conservation of Apoptotic Pathway

• Example: Caenorhabditis elegans:

  • Hermaphrodites have 959 somatic cells, of which 131 undergo apoptosis during development.

• Identification of four genes that control apoptosis provides evidence for a genetic programme governing the process.

• Caspases: Same genes found in humans and play similar roles, driving  apoptosis in multicellular eukaryotes.

Function of Caspases

• Caspases are proteases with cysteine at their active sites that cleave substrates at specific aspartate sites.

• Over 10 caspase genes exist in the human genome.

• Examples of caspase targets include:

  • ICAD: Inhibitor of caspase-activated DNase (CAD), when cleaved, activates the DNAase.

  • Structural proteins:

  • Lamin: Cleavage leads to nuclear shrinkage and fragmentation.

  • Gelsolin: Cleavage causes membrane blebbing.

Control and Activation of Caspases

• Caspases cause rapid cell death; are found in all mammalian cells.

• Control measures include:

  • Premature activation would be lethal.

  • Synthesis as inactive zymogens.

  • Upstream regulatory pathways and endogenous inhibitors are highly evolved.

Apoptotic Pathways

I. Extrinsic Pathway

• Members of this receptor family bind to extrinsic ligands to activate caspases.

• This pathway responds to extracellular signals to indicate the non-necessity of a specific cell for the organism's well-being.

• Involves transmembrane death receptors belonging to the TNF receptor superfamily; often referred to as the death receptor pathway.

II. Intrinsic Pathway

• Also known as the mitochondrial pathway.

• Triggered by DNA damage or exposure to cytotoxic drugs entering the cell.

• If DNA damage is irreparable, the responsible cell must undergo apoptosis to prevent the risk of tumor development.

• Example: Apoptosis due to excessive DNA damage from UV irradiation (Sunburn).

UV Irradiation Impact

• UV-C (180-290 nm): Highly energetic and lethal; used as a sterilizing agent.

• UV-B (290-320 nm): Major mutagenic factor, induces chemical bonds creating thymine dimers, distorting DNA and resulting in mutations.

Apoptosis in Disease

• Research in apoptosis is crucial due to either excessive or insufficient apoptosis contributing to diseases:

  • Excessive Apoptosis:

  • E.g., Type I diabetes mellitus: Characterized by pancreatic beta cell apoptosis leading to loss of insulin production.

  • Insufficient Apoptosis:

  • Tumor cells often exhibit defective apoptosis, facilitating cancer progression.

Excitotoxicity

• Glutamate: Most abundant neurotransmitter in the brain, pivotal in neuronal cell death pathogenesis.

• In 1969, Olney coined the term excitotoxicity, describing cell death caused by excessive glutamate acting on excitatory receptors, resulting in an increase in intracellular $Ca^{2+}$ levels.

Synthesis and Reuptake of Glutamate

• Glutamate synthesized in two ways:

  1. From precursors in the Krebs cycle.

  2. After use as a neurotransmitter, glutamate is taken back by exocytic vesicles.

• Reuptake Process:

  • Nerve terminals and glial cells utilize membrane transporters (1 & 3) to take back released glutamate.

  • In glial cells, glutamate is converted to glutamine.

  • Glutamine is transported to neuronal terminals via transporters across glial and neuronal membranes.

  • Glutamine is then converted back to glutamate in neuronal terminals.

  • Glutamate is stored in vesicles, released by exocytosis.

Disturbances Leading to Excitotoxicity

• Typically occurs during conditions like hypoxia or hypoglycemia, causing excessive glutamate release.

• Under normal conditions, intracellular glutamate levels are $10,000$ times greater than extracellular levels due to vesicle sequestration, protecting cells from excessive activation of glutamate receptors.

• Prolonged activation of receptors due to excessive glutamate leads to cell death.

Mechanisms of Receptor Activation

• Glutamate Interaction:

  • Binds to NMDA and AMPA receptors.

  • Dislodges Mg^{2+} from NMDA receptors, allowing for $Ca^{2+}$ entry.

  • Activated AMPA receptors permit $Na^{+}$ entry, depolarizing the plasma membrane.

• Prolonged glutamate exposure results in sustained $Ca^{2+}$ entry, which activates:

  • Ca^{2+} dependent enzymes breaking down:

  • Proteins.

  • Phospholipids.

  • Nucleic acids.

  • Elevated reactive oxygen species (ROS) levels interacting with biomolecules causing further damage.

Disorders Linked to Excitotoxicity

• Associated with various disorders:

  • Stroke.

  • Trauma.

  • Epilepsy.

  • Neurodegenerative disorders:

  • Huntington’s disease.

  • Parkinson’s disease.

  • Alzheimer’s disease.

Summary of Cell Death Forms

• Different forms of cell death exist, including necrosis and apoptosis.

• Apoptosis is highly regulated, resulting in the death of unwanted cells.

• Excitotoxicity is a neural tissue-specific cell death type.

• Necrosis results from acute cell injury, notably through elevated levels of glutamate causing prolonged activation of receptors, which in turn heightens intracellular $Ca^{2+}$ levels.