Deafness and retinal degeneration in a novel USH1C knock-in mouse model

Abstract

Usher syndrome is the leading cause of combined deaf-blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction, and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in Exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock-in mice. Absent auditory-evoked brainstem responses indicated that the mutant mice are deaf at 1 month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock-in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.

Figure 1

Representative audiograms at 16 kHz (A) of P30 wild type CBA controls (left panel) and Ush1c216AA mutant mice (right panel). CBA control mice hearing thresholds at 16 kHz (15 dB SPL) is circled. B Average ABR thresholds (dB SPL) to pure tones ranging in frequency from 3.5 to 40 kHz in Ush1c216AA mutant mice and CBA controls at P30. Error bars represent standard deviation. Caret marks indicate no detectable response to 110 dB SPL tones.

Figure 2

Cochlear histology of wild type and Ush1c216AA mutant mice at P30. Figures A-C show whole-mount preparations of the organ of Corti at the apex of wild type (A) and the apex (B) and middle turns (C) of mutant mice. All images are brightest point projections from a confocal Z series spanning the full depth of the sensory epithelium. The cytoplasm of inner (IHCs) and outer (OHCs) hair cells is immunolabeled with parvalbumin (red) and nuclei are counterstained with DAPI (blue). White arrows and bracket indicate missing OHCs. Figures D, E show cross sections of the basal turn of the organ of Corti from plastic embedded cochleae from wild type (D) and mutant (E) mice. Both images are brightest point projections from a confocal Z series spanning the depth of 2-3 outer hair cells. Nerve fibers (Neurofilament, green) are visible innervating IHCs and OHCs in the wild type mice, while OHCs are missing entirely from this region of the cochlea in mutant mice. Additionally, IHCs appear abnormal, and few nerve fibers cross the tunnel of Corti. Scale bars in all figures are 25 μm.

Figure 3

Average number of inner and outer hair cells per 100 μm longitudinal section of organ of Corti for wild type and Ush1c216AA mutant mice at P30. A Illustration of the region where cochlear hair cells were counted, scale bar = 100 μm. B,C Graphs show significantly fewer IHCs (B, p=.02) and OHCs (C, p=.001) in more basally located regions in Ush1c216AA mutant mice as compared with their wild type littermates (two-way Anova, Origin 8.0). (IHC, inner hair cells, OHC, outer hair cells) Error bars represent the standard error.

Figure 4

Scanning electron micrographs of cochleae from Ush1c216AA mutant and CBA controls at P30. A OHCs in the middle turn of the cochlea from a wild type mouse. The most medial and lateral rows of OHCs are at the top and base of the figure, respectively. B Disorganized OHC rows with missing hair cells in the middle turn from a mutant mouse. Hair cell stereociliary bundles are abnormal. C Higher magnification (15,000× original magnification) of an OHC stereociliary bundle from a wild type mouse. D Higher magnification of an abnormal OHC stereociliary bundle from a mutant mouse (15,000× original magnification). Scale bars = 1 μm.

Figure 5

ERG analysis of wild type and Ush1c216AA mutant mice at 1, 3, 6.5 and 12 months of age. A,B Representative ERGs of mutant (Ush1c216AA) and control (wild type) at 6.5 and 12 months of age. C,D Averaged a- and b-wave amplitudes at 1, 3, 6.5 and 12 months of age of control mice (black line) and mutant mice (red line). At all ages tested, mutant a- and b-wave maximum amplitudes are lower (1 month: a-wave Vmax=382.48 μV, b-wave Vmax=747.48 μV; 3 month: a-wave Vmax=302.6, b-wave Vmax=608.62 μV; 6.5 month: a-wave Vmax=304.56 μV, b-wave Vmax=676.58 μV; 12 months: a-wave Vmax=136.36 μV, b-wave Vmax=324.14 μV) than controls (1 month: a-wave Vmax=546.68, b-wave Vmax=1143.73 μV; 3 month a-wave Vmax=362.13 μV, b-wave Vmax=765.87 μV; 6.5 month: a-wave Vmax=374.54 μV, b-wave Vmax=870.96 μV; 12 month: a-wave Vmax=199.72 μV, b-wave Vmax=415.89 μV). By 12 months of age, mutant a- and b-wave thresholds are higher compared to controls (WT a-wave threshold=0.005 Cd s/m² vs. Mut a-wave threshold=0.0075; WT b-wave threshold=0.0001 Cd s/m² vs. Mut b-wave threshold=0.0005). Error bars represent the standard error. E,F b/a-wave ratio of wild type (black line) and Ush1c216AA mutant mice (red line) at 6.5 (left graph) and 12 months (right graph) of age. Graphs show that at one year of age mutant and wild type mice have significantly different b/a wave ratios at lower light intensities (p=0.002, two-way Anova, Origin 8.0). Light intensity is shown in log scale with x=log cycle number. Error bars represent the standard error.

Figure 6

Histology (100× and 400× original magnification) of superior peripheral retina of wild type and Ush1c216AA mutant mice at 6.5 months and 1 year of age. Figures A,B show 6.5 month old wild type (A) and mutant (B) sections from the margin of the superior retina. Figures C-F show sections from the margin (C,D) and central (E,F) regions of the superior retina of wild type and mutant 1 year old mice. (RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.) Scale bars in superior meridians (100× magnification) are 100 μm, inside panels (400× magnification) are 25 μm.

Figure 7

Progressive retinal degeneration in Ush1c216AA mutant mice from 6.5 months (6.5 mo) to 1 year of age (1 yr). A Diagram of hemisected retina and regions (marginal, peripheral, central, medial) where number of rods were counted. B Average number of nuclei in the ONL of the superior and inferior meridians of 6.5 month old wild type (black line) and Ush1c216AA mutant (red line) mice. C Average number of nuclei in the ONL of the superior and inferior meridians of 1 year old wild type (black line) and Ush1c216AA mutant (red line) mice. There are significantly fewer ROD nuclei across the entire retina in mutants compared with age-matched littermates (p=0.00035, one-way Anova, Origin 8.0). Error bars represent the standard error.