Requirement of nicotinic acetylcholine receptor subunit beta2 in the maintenance of spiral ganglion neurons during aging

Abstract

Age-related hearing loss (presbycusis) is a major health concern for the elderly. Loss of spiral ganglion neurons (SGNs), the primary sensory relay of the auditory system, is associated consistently with presbycusis. The causative molecular events responsible for age-related loss of SGNs are unknown. Recent reports directly link age-related neuronal loss in cerebral cortex with the loss of high-affinity nicotine acetylcholine receptors (nAChRs). In cochlea, cholinergic synapses are made by olivocochlear efferent fibers on the outer hair cells that express alpha9 nAChR subunits and on the peripheral projections of SGNs that express alpha2, alpha4-7, and beta2-3 nAChR subunits. A significantly decreased expression of the beta2 nAChR subunit in SGNs was found specifically in mice susceptible to presbycusis. Furthermore, mice lacking the beta2 nAChR subunit (beta2-/-), but not mice lacking the alpha5 nAChR subunit (alpha5-/-), have dramatic hearing loss and significant reduction in the number of SGNs. Our findings clearly established a requirement for beta2 nAChR subunit in the maintenance of SGNs during aging.

Figure 1.

Expressions of nAChR subunits in SGNs. Expression of nAChR α2, α4-7, and β2-3 subunits in SGNs were detected by RT-PCR, and samples from two mice of each age group (4 and 8 months of age) are shown (A). Expression of the neuronal marker snap-25 (internal control) is shown from two mice from each age group (B), and α5 and β2 protein products detected by Western blots are also shown from two mice (C). Levels of nAChR subunit mRNAs from each age group (6 mice per group) were quantified relative to snap-25 mRNA by RT-PCR (D). Expression levels of α4, α5, and β2 subunit mRNAs are significantly different between the two age groups (t test; ^*p < 0.01). Level of β2 subunit mRNA was compared further between 4- and 8-month-old groups (4 mice per group) from C57BL/6J and B6.CAST by real-time RT-PCR (E). Expression level of β2 subunit mRNA is significantly different between the two age groups for both C57BL/6J and B6.CAST (t test; ^*p < 0.01). Error bars represent mean ± SD.

Figure 2.

Mean ± SD auditory brainstem response (ABR) thresholds in young and old B6.CAST mice with or without the β2 subunit. ABR thresholds were measured for 2-month-old β2+/+ (6 male and 6 female) and β2-/- (4 male and 8 female) mice and also for 8-month-old wild-type AHL allele with β2+/+ (5 male and 5 female) and β2-/- (8 male and 6 female) mice. Significant differences in 8-month-old mice between β2+/+ and β2-/- mice were found across all five frequencies tested (two-way ANOVA on ranks with Dunn multiple comparisons; p < 0.01).

Figure 3.

Histology of spiral ganglia from α5 or β2 null mice. The top shows mid-modiolar sections of spiral ganglion from one 8-month-old mouse lacking α5 (right) and one 8-month-old α5+/+ control mouse (left) with the same genetic background (A). The bottom consists of mid-modiolar sections of spiral ganglion from one 8-month-old mouse lacking β2 (right) and one 8-month-old β2+/+ control mouse (left) with the same genetic background. Dramatic loss of SGNs can be observed in the section of spiral ganglia from 8-month-old mice lacking the β2 subunit (A). In B, the right basal area of the spiral ganglion and the number of SGNs were quantified in every other section using Image Pro Plus (Media Cybernetics). Each group contains three 8-month-old mice. The density of SGNs in β2-/- mice was significantly decreased compared with genetic-matched controls (^*p < 0.01). Error bars represent SEM.