Pathology 10-Cell Injury II

Apoptosis in Health and Disease: Dysregulated Apoptosis

Dysregulated apoptosis ("too little or too much") has been postulated to explain aspects of a wide range of diseases.

Apoptosis in Health and Disease: DNA Damage

Exposure of cells to radiation or chemotherapeutic agents induces apoptosis by a mechanism that is initiated by DNA damage (genotoxic stress), and that involves the tumor-suppressor gene *TP53*. -p53 protein accumulates in cells when DNA is damaged, and it arrests the cell cycle (G1 phase) to allow time for repair. -*If the damage is too great to be repaired successfully, p53 triggers apoptosis*.

Apoptosis in Health and Disease: Protein Misfolding

Normally, chaperones in the endoplasmic reticulum (ER) control the proper folding of newly synthesized proteins, and misfolded polypeptides are ubiquitinated and targeted for proteolysis in proteasomes.

Removal of Dead Cells

*Apoptotic cells* and their fragments undergo several changes in their membranes that actively promote their *phagocytosis* so they are cleared *before* they undergo secondary necrosis and release their cellular contents. -Eg, in healthy cells, phosphatidylserine is present on the inner leaflet of the plasma membrane, but in apoptotic cells this phospholipid "flips" out and is expressed on the outer layer of the membrane, where it is recognized by several macrophage receptors.

Morphology of Apoptosis

*Cell shrinkage* -The cell is smaller in size, the cytoplasm is dense. *Chromatin condensation* -This is the *most characteristic* feature of apoptosis. -The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. -The nucleus itself may break up, producing two or more fragments. *Formation of cytoplasmic blebs and apoptotic bodies* -The apoptotic cell first shows extensive surface blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments *Phagocytosis, usually by macrophages* -The apoptotic bodies are rapidly ingested by phagocytes and degraded by the phagocyte's lysosomal enzymes.

The Intrinsic (Mitochondrial) Pathway of Apoptosis: Growth Factors

*Growth factors and other survival signals* stimulate the production of anti-apoptotic proteins such as *BCL2*, thus preventing the leakage of death-inducing proteins, eg cytochrome c, from the outer mitochondrial membrane.

Protein Misfolding: Neurodegenerative Diseases

*Intracellular accumulation of abnormally folded proteins*, caused by genetic mutations, aging, or unknown environmental factors, is now recognized as a feature of a number of *neurodegenerative diseases*, including Alzheimer, Huntington, and Parkinson diseases, and possibly type 2 diabetes.

The Execution Phase of Apoptosis: Executioner caspases

-*Executioner caspases*, once activated, cleave, for example, an *inhibitor* of a cytoplasmic DNase and thus make the DNase enzymatically active, inducing cleavage of DNA. Executioner caspases also degrade structural components of the nuclear matrix, and thus promote fragmentation of nuclei.

Dysregulated Apoptosis in Embryogenesis

-*Failure* of apoptosis is a factor in the development of syndactyly (webbed fingers). -*Exaggeration* of apoptosis is a factor in the development of cleft palate and spina bifida.

Apoptosis

-A pathway of cell death that is induced by a tightly regulated "suicide program", where cells destined to die activate intrinsic enzymes that degrade the cells' own nuclear DNA, nuclear and cytoplasmic proteins. -Because it is genetically regulated, it is sometimes referred to as "*programmed cell death*".

Apoptosis in Embryogenesis

-Apoptosis allows for the destruction of cells during embryogenesis, including implantation, organogenesis, developmental involution, and metamorphosis. -Embryogenesis involves the sequential appearance and *regression*, via apoptosis, of many anatomical structures. -The mesonephros regresses in favor of the metanephros. -Interdigital tissues disappear to allow development of discrete fingers and toes. -Excess neurons are pruned from the developing brain. -Redundant epithelium is removed following fusion of the palatine processes during development of the roof of the mouth. -Redundant tissue is removed during the closure of the neural tube.

Causes of Apoptosis

-Apoptosis occurs *normally* both during development and throughout adulthood, and serves to remove unwanted, aged, or potentially harmful cells. -Apoptosis is also a *pathologic* event when diseased cells become damaged beyond repair and are eliminated.

Apoptotic Bodies

-Apoptotic cells break up into fragments, called apoptotic bodies, which contain portions of the cytoplasm and nucleus. -The plasma membrane of the apoptotic cell and bodies remains *intact*, but its structure is altered in such a way that these become targets for phagocytes. -The dead cell and its fragments are rapidly phagocytized, before the contents have leaked out, and therefore cell death by this pathway does *not* elicit an inflammatory reaction.

Apoptosis in Health and Disease: Apoptosis Induced by the TNF Receptor Family

-FasL on T cells binds to Fas on the same or neighboring lymphocytes. -This interaction plays a role in the elimination of lymphocytes that recognize self antigens. -Mutations affecting Fas or FasL result in *autoimmune diseases* in humans and mice.

Apoptosis in Health and Disease: Growth Factor Deprivation

-For example, hormone-sensitive cells *deprived* of the relevant hormone, lymphocytes that are *not* stimulated by antigens and cytokines, and neurons *deprived* of nerve growth factor die by apoptosis. -In these situations, apoptosis is triggered by the intrinsic (mitochondrial) pathway.

Apoptosis Distinct From Necrosis

-The process was recognized in 1972 by the distinctive morphologic appearance of membrane-bound fragments derived from cells, and was named "apoptosis" after the Greek for "falling off." -It was appreciated that apoptosis was a unique mechanism of cell death, *distinct* from necrosis, characterized by loss of membrane integrity, enzymatic digestion of cells, leakage of cellular contents, and frequently a host reaction (inflammation).

The Intrinsic (Mitochondrial) Pathway of Apoptosis: BAX and BAK

-When cells are *deprived* of survival signals, or their DNA is damaged, or misfolded proteins induce ER stress, the *BH3-only proteins* are activated. -*These sensors activate the two critical (pro-apoptotic) effectors, BAX and BAK*, which form oligomers that insert into the mitochondrial membrane and allow proteins from the inner mitochondrial membrane, eg *cytochrome c*, to leak out into the cytoplasm. -Once released into the cytosol *cytochrome c* can activate initiator caspases, leading to apoptosis.

Apoptosis in Pathologic Conditions

Apoptosis eliminates cells injured beyond repair without eliciting a host reaction in the following: -*DNA damage* -*Accumulation of misfolded proteins* -*Cell death in certain infections* -*Pathologic atrophy in parechymal organs after duct obstruction*

Apoptosis in Physiologic Situations

Apoptosis is a normal phenomenon that serves to eliminate cells that are no longer needed, and to maintain a steady number of various cell populations in tissues, as in these physiologic situations: 1. *Involution of hormone dependent tissues upon hormone withdrawal*. -Eg. Endometrial cell breakdown during the menstrual cycle. 2.*Cell loss in proliferating cell populations to maintain a constant number*. -Eg. B cells lymphocytes in germinal centers of lymphoid tissue. 3. *Elimination of self-reactive lymphocytes*. 4. *Death of cell that have served their useful purpose*. -Eg. Neutrophils in an acute inflammatory response. 5. *Embryogenesis*.

Mechanisms of Apoptosis

Apoptosis results from the activation of enzymes called *caspases* (cysteine proteases that cleave proteins after aspartic residues). -Like many proteases, caspases exist as inactive proenzymes, and must undergo enzymatic cleavage to become active. -The presence of cleaved, active caspases is a marker for cells undergoing apoptosis

Morphology of Apoptosis: H&E Staining

Histologically, in tissues stained with H&E, the apoptotic cell appears as a round or oval mass of intensely eosinophilic cytoplasm with fragments of dense nuclear chromatin -Because the cell shrinkage and formation of apoptotic bodies are *rapid* and the pieces are quickly phagocytosed, considerable apoptosis may occur in tissues before it becomes apparent in sections. -Also, apoptosis—in contrast to necrosis—does *not* elicit inflammation, making it more difficult to detect histologically.

Protein Misfolding

If unfolded or misfolded proteins accumulate in the ER because of inherited mutations or stresses, they trigger a number of cellular responses, collectively called the *unfolded protein response*. -The unfolded protein response activates signaling pathways that increase the production of chaperones, enhance proteasomal degradation of abnormal proteins, and slow protein translation, reducing the load of misfolded proteins in the cell If this cytoprotective response is *unable* to cope with the accumulation of misfolded proteins, the cell activates caspases and *induces apoptosis*.

Mechanisms of Apoptosis: Initiation and Execution Phases

The process of apoptosis may be divided into an *initiation phase*, during which some caspases become catalytically active, and an *execution phase*, during which other caspases trigger the degradation of critical cellular components.

The Intrinsic (Mitochondrial) Pathway of Apoptosis

The *mitochondrial pathway* is the *major* mechanism of apoptosis in mammalian cells. -It results from increased permeability of the mitochondrial outer membrane with consequent release of death-inducing (pro-apoptotic) molecules from the mitochondrial intermembrane space into the cytoplasm. -Eg, cytochrome c, when released into the cytoplasm (an indication that the cell is not healthy), initiates apoptosis. -The release of mitochondrial pro-apoptotic proteins, like cytochrome c, is tightly controlled by the "BCL2 family of proteins".

The Execution Phase of Apoptosis

The *two* initiating pathways converge to a cascade of caspase activation, which mediates the *final* phase of apoptosis. -After an initiator caspase is cleaved to generate its active form, the enzymatic death program is set in motion by rapid and sequential activation of the *executioner caspases*.

Extrinsic (Death Receptor-Initiated) Pathway of Apoptosis

The extrinsic pathway is initiated by engagement of plasma membrane *death receptors* on a variety of cells. -The best known death receptors are the *type 1 TNF receptor (TNFR1)* and a related protein called *Fas (CD95)*. -Death receptors contain a *cytoplasmic* domain involved in protein-protein interactions that is called the death domain because it is essential for delivering apoptotic signals.

BCL Family

There are more than 20 members of the BCL family of proteins, which can be divided into three groups. 1. *Anti-apoptotic*, BCL2, BCL-XL, and MCL1 are the principal members of this group. 2. *Sensors (BH3-only proteins)*, including BAD, BIM, and BID. 3. *Pro-apoptotic*, Eg, BAX and BAK.

Mechanisms of Apoptosis: Mitochondrial and Death Receptor Pathways

Two distinct pathways converge on caspase activation in the *initiation* phase: -*The mitochondrial pathway* -*The death receptor pathway* -These two pathways are generally induced under different conditions, involve different molecules, and serve distinct roles in physiology and disease.

Apoptosis in Health and Disease: TP53

When TP53 is mutated or absent (as in many cancers), cells with damaged DNA fail to undergo p53-mediated apoptosis, and instead survive. -In such cells, the DNA damage may result in mutations of various types that lead to neoplastic transformation.

Extrinsic (Death Receptor-Initiated) Pathway of Apoptosis:FasL and Fas

When the ligand for Fas (*FasL*) binds to Fas, three or more molecules of *Fas* are brought together, and their cytoplasmic death domains form a binding site for an adaptor protein, *FADD.* -FADD in turn binds an inactive form of *caspase-8*. -Multiple pro-caspase-8 molecules are brought into proximity, and they cleave one another to generate active (initiator) *caspase-8*. -The subsequent events are the same as in the mitochondrial pathway, and result in the activation of multiple *executioner caspases*.