Current Concepts and State of Knowledge

Characteristics of Apoptosis

Apoptosis may be triggered by signals from within the cell or proximal and distal cells, as well as exogenous agents; apoptotic cell death may also result from the withdrawal of trophic factors (► nerve growth factors). All of these triggers initiate a cascade of events that results in the elimination of cells without releasing harmful substances into the surrounding areas. Apoptosis is not associated with an inflammatory response; in situ, apoptotic cells are phagocytosed by macrophages or neighboring epithelial cells, whereas the fragments of cells that have undergone apoptosis in vitro are eventually lysed.

In its original sense, the term apoptosis was used to refer to a form of "programmed cell death" that depends on the activation and/or repression of ► gene transcription to delete biologically redundant or dysfunctional cells. Growing knowledge of how cell death is triggered and of the processes that lead up to it indicate a degree of overlapping mechanisms in different forms of cell death; this has resulted in a blurring of how apoptosis should be defined. However, the consensus is that apoptosis can only be ascertained through the morphological observation of chromatin condensation with evidence of DNA cleavage, nuclear fragmentation, cell shrinkage, blebbing of the nuclear and plasma membranes, and formation of membrane-bound apoptotic bodies in cells that otherwise have an integral plasma membrane. At the biochemical level, apoptotic cells show signs of mitochondrial dysfunction. Specifically, the mitochondrial membrane potential is perturbed and results in a leakage of mitochondrial proteins, such as cytochrome c, into the cytoplasm; cytochrome c is a soluble protein whose ability to transfer electrons plays a key role in energy generation.

Apoptosis Versus Necrosis

In contrast to apoptosis, cell death through necrosis maybe considered to occur "accidentally" following direct disruption of cellular homeostasis of cellular functions by a toxin or noxious stimulus (e.g., hypoxia, hypothermia), resulting in an influx of water and extracellular ions. Morphologically, this form of cell death, which differs from apoptosis, is characterized by swelling of the cytoplasm and mitochondria and the ultimate disintegration of the plasma membrane, leading to the leakage of lysosomal enzymes that lyse a group of contiguous cells (typically apoptosis affects only individual cells), usually followed by an inflammatory response. Other important features that distinguish apoptosis and necrosis are (1) DNA fragmentation in apoptosis occurs through the actions of specific DNA-cleaving enzymes (endonucleases) while the cell is still intact, whereas DNA breakdown in necrotic cells occurs only after cell lysis and the DNA fragments are of random size; (2) apoptosis is an energy (ATP)-dependent process, whereas necrosis is a passive process; and (3) whereas necrosis results from a massive influx of calcium in the cell, apoptosis is triggered by moderate increases in calcium influx. Various assays that are based on the above-mentioned morphological and cytological descriptions serve to discriminate between apoptotic and necrotic cells both in vivo and in vitro; in addition, researchers increasingly rely on other biochemical and molecular markers to detect early- and late-stage apoptosis.

Apoptotic Mechanisms

An understanding of the molecular and cellular basis of apoptosis has grown immensely in the last decade. While the molecular processes that lead to apoptosis are initiated in the cytoplasm or the membranes of organelles, the final execution of the apoptotic process takes place in the nucleus where irreversible, Ca2+/Mg2+-dependent inter-nucleosomal DNA fragmentation occurs in a sequential fashion, mediated by endonucleases.

In general, apoptosis results through one of two cellular pathways, the intrinsic and extrinsic pathways; however, growing evidence points to the possibility that these pathways may converge under certain circumstances. The sequential activation of caspases is central to both pathways; the activation of caspase 3, a so-called executor caspase serves as a good biochemical marker of apoptosis. In addition, apoptosis may occur independently of caspase activation. Several stimuli, including DNA damage, and oxidative and excitotoxic stress, cause the translocation of apoptosis-inducing factor (AIF) from the mito-chondrial intermembranous space to the nucleus where AIF binds to DNA and initiates apoptosis by promoting chromatin condensation (Fig. 1).

The intrinsic pathway is initiated by the perturbation of the mitochondrial transmembrane potential, which is rheostatically regulated by the availability of pro- (e.g., Bax, Bid) and anti-apoptotic (e.g., Bcl-2, Bcl-XL) members of the Bcl-2 family of proteins. A shift in the ratio of these proteins in favor of the pro-apoptotic members leads to the release of mitochondrial cytochrome c (a heme-containing protein involved in electron transfer) which, in turn, binds to apoptosis protease activator factor-1 (Apaf-1), forming an apoptosome that subsequently cleaves and activates pro-caspase 3. In the extrinsic pathway, the binding of either Fas ligand, tumor necrosis factor (TNF) a, or tumor necrosis factor-related apopto-sis-inducing ligand (TRAIL) to their corresponding membrane receptors results in the formation of a complex between liganded and pro-caspase 8. When cleaved, caspase 8 can activate caspase 3 directly, or indirectly by cleaving the pro-apoptotic protein Bid in the cytoplasm; truncated Bid (tBid) translocates to the mitochondrion where it disrupts that organelle's permeability transition pore, resulting in cytochrome c release and the ultimate activation of caspase 3.

Neuronal apoptosis may be triggered by both, pro-apoptotic signals as well as limited availability of (neuro) trophic molecules (nerve growth factors); the latter mechanism is important in both physiological and pathological contexts and led to the discovery of pro-survival signaling pathways that act to suppress the cell death machinery. Among the best-characterized survival pathways are the Ras-MAPK (mitogen-activated protein kinase) and the PI3K (phosphatidylinositide-3'-OH kinase)-Akt pathways.

It is estimated that neurons undergo apoptotic death within 6-12 h from first being exposed to a pro-apoptotic stimulus. Under normal circumstances, apoptosis occurs widely in the developing brain; it also occurs throughout postnatal life, albeit at a much reduced rate. It is estimated that apoptosis results in the elimination of up to 70% of neurons during early brain development; accordingly, it is thought to serve a physiological role, serving to ensure appropriate structure and function of the brain and the

Translocation

Apoptosis. Fig. 1. The pathways to apoptotic cell death. Two pathways, the so-called intrinsic and extrinsic pathways, regulate apoptosis. In both pathways, caspases, a family of cysteine proteases, play a central role. The arrival of apoptotic signals leads to the activation of initiator caspases (e.g., caspase-8, -9) and sequential cleavage/activation of downstream effector caspases; caspase-3 is the last caspase in this cascade and is known as the ''executor caspase'' since the process cannot be reversed once this caspase is activated. The extrinsic pathway is triggered after the binding of FasL or TNF to their respective cognate receptors (Fas and TNFR), followed by the activation of caspase-8 through the adaptor protein FADD/TRADD. Activated caspase-8 can stimulate apoptosis through one of two cascades: direct activation of caspase-3 or cleavage of Bid. Truncated Bid (tBid) translocates to the mitochondrion where the extrinsic pathway converges with the intrinsic pathway in which the formation of pores in the outer mitochondrial membrane and the leakage of cytochrome c is a central event. Cytochrome c binds Apafl to form an activation complex with caspase-9, an event critical to the activation of the intrinsic pathway. Members of the BCl-2 family of proteins, including the anti-apoptotic proteins Bcl-2 and Bcl-xL and pro-apoptotic protein Bax, rheostatically control apoptosis by determining the integrity and permeability of mitochondrial membranes and thus, cytochrome c release. The tumor suppressor protein p53, which is activated following DNA damage, induces the transcription of Bax, which, in turn, disrupts the mitochondrial potential. More recently, another apoptotic mechanism involving mitochondrial release of apoptosis inducing factor (AIF) was identified. AIF translocates to the nucleus and directly triggers the internucleosomal degradation of DNA. Reactive oxygen species (ROS) can also directly induce apoptosis by damaging DNA strands. On the other hand, anti-apoptotic signals, including growth factors and cytokines, act by inducing the phosphorylation of signaling molecules such as Erk1/2 and Akt. Erk1/2 activates p90RSK, which upregulates the expression of the anti-apoptotic proteins Bcl-xL and BCl2 and inhibits the Fas pathway; similarly, activated Akt regulates the expression of members of the Bcl2 family and of Fas while inhibiting GSK-3 signaling.

ability of the brain to adapt to changing demands. As neuronal apoptosis is essential to normal brain development and function, it is conceivable that it may contribute to brain pathology and pathophysiology if it arises at an inappropriate time or location, or in excess or an insufficient extent.

Neuronal Birth, Death, and Plasticity

Ensuring the correct number of functional neurons in a given brain area depends on the coordinated birth and death of neurons. Extensive neuronal birth (► neurogenesis) in the mammalian brain ceases during postnatal life; two areas, the ► hippocampus and

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