Caspases, the enemy within, and their role in oxidative stress-induced apoptosis of inner ear sensory cells

Caspases, the enemy within, their role in oxidative stress-induced apoptosis of

inner ear sensory cells

1,2

_{TR Van De Water,}

_{F Lallemend,}

_{AA Eshraghi,}

_{S Ahsan,}

_{J He,}

_J

Guzman,

_{M Polak,}

_{B Malgrange,}

_{PP Lefebvre,}

_{H Staecker,}

_{TJ Balkany}

School of Medicine, Miami, FL; 2_{Center for Molecular and Cellular Neuroscience,}

3_{Department of Otolaryngology and Audiophonology, University of Liege, Liege, Belgium}

and 4_{Division of Otolaryngology, University of Maryland School of Medicine, Baltimore,}

MD.

Running title: Activated caspases execute damaged inner ear sensory cells

Corresponding author: Thomas R. Van De Water, Ph.D. Cochlear Implant Research Program University of Miami Ear Institute 1600 N.W. 10th _{Avenue, RMSB 3160}

Miami, FL 33136

tvandewater@med.miami.edu

Tel. 305-243-4641; Fax 305-243-5552

Abstract

This review covers the general roles of members of the cysteine protease family of caspases in the process of apoptosis (programmed cell death) looking at their participation in both

the“Extrinsic” and the “Intrinsic” cell death pathways. It defines the difference between initiator and effector caspases and shows the progression of caspase activations that ends up in the apoptotic cell death and elimination of a damaged cell. The review then presents what is currently know about the participation of caspases in the programmed cell death of inner ear cells during the process of normal development and maturation of the inner ear and there importance in this process as illustrated by gene knockout experiments. The participation of specific caspases and the sequence of their activation in the elimination (apoptosis) of damaged sensory cells from adult inner ears after an injury that generates oxidative stress are reviewed. Both the possibility and the potential efficacy of caspase inhibition as an interventional therapy to treat and rescue damaged inner ear sensory cells from apoptosis are presented and discussed.

Key words: oxidative stress, inner ear sensory cells, apoptosis, caspases, otoprotective therapy,

caspase inhibition

The role of caspases in cellular apoptosis (programmed cell death): Caspases are a family of

cysteine proteases that are present within the cells of normal healthy tissue in inactive

(procaspase) forms (1,2). The procaspase configuration of a caspase is quiescent and in order for a caspase to become active it requires cleavage of its prodomain. After the prodomain has been cleaved from the procaspase molecule, the caspase molecule is activated and can now act upon and cleave a specific tetrapeptide sequence (this sequence varies for each member of the caspase family) in targeted proteins, which can include both cytoskeletal, and nuclear proteins of an affected cell (3). There are currently fourteen known members of the caspase family of proteases (i.e. Caspases-1 to -14), which have been conserved throughout evolution (2). The first member of the family of caspases (i.e. caspase-1) was originally identified during genetic studies of neural development in the nematode, C. elegans (1). Not all members of the caspase family participate in programmed cell death (apoptosis) of cells, for example caspases-1 and -11

function mainly in the processing of cytokines, e.g. interleukin 1 beta (2). However, many of the members of the caspase family of proteases (e.g. caspases-2, -3 and -6 to -10) have been proven to participate in the regulation and execution of apoptosis of affected cells, which can be either the result of a normal developmental event (e.g. neuronal competition for trophic support from a target tissue) or a cell’s response to a high level of internal injury (e.g. membrane lipid

peroxidation) that results from exposure to oxidative stress (4-6). The members of the caspase family that participate in apoptosis can be separated into two general groups: 1) initiator caspases (e.g. caspases-8 and -9) (5); and 2) effector caspases (e.g. caspases-3, -6 and -7) (4). Initiator caspases can be activated by either an “Extrinsic” Cell Death Pathway (e.g. Fas ligand-Fas receptor/procaspase-8 interactions) or an “Intrinsic” Cell Death Pathway (e.g.

MAPK/mitochondria/cytochrome-c/procaspase-9 interactions) as presented in figure 1. Initiator caspases (e.g. caspase-9) then activate downstream effector caspases (e.g. caspase-3) which in turn act as executioners in the process of apoptosis of an affected cell (see figure 1) (5). The “Extrinsic” cell death pathway involves the binding and dimerization of cell death receptors from members of the tumor necrosis factor (TNF) family of receptors and ligands (e.g. Fas ligand/Fas) which brings procaspase-8 molecules into close apposition initiating dimerization of the

procaspase-8 molecules and thereby causing catalytic cleavage of the prodomains resulting in actived caspase-8 molecules (2). Activated caspase-8 can then participate in either the

“Extrinsic” (high caspase-8 concentration) cell death pathway with caspase-8 directly activating quiescent effector caspases such as procaspase-3 or the “Intrinsic” (low caspase-8 concentration) cell death pathway by cleaving BID which then acts on the mitochondrial cell death pathway by facilitating the release of cytochrome-c thereby indirectly participating in the activation of another quiescent initiator caspase, i.e. procaspase-9. Activation of the “Intrinsic” cell death pathway generally involves an insult that generates oxidative stress with the creation of reactive oxygen species (ROS) and other free radicals (e.g. OH ._{) that damage the cell’s organelles and}

internal membranes resulting in mitochondrial membrane damage and loss of membrane

potential. This results in a release of cytochrome c (Cyto C) from the damaged mitochondria into the cell’s cytoplasm where it combines with apoptotic protease-activating factor 1 (APAF-1), dATP, and procaspase-9 to form the Apoptosome (also known as the Aposome) to generate activated caspase-9 (2,5,7). Smac/Diablo is a small mitochondrial protein that is thought to be required for activation of a competence to die reaction within an affected cell by inhibiting some of the damaged cell’s naturally occurring caspase inhibitory molecules (e.g. NIAP inhibitiory protein in neurons) (8-10). Once procaspase-9 has been activated its downstream targets are the

effector caspases, e.g. caspases-3, -6, -7 (4). The cell’s naturally occuring apoptosis inhibitory proteins (IAPs) are thought to target activated effector caspases such as caspases-3 and -7 (3,11). However these IAPs are themselves natural targets of the effector caspases (3). The effector caspases interact with a large number of targets (i.e. >280) within an affected cell to bring about its destruction (3). Some of the cellular molecules targeted by effector caspases are: PARP-1 ( a DNA repair enzyme); DNA; nuclear lamins A, B and C; DNA fragmentation factor 45; inhibitor of caspase-activated DNase; receptor interacting protein (RIP); topoisomerase; signal transducer and activator of transcription-1; Rb; X-linked inhibitor of apoptosis (XIAP); U1 small

nucleoprotein; fodrin; vimentin; and procaspases-2 , -6 and -10.

Role of caspases in the inner ear:

Development of the membranous labyrinth: Programmed cell death cell death is a normal event

in the formation and differentiation of most organ systems within the embryo and it has been shown to be required for the normal development and maturation of the sensory epithelium of the inner ear (12). Caspase-3 has been suggested to be the primary executioner caspase in most cellular apoptosis during both normal developmental cell death and the removal of damaged cells after injury (1,2). The results of gene knock out (null mutation) experiments in mice clearly show that the presence of a functional caspase-3 gene is required for the development of normal hearing (13,14). In one analysis the cochlear ducts of caspase-3 (-/-) deficient mice were found to be immature in 2 week-old neonates with hyperplasia of supporting cells and progressive

degeneration of sensory hair cells that resulted in a severe loss of hearing acuity (13). Analysis of caspase-3 null mutant mouse cochleae by another group pointed out that in addition to the progressive loss of both inner and outer hair cells that there was also a progressive postnatal loss

of the auditory neurons from the spiral ganglion (14). This group also pointed out that the human caspase-3 gene and a locus for a nonsyndromic form of autosomal deafness, i.e. DFNA 24, both map to the q35 area on chromosome-4 suggesting a relationship and that caspase-3 null mutant mice may serve as a useful model for this type of autosomal deafness. A more recent study (14) examined the apposing roles of nerve growth factor and insulin like growth factor type 1 in the initiation of programmed cell death in the developing otocyst, the results of this in vivo study showed that both a pancaspase inhibitor (i.e. VAD-fmk) and a caspase-3/-7 inhibitor (i.e. z-DEVD-fmk) could completely block NGF/p75ngfr /ceramide-initiated cell death in both the otocyst epithelium and the cochleo-vestibular ganglion. This result demonstrated that caspases and more specifically caspase-3 and possibly caspase-7 participate in NGF evoked programmed cell death in the developing inner ear.

Caspase-3 appears to participate in the normal development and maturation of the membranous labyrinth and a loss of function mutation of the gene for this caspase could result in

maldevelopment of the inner ear and a hearing deficit.

Ototoxicity: The inital demonstration that caspases were involved in the apoptosis of

ototoxin-damaged auditory sensory cells came from a series of in vitro experiments where auditory sensory cells were exposed to ototoxic levels of the chemotherapy drug, cisplatin. TUNEL staining (an indicator of DNA damage and therefore apoptosis) indicated that both auditory hair cells and auditory neurons were undergoing apoptotic cell death as a result of cisplatin exposure and that treatment of both cisplatin exposed dissociated spiral ganglion cell cultures and organ of Corti explants with a pancaspase inhibitor, i.e. BOC-fmk (also known as BAF), prevented the apoptosis and loss of both of these types of auditory sensory cells (16). Caspases were also found to participate in the apoptotic cell death of gentamicin-damaged vestibular hair cells.

Explants of utricular maculae excised from both adult guinea pigs and adult gerbils were exposed to ototoxic levels of gentamicin and protected by addition of a broad-spectrum pancaspase inhibitor, i.e. either z-VAD-fmk or BAF, to the culture medium (17). The explants with the pancaspase inhibitor treatment showed a significant level of protection against gentamicin-damage initiated loss of vestibular hair cells and a significant reduction in the number of

apoptotic hair cell nuclei when gentamicin exposed, pancaspase treated cultures were compared to gentamicin exposed, untreated cultures. This finding of pancaspase protection of vestibular hair cells from aminoglycoside-damage initiated apoptosis was confirmed in another in vitro study that used post hatch white leghorn chickens for a source of utricular maculae explants and neomycin as the ototoxic drug (18). The pancaspase inhibitors added to the culture medium were the same used in the previous study, i.e. z-VAD-fmk and BAF. The efficacy of treatment with these pancaspase inhibitors was evaluated with hair cell counts using: 1) hair cell specific immunostaining; 2) counts of pycnotic nuclei; and 3) TUNEL staining. A unique observation made in this vitro study was that in addition to reducing the level of aminoglycoside-induced hair cell loss there was also a dose dependent reduction in the proliferation of supporting cells in the neomycin exposed, pancaspase inhibitor treated explants. This reduction in supporting cell proliferation was not due to a direct action of the pancaspase inhibitors on the support cells but rather appeared to be a consequence of the prevention of hair cell death and that it was the aminoglycoside-induced death of the hair cells that acted as a stimulus for the proliferation of the support cells within the neomycin exposed utricles. The first proof that specific members of the caspase family of cysteine proteases are involved in the apoptosis of both auditory and vestibular hair cells after an ototoxic insult come from two recent in vitro studies, i.e. avian basilar papilla explants and mouse utricular explants, respectively. The study of chick basilar papilla explants

exposed to an ototoxic level of gentamicin utilized fluorescent-labeled peptide substrates for caspases-3, -8 and -9 to detect their activation within the ototoxin exposed basilar papilla

explants in comparison to untreated, control explants (19). The results of this in vitro study show that gentamicin-damaged hair cells degenerate and undergo apoptosis in a caspase-dependent manner and that the initiator caspases-8 and -9 and a downstream effector caspase, i.e. caspase-3, are activated in aminoglycoside-damaged auditory hair cells (figure 2). This study used the general caspase inhibitor, z-VAD-fmk, to prevent caspase activation so no conclusions could be drawn about the action or efficacy of specific caspase inhibitors in this ototoxin-damage model system. However, the adult mouse utricular explant/neomycin study did use a combination of: 1)

in situ substrate detection for specific caspases; 2) immunolabeling of activated caspases; and 3)

caspase inhibitors with known specificity (20). Initially, a pancaspase inhibitor was used to confirm that caspases were involved in the apoptosis of these ototoxin-damaged vestibular hair cells. The results of this study confirm the activity of casapses-8, -9 and -3 in the apoptosis of ototoxin-damaged vestibular hair cells additionally this study showed that caspase-8 plays only a minor role in the process of apoptosis while both caspases-9 and -3 were found to have major roles in the apoptosis of these damaged hair cells (see figure 2). When specific caspase inhibitors were used to prevent hair cell death the caspase-8 inhibitor (z-IETD-fmk) had no significant effect on either preventing neomycin-induced hair cell death or downstream

activation of procaspase-3. In contrast, the caspase-9 specific inhibitor (z-LEHD-fmk) prevented both neomycin-induced hair cell death and downstream activation of procaspase-3 in these ototoxin-exposed utricular explants. Currently there are no reported studies to determine

whether downstream effector caspases-6 and -7 participate in the process of apoptotic cell death, which eliminates ototoxin-damaged sensory hair cells. All of the caspase inhibitor studies

discussed in this section have been performed in vitro so it not currently know whether these irreversible inhibitors of caspases (e.g. z-VAD-fmk; a pancaspase inhibitor) will be effective when delivered in vivo (e.g. perfused into the scala tympani).

Caspases-9 and –3 appear to be essential components in the apoptotic cell death of ototoxin-damaged inner ear hair cells whereas caspase-8 has been shown to play only a minor role in this cell death process (figure 2).

Bacterial endotoxin: Transtympanic injection of the bacterial toxin, lipopolysaccharide (LPS)

into the middle ear cavity of a healthy guinea pig caused elevation of hearing threshold measurements, fragmentation of DNA (as determined by immunostaining for single stranded DNA) and activation of caspase-3 suggesting that exposure to a bacteria generated toxin (i.e. LPS) can result in the apoptosis of cells within the lateral wall (e.g. spiral ligament) and organ of Corti (21). This is an interesting preliminary observation and needs both confirmation and a LPS dose response study.

The results of this study suggest caspase-3 may be involved in the apoptosis of bacterial toxin-damaged cochlear cells located in both lateral wall tissues and within the organ of Corti.

Acoustic trauma: A double label study of sound trauma-initiated apoptosis of cochlear outer hair

cells in the chinchilla localized activated caspase-3 to the cell bodies of the apoptotic hair cells (22). The results of this study clearly show a relationship between post-noise exposure

progression of hair cell loss, apoptosis, and activation of caspase-3 but did not test the efficacy of a caspase inhibitor therapeutic strategy. There is some mention of an attempt to use such a protective strategy in the discussion but no details are provided so no evaluation is possible.

There is a correlation between post-exposure loss of noise-damaged outer hair cells, apoptotic changes in the outer hair cell nuclei and the presence of activated caspase-3 in the cell bodies of these damaged cells.

Aging: In an immunolabeling study of apoptosis related proteins present within the cochleae of

aged (i.e. 24 months) versus young (i.e. 6 months) Mongolian gerbils it was found that changes in the levels of two of these proteins correlated with decreases in cochlear function as measured by distortion product otoacoustic emissions (DPOAE) (23). The two apoptotic proteins that correlated with a decrease in DPOAEs were: 1) an anti-apoptotic molecule, bcl-2, which was decreased in the tissues of the aged cochlea; and 2) activated casapse-3 which increased in the tissues of the aged cochlea when these tissues were compared to the tissues obtained from the young cochleae. The levels of immunostaining within cochlear tissues for procaspase-3 (i.e. the non-active form) and a pro-apoptotic member of the bcl-2 family, i.e. bax, did not show any aging-related increases or decreases.

This is an important finding because both bcl-2 and caspase-3 are involved in the control and execution of the “Intrinsic” cell death pathway (see figure 1) which is thought to be the primary mediator of oxidative stress-induced apoptosis. Activation of caspase-3 may be involved in age-related hearing loss in the Mongolian gerbil.

Summary and hypothetical applications: Several caspase molecules (e.g. caspases-8, -9 and -3)

have been shown to participate in both the “Extrinsic” and “Intrinsic” cell death pathways (figure 1). Inner ear hair cells exposed to aminoglycoside ototoxic-damage within in vitro experimental conditions have been shown to activate caspases-8, -9 and -3 while in these

experiments caspases-9 and –3 have been demonstrated to be major participants in the process of apoptosis to eliminate the damaged hair cells (1-3). Caspase-3 activity has also been implicated in the apoptotic cell death of inner ear sensory cells in response to: 1) bacterial toxin exposure; 2) sound trauma damage; and 3) aging related changes within cochlear tissues. It is therefore probable that these three caspases and perhaps other members of this family of cysteine proteases (e.g. caspase-7) are involved in the loss of inner ear sensory cell that have been exposed to insults that damage these sensitive and at present irreplaceable sensory cells (see figure 2). The use of specific caspase inhibitors may or may not be effective, for instance if a caspase 9 specific inhibitor is used it would be effective in blocking the “Intrinsic” pathway but ineffective in blocking the “Extrinsic” pathway if a high level of activated caspase-8 is present (see figure 1) and a similar but converse argument could be made for the use of a caspase-8 specific inhibitor (1-3). However there are highly effective pancaspase inhibitors (e.g. z-VAD-fmk) that can block all of the activated caspases that are involved in both of these cell death pathways (24).

Application of a pancaspase inhibitor should be local or if administered in a systemic application it should be of a very short duration because apoptosis is an important process by which the body eliminates cells that could be dangerous to the overall health of the individual. Caspases have already been suggested as treatment targets in both stroke and in neurodegenative diseases of the central nervous system (25). Because to inner ear tissues are accessible via the round window membrane and the round window membrane is already being used to deliver some drugs to the inner ear (26) there may be an application for a broad acting pancaspase inhibitor delivered via this local route of administration in the treatment of inner ear disorders and trauma that involves the generation of oxidative stress and apoptotic death of inner ear sensory cells (27) (e.g.

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Figure Legends

Figure 1. A flow chart of caspase activation depicting both the “Extrinsic” and “Intrinsic” cell death pathways that lead to the apoptosis of an injured cell.

Figure 2. An illustration of the caspases that are known to be active in the process of apoptosis of an aminoglycoside-injured inner ear hair cell.