Alström Syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity

Abstract

Alström Syndrome is a life-threatening disease characterized primarily by numerous metabolic abnormalities, retinal degeneration, cardiomyopathy, kidney and liver disease, and sensorineural hearing loss. The cellular localization of the affected protein, ALMS1, has suggested roles in ciliary function and/or ciliogenesis. We have investigated the role of ALMS1 in the cochlea and the pathogenesis of hearing loss in Alström Syndrome. In neonatal rat organ of Corti, ALMS1 was localized to the basal bodies of hair cells and supporting cells. ALMS1 was also evident at the basal bodies of differentiating fibrocytes and marginal cells in the lateral wall. Centriolar ALMS1 expression was retained into maturity. In Alms1-disrupted mice, which recapitulate the neurosensory deficits of human Alström Syndrome, cochleae displayed several cyto-architectural defects including abnormalities in the shape and orientation of hair cell stereociliary bundles. Developing hair cells were ciliated, suggesting that ciliogenesis was largely normal. In adult mice, in addition to bundle abnormalities, there was an accelerated loss of outer hair cells and the progressive appearance of large lesions in stria vascularis. Although the mice progressively lost distortion product otoacoustic emissions, suggesting defects in outer hair cell amplification, their endocochlear potentials were normal, indicating the strial atrophy did not affect its function. These results identify previously unrecognized cochlear histopathologies associated with this ciliopathy that (i) implicate ALMS1 in planar cell polarity signaling and (ii) suggest that the loss of outer hair cells causes the majority of the hearing loss in Alström Syndrome.

Figure 1.

ALMS1 localizes to basal bodies in cochlear tissues. (A and B) DIC photo-micrographs of a cross-section of the basal turn region of a P1 rat cochlear slice. (A) The major tissue types are shown, including the organ of Corti (oC), Kölliker's cells (Kc), spiral ganglion (sg), stria vascularis (sv), spiral ligament (sl) and Reissner's membrane (Rm). (B) Detail of cells in the developing organ of Corti. Laterally, there are three rows of outer hair cells (ohc1–3), and medial to these are a single row of inner hair cells (ihc). Medial to the inner hair cells are Kölliker's cells (Kc), which are columnar epithelial supporting cells. Basilar membrane lining cells (bmlc) form a multi-cellular layer beneath the organ of Corti. The arrow depicts the direction of view for whole-mount preparations as in (D). (C and D) Projections of confocal image stacks of cochlear slices or whole-mount preparations labeled with antibodies against acetylated tubulin (red), which labels cytoplasmic microtubules and primary cilia, and ALMS1 (green). (C) Organ of Corti in a cochlear slice. ALMS1 localized to ciliary basal bodies at the apical poles of hair cells and supporting cells, and also in basilar membrane lining cells. (D) Surface view of a whole-mount preparation of the organ of Corti labeled with antibodies against acetylated tubulin and ALMS1. Basal body pairs showed a regular arrangement in hair cells (large arrow) and supporting cells (small arrow). (E) In a comparable surface view of a different whole-mount preparation, the ALMS1 immunofluorescence (green) was coincident with that of γ-tubulin (red), confirming the specificity of the ALMS1 antibody labeling to basal bodies. (F) Projection of a confocal stack through the lateral wall in a cochlear slice, labeled with antibodies against acetylated tubulin (red) and ALMS1 (green). The large arrow shows ALMS1 labeling of basal bodies in a marginal cell (mc) in stria vascularis, the small arrow shows ALMS1 labeling of basal bodies of a mesenchymal fibrocyte (fc) in spiral ligament. (G) Montage image showing details of the apices of apical turn hair cells in a P30 cochlear slice labeled with phalloidin (red) that labels filamentous actin in hair cell stereociliary bundles and apices of supporting cells and the antibody against ALMS1 (green). ALMS1 localized to centrioles in hair cells (large arrows) and supporting cells (small arrows). (H) DIC photo-micrograph of the cochlear lateral wall, showing Reissner's membrane (Rm), stria vascularis (sv) and spiral ligament (sl). (I) In the lateral wall, ALMS1 localized to centrioles of basal cells (bc), intermediate cells (ic) and fibrocytes (fc). Labeling was not apparent in marginal cells (mc). (J) ALMS1 (green) localized to basal bodies at the base of the primary cilium (acetylated tubulin, red) in a Reissner's membrane epithelial cell facing scala media. Scale bars, 10 µm.

Figure 2.

Planar polarity abnormalities in neonatal Alms1^−/− mice hair cell stereociliary bundles. (A) Mid-apical turn organ of Corti whole-mount from a P2 Alms1^+/− mouse stained with phalloidin (red) and an anti-acetylated tubulin antibody (green), demonstrating the regular arrangement of stereociliary bundles of inner hair cells (ihc) and outer hair cells (ohc1–3), and primary cilia of hair cells and supporting cells. The tubulin-rich kinocilia of outer hair cells were located at the vertex of the V-shaped bundles in all cells. (B) In the mid-apical turn of an Alms1^−/− mouse organ of Corti some bundles were irregularly shaped (denoted *) and some were noticeably mis-oriented (arrows). Several kinocilia were not located at the vertex of the bundles. In mis-oriented bundles, the kinocilia were mis-localized relative to those in unaffected neighboring cells. (C) In the mid-basal turn organ of Corti of a P2 Alms1^+/− mouse, the outer hair cell bundles were wider than in the apical turn (A), but were also regular in shape and the kinocilia were properly located at the bundle vertex. (D) In Alms1^−/− mouse mid-basal turn organ of Corti, there were irregularly shaped bundles, mis-oriented bundles and mis-located kinocilia. (E) A scanning electron micrograph of a whole-mount of the mid-turn organ of Corti in a P2 Alms1^+/− mouse demonstrated the regularity of the outer hair cell bundles. (F) In Alms1^−/− mouse mid-turn organ of Corti, there were irregularly shaped and mis-oriented bundles (*) and mis-located kinocilia (arrows). Scale bars, 5 µm.

Figure 3.

Planar polarity abnormalities of hair cell stereociliary bundles in juvenile Alms1^−/− mice. (A) Scanning electron micrograph of an apical turn organ of Corti whole-mount from a P22 Alms1^+/− mouse. Outer hair cell stereociliary bundles were arranged in a regular pattern, although occasional mis-shapen bundles could be identified (arrows). (B) In the P22 Alms1^−/− mouse organ of Corti, there were various examples of mis-shapen bundles in all three rows of outer hair cells (arrows). (C) Basal turn organ of Corti whole-mount from a P22 Alms1^+/− mouse. (D) Basal turn organ of Corti whole-mount from a P22 Alms1^−/− mouse, showing various bundle abnormalities (arrows). (E–G) Detail of outer hair cells with asymmetric (E), flattened/asymmetric (F) and mis-oriented/asymmetric (G) bundles. Scale bars: (A–D) 20 µm, (E–G) 5 µm.

Figure 4.

Proportion of outer hair cell stereociliary bundles displaying planar polarity abnormalities in juvenile control and Alms1^−/− mice. (A) Number of noticeably abnormal outer hair cell bundles as a proportion of total cells, within the apical, mid-turn and basal regions of the cochlea. (B) Counts for the basal turn region, within the innermost cell row (row 1), middle row (row 2) and outermost row (row 3). (C) Counts for the mid-turn region. (D) Counts for the apical turn region.

Figure 5.

Outer hair cell function in juvenile Alms1^−/− mice was normal. (A) ABR thresholds of 1-month-old Alms1^−/− mice (red) were comparable with those of control animals (blue). (B and C) Comparable DPOAEs in a single control mouse (B) and an Alms1^−/− littermate (C) in response to low (12 kHz) and mid (24 kHz) frequency tones. DPOAEs were not activated in the Alms1^−/− mouse at 48 kHz, and were only activated at sound levels >60 dB in the control mouse. (D–E) Group data showed the similarities between DPOAEs in both sets of mice at 12 kHz (D) and 24 kHz (E).

Figure 6.

Outer hair cell loss in older control and Alms1^−/− mice. (A)–(F) show scanning electron micrographs of organ of Corti whole-mount preparations from P213 Alms1^+/+ (A, C, E) and Alms1^−/− (B, D, F) mice. (A) In the apical turn of the Alms1^+/+ mouse cochlea, there were few outer hair cells missing. (B) In the apical turn of the Alms1^−/− mouse cochlea, there were few missing cells but surviving cells displayed bundle abnormalities. (C) In the mid-turn region of the Alms1^+/+ mouse, there was scattered cell loss. (D) In the mid-turn region of the Alms1^−/− mouse, the cell loss was more extensive. (E) In the upper basal region of the Alms1^+/+ mouse, there was scattered cell loss. (F) In the basal turn of the Alms1^−/− mouse, the cell loss was extensive, with only few outer hair cells remaining. (G)–(I) show transmission electron micrographs of organ of Corti from P191 Alms1^−/− mice. (G) Surviving outer hair cells (ohc) in the apical turn. (H) Detail of the sub-nuclear synaptic region of a surviving outer hair cell. An efferent nerve ending (eff) attached to the outer hair cell appeared normal. (I) In the basal turn, outer hair cells had been lost, but an inner hair cell (ihc) survived. Supporting cells migrated to fill the spaces arising from outer hair cell loss. Scale bars: (A–F, I) 10 µm, (G) 2 µm, (H) 1 µm.

Figure 7.

Abnormal outer hair cell function in older Alms1^−/− mice. (A) ABR thresholds of 6–7-month-old Alms1^−/− mice (red) were higher than those of control animals (blue) at low (12 kHz) and mid (24 kHz) frequencies. Thresholds were high in all animals at 48 kHz. (B) In a single control mouse, DPOAEs were recorded in response to low and mid frequency tones (>35 dB), but not at 48 kHz. (C) In an Alms1^−/− littermate, DPOAEs were impaired at all frequencies. (D and E) Group data reflected the differences between DPOAEs in the control (blue) and Alms1^−/− (red) mice at 12 kHz (D) and 24 kHz (E).

Figure 8.

Ultra-structural changes in stria vascularis of Alms1^−/− mice. (A–D) Photomicrographs of cochlear sections from P191 Alms1^+/+ and Alms1^−/− mice. (A) DIC photomicrograph detailing the basal turn region of the cochlea (Rm, Reissner's membrane; oC, organ of Corti; sv, stria vascularis, sv; sl, spiral ligament). (B) The basal turn region of Alms1^+/+ cochlea. (C) The basal turn region of Alms1^−/− cochlea. There were defects in the ultra-structure of the upper area of stria vascularis (arrow). (D) DIC photomicrograph detailing the tissues of the cochlear lateral wall (mc, marginal cell layer; ic, intermediate cell layer; bv, blood vessel; bc, basal cell layer; fc, spiral ligament fibrocytes). (E) Detail of the stria vascularis of Alms1^+/+ mouse, showing homogenous structure with basal cell, intermediate cell and marginal cell layers all present. (F) Detail of the stria vascularis of Alms1^−/− mouse, showing large spaces within the intermediate cell layer (denoted *). There were large blebs on the apical (lumenal) membranes of marginal cells (arrow). (G and H) Projections of confocal image stacks of cochlear cryo-sections from an Alms1^−/− mouse, labeled with an antibody against acetylated tubulin (green) that labels cytoplasmic microtubules in marginal cells, and phalloidin (red) that labels the basal cell layer and the apical region of marginal cells. (G) Large intercellular spaces in stria vascularis (arrows) were apparent in all cochlear turns. (H) Detail of very large lesions in the lower region of stria vascularis in the apical turn region. (I) Endocochlear potential (EP) values for individual control mice and Alms1^−/− mice were within the normal range. Dashed line shows the +80 mV level for reference. Scale bars 20 µm.

Figure 9.

Formation of membrane-bound intracellular vacuoles in Alms1^−/− mice. (A–I) Transmission electron micrographs of stria vascularis from P191 Alms1^+/+ and Alms1^−/− mice. (A) Normal appearance of Alms1^+/+ mouse stria vascularis, including marginal cells (mc), intermediate cells (ic), basal cells (bc) and a blood vessel (bv). (B) Detail of a single marginal cell. (C) Detail of a blood vessel. (D) Abnormal appearance of Alms1^−/− mouse stria vascularis. Large spaces were apparent in the intermediate cell layer, and nuclei in marginal cells and basal cells were atypical in shape. (E) Detail of a single marginal cell, showing a sub-cellular vacuole (marked *) above the nucleus and a bleb extending from the apical membrane (arrow). (F) Detail of the apical membrane of a marginal cell, showing amorphous material in the apical bleb, and microfilament assemblies bordering the bleb (arrows). (G) Large membrane-bound intracellular vacuoles (*) in the intermediate cell layer, above the basal cell layer. The vacuoles contained cytoplasmic material. (H) Large membrane-bound intracellular vacuoles (*) inside an intermediate cell causing deformation of the nucleus. (I) Normal appearance of a blood vessel. Scale bars, 2 µm.