ATP8B1 is essential for maintaining normal hearing

Abstract

ATP8B1 deficiency is caused by autosomal recessive mutations in ATP8B1, which encodes the putative phospatidylserine flippase ATP8B1 (formerly called FIC1). ATP8B1 deficiency is primarily characterized by cholestasis, but extrahepatic symptoms are also found. Because patients sometimes report reduced hearing capability, we investigated the role of ATP8B1 in auditory function. Here we show that ATP8B1/Atp8b1 deficiency, both in patients and in Atp8b1(G308V/G308V) mutant mice, causes hearing loss, associated with progressive degeneration of cochlear hair cells. Atp8b1 is specifically localized in the stereocilia of these hair cells. This indicates that the mechanosensory function and integrity of the cochlear hair cells is critically dependent on ATP8B1 activity, possibly through maintaining lipid asymmetry in the cellular membranes of stereocilia.

Fig. 1.

Hearing loss in BRIC type 1 patients and Atp8b1^G308V/G308V mutant mice. (A) Mean age-corrected hearing loss expressed in decibel hearing loss (dBHL) for six octave frequencies (250 to 8000 Hz) of the BRIC type 1 patient group (n = 10) and PSC patient group (n = 7). (B) Mean auditory brainstem thresholds expressed in decibel sound pressure level (dBSPL) for wild-type and Atp8b1^G308V/G308V mutant mice at different ages (16 days, 1, 3 and 6 months) at three octave frequencies (8–32 kHz) and a click stimulus. d, days; m, months; BRIC, benign recurrent intrahepatic cholestasis; PSC, primary sclerosing cholangitis. Asterisks indicate significant differences between BRIC type 1 versus PSC patients or mutant mice of 1, 3, and 6 months versus age-matched wild-type mice (*P < 0.05; **P < 0.01). The 16-day-old mutant mice display a normal hearing just like the age-matched wild-type mice. Bars indicate 2× standard error (SE).

Fig. 2.

Localization of Atp8b1 in cochlea. (A–C) Fluorescence images of paraffin sections of 4-day-old wild-type murine inner ear (basal turn) incubated with anti-ATP8B1-C6 or anti-calbindin-D_28k antibody (green or white signal) and counterstained with propidium iodide (red signal). (A–Aii) For orientation anti-calbindin-D_28K was used as a marker for hair cells in the organ of Corti. (B–Bii) Atp8b1 expression was seen in apical region of these hair cells. (C–Cii) Serial sections were incubated with preimmune serum as a negative control. Arrow points to row of inner hair cells; arrowhead points to three rows of outer hair cells. sg, spiral ganglion; oc, organ of Corti. Scale bar, (Ai–Ci) 50 μm, (Aii–Cii) 10 μm.

Fig. 3.

Localization of Atp8b1 in cochlear hair cell stereocilia. Confocal images of cochlear whole mounts of 3-month-old wild-type mice incubated with anti-ATP8B1. Confocal images overlay (A) or at level 0.0 μm (B), 1.9 μm (C), 3.5 μm (D), and 5.0 μm (E) showing Atp8b1 staining from top to base in the stereocilia of the cochlear basal turn. The arrow points to the row of inner hair cells, the arrowhead points to the (three) rows of outer hair cells. Scale bar, 6 μm.

Fig. 4.

Degeneration of hair cells and spiral ganglion neurons in Atp8b1^G308V/G308V mutant mice. Light micrographs of cochlear morphology of the organ of Corti (A, C, E, G, I, K, M) and spiral ganglion (B, D, F, H, J, L, N) in wild-type (A–D) and mutant (E–N) mice at 4 days (A, B, E, F), 16 days (C, D, G–J), 1 month (K, L) and 6 months (M, N) of age. All sections are from the basal turn except for (G, H), which represent the medial turn. Although morphologically intact looking inner hair cells could be seen in the mutant mice till the age of 1 month (E, G, I, K), the outer hair cells show signs of degeneration from 16 days on (I, K, M), and the number of neurons in the spiral ganglion is decreased from 1 month of age (L). Complete degeneration of the organ of Corti and nearly all connected spiral ganglion cells and nerve fibers is observed in 6-month-old mutant mice (M, N). Scale bar, 24 μm.

Fig. 5.

Ultrastructural features of inner hair cell degeneration in Atp8b1^G308V/G308V mutant mice. Transmission electron micrographs of inner hair cells in the upper basal turn of the cochlea of wild-type (A) and mutant (B–G) mice at 1 month of age. The inner hair cells of the mutant mice display a normal cell shape and nucleus (B) and largely intact stereocilia (B, F). However, signs of degeneration are already observed, such as condensed cytoplasm (B), membrane-bound vesicles enclosing cellular debris (D, arrow), multivesicular bodies (D, double arrow), degenerating mitochondria (E, arrowhead) and vacuoles with cellular debris (E, asterisk). More severe degeneration aspects can also be seen in hair cells of the same turn (C) especially using higher magnification (G). Scale bars: A, B, 2 μm; C–G, 400 nm.

Fig. 6.

Degeneration of hair cells and stereocilia in Atp8b1^G308V/G308V mutant mice. Scanning electron microscopy images of the organ of Corti in wild-type (A, C) and mutant mice (B, D–F) at 4 days (A, B), 16 days (C, D), 1 month (E) and 6 months (F) of age, all in the basal turn. There is normal development of stereocilia in the 4-day-old mutant mice (B). Loss of the stereocilia starts in some outer hair cells in 16-day-old mutant mice (D) and progresses to complete absence, whereas stereocilia on inner hair cells do not show detectable morphological aberrations in 1-month-old mutant mice (E). The total organ of Corti has degenerated at 6 months in mutant mice, resembling merely a simple nonciliated epithelial layer (F). Scale bar, 1 μm.