Why do hair cells and spiral ganglion neurons in the cochlea die during aging?

Abstract

Age-related decline of cochlear function is mainly due to the loss of hair cells and spiral ganglion neurons (SGNs). Recent findings clearly indicate that survival of these two cell types during aging depends on genetic and environmental interactions, and this relationship is seen at the systemic, tissue, cellular, and molecular levels. At cellular and molecular levels, age-related loss of hair cells and SGNs can occur independently, suggesting distinct mechanisms for the death of each during aging. This mechanistic independence is also observed in the loss of medial olivocochlear efferent innervation and outer hair cells during aging, pointing to a universal independent cellular mechanism for age-related neuronal death in the peripheral auditory system. While several molecular signaling pathways are implicated in the age-related loss of hair cells and SGNs, studies with the ability to locally modify gene expression in these cell types are needed to address whether these signaling pathways have direct effects on hair cells and SGNs during aging. Finally, the issue of whether age-related loss of these cells occurs via typical apoptotic pathways requires further examination. As new studies in the field of aging reshape the framework for exploring these underpinnings, understanding of the loss of hair cells and SGNs associated with age and the interventions that can treat and prevent these changes will result in dramatic benefits for an aging population.

Presbycusis is the predominant neurodegenerative disease of aging [1]. Clinically, presbycusis refers to permanent hearing loss coincident with the aging process, yet this loss is typically of multifactorial etiology [2]. This condition can dramatically affect an individual’s sense of isolation – often leading to depression – and has even been suggested to accelerate dementia [2, 3]. Current treatment options primarily involve the use of hearing aids, which only partially restore auditory function. Given the prevalence and debilitating effects of presbycusis, lack of effective prevention and treatment strategies will become an even more significant public health issue as the population ages and life expectancy increases.

Presbycusis in part reflects age-related neuronal changes in both the peripheral and central nervous systems (PNS and CNS) [4–10]. It is often characterized by a progressive bilateral increase in hearing thresholds, typically beginning with high frequencies, histologically corresponding to the loss of hair cells and SGNs at the base of the cochlea. Hearing tests and postmortem histological examinations [11, 12] led to the classification of presbycusis into the following four types: sensory (hair cell loss), neural (SGN loss), metabolic (strial dysfunction), and cochlear conductive (changes in the stiffness of the basilar membrane). These classifications imply that age-related loss of hair cells, SGNs, and stria vascularis can occur independently (Figure 1). In practice, most cases show mixed pathology that affects many cell types in the same cochlea, reflecting the importance of both genetic and environmental factors in causing presbycusis [13–17]. Because exposure to some level of noise is a reality of all human environments, the contribution of this environmental noise to presbycusis has been studied extensively. The prevalence of presbycusis increased 150% between 1965 and 1994 among people in their fifties [1]. The baby boomers who currently comprise this demographic were raised in a dramatically noisier environment than previous generations, reflecting increased industrial noise and widespread use of personal audio-devices. One longitudinal study found that presbycusis was exacerbated in people who were presumed to have cochlear damage from early noise exposure [18]. This finding is consistent with analyses of the annual decline of pure tone hearing thresholds in aging men with reported histories of occupational noise exposure versus cohorts without occupational noise exposure [19]. While presbycusis due to early noise exposure is difficult to study in humans because of confounding factors like socioeconomic status [20], Kujawa and Liberman [21, 22] have clearly demonstrated in mice that early noise exposure can cause a reversible temporary hearing threshold increase with irreversible SGN loss. Therefore, most cases of permanent hearing loss during aging could be the result of a close interaction between aging and early noise exposure. Although the exact cellular and molecular mechanisms underlying age-related loss of hair cells and SGNs are not completely understood, the past few decades have produced a number of studies that identify several key mechanisms likely contributing to the death of these two cell types during aging.

Figure 1.

Age-related loss of SGNs and hair cells. (A) Schematic drawing of spiral ganglion and organ of Corti, which contains one IHC and three OHCs (blue). (B) Histological sections of C57BL/6J mouse cochleae showing spiral ganglion (left) and organ of Corti (right) at 2 (top) and 20 (bottom) months old. The organ of Corti is absent at 20 months old in this case.

Cellular mechanisms

Which die first, hair cells or SGNs?

Studies of aging human and animal cochleae have typically shown mixed pathology in the organ of Corti, SGNs, and lateral wall [11, 12, 23–26]. This co-degeneration of distinct cochlear cells and structures obfuscates the important question of whether, and under what conditions, the pathology of hair cells and neurons are causally linked [9]. Loss of hair cells may often be the main cause of age-related SGN loss, but distinguishing between neuronal loss as a primary versus secondary degeneration is imperative [27, 28]. After chemical or mechanical damage to hair cells, SGNs begin to die, albeit at a highly species-dependent rate. The fact the SGNs reliably disappear is consistent with the notion that hair cells provide SGNs with trophic support [29–31]. Loss of SGNs without associated loss of hair cells, however, is common among mammals during aging [32–37]. Moreover, apparent primary and secondary degeneration of SGNs may coincide in the same cochlea [38]. Thus, age-related losses of SGNs and hair cells could occur in parallel by independent mechanisms. Verifying this hypothesis requires definitively ruling out hair cell causes of SGN degeneration, a task that has proven difficult. For example, C57BL/6 mice, which are commonly used models, carry a mutation (Cdh23^Ahl) that promotes progressive hair cell loss [6]. Ultrastructural signs of synaptic pathology – a likely precursor to neuronal loss – can be found in these mice prior to overt hair cell loss [39]. Yet, this change may just reflect an early and subtle aspect of Cdh23^Ahl–related hair cell degeneration. Our group has addressed this question by examining mice genetically engineered to over- or under-express neuregulin-1 (NRG1), a direct modulator of synaptic transmission [40, 41]. Transgenic mice over-expressing NRG1 in SGNs show improvements in hearing thresholds, whereas NRG1−/+ mice show a complementary worsening of thresholds. However, no significant change in age-related loss of SGNs in either NRG1 −/+ mice or mice over-expressing NRG1 is observed, while a negative association between NRG1 expression level and survival of inner hair cells (IHCs) during aging is observed. We have also found that modulating NRG1 levels changes synaptic transmission between SGNs and hair cells. These data demonstrate for the first time that synaptic modulation between hair cells and SGNs is unable to prevent age-related SGN loss and that IHC loss does not necessarily lead to the loss of SGNs during aging [41]. Interestingly, the loss of SGNs has also been observed in the cochlea of CBA/CaJ mice after mild noise exposure without a significant loss of hair cells [21, 22]. Thus, although age-related loss of SGNs is often closely associated with the loss of hair cells, cellular interactions between these two types of cells plays no major role in their death during aging.

Is this independent mechanism unique to hair cells and SGNs?

Previous studies have also identified an age-related functional decline in the medial olivocochlear (MOC) efferent system prior to age-related loss of outer hair cells (OHCs) [42–44]. We have recently evaluated whether this functional decline of the MOC efferent system is due to age-related synaptic loss of the efferent innervation of the OHCs [45]. To this end, we used a newly-identified transgenic mouse line in which the expression of yellow fluorescent protein (YFP), under the control of neuron-specific elements from the thy1 gene, permits the visualization of the synaptic connections between MOC efferent fibers and OHCs. In this model, there is a dramatic synaptic loss between the MOC efferent fibers and the OHCs in older mice (Figure 2). However, age-related loss of efferent synapses is independent of OHC status. These data demonstrate that age-related loss of efferent synapses contributes to the functional decline of the MOC efferent system, but an independent mechanism must exist for the OHC loss and the synaptic loss of the medial olivocochlear fibers [45]. Thus, this independent mechanism may be a common cellular pathway for age-related neuronal changes in the PNS.

Figure 2.

Age-related loss of MOC terminals in the cochlea. (A) Schematic drawing of the organ of Corti, which shows the MOC innervations (red). (B) Histological cochlear sections of Thy-1-YFP transgenic mice at 2 (top) and 12 (bottom) months old. The OHCs are located by the nuclei staining (left, blue), and the MOC terminals by YFP signal (right, red).

Molecular mechanisms

Involvement of the reactive oxygen species (ROS) pathway

Recent studies suggest the involvement of the ROS pathway in the development of presbycusis [16, 46–48]. Although the vast majority of life on earth depends on oxygen for its survival, failure to effectively regulate ROS leads to damaged cellular components. Both hair cells and SGNs are protected against ROS by an interacting network of enzyme systems and antioxidants [16, 46–48]. Because ROS are used in signaling, the normal function of this network is not to remove oxidants completely, but instead to maintain them at appropriate levels [49]. ROS inside cells are first converted to hydrogen peroxide by superoxide dismutases (SOD) and then further reduced to water by catalase and various peroxidases. Mice lacking glutathione peroxidase display accelerated presbycusis and increased susceptibility to noise-induced hearing loss (NIHL) [50]. Similar results are also seen in mice with genetic deletion of Cu/Zn superoxide dismutase 1 (SOD1) [4, 7]. Notably, the progression of presbycusis does not differ between wild type mice and those heterozygous for SOD1, or for those over-expressing SOD [7, 51, 52], supporting the importance of maintaining antioxidant/oxidative balance during aging [53]. Studies examining exogenous antioxidants and presbycusis have produced mixed results [16]. Seidman [46], and recently Someya et al. [47] showed that oral supplementation of antioxidants could significantly delay presbycusis in rodents, yet other studies found no anti-presbycusis activity from the same or similar antioxidants [54–56]. Dosing issues could be a major underlying cause of these disparate outcomes given the likelihood that, for any exogenous antioxidant agents, there will exist both minimum and maximum beneficial doses. Thus, future pharmocodynamic studies are needed to address these issues.

Because age-related changes in the mitochondrial electron transport chain can increase free radical generation, many studies have focused on the role of mitochondria in presbycusis [57]. Generation of ROS by mitochondria can promote injury throughout the entire cell but may critically promote further injury to the mitochondria themselves as part of an accelerating destructive process. An increase in mutations in mitochondrial DNA is found in samples from people with presbycusis [58]. In knock-in mice with base substitutions that impair the proofreading ability of mitochondrial DNA polymerase, age-related loss of hair cells and SGNs is significantly more severe than in control mice [59–60]. Interestingly, caloric restriction (CR) retards the deterioration of mitochondrial respiratory functions during aging [61] and has been shown to delay presbycusis in mice [62]. In addition to ROS generation, mitochondria also play a key role in apoptosis and cell calcium signaling, so mitochondrial injury may promote presbycusis via pathways besides that of ROS.

A role for calcium signaling

Disturbance in calcium homeostasis has long been suspected to be a contributing factor in age-related neuronal loss and trauma-induced neuronal injury [63–65]. Calcium levels in hair cells and SGNs can be regulated by several types of channels, including those with receptors for the neurotransmitters alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) [66, 67], N-methyl-D-aspartic acid (NMDA) [68], and acetylcholine [69, 70], as well as L-type and T-type voltage-gated calcium channels [71–73]. One study showed a delay of hearing loss in elderly women taking calcium channel blockers [74]. We reported that blockers for T-type calcium channels, such as trimethadione and ethosuximide, can effectively prevent and treat NIHL in vivo [72], and protect neurons in vitro [75]. Therefore, there is compelling evidence to suggest that calcium signaling could contribute to age-related loss of hair cells and SGNs.

Contributions from other mechanisms

Among the many other mechanisms that could contribute to presbycusis, there is data to suggest roles for glucocorticoid signaling pathways and protein quality control systems. An accelerated loss of SGNs is observed in mice lacking NFκB, a key molecule in both calcium and glucocorticoid signaling pathways [76]. In addition, our group has found a significant SGN loss after early noise exposure in mice lacking the β2 subunit of nicotinic acetylcholine receptor. This knock-out mouse line has age-related increases in corticosterone (see Figure 6 in [77]). Thus, it is possible that the accelerated SGN loss during aging is linked to the activation of glucocorticoid signaling pathways [78, 79]. That being said, significant protection of IHCs after noise exposure is observed in the same knock-out animals. The question of why a chronically elevated corticosterone level is associated with both an accelerated SGN loss during aging and a strong protection of IHCs after acoustic trauma certainly merits further investigation. In addition, a selective loss of support cells, hair cells, and SGNs is found in mice lacking Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoprotein [80]. This suggests that the components for monitoring protein structural integrity play an essential role in the survival of SGNs during aging. The current research demonstrates that multiple molecular pathways are involved in the age-related loss of hair cells and SGNs, and future studies will likely uncover contributions from additional sources.

Death pathways for hair cells and SGNs

In general, cells may die by either passive or active processes [81]. Clearly distinguishing between forms of cell death in vivo during aging proves difficult in certain cases [82–84]. Necrosis is a passive process characterized histologically by swelling and rupture of the cell body and release of intracellular contents. Apoptosis is an active form of cell death characterized by a shrunken cell body and masses of condensed DNA. Recently, a third type of cell death has been proposed for OHCs [82]. These neurons may display most of the hallmarks of apoptosis but fail to show key properties, such as DNA laddering or condensation during death [85, 86]. Nevertheless, the current view holds that most of the cell death during aging occurs via apoptosis, whether in the brain [87, 88] or cochlea [89, 90].

Active cell death requires synthesis of new proteins and a programmed biochemical cascade. Elegant studies in Caenorhabditis elegans have elucidated this cascade and identified several key cell-death (CED) genes [91–94]. Bcl-2 is the mammalian counterpart of one such cell-death gene, CED-9, and has demonstrated relevance to cell death and acoustic processing. In the auditory system, over-expression of Bcl-2 in transgenic mice prevents apoptosis in afferent deprivation-induced neuronal death of the anteroventral cochlear nucleus and aminoglycoside-induced hair cell death [95, 96]. In contrast, deletion of Bax, a proapoptotic member of the Bcl-2 family [97–99], reduces the incidence of naturally occurring neuronal apoptosis during development [99]. Since it is still uncertain whether age-related neuronal loss occurs in the central nervous system, interpreting the meaning of changing expression levels of various apoptotic genes in the brain during aging poses a challenge [88,100]. In the cochlea, however, age-related loss of hair cells and SGNs is consistently observed across species [37, 101]. Furthermore, several studies implicate apoptosis in this age-related loss of hair cells and SGNs.

Evaluation of a role for apoptosis in hair cell and SGN loss with age has occurred at several levels. Using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, studies have found the presence of DNA fragmentation in hair cells and SGNs during aging [62, 102,103]. Further evidence to support age-related loss of hair cells and SGNs through apoptosis has come from an association between aging and the expression of apoptosis-related proteins in the cochlea [89, 90]. An indirect piece of evidence bolstering this hypothesis is the significant reduction in the number of TUNEL-positive cells and cleaved caspase-3-positive cells in the cochleae from mice under CR [62], and a significant increase in the number of TUNEL-positive cells and activated caspase-3-positive cells in mice with the mutated mitochondrial DNA polymerase [59, 60]. However, TUNEL is not absolutely specific for apoptotic cells, as nuclear fragments from necrotic or autolytic cells may also be TUNEL-positive [104–105]. Furthermore, loss of hair cells and SGNs is found in one-month-old mice lacking caspase-3, a key downstream caspase in the apoptotic cascade. This suggests that activated caspase-3 may not be essential for the death of hair cells and SGNs [106]. Using both Bax knockout (Bax−/−) and Bcl-2 over-expressing mice, we failed to find any significant difference in age-related loss of hair cells and SGNs between controls and transgenic mice [78]. These findings suggest that age-related hearing loss does not occur through an apoptotic pathway involving key members of the Bcl-2 family. However, a new study has suggested the involvement of Bak in age-related hearing loss [47]. Thus, apoptosis involved with Bak may contribute to the loss of hair cells and SGNs with age.

Conclusions

Although age-related hearing loss is consistently observed in humans and animals, its underlying mechanisms remain only partially understood. At cellular and molecular levels, age-related loss of hair cells and SGNs can occur independently, suggesting distinct mechanisms for the death of each during aging. This mechanistic independence is also observed in the loss of MOC efferent innervation and OHC loss during aging, suggesting a universal independent cellular mechanism for age-related neuronal death in the PNS. Several molecular signaling pathways, including ROS, calcium, glucocorticoid, and protein quality controls (e.g, ubiquitins), are implicated in the age-related loss of hair cells and SGNs (Figure 3). However, studies with the ability to locally modify gene expression in these cells, such as those employing transgenic models, are needed to address whether these signaling pathways have direct effects on hair cells and SGNs during aging. Finally, the issue of whether age-related loss of hair cells and SGNs occurs via typical apoptotic pathways requires further examination. As new studies in the field of aging reshape the framework for exploring these underpinnings, understanding of the loss of hair cells and SGNs associated with age and the interventions that can treat and prevent these changes will result in dramatic benefits for an aging population.

Figure 3.

Flow chart for possible causes of age-related loss of hair cells and SGNs. The top half summarizes possible contributions from reactive oxygen species to age-related hearing loss, and the bottom half summarizes other possible contributors.