Like aminoglycosides, cisplatin results in the formation of reactive oxygen species in hair cells, including superoxide. Some thiol antioxidants, including sodium thiosulfate, D-methionine, and lipoic acid, can inhibit cisplatin-induced ototoxicity. However, some of these thiols, including sodium thiosulfate, diminish cisplatin’s tumoricidal activity by the formation of inactive platinum–thiol conjugates.

3.4. Therapeutic strategies to prevent hair cell death

Several cotherapies have been shown to inhibit ototoxic hair cell death in animal model systems. As mentioned previously, a variety of antioxidants can inhibit both aminoglycosideand cisplatin-induced hair cell apoptosis. Similarly, ototoxicity is inhibited by either inhibition of caspase activity or upregulation of antiapoptotic Bcl-2 family members. Interest is emerging in intrinsic protective mechanisms in the inner ear that, if activated, may be able to inhibit hair cell death. One such intrinsic protective mechanism is the activation of heat shock proteins (HSPs). HSP induction is one of the most ubiquitous and highly conserved stress responses in biology. Stress-induced HSP expression promotes cellular survival in a large number of systems, and HSPs can directly inhibit apoptotic signaling. One well-characterized HSP inducer is heat stress, which induces most HSPs and protects cells against a number of stresses. For example, short-term total-body hyperthermia has been shown to protect the retina against light-induced damage and to prevent ischemia-induced death in both cardiomyocytes and hippocampal neurons. Induction of HSPs via either total-body hyperthermia or local hyperthermia inhibits noise-induced hearing loss. Induction of HSPs via heat shock inhibits both cisplatinand aminoglycoside-induced hair cell death in vitro. Hsp70 is both necessary and sufficient to account for this protective effect of heat shock against aminoglycoside-induced hair cell death. Geranylgeranyl acetone, a chemical HSP inducer, inhibits aminoglycoside-induced hair cell death in vitro. Constitutive over-expression of Hsp70 in transgenic mice inhibits aminoglycoside-induced cochlear hair cell death and hearing loss. It is likely that clinical strategies aimed at inhibiting ototoxic hearing loss will involve both inhibition of apoptotic signaling and upregulation of intrinsic protective mechanisms, possibly including HSP induction.

3.5. Challenges to studies of hair cell death

inhibited in animal studies by several cotherapies, including antioxidants and caspase inhibition. However, there is currently no commonly used cotherapy for the prevention of either cisplatinor aminoglycosideinduced hair cell toxicity. Studies of apoptosis in the inner ear are complicated by a lack of suitable model systems in which to examine apoptotic signaling. Although several cell lines have been developed from inner ear tissue, no cell line has yet been identified that develops morphological features of hair cells (i.e., stereocilia) and none that is sensitive to both aminoglycosideand cisplatin-induced death. Furthermore, adult mammalian cochlear hair cells do not survive in culture for more than a few hours. Therefore, research into sensory hair cell apoptosis is usually carried out in whole organ cultures, either of the organ of Corti from neonatal rodents or in the macular organs (utricle and/or saccule) from mature rodents. Both of these systems have limitations. First, both yield a mixture of cell types that includes hair cells, supporting cells, stromal cells, and neuronal processes. This nonhomogeneity of the culture makes it difficult to be certain that changes observed by quantitative methods such as real-time reverse-transcriptase polymerase chain reaction and Western blotting occur in the hair cells themselves. Second, results obtained in cultures from neonatal animals may not reflect the degenerative changes that occur in mature hair cells, and differences also may exist between cochlear and vestibular hair cells in their responses to stress. Third, both systems yield very small numbers of hair cells: the mouse organ of Corti and the adult mouse utricle each contain only approximately 3,000 hair cells. This is an extremely small amount of tissue, and this limitation restricts the feasibility of many biochemical and molecular biology techniques that require large numbers of cells. Despite these limitations, significant progress has been made in recent years toward understanding the mechanisms underlying sensory hair cell death and survival.

4. SPIRAL GANGLION NEURON DEATH

When hair cells are destroyed by noise trauma or ototoxic drugs such as aminoglycoside antibiotics and cisplatin, the SGNs show signs of apoptosis within days and continue to degenerate over a lengthy period of time (Figure 17–3). In certain cases, genetic mutations that cause atrophy of the organ of Corti also lead to a corresponding loss of SGNs. Emerging evidence supports the concept that cells lying close to inner and outer hair cells complement the function of the sensory hair cells in promoting SGN survival.

4.1. Neurotrophic support from sensory hair cells and supporting cells

Neurotrophins are a major family of molecules that are required for SGN development and survival. The neurotrophins – nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4/5 – constitute a family of secreted molecules that provide target-derived guidance cues for neurons and are essential for neuronal survival and function. During development, inner and outer hair cells as well as supporting cells of the organ of Corti express BDNF, NT3, and another type of neurotrophic factor called glial cell–derived neurotrophic factor (GDNF) to varying degrees and in a spatio-temporal pattern. The importance of BDNF and NT3 in inner ear development and SGN survival is demonstrated by knockout mouse models in which deletion of these neurotrophic genes in mice results in both loss of SGNs and retraction or retardation of their peripheral processes to the organ of Corti. Thus BDNF and NT3 can function as target-derived factors to regulate the survival of SGNs and guide their innervation to hair cells in the organ of Corti during development. Recently, it has been suggested that neurotrophin expression in sensory hair cells and supporting cells is increased by another trophic signaling mechanism involving neuregulins produced by SGNs and their receptors, erbB2, present in hair cells. This reciprocal signaling between SGNs and hair cells is thought to increase neurotrophin expression in either supporting cells or hair cells. Consistent with this reasoning, it has been demonstrated that transgenic mice with disrupted erbB signaling demonstrate dramatic loss of SGNs and reduced NT3 expression.

Because neurotrophins need to bind to their cognate receptors to mediate survival, it is reasonable to speculate that mice deficient in these receptors would show corresponding SGN loss. Among these receptors, the tropomyosin-related kinase (Trk) receptor tyrosine kinase B binds selectively to BDNF, whereas TrkC preferentially interacts with NT3. These receptors are expressed in SGNs, and mice deficient in TrkB or TrkC show significant SGN loss and innervation defects at the organ of Corti. Furthermore, TrkB and TrkC double knockout mice display an even more severe SGN loss than either of the single knockout mouse models, underscoring the requirement of both neurotrophins for SGN survival. The importance of these neurotrophins in the inner ear does not appear to be restricted to this developmental window, because sensory hair cells and supporting cells of the adult organ of Corti continue to express BDNF and NT3. In addition, TrkB and TrkC expression

Deafened

Organ of Corti

SGNs

Osseous Spiral

Basilar Lamina

Membrane

Figure 17-3. Schematic of the normal and degenerating cochlea. In a cross-section of the mammalian cochlea, inner (labeled with a white dot) and outer (highlighted with asterisks) hair cells in the organ of Corti are innervated by peripheral processes of spiral ganglion neurons (SGNs). Deafness induced by ototoxic drugs or noise results in death of these hair cells in the organ of Corti, leading to secondary degeneration and loss of SGNs (see inset). Adapted with permission from Hurley et al., 2007.

persist in adult SGNs, suggesting an ongoing physiologic role for neurotrophin signaling in adulthood (Figure 17-3).

4.2. Afferent activity from hair cells

In addition to trophic support, SGNs require afferent activity from sensory hair cells for survival. In the organ of Corti, inner hair cells stimulate SGNs by secreting glutamate, which activates two classes of receptors: the N-methyl-D-aspartate and α-amino-3-hy- droxy-5-methylisoxazole-4-propionate receptors. These ligand-gated receptors are ion channels that open on activation, causing an influx of cations into the SGN. This gradually depolarizes the neuron, activating another class of voltage-gated ion channels – the L- type Ca2+ channels. Blockade of L-type Ca2+ channels with inhibitors (nifedipine and verapamil) abolishes the trophic effect of depolarization in SGN cultures, demonstrating that Ca2+ entry is necessary for SGN survival. Furthermore, both glutamate receptors and L-type Ca2+ channels are present in SGNs, suggesting that these ion channels mediate afferent activity initiated by hair cells.

A regulated influx of Ca2+ in neurons is essential to trigger intracellular survival signaling cascades linked to

SGN survival. Considering the diversity and cross-talk among signaling cascades, one approach to understanding SGN physiology has been to identify the downstream targets that promote survival and then examine the corresponding upstream signaling cascades. Degenerating SGNs show a decline in phosphorylation of the nuclear transcription factor, cyclic adenosine monophosphate response element binding protein (CREB). CREB is a downstream target that is indirectly activated by Ca2+ influx. CREB regulates the expression of many genes necessary for neuronal function and survival. Phosphorylation of CREB can be induced by at least two pathways: the Ras-MAPK pathway and the Ca2+/calmodulindependent kinases, both of which are activated by Ca2+ influx. MAPK and Ca2+/calmodulin-dependent kinases phosphorylate CREB at specific amino acid residues, enabling CREB to bind to distinct nucleotide sequences and promote transcription of selected genes such as BDNF. In primary cultures of postnatal SGNs, inhibitors of either MAPK or Ca2+/calmodulin-dependent kinases reduce SGN survival, suggesting that these pathways are necessary for SGN survival. Because BDNF mRNA transcripts in SGNs are expressed in a manner that is dependent on neuronal activity, it remains likely that BDNF produced by SGNs can promote survival via an autocrine signaling loop involving their TrkB receptors. Another mechanism by which activityinduced depolarization can promote SGN survival is via phosphorylation (inactivation) of proapoptotic Bad by Ca2+ /calmodulin-dependent kinase II.

4.3. Molecular manifestations of spiral ganglion neuron death

The death of SGNs is not remarkably different from that of most other neurons, but we will highlight certain features that are manifested in degenerating SGNs. When aminoglycoside antibiotics are administered to rats to induce hair cell death, a dramatic reduction in TrkB expression in SGNs and their peripheral processes is observed. Concomitantly, increased expression of the p75 neurotrophic receptor (p75NTR) occurs in both SGNs and Schwann cells. Although classified as a neurotrophic receptor like TrkB and TrkC, p75NTR has diverse roles in the nervous system depending on the pathological status of the cells. In healthy cells, the p75NTR receptor can increase the affinity of binding between NGF and its cognate TrkA receptor, or BDNF and TrkB. However, under conditions of trauma and inflammation, p75NTR can trigger apoptosis.

The mechanisms underlying the proapoptotic activity of p75NTR are largely unknown. However, this

signaling may be related to whether the ligand is in a mature or immature form. In their mature forms, neurotrophins BDNF and NT3 support SGN survival. Like many hormones and enzymes, neurotrophins are produced initially as pro-neurotrophin forms consisting of a pro-domain linked to the mature neurotrophin. Proteolytic cleavage releases the mature neurotrophins, enabling them to mediate most of their biological functions, including survival. However, proneurotrophins can bind to p75NTR at subnanomolar concentrations to induce cell death, illustrating the dependence of signaling by neurotrophins on their state (whether proor mature neurotrophin). In rat cochleae exposed to aminoglycoside antibiotics, the accumulation of uncleaved pro-BDNF and the augmented expression of p75NTR in the cochleae suggest that this mechanism may contribute to SGN degeneration. Although it remains to be determined whether this mechanism contributes to SGN death in humans, new lines of evidence support this hypothesis. For example, pro-neurotrophin forms of NGF accumulate in brains of humans with Alzheimer’s disease. Furthermore, pro-NGF isolated from these diseased brains rapidly triggers death in cultured neurons. It is unclear how p75NTR signals cell death, but an increase in JNK activity has been associated with p75NTRdependent apoptosis. In addition, degenerating SGNs show increased phosphorylation of c-Jun, indicating activation of the JNK signaling pathway. Thus, similar to what is known about death of sensory hair cells, activation of the JNK signaling pathway may contribute to SGN apoptosis.

Hair cells do not always perish immediately after ototoxic drug exposure; they sometimes undergo a progressive atrophy with time. In particular, aminoglycoside antibiotics, which have a half-life in serum of approximately 3 to 5 hours, are not efficiently cleared from the inner ear, resulting in an extended half-life in inner ear tissues and fluids that may exceed 30 days. This protracted retention in the cochlea leads to hair cell death, followed by gradual death of SGNs. Apoptotic features can be observed within somas of degenerating SGNs weeks and months after the initial traumatic insult, in part because of the gradual nature of hair cell death. Some of the apoptotic features in degenerating SGNs include increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, cytochrome c release, activation of caspase-9, and increases in the caspase-cleaved fragment of poly (ADP-ribose) polymerase. These features suggest the involvement of members of the mitochondrial cell death pathway in regulating apoptosis in SGNs, and over-expression of Bcl-2 in