Development and Protein Composition of the Striated Organelle and Spectrin Localization in the Inner Ear

The striated organelle (SO) is an intriguing cytoskeletal structure that has consistently been observed to occur in the subcuticular region of inner ear vestibular type I and type II hair cells, and cochlear inner (but not outer) hair cells, in most vertebrates. Its function is still unknown, and what little has been reported of its protein composition consists largely of multifunctional, common, everyday proteins (such as α-II-spectrin and F-actin) whose inclusion in the SO make-up offers no obvious clues from which we can infer function. What is known from previous studies, however, is that the SO has been observed to occur in both normal and diseased hair cells; is intricately linked to microtubules, stereociliar rootlets and ‘giant’ apical mitochondria; and is composed of alternating bands of filaments 10nm and 35nm thick that lie 65nm apart. Going forward the primary objectives of this work were to: 1) Determine some of the major proteins making up the organelle by identifying those interacting with alpha-II-spectrin; 2. Determine the timeline of the SO appearance in the hair cells by tracking the developmental expression of αII spectrin; and 3) map all the beta spectrins to identify the expression patterns and the β-spectrin partner for αII-spectrin, which could then be incorporated into 1) and 2) above. To accomplish these aims, I primarily relied on and performed immunohistochemistry (confocal and electron microscopy), western blots, immunoprecipitations, and liquid chromatography-mass spectroscopy for adata acquisition. From my data it appears that βII-spectrin is the pairing partner to αII-spectrin in the cuticular plate, striated organelle and lateral membranes of all vestibular cell types except type I hair cells; striated organelle biogenesis is postnatal, occurring between postnatal days 3 and 4 (P3-P4), and is preceded by maturation of the cuticular plate; all seven mammalian spectrin subunits occur in the inner ear sensory epithelium. Our work was limited to structural characterization and function of the SO still remains unresolved

Glutamate transporters EAAT4 and EAAT5 are expressed in vestibular hair cells and calyx endings.

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.</p

EAAT4 and EAAT5 protein localization in mouse tissue.

<p>Electron micrographs of EAAT4 (<b>A</b>) and EAAT5 (<b>B</b>) immunogold labeling particles on hair-cell membrane <i>(arrows),</i> calyx inner-face membrane <i>(arrowheads)</i> and calyx outer-face membrane <i>(arrow</i>, lower right in <i>A).</i> In both panels from top to bottom, the darkened area is a hair-cell nucleus rimmed by hair-cell cytoplasm, hair-cell and calyx inner-face membranes. The lightened area with gray mitochondria is a calyx ending whose outer-face abuts supporting cells. Scale bars: 0.5 µm.</p

Schematic representation of EAAT4 and EAAT5 expression in the mammalian vestibular neuroepithelium.

<p>EAAT4 and EAAT5 are expressed in type I (<i>right</i>) and type II (<i>left</i>) hair cells and on the calyx inner face. EAAT4, but not EAAT5, is also expressed on the calyx outer face. EAAT5 may have a higher expression in type I versus type II hair cells. Both transporters are preferentially expressed in the subnuclear region of hair cells. EAAT1 is expressed in supporting cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Takumi1" target="_blank">[22]</a>.</p

In the EAAT4-eGFP mouse, the expression of eGFP is under the control of the EAAT4 promoter[23].

<p>The GFP fluorescence is observed in type I <i>(white arrows)</i> and type II hair cells using an anti-eGFP antibody with phalloidin red labeling of hair bundles. Scale bar: 10 µm.</p

EAAT4 and EAAT5 mRNA expression in mouse tissue.

<p>(<b>A</b>) Retina (Ret), vestibular epithelia (VE), or vestibular ganglion (VG) were pooled from ten mice. RT-PCR using substrate-specific primers show expression of both EAAT4 (first panel, 329 bp) and EAAT5 (second panel, 246 bp) in various tissues. Actin PCR was used as a control (third panel, 539 bp). (<b>B, D</b>) <i>In situ</i> hybridization using EAAT4- or EAAT5-specific antisense probes in the utricular macula. Both EAAT4 and EAAT5 mRNA are found in the hair-cell layer (<i>HC</i>), but not in the supporting-cell layer (<i>SC</i>). (<b>C, E</b>) Specific sense controls showed no labeling. Scale bars, 20 µm (B–E).</p

Transporter-mediated chloride conductance in partially isolated type I vestibular hair cells (<i>VHCs</i>).

<p>(<b>A</b>) Differential interference contrast micrograph of a recording electrode tip approaching the base of an amphora-shaped, type I utricular hair cell. (<b>B</b>) Electrophysiological protocol used to identify type I hair cells (<i>upper traces</i>) versus type II hair cells (<i>bottom traces</i>). As previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Gaboyard1" target="_blank">[27]</a>, only type I cells exhibit <i>I_KL</i>, an outwardly rectifying current activated at rest that was evidenced by an instantaneous current upon stepping to higher voltages (<i>leftmost arrow</i>) and deactivated by hyperpolarization <i>(rightmost arrow)</i>. (<b>C</b>) Inward current evoked in different type I VHCs voltage-clamped at −80 mV upon application of glutamate <i>(uppermost trace)</i>; inward current enhanced by substitution of thiocyanate (<i>SCN^–</i>) for chloride <i>(second trace)</i>; the glutamate-evoked current was blocked following application of dl-TBOA <i>(third trace)</i>. Application of glutamate did not evoke a current in type II VHCs <i>(bottom trace)</i>. (<b>D</b>) Glutamate activated current under different conditions (glutamate or aspartate at various concentrations, chloride or thiocyanate anion intracellularly, and type I or type II VHCs). Each bar represents mean ± sd. Brackets with asterisk indicate <i>Mann-Whitney U</i> comparisons (<i>p</i><0.05 for glutamate, 100 µM vs 1 mM and 1 mM Cl^- vs 1 mM SCN^-); sample sizes (<i>n</i>) in parentheses. (<b><i>E</i></b>) Currents evoked by 1 mM glutamate application at various holding potentials on a type I VHC, from −120 to +20 mV in 20 mV steps. (<b>F</b>) Current-voltage relationship of glutamate-evoked responses obtained with <i>E</i>_Cl  =  −0.5 mV (n = 7) and <i>E</i>_Cl  =  −30.3 mV (n = 5). Peak currents at each holding potential were normalized to responses at −80 mV. Dots represent mean ± sem. When no bar is shown, the SEM was too small.</p

EAAT4 (<i>A, C, E</i>) and EAAT5 (<i>B, D, F</i>) protein localization in mouse tissue.

<p>Western blots using antibodies against EAAT4 (<b>A</b>, ∼61 kD) or EAAT5 (<b>B</b>, ∼62kD). EAAT4 is expressed in the cerebellum (<i>Cbl</i>), vestibular ganglia (<i>VG</i>) and vestibular organs (<i>VE</i>). EAAT5 is expressed in the retina (<i>Rt</i>), vestibular ganglia (<i>VG</i>) and vestibular organs (<i>VE</i>). Immunohistochemistry on utricular sections shows that EAAT4 (<b>C</b>) and EAAT5 (<b>D</b>) labeling (red, top and bottom panels) is conspicuous below the nuclei of both types of hair cells (<i>I, II</i>), where ribbon synapses are abundant. Calretinin labeling (green, top panels) marks type II hair cells (II) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Lysakowski2" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Desai1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Desai2" target="_blank">[46]</a>. Immunohistochemistry of vestibular ganglion cells shows weak EAAT4 (<b>E</b>) and EAAT5 (<b>F</b>) labeling. Scale bars: 10 µm.</p