Caspases, the enemy within, their role in oxidative stress-induced apoptosis of
inner ear sensory cells
1,2
TR Van De Water, 2F Lallemend, 1AA Eshraghi, 1S Ahsan, 1J He, 1J
Guzman, 1M Polak, 2,3B Malgrange, 2,3PP Lefebvre, 4H Staecker, 1TJ Balkany
1
Department of Otolaryngology, University of Miami Ear Institute, University of Miami
School of Medicine, Miami, FL; 2Center for Molecular and Cellular Neuroscience,
3
Department of Otolaryngology and Audiophonology, University of Liege, Liege, Belgium
and 4Division 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
Supported by a grant from the MedEl Corporation to TRV and TJB.
1
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
2
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.
3
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
4
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
5
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. z-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.
6
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
7
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
8
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.
9
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
10
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.
placement or removal of a cochlear implant electrode arrays within the scala tympani).
11
References
1. Vaux DL, Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci USA
1996;93:2239-44.
2. Budihardjo I, Oliver H, Lutter M, et al. Biochemical pathways of caspase activation
during apoptosis. Ann Rev Cell Devel Biol. 1999;15:269-90.
3. Fisher U, Jaenicke RU, Schulze-Osthoff K. Many cuts to ruin: a comprehensive
update of caspase substrates. Cell Death Differ. 2003;10:76-100.
4. Slee EA, Adrain C, Martin SJ, et al. Executioner caspase-3, -6, and -7 perform
distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem.
2001;276:7320-26.
5. Cao G, Luo Y, Nagayama T, et al. Cloning and characterization of rat caspase-9:
implications for a role in mediating caspase-3 activation and hippocampal cell death
after transient cerebral ischemia. J Cereb Blood Flow Metab. 2002;22:534-46.
6. Renatus M, Stennicke HR, Scott FL, et al. Dimer formation drives the activation of the
cell death protease caspase 9. Proc Natl Acad Sci U S A. 2001;98:14250-55.
7. Lesuisse C, Martin LJ. Immature and mature cortical neurons engage different
apoptotic mechanisms involving caspase-3 and the mitogen-activated protein kinase
pathway. J Cereb Blood Flow Metab. 2002;22:935-50.
8. Cain K, Brown DG, Langlais C, et al. Caspase activation involves the formation of the
aposome, a large (approximately 700 kDa) caspase-activating complex. J Biol Chem.
1999;274:22686-92.
9. Deshmukh M, Du C, Wang X, et al. Exogenous smac induces competence and
permits caspase activation in sympathetic neurons. J Neurosci. 2002;22:8018-27.
10. Maier JK, Lahoua Z, Gendron NH, et al. The neuronal apoptosis inhibitory protein is
a direct inhibitor of caspases 3 and 7. J Neurosci. 2002;22:2035-43.
12
11. Keane RW, Kraydieh S, Lotocki G, et al. Apoptotic and antiapoptotic mechanisms
after traumatic brain injury. J Cerebral Blood Flow & Metab.2001;21:1189-98.
12.Fekete DM, Hamburger SA, Waring MT, et al. Involvment of programmed cell death
in morphogenesis of the vertebrate inner ear. Development. 1997;124:2451-61.
13. Takahashi K, Kamiya K, Urase K, et al. Caspase-3-deficiency induces hyperplasia
of supporting cells and degeneration of sensory cells resulting in the hearing loss. Brain
Res 2001;894:359-67.
14. Morishita H, Makishima T. Kaneko C, et al. Deafness due to degeneration of
cochlear neurons in caspase-3-deficient mice. Biochem Biophys Res Comm.
2001;284:142-49.
15. Frago LM, Canon S, de la Rosa EJ, et al. Programmed cell death in the developing
inner ear is balanced by nerve growth factor and insulin-like growth factor I. J Cell Sci.
2003;116:475-86.
16. Liu W, Staecker H, Stupak H, et al. Caspase inhibitors prevent cisplatin-induced
apoptosis of auditory sensory cells. Neuroreport. 1998;9:2609-14.
17. Forge A, Li L. Apoptotic death of hair cells in mammalian vestibular sensory
epithelia.
Hear Res. 2000;139:97-115.
18. Matsui JI, Ogilvie JM, Warchol ME, et al. Inhibition of caspases prevents ototoxic
and ongoing hair cell death.
J Neurosci. 2002;22:1218-27.
19. Cheng AG, Cunningham LL, Rubel EW, et al. Hair cell death in the avian basilar
papilla: characterization of the in vitro model and caspase activation. J Assoc Res
Otolaryngol. 2003;4:91-105.
20. Cunningham LL, Cheng AG, Rubel EW, et al. Caspase activation in hair cells of the
mouse utricle exposed to neomycin. J Neurosci. 2002;22:8532-40.
21. Watanabe K, Jinnouchi K, Hess A, et al. Detection of apoptotic change in the
lipopolysaccharide (LPS)-treated cochlea of guinea pigs. Hear Res. 2001;158:116-22.
22. Hu BH, Henderson D, Nicotera TM, et al. Involvement of apoptosis in progression of
cochlear lesion following exposure to intense noise. Hear Res. 2002;166:62-71.
23. Alam SA, Oshima T, Suzuki M, et al. The expression of apoptosis-related proteins in
the aged cochlea of Mongolian gerbils. Laryngoscope. 2001;111:528-34.
13
24. Iwata A, Harlan JM, Vedder NB, et al. The caspase inhibitor z-VAD is more effective
than CD18 adhesion blockade in reducing muscle ischemia-reperfusion injury:
implication for clinical trials. Blood. 2002;100:2077-80.
25. Schulz JB, Weller M, Moskowitz MA, et al. Caspases as treatment targets in stroke
and neurodegenerative diseases. Ann Neurol 1999;45:421-29.
26. Seidman MD, Van De Water TR. Pharmacologic manipulation of the labyrinth with
novel and traditional agents delivered to the inner ear. Ear Nose Throat J. 2003;82:276-
300.
27. Lefebvre PP, Malgrange B, Lallemend F, et al. Mechanisms of cell death in the
injured auditory system: Otoprotective strategies. Audiol Neurootol. 2002;7:165-70.
14
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.
15
16