Adenosine kinase inhibition in the cochlea delays the onset of age-related hearing loss
Highlights
► Age-related hearing loss (ARHL) is the most common sensory deficit. ► We investigated the role of adenosine signalling in the development of ARHL in mice. ► Adenosine kinase (ADK) is the primary route for adenosine metabolism in the cochlea. ► ADK inhibition provides partial protection of C57BL/6J mice from ARHL. ► ARHL can be mitigated by enhancing adenosine signalling in the cochlea.
Introduction
Age-related hearing loss (ARHL), or presbyacusis, is the most common sensory deficit (Morton, 1991). It is characterised by a decline in hearing sensitivity and speech discrimination, delayed central processing of acoustic information, and impaired localisation of sound sources (Gates and Mills, 2005). Various cochlear pathologies related to ARHL have been observed, including loss of sensory hair cells and spiral ganglion neurones, and degeneration of secretory and supporting tissues (stria vascularis and spiral ligament) that are responsible for maintenance of electrochemical homeostasis of cochlear fluids (Schuknecht and Gacek, 1993). Mechanical changes to the basilar membrane have also been proposed to explain the deterioration in the audiogram (Schuknecht and Gacek, 1993). Secondary changes in the central auditory pathways also contribute to the hearing deficits (Syka, 2002).
Multiple mechanisms have been proposed for age-related cochlear degeneration, and it appears that both genetic and environmental factors play a role (Van Eyken et al., 2007). A dominant theory is that accumulated oxidative stress causes cochlear damage (Seidman et al., 1999, Staecker et al., 2001, Jiang et al., 2007), possibly as a consequence of impaired blood flow (Dai et al., 2004) or environmental factors such as excessive noise (Ohlemiller et al., 2000a). This results in membrane and mitochondrial DNA damage in cochlear tissues leading to cell death and hearing loss (Fischel-Ghodsian et al., 1997, Ueda et al., 1998, Seidman et al., 1999, Fischel-Ghodsian, 2003, Pickles, 2004, Yin et al., 2007, Niu et al., 2007).
Much of our understanding of the mechanisms of ARHL comes from animal models (Zheng et al., 1999, Ohlemiller, 2006, Bielefeld et al., 2008). The variance seen in susceptibility to hearing loss both within and between genetic models is similar to that seen in susceptibility to both noise- and age-related hearing loss in humans, which may include individual differences in resistance to environmental stress (Duvdevany and Furst, 2007). Inbred mice often show early onset ARHL (Zheng et al., 1999), and the C57BL/6J mouse is the most established model of accelerated ARHL exhibiting behavioural and functional changes similar to those in the ageing human ear (Prosen et al., 2003, Francis et al., 2003). Akin to humans, the hearing loss progresses from the high to low frequencies and the damage comprises loss of sensory cells and neurons starting in the base and progressing to the apex of the cochlea (Spongr et al., 1997, Bartolomé et al., 2002). Other studies have shown that cochlear pathology also includes degeneration of the stria vascularis and fibrocytes of the spiral ligament (Ichimiya et al., 2000, Hequembourg and Liberman, 2001), reinforcing the utility of the C57BL/6J mouse as a model of mixed presbyacusis in humans (Ohlemiller, 2006). The C57BL/6J mouse is also more susceptible to the damaging effects of noise exposure, ototoxic drugs and hypoxia (Ohlemiller et al., 2000a, Ohlemiller, 2006), suggesting diminished protective or reparative processes compared to mouse strains that do not show early onset ARHL.
The ARHL locus (ahl) that contributes to the hearing loss in the C57BL/6J mouse has been mapped to chromosome 10 (Erway et al., 1993, Johnson et al., 1997). It has been shown that strains susceptible to early onset ARHL carry a specific mutation in the cadherin 23 gene (Cdh23753A), which encodes a component of the hair cell stereocilia tip-link associated with the mechanical-to-electrical transduction channels (Noben-Trauth et al., 2003). There is also evidence that the ahl locus on chromosome 10 is not the only region involved in the development of hearing loss in inbred mice (Johnson and Zheng, 2002, Keithley et al., 2004, Mashimo et al., 2006, Zheng et al., 2009). Ahl3 on chromosome 17, for example, contributes to susceptibility of C57BL/6J mice to age- and noise-induced hearing loss (Morita et al., 2007). In keeping with the mitochondrial theory of ageing (Loeb et al., 2005), it was proposed that the ahl locus at mouse chromosome 10 mediates a decrease in protective anti-oxidant enzymes and consequently increased impact of oxidative stress on tissues (Staecker et al., 2001). This notion has been confirmed in a recent study (Someya et al., 2009) demonstrating that Bak-mediated mitochondrial apoptosis in response to oxidative stress is a key mechanism of ARHL in C57BL/6J mice. The Cdh23753A allele thus affects the age of onset of ARHL, but the basic mechanisms of cochlear ageing such as oxidative imbalance appear to be similar in early and late onset ARHL mouse strains (Someya et al., 2009).
As the prevalence of hearing impairment increases with an ageing population (Gates and Mills, 2005), there is a demand for novel treatment strategies that would target the principal mechanisms of ARHL and reduce the impairment. We and others have shown that the adenosine signalling system in the cochlea has an important role in its protection from oxidative stress (for review, see Vlajkovic et al., 2009). For example, the administration of A1 adenosine receptor agonists onto the round window membrane (a membrane separating the middle ear from the perilymph of the cochlea) can prevent cochlear injury from noise (Hu et al., 1997, Hight et al., 2003) or partially reverse hearing loss after noise exposure (Wong et al., 2010, Vlajkovic et al., 2010a). In addition, selective A1 adenosine receptor agonists can reduce cisplatin-induced auditory threshold shifts (Whitworth et al., 2004), most likely by promoting the antioxidant defence system (Ford et al., 1997).
Adenosine signalling is known to decline in the ageing brain (Cunha, 2005), and a similar process has been postulated to occur in the ageing cochlea (Vlajkovic et al., 2009). Given the evidence of an otoprotective effect of adenosine described above, restoring adenosine signalling may protect the cochlea from age-related degeneration. Adenosine kinase (ADK) is the primary route for adenosine metabolism and the principal negative regulator of intracellular and extracellular adenosine concentrations in the brain (Boison, 2006) and the cochlea (Vlajkovic et al., 2010b). We have previously demonstrated that physiological reduction of ADK expression is associated with an increase in endogenous adenosine in the brain (Pignataro et al., 2008); conversely, experimental overexpression of ADK in the brain is associated with a reduced concentration of adenosine (Fedele et al., 2005). Subsequently, we demonstrated that ADK-expression levels are key determinants for adenosine-based neuroprotection in the brain (Li et al., 2008, Pignataro et al., 2007, Theofilas et al., 2011). Drawing on this background, it is reasonable to speculate that activity of ADK and the resultant enhancement of endogenous adenosine levels in the cochlea has potential to ameliorate ARHL. In this study, we measured auditory thresholds and hair cell loss in C57BL/6J mice in the period spanning 3–9 months of age (by which point this strain of mice develops significant ARHL) to investigate the otoprotective potential of the selective ADK inhibitor ABT-702. This study provides the first evidence that a manipulation of the adenosine signalling system in the cochlea can delay the onset of ARHL.
Section snippets
Animals
Male C57BL/6J inbred mice were used in this study. The mice were housed under standard conditions at the animal unit at the University of Auckland for the duration of the study (up to 6 months). All experimental procedures described in this study were approved by the University of Auckland Animal Ethics Committee.
Adenosine kinase immunohistochemistry
Adenosine kinase (ADK) immunostaining in cochlear tissues of 3-month-old C57BL/6 mice was visualised by laser scanning confocal microscopy. Mice were euthanised with sodium
ADK distribution in the adult mouse cochlea
There was extensive distribution of ADK immunofluorescence in the adult C57BL/6J mouse cochlea, which was reminiscent of the nuclear/cytoplasmic ADK immunolabelling seen in the adult rat cochlea (Vlajkovic et al., 2010b). ADK immunofluorescence was observed in the spiral ganglion neurones and satellite cells, fibrocytes and interdental cells in the spiral limbus, epithelial cells of the auditory sensory organ (Deiters' cells, inner and outer sulcus cells), fibrocytes in the spiral ligament
Discussion
This study demonstrates that treatment with a selective adenosine kinase inhibitor ABT-702, thereby enhancing extracellular adenosine, can provide partial protection from age-related hearing loss in C57BL/6J mice. Chronic treatment with ABT-702, commencing at the age of 3 months or 6 months, improved auditory thresholds, suprathreshold responses and hair cell survival in ageing (9-month-old) mice. This implies that ABT-702 enhances the survival of both sensory hair cells and spiral ganglion
Acknowledgements
This study was supported by the NZ Lottery Grants Board, Royal National Institute for Deaf People (RNID, UK), Deafness Research Foundation (NZ), Lodge Discovery 501 (NZ) and grant NS061844 from the National Institutes of Health (NIH, USA).
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