Absence of plastin 1 causes abnormal maintenance of hair cell stereocilia and a moderate form of hearing loss in mice

Abstract

Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals. Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness. One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed. Here, we found that plastin 1 knock-out (Pls1 KO) mice have a moderate and progressive form of hearing loss across all frequencies. Auditory hair cells developed normally in Pls1 KO, but in young adult animals, the stereocilia of inner hair cells were reduced in width and length. The stereocilia of OHCs were comparatively less affected; however, they also showed signs of degeneration in ageing mice. The hair bundle stiffness and the acquisition of the electrophysiological properties of hair cells were unaffected by the absence of plastin 1, except for a significant change in the adaptation properties, but not the size of the mechanoelectrical transducer currents. These results show that in contrast to other actin-bundling proteins such as espin, harmonin or Eps8, plastin 1 is dispensable for the initial formation of stereocilia. However, the progressive hearing loss and morphological defects of hair cells in adult Pls1 KO mice point at a specific role for plastin 1 in the preservation of adult stereocilia and optimal hearing. Hence, mutations in the human PLS1 gene may be associated with relatively mild and progressive forms of hearing loss.

Figure 1.

The organ of Corti and expression of plastin 1 in the mouse inner ear. (A) Schematic transverse view of the organ of Corti. The two types of auditory hair cells, the IHCs and OHCS, rest on supporting cells, and their stereociliary bundles are in contact with the tectorial membrane (tm). The IHCs are contacted by the majority of afferent nerve fibres and are the primary receptors of auditory signals conveying information to the brain. The OHCs have unique electromotile properties and play an important role in hearing sensitivity and frequency discrimination by locally amplifying sound-elicited vibrations of the organ of Corti. (B–B′) Low-magnification transverse view of the adult mouse organ of Corti. Plastin 1 immunoreactivity is only detected at the apical surfaces of hair cells (arrowheads). (C–E′) Surface preparation of the organ of Corti of P2 (C–C′) and adult (D–E′) mice immunostained for plastin 1 and counterstained with fluorescently labelled phalloidin. Plastin 1 is present in immature stereocilia, and its expression is maintained in the stereocilia and cuticular plate of both types of auditory hair cells at adult stages.

Figure 2.

Pls1 KO mice have a moderate and progressive form of hearing loss. Auditory brainstem responses to click (A) stimuli and to tone pips (B–D) at 8, 12, 24, 32 and 40 kHz at different ages in control (wt), het and Pls1 KO mice. P-values for Tukey–Kramer multiple comparisons test, P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***). Significant differences between Pls1 KO and wt thresholds are indicated on (B–D). Error bars represent SEM.

Figure 3.

Hair cell loss is not responsible for the hearing deficit in Pls1 KO mice. (A) Whole-mount, surface views of the organ of Corti of 6-week-old wt and Pls1 KO mice immunostained for parvalbumin. The vast majority of hair cell bodies are present in the medial and basal turn of the cochlea. Arrowhead points to the site of one missing OHC in the Pls1 KO sample. (B) Surface views of the organ of Corti of 4-month-old wt and Pls1 KO mice immunostained for myosin-VIIa . The IHC are well preserved in both the Pls1 KO and wt mice, but some OHCs are missing in wt and Pls1 KO mice (arrowheads), with increased frequency of OHC losses in the basal turns of the cochlea.

Figure 4.

Auditory hair cells develop normally in young Pls1 KO mice. (A and B) Scanning electron micrographs of the surface of the organ of Corti in the medial turn of the cochlea of P12 wt (A) and Pls1 KO (B) mice. (C–F) Transmission electron micrographs of the apical region of the IHC and OHC in P12 wt and Pls1 KO mice. The contour of the cuticular plate (CP) is outlined by a white dotted line. (G–J) Surface views of the IHCs and OHCs of P12 wt and Pls1 KO mice immunostained for spectrin, a marker of the CP. (K–N) Surface views of the IHCs and OHCs of P30 wt and Pls1 KO mice stained with fluorescent phalloidin. The F-actin content of stereocilia and the CP appears similar in wt and Pls1 KO hair cells.

Figure 5.

Morphological defects of stereocilia in adult Pls1 KO mice. (A–E) Scanning electron micrographs of IHCs in wt and Pls1 KO mice. Compared with those of P30 wt littermates (arrowheads in A), the stereocilia of Pls1 KO mice show a reduced width and abnormal bending (arrows in B). Similar defects are visible at 8 weeks (C–E). Note that the same IHC can have a mixture of thin (arrow) and normal (arrowhead) stereocilia (D). A distal tapering is also visible in some of the stereocilia of Pls1 KO (arrows in E). (F–J) Scanning electron micrographs of OHCs in wt and Pls1 KO mice. (F and G) At P30, the stereocilia of OHCs were very much less affected than those of IHCs in Pls1 KO mice and looked normal. There was no evidence of reduced width or abnormal bendiness. (H) At 8 weeks, some defects such as fusion of neighbouring stereocilia (arrow) were occasionally visible. (I and J) At 12 weeks, the stereocilia of Pls1 KO OHCs exhibited increased fusion and defects in organization (arrows) compared with those of wt OHCs; both images were taken from the basal turn of the cochlea. Scale bars = 1 µm.

Figure 6.

Organization of the actin filaments in the stereocilia of Pls1 KO mice. (A and B) Transmission electron micrographs of the top and shaft region of the IHC stereocilia in adult (4 month and older) het and Pls1 KO mice. (C) Mean values of the interfilament distance within the stereocilia of het and Pls1 KO mice. Mean values are shown for all measurements, and those obtained from either the shaft or the top region (within 300 nm of the tip) only; 250 measurements were taken from 5 different stereocilia for each genotype; error bars represent the standard error of the mean. The interfilament distance was significantly increased in the top region of stereocilia of Pls1 KO mice compared with het mice (P-value = 0.004; unpaired T-test with Welch's Correction).

Figure 7.

Current and voltage responses from IHCs of plastin 1 mice. (A and B) K⁺ currents recorded from mature het and Pls1 KO P24 IHCs were elicited by depolarizing voltage steps (10-mV nominal increments) from –144 mV to more depolarized values from the holding potential of –64 mV. The K⁺ currents characteristic of adult IHCs, I_K,f and I_K,n were similarly expressed in both genotypes. (C) Steady-state current–voltage curves for the total K⁺ current in het (n = 5) and Pls1 KO (n = 5) P24 IHCs. (D and E) Voltage responses to different current injections recorded from a het and a Pls1 KO IHC.

Figure 8.

Mechanotransducer currents in OHCs from Pls1 KO mice. (A, B) Saturating transducer currents recorded from a P5 het (A) and Pls1 KO (B) apical-coil OHC by applying a 50-Hz sinusoidal force stimuli to the hair bundles at the potential of −121 mV and +99 mV. The driver voltage (DV) signal of ±40 V to the fluid jet is shown above the traces (negative deflections of the DV are inhibitory). The arrows indicate the closure of the transducer channels, i.e. disappearance of the resting current, during inhibitory bundle displacements at −121 mV and +99 mV, respectively. Dashed lines indicate the holding current. (C) Peak-to-peak current–voltage curves were obtained from 7 het and 12 Pls1 KO OHCs (P5–P8) using 1.3 mm extracellular Ca²⁺. The fits through the data are according to Equation 1 (see Material and methods) with values: het k = 418 ± 33, V_r = 1.3 ± 0.3 mV, V_s = 38 ± 2 mV and γ = 0.42 ± 0.01; Pls1 KO k = 418 ± 40, V_r = 1.1 ± 0.4 mV, V_s = 40 ± 3 mV and γ = 0.41 ± 0.01.

Figure 9.

Adaptation properties of the MET current in Pls1 KO OHCs. (A and B) Driver voltages to the fluid jet (top) and transducer currents recorded at –81 mV (bottom) from a het and a Pls1 KO OHC, respectively. At –81 mV, positive DVs (excitatory direction) elicited inward transducer currents that declined or adapted over time in OHCs (arrows). Current decline was best fitted with two time constants (thick line superimposed on the currents): control τ_fast 1.2 ms, τ_slow 18.6 ms; knock-out τ_fast 1.4 ms, τ_slow 12.8 ms. A small transducer current was present at rest (before t = 0) and inhibitory bundle displacements turned this off. Upon termination of the inhibitory stimulus, the transducer current in het and Pls1 KO OHCs showed evidence of rebound adaptation (arrowheads). (C and D) Driver voltages to the fluid jet (top) and transducer currents recorded at +99 mV (bottom) from a het and a Pls1 KO OHC, respectively. Note that all manifestations of transducer current adaptation (current decline during excitatory stimuli and rebound following inhibitory stimuli) were absent at +99 mV and the resting current increased. (E) Average fast and slow time constants (τ) used to fit the onset adaptation to excitatory displacement at –81 mV in het and Pls1 KO OHCs (P5–P8), including the cells shown in (A) and (B). (F) Extent of adaptation to excitatory displacement from the same cells used in (E).