Учебники / Hair Cell Regeneration, Repair, and Protection Salvi 2008

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integrity of the plasma membrane; cellular swelling, as a consequence of osmotic imbalance resulting from loss of membrane integrity; swelling of the nucleus and other organelles; rupture of the plasma membrane; and spillage of the necrotic cell’s contents into the local microenvironment. The exposure of the cell contents to the extracellular environment provokes an inflammatory response. This in itself may cause further localized tissue disruption as inflammatory cells are recruited to the lesion site. Apoptosis (type 1 cell death) and autophagy (type 2) are different manifestations of “programmed cell death,” active processes that involve a cascade of biochemical events triggered within a cell such that the affected cell essentially destroys itself (colloquially known as “cell suicide”). During programmed cell death, the integrity of the plasma membrane is largely retained so that, unlike in necrosis, an inflammatory response is not initiated. However, it is becoming increasingly apparent that a clear distinction between these active cell death processes and a passive one is somewhat blurred as a necrotic-like morphology may be the end stage of a programmed cell death pathway, and cells that initiate the program that normally leads to apoptosis may become necrotic (Danial and Korsmeyer 2004).

Of the defined programmed cell death pathways, autophagy (“self-eating”) has been recognized only relatively recently as a distinctive process that can lead to cell death, rather than just a mechanism for turnover and recycling of cell components which is meant to protect the cell rather than eliminate it (Debnath et al. 2005; Meijer and Codogno 2006). It involves the enclosure of cellular components within specialized vacuoles and their delivery to lysosomes where they are destroyed (Reggiori and Klionsky 2005). Under normal circumstances, this serves to recycle cellular material and provide energy sources alternative to those obtained by normal nutrition. The double-membraned autophagic vacuoles are the most characteristic morphological feature (Reggiori and Klionsky 2005). There is no evidence as yet that autophagy is a significant contributing factor in the loss of hair cells or neurons from the inner ear, and therefore it is not considered further in this chapter. However, with the recent indications of the contribution of autophagy to some neurodegenerative diseases and in aging (Reggiori and Klionsky 2005; Rubinzstein et al. 2005; Bergamini 2006; Boland and Nixon 2006) it may be that it does play a role in the removal of cells at a site of damage within the inner ear.

Apoptosis is the end stage of a biochemical pathway that produces an orderly destruction of the cell from within (Danial and Korsmeyer 2004; Green and Kroemer 2004). Activation of the pathway either through external signals (the extrinsic pathway) or by intracellular stress (the intrinsic pathway) leads to distinctive morphological features that include condensation of nuclear chromatin accompanied by shrinkage of the nucleus (“pyknotic” nuclei); nuclear fragmentation; cell shrinkage; membrane blebbing; and breakdown of the cell into fragments, so-called “apoptotic bodies.” The apoptotic bodies are phagocytosed by “professional” phagocytic cells such as circulating macrophages and/or by neighboring undamaged cells within the tissue (Thornberry and Lazebnik 1998; Savill and Fadok 2000; Monks et al. 2005). In this way, a dying apoptotic cell is

removed without compromising tissue integrity or stimulating an inflammatory process. The biochemical cascade within an apoptotic cell involves the orderly destruction of DNA by endonucleases that results in a distinct “ladder” pattern of different size DNA fragments that are evident on an agarose gel (Fraser et al. 1996). The fragmentation of DNA by those endonucleases activated during apoptotic cell death also leads to the formation of single-strand ends at the sites where the DNA double strand has been cleaved, i.e. the “nick ends.” These offer multiple sites for binding a nucleotide that can be labeled during preparation of tissue for examination by microscopy, providing a means to mark nuclei in which the orderly destruction of DNA has occurred. Such Terminal deoxynucleotide–UTP-Nick-End Labeling (TUNEL) provides for the objective identification of cells dying by apoptosis. In necrotic cells, although DNA is also broken down, several different cleavage events may occur. Consequently, as the destruction is more random than with apoptosis, the DNA gel pattern of a cell dying by necrosis consists of a smear of DNA fragments without any definitive size order. Further, although some nick ends may be randomly generated during necrosis, they are usually relatively few so that TUNEL staining, if present at all, is quite weak. Thus, shrunken condensed nuclei and positive TUNEL staining distinguish apoptotic cells from cells undergoing necrosis, which are TUNEL negative (or weakly positive) and which have swollen nuclei (see Fig. 6.1). One link between autophagic cell death and apoptosis in that both of these types of programmed cell death can be inhibited by the anti–cell-death action of Bcl-2 (Miller and Girgenrath 2006). In addition, there is now evidence that calpainmediated cleavage of the autophagy-related gene-5 product (Atg-5) in a cell dying by the process of autophagy can switch the cell over to apoptosis (Yousefi et al. 2006).

1.2 Apoptotic Pathways

Apoptosis occurs naturally in many tissues to control cell numbers and in the processes that shape tissues and organs during their development. Extracellular signaling molecules can induce cell death on binding to their cognate receptors, thereby activating the extrinsic cell death pathway. Apoptosis may also be triggered when cells are damaged to remove such cells without disrupting tissue integrity. When cells are stressed or when DNA is damaged several biochemical pathways that constitute the “intrinsic” cell death program are activated. These cell death pathways are under tight regulation within each cell. Details of the gene expression patterns and the complex series of interactions that promote or act to inhibit the cell death pathways are described in several reviews (Danial and Korsmeyer 2004; Green and Kroemer 2004), and only a brief description of some features salient to the present discussion is presented here.

The “classical” apoptotic pathway is mediated by caspases (cysteine proteases with aspartate specificity), a family of proteases that have a role specifically in programmed cell death. Caspases are normally present as inactive procaspases within a cell. Triggers of cell death lead to activation of caspases

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Figure 6.1. Morphology of necrosis and apoptosis. (a, b) necrosis. (a) Toluidine blue– stained section of plastic embedded mouse organ of Corti. Hair cell nucleus is enlarged and less dense in comparison with normal nuclei in neighboring cells. Scale bar = 10 m. (b) Transmission electron microscopy (TEM) of thin section of mouse organ of Corti. Outer hair cell with swollen nucleus, disrupted organelles, and loss of cytoplasmic density suggesting ruptured plasma membrane. Scale bar = 5 m. (c–f) apoptosis. (c) Propidium iodide labeling of nuclei in whole-mount preparation of mouse organ of Corti affected by aminoglycoside (kanamycin). The morphologies of nuclei during progression of apoptosis are revealed: 1, margination and initial condensation of chromatin, with some shrinkage of nucleus; 2, more advanced margination and condensation of chromatin; 3, shrunken nucleus with condensed chromatin; 4, fragmented nuclei. Scale bar = 10 m. (d) TUNEL in whole-mount preparation of guinea pig utricular macula affected by aminoglycoside (gentamicin). The labeled, TUNEL-positive, nuclei have a variety of morphologies similar to those shown in (c), and most are shrunken and irregular in shape in comparison with the TUNEL-negative nuclei (arrows) in undamaged, normal cells. Scale bar = 10 m. (e) Nucleus in apoptotic hair cell in gentamicin affected utricular macula exhibiting condensation of chromatin. Scale bar = 5 m. (f) Apoptotic body of hair cell taken up by and enclosed within a supporting cell. Utricular macula damaged by gentamicin. Scale bar = 5 m.

through a series of regulated interactions that cleave off their pro-domains (see Fig. 6.2a). Activated “initiator” caspases, which include caspases-8, -9 and -10, in combination with other regulators, activate the “effector” caspases. The effector caspases, most notably caspases-3 and/or -7, lead to the activation of proteases that destroy cell proteins and to enzymes that fragment DNA. Different stimulators of apoptosis activate differing sets of caspases and/or different pathways depending on the cellular environment and cell type (Fig. 6.2a). For example, tumor necrosis factor, an extracellular death signaling molecule, activates initiator caspase-8 to directly activate effector caspase-3 (the “extrinsic” pathway). An excess of intracellular free radicals, meanwhile, activate caspase-3 through the activation of initiator caspase-9 (the “intrinsic” pathway)