Glial-Specific Deletion of Med12 Results in Rapid Hearing Loss via Degradation of the Stria Vascularis

Abstract

Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult CNS results in region-specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiologic analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis.SIGNIFICANCE STATEMENT Mutations in Mediator protein complex subunit 12 (Med12) are associated with X-linked intellectual disability syndromes and hearing loss. Using temporal-conditional genetic approaches in CNS glia, we found that loss of Med12 results in severe hearing loss in adult animals through rapid degeneration of the stria vascularis. Our study describes the first animal model that recapitulates hearing loss identified in Med12-related disorders and provides a new system in which to examine the underlying cellular and molecular mechanisms of Med12 function in the adult nervous system.

Keywords: astrocyte; hearing loss; mediator complex; stria vascularis.

Figure 1.

Deletion of Med12 in mature astrocytes selectively reduces morphologic complexity. A–H, Coexpression of Med12 and Aldh1l1-GFP in AG12-Con and AG12-KO mice. Filled arrowheads show coexpression of Aldh1l1-GFP and Med12; unfilled arrowheads show Aldh1l1-GFP cells that do not express Med12. I, Tamoxifen treatment paradigm. J–Q, High-magnification confocal images of Aldh1l1-GFP from AG12-Con or AG12-KO mice showing reduced complexity in the cortex and hippocampus. R, Quantification of Med12/Aldh1l1-GFP coexpression in AG12-Con and AG12-KO mice. Data are derived from three mice per genotype, three slides per region, per mouse: at least 500 cells per region, per mouse. One-way ANOVA. S–V, Scholl analysis of astrocyte complexity in the cortex, olfactory bulb, brainstem, and hippocampus. Data are derived from three mice per genotype, three slides per region, per mouse, with at least 30 cells per region per genotype; two-way repeated-measures ANOVA. W, Quantification of Aldh1l1-GFP/DAPI coexpression in AG12-Con and AG12-KO mice. Data are derived from three mice per genotype, three slides per region, per mouse: at least 500 cells per region, per mouse. One-way ANOVA. NS: not significant. *p < 0.05.

Figure 2.

Deletion of Med12 results in hearing loss and no changes in synaptic plasticity in the hippocampus. A, B, Quantification of freezing in cued fear conditioning (A) and startle responses in acoustic startle (B). In A, four AG12-Con and five AG12-KO mice were used; in B, three AG12-Con and four AG12-KO mice were used. Two-tailed unpaired t test. AG12-Con denotes Med12^fl/fl; Aldh1l1-GFP and Med12fl/y; Aldh1l1-GFP. AG12-KO denotes Med12^fl/fl; Aldh1l1-CreER; Aldh1l1-GFP and Med12fl/y; Aldh1l1-CreER; Aldh1l1-GFP. *p < 0.05; **p < 0.01. C, Schematic of LTP recording experimental setting. D, LTP traces from AG12-Con and AG12-KO hippocampal slices. E, Quantification of LTP. All electrophysiological experiments are derived from three mice from each genotype, ranging from 7 to 13 cells total for each experiment. NS, No significance. *p < 0.05; **p < 0.001; Student's two-tailed paired (L) t test. AG12-Con denotes Med12^fl/fl; Aldh1l1-GFP and Med12fl/y; Aldh1l1-GFP. AG12-KO denotes Med12^fl/fl; Aldh1l1-CreER; Aldh1l1-GFP and Med12fl/y; Aldh1l1-CreER; Aldh1l1-GFP. Stim., Stimulus; Rec., recording; Ctrl, control.

Figure 3.

Med12 deletion causes rapid peripheral hearing loss in adult mice. A, Tamoxifen treatment paradigm for weekly ABR tests. B–E, ABR thresholds at different frequencies before (B), after 1 week (C), after 2 weeks (D), and after 3 weeks (E) of the tamoxifen treatment. Dashed lines mark the maximum pressure, 90 dB, used in the tests. In B, Nine AG12-Con and 8 AG12-KO mice were recorded; in C, 6 AG12-Con and 7 AG12-KO mice were recorded; in D, 4 AG12-Con and 6 AG12-KO mice were recorded; in E, 9 AG12-Con and 10 AG12-KO mice were recorded. F, G, ABR to stimuli of 60–90 dB SPL (8 kHz) in AG12-Con (F) and AG12-KO (G) mice. AG12-Con denotes Med12^fl/fl; Aldh1l1-GFP and Med12fl/y; and Aldh1l1-GFP. AG12-KO denotes Med12^fl/fl; Aldh1l1-CreER; Aldh1l1-GFP and Med12fl/y; Aldh1l1-CreER; Aldh1l1-GFP. Two-way repeated-measures ANOVA. *p < 0.05; **p < 0.01.

Figure 4.

Deletion of Med12 has latent effects on the structure of organ of Corti. A–X, Detection of the markers of supporting cells, Sox2, and hair cells, Myo7a, by immunofluorescence on midmodiolar sections of cochleae of AG12-Con and AG12-KO mice. Figures show the organ of Corti in the basal turn. A comparison of midmodiolar sections of cochleae of AG12-Con and AG12-KO mice 3 weeks post-TM (A–H), 5 weeks post-TM (I–P), and 18 weeks post-TM (Q–X). AG12-Con denotes Med12^fl/fl; Aldh1l1-GFP and Med12fl/y; Aldh1l1-GFP. AG12-KO denotes Med12^fl/fl; Aldh1l1-CreER; Aldh1l1-GFP and Med12fl/y; Aldh1l1-CreER; Aldh1l1-GFP. Scale bar, D, 20 µm.

Figure 5.

Med12 expression in basal cells is required to maintain the stria vascularis. A–P, Detection of the stria vascularis marker, ATP1A1, by immunofluorescence on midmodiolar sections of cochleae of AG12-Con and AG12-KO mice. Figures show the stria vascularis in the apical turn. Yellow arrowheads mark the regions of stria vascularis. A comparison of midmodiolar sections of cochleae of AG12-Con and AG12-KO mice 3 weeks post-TM (A–H) and 5 weeks post-TM (I–P). Q–V, Immunostaining of Med12 in stria vascularis of midmodiolar sections of cochleae of AG12-Con and AG12-KO mice at 2 weeks post-TM. Figures show the stria vascularis in the middle turn. R–S and U–V are the regions from dashed squares in Q and T. Unfilled arrowheads mark the sides of intermediate cells; yellow arrowheads mark the sides of basal cells and the spiral ligament. AG12-Con denotes Med12^fl/fl; Aldh1l1-GFP and Med12fl/y; Aldh1l1-GFP. AG12-KO denotes Med12^fl/fl; Aldh1l1-CreER; Aldh1l1-GFP and Med12fl/y; Aldh1l1-CreER; Aldh1l1-GFP. W, Quantification of Med12 expression in Aldh1l1-GFP basal cells; *p < 0.05. Scale bars: D, 50 µm; Q, 20 µm.

Figure 6.

Med12 is required to maintain expression of cell adhesion proteins. A–H, Detection of Glut1, an endothelial and basal cell marker, by immunofluorescence on midmodiolar sections of cochleae of AT12-Con and AT12-KO mice at 2 weeks post-TM. Figures show the stria vascularis in the middle turn. Yellow arrowheads denote Glut1-positive basal cells, labeled by AT12-Con tdTomato reporter (B, C); unfilled arrowheads denote disorganized Glut-1/tdTomato basal cells in AT12-KO (F, G). I–T, Detection of ZO1, E-cadherin, and Connexin-31 proteins by immunofluorescence on midmodiolar sections of cochleae of AG12-Con and AG12-KO mice at 2 weeks post-TM. I, M, Q, Filled arrowheads denote AG12-Con expression of makers. K, O, S, Unfilled arrows denote altered expression of markers in AG12-KO. U, Quantification of immunostaining from I to T; *p < 0.05. Scale bars: D, L, 50 µm.

Figure 7.

Loss of Med12 results in stria vascularis-specific defects. A, ABR thresholds were significantly higher in KOs versus controls (two-way ANOVA, F = 46.188, df = 6, p < 0.001) with no significant interactions. B, EPs were significantly lower in KOs (t test for unequal variance, df = 11, t = −7.23, p < 0.001). C, 2f1-f2 maximum values were not significantly different by group (t test for unequal variance, df = 60, t = −1.84, p < 0.07). D, Difference in ABR versus DPOAE threshold (ABR-DPOAE for −15 dB SPL criterion) was significantly higher in KOs (t test for unequal variance, df = 59, t = 2.32, p < 0.02). DPOAE metrics were derived from input–output curves for f2 = 12, 18, and 24 kHz (see horizontal bar in A). Since these curves did not differ by f2 (data not shown), data from all three frequencies were included in C and D.