A lack of immune system genes causes loss in high frequency hearing but does not disrupt cochlear synapse maturation in mice

Abstract

Early cochlear development is marked by an exuberant outgrowth of neurites that innervate multiple targets. The establishment of mature cochlear neural circuits is, however, dependent on the pruning of inappropriate axons and synaptic connections. Such refinement also occurs in the central nervous system (CNS), and recently, genes ordinarily associated with immune and inflammatory processes have been shown to play roles in synaptic pruning in the brain. These molecules include the major histocompatibility complex class I (MHCI) genes, H2-K(b) and H2-D(b), and the complement cascade gene, C1qa. Since the mechanisms involved in synaptic refinement in the cochlea are not well understood, we investigated whether these immune system genes may be involved in this process and whether they are required for normal hearing function. Here we report that these genes are not necessary for normal synapse formation and refinement in the mouse cochlea. We further demonstrate that C1qa expression is not necessary for normal hearing in mice but the lack of expression of H2-K(b) and H2-D(b) causes hearing impairment. These data underscore the importance of the highly polymorphic family of MHCI genes in hearing in mice and also suggest that factors and mechanisms regulating synaptic refinement in the cochlea may be distinct from those in the CNS.

Figure 1. K^bD^b−/− mice have normal cochlear synaptic refinement.

A) Whole cochlear tissue was isolated from mice during ages of synaptic refinement and maturation and H2-K^b mRNA expression was analyzed. n = 3–5 mice per age group. Error bars show standard error of the mean. B) Double labeling fluorescent images showing RIBEYE (green) and SHANK1 (red) puncta to mark presynaptic and postsynaptic ribbons, respectively in mice P29 of age in both Control and K^bD^b−/− mice. Scale bar 10 um. C) and D) Quantification of RIBEYE and/or SHANK1 puncta beneath IHCs and OHCs of Control wild type and K^bD^b−/− mice, respectively, showed no significant differences between the two groups. p = 0.46 for RIBEYE, p = 0.9 for SHANK, and p = 0.70 for colocalized synaptic puncta in IHCs and p = 0.06 for RIBEYE in OHCs). E) Double labeling fluorescent images showing SYNAPTOPHYSIN (green) to mark efferent synapses or TUJ1 (green) for efferent and afferent fibers and MYOVIIa (red) to mark IHCs and OHCs in Control and K^bD^b−/− mice at P29 of age. Scale bar 10 um.

Figure 2. C1qa^−/− mice have no alteration in synaptic refinement.

A) Whole cochlea tissue was isolated from mice during ages of synaptic refinement and maturation and C1qa mRNA expression was analyzed. n = 3–5 mice per age group. Error bars show standard error of the mean. B) Double labeling fluorescent images showing RIBEYE (green) and SHANK1 (red) puncta to mark presynaptic and postsynaptic ribbons, respectively in mice P29 of age in both Control and C1qa^−/− mice. Scale bar 10 um. C) and D) Quantification of RIBEYE and/or SHANK1 puncta beneath IHCs and OHCs of Control wild type and C1qa^−/− mice, respectively, showed no significant differences between the two groups. p = 0.27 for RIBEYE, p = 0.8 for SHANK, and p = 0.22 for colocalized synaptic puncta in IHCs and p = 0.517 for RIBEYE in OHCs. E) Double labeling fluorescent images showing SYNPATOPHYSIN (green) to mark efferent synapses or TUJ1 (green) for efferent and afferent fibers and MYOVIIa (red) to mark IHCs and OHCs in Control and C1qa^−/− mice at P29 of age. Scale bar 10 um.

Figure 3. ABR and DPOAE thresholds are increased in K^bD^b−/− mice.

Average ABR (A–B) and DPOAE (E) thresholds are shown for mice at 8-weeks of age. Significantly increased ABR thresholds were recorded in K^bD^b−/− mice with pure-tone 32 kHz stimuli (A) and with broad-band clicks (B). *p = 2.5E-07 and p = 0.0001 for 32 kHz and broad-band clicks, respectively. ABR waveform amplitude of peaks 1 and 2 (C) and latency analysis of peak 1 (D) measured at the 16 kHz frequency stimulus and at 40 dB demonstrated no significant differences between Control and K^bD^b−/− cohorts. p = 0.44, p = 0.67 and p = 0.57 for peak 1 and peak 2 amplitudes and peak 1 latency, respectively. Increased DPOAE thresholds were recorded at 8, 11.3, 16.1, and 23 kHz frequencies (E). n = 10 (Control) and 9 (K^bD^b−/−) mice per group. *p = 0.007, p = 0.04, p = 0.003 and p = 1.25E-07 for 8, 11.3, 16 and 23 kHz, respectively. Error bars show SDs in (A), (B), (C), (D), and SEMs in (E). *p<0.05 for (E).

Figure 4. Average ABR and DPOAE thresholds continue to elevate with age in K^bD^b−/− mice.

Average ABR (A–B) and DPOAE (C) thresholds are shown for mice at 16-weeks of age. Significantly increased ABR thresholds were recorded in K^bD^b−/− mice with pure-tone 16, and 32 kHz stimuli (A) and with broad-band clicks (B). *p = 0.017, p = 0.001 and p = 0.032 for 16 kHz, 32 kHz and broad-band clicks, respectively. Increased DPOAE thresholds were recorded at 11.3, 16, and 23 kHz frequencies. *p = 0.006, p = 0.005 and p = 0.005 for 11.3, 16 and 23 kHz, respectively. n = 6 (Control) and 5 (K^bD^b−/−) mice per group (C). Error bars show SDs in (A), (B) and SEMs in (C).

Figure 5. ABR and DPOAE thresholds are not altered in C1qa^−/− mice.

Average ABR (A–B) and DPOAE (D) thresholds are shown for mice at 8-weeks of age. Student’s t-test did not reveal significant differences in ABR thresholds that were recorded in C1qa^−/− mice with pure-tone 8, 16, and 32 kHz stimuli (A) or broad-band clicks (B) compared to controls. ABR waveform amplitude of peaks 1 and 2 (C) and latency analysis of peak 1 (D) measured at the 16 kHz frequency stimulus and at 40 dB demonstrated no significant differences between Control and C1qa^−/− cohorts. There were no differences in DPOAE thresholds at any frequency tested between the cohorts (E). p>0.05 for all comparisons. n = 4 mice per group. Error bars show SDs.

Figure 6. C1qa^−/− mice are not protected from progressive or age-related hearing loss.

A) Average ABR thresholds for DBA/2J-C1qa^+/+ controls (n = 5) and congenic D2.B6-C1qa^+/+ (n = 5), D2.B6-C1qa^+/− (n = 8), and D2.B6-C1qa^−/− (n = 9) mice at 1-month of age are shown. Average ABR (B) and DPOAE (C) thresholds are shown for B6-C1qa^−/− (n = 4) and B6 control (n = 5) mice at 10-months of age. *(p = 0.01). Error bars show SDs.