Immunocytochemical Localization of the Translocase of the Outer Mitochondrial Membrane (Tom20) in the Human Cochlea

Authors

  • Ashley E. Balaker,

    1. Department of Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1624, USA
    Search for more papers by this author
  • Paul Ishiyama,

    1. Department of Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1624, USA
    Search for more papers by this author
  • Ivan A. Lopez,

    Corresponding author
    1. Department of Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1624, USA
    • Department of Head and Neck Surgery, UCLA School of Medicine, 35-64 Rehabilitation Center, 1000 Veteran Avenue, Los Angeles CA 90095
    Search for more papers by this author
    • Fax: 310-206-1513.

  • Gail Ishiyama,

    1. Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1624, USA
    Search for more papers by this author
  • Akira Ishiyama

    1. Department of Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1624, USA
    Search for more papers by this author

Abstract

Mitochondrial degeneration in the inner ear is likely a contributing factor in age-related hearing loss and other otopathologies such as Meniere's disease. Most mitochondrial proteins are synthesized in the cytosol and imported through the mitochondrial membranes by translocators. The translocase of the outer membrane (Tom) is the universal entry gate for all proteins that are imported into mitochondria. Altered function of the translocator could alter protein transport into the mitochondria, and disrupt function. In this study, we determined the immunolocalization of Tom20, a major mitochondrial protein import receptor, in microdissected human cochlea frozen sections obtained from postmortem autopsy and celloidin-embedded archival specimens. We used affinity purified rabbit polyclonal antibodies against Tom20. We also determined the Tom20 immunolocalization in the mouse inner ear. In the human and mouse cochlea, Tom20 was ubiquitously distributed in the organ of Corti, allowing well-delineated visualization of inner and outer hair cells. Tom20 immunoreactivity localized in the cytoplasm of spiral ganglia neurons. In the inner ear of aged subjects with Meniere's disease, there was decreased expression of Tom20. These results suggest that Tom20 can be used in the inner ear as a marker for mitochondrial protein import. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Mitochondria are essential energy producing organelles in most eukaryotic cells and participate in cellular processes ranging from energy production to apoptosis (Endo et al., 2003). A large number of genetic syndromes associated with hearing loss are due to mitochondrial defects (Someya and Prolla, 2010). Experimental evidence suggests that mitochondrial dysfunction associated with reactive oxygen species (ROS) plays a central role in the aging process of cochlear cells (Someya and Prolla, 2010, Kujot et al., 2005). Mitochondrial dysfunction in the inner ear may contribute to age-related hearing loss and the development of pathological conditions such as Meniere's disease, ototoxicity, and otosclerosis.

Mitochondria are bounded by two membranes, the outer and inner membranes, and consist of 500–1,000 proteins (Endo and Kohda, 2002). Most mitochondrial proteins are synthesized in the cytosol, and imported through the mitochondrial membranes by translocators (Schmidt et al., 2010). The translocator, an assembly of multiple membrane protein subunits, performs multiple functions (Schleiff and Becker, 2011). The translocase of the outer membrane (Tom) is the universal entry gate for all proteins that are imported into mitochondria (Baker et al., 2007; Perry et al., 2008). A recent study by Someya et al. (2007) showed that the genes encoding mitochondrial respiratory components are profoundly down regulated with age in the cochlea of DBA/2J mice. Furthermore, among the genes involved in mitochondrial transport, four of nine genes were significantly down regulated. Dysfunction in protein transport by Tom could lead to deterioration in normal mitochondrial function (Li et al., 2010). To our knowledge, there are no studies addressing the localization or altered expression of mitochondrial transporters in the inner ear in normal and pathological conditions.

In this study, we determine the localization of Tom20 (the major mitochondrial import receptor) by immunocytochemistry in formalin fixed-frozen microdissected human cochlea, and in celloidin-embedded temporal bone specimens from patients with normal auditory function, patients diagnosed with Meniere's disease, and other otologic and non-otologic diseases. We also determined the Tom20 immunolocalization in the mouse inner ear. In the human and mouse cochlea, Tom20 was ubiquitously distributed in the organ of Corti, allowing well-delineated visualization of inner and outer hair cells. Tom20 immunoreactivity (IR) localized in the cytoplasm of spiral ganglia neurons (SGNs). In the inner ear of aged subjects with Meniere's disease, there was decreased expression of Tom20.

MATERIALS AND METHODS

The Institutional Review Board of UCLA has approved this study. The temporal bone donors in this study were part of a National Institute of Health funded Human Temporal Bone Consortium for Research Resource Enhancement. Temporal bones were obtained at autopsy from three subjects with a documented history of normal auditory and vestibular function (age 75-year-old male, 82-year-old female, and 85-year-old female). In addition celloidin-embedded sections from archival temporal bones from 16 patients were used for Tom20 immunocytochemistry (Table 1).

Table 1. Tom20 immunoreactivity in the human spiral ganglia
TBAgeSexDiseaseB versus MB versus AM versus A
  1. Comparisons of Tom20-IR were made by one-way ANOVA. Base (B) versus medial (M), base (B) versus apical (A), Medial (M) versus Apical (A). * = P ≤ 0.05 difference in immunoreactivity was considered statistically significant different; ¶: not statistically significant. Quantification of Tom20-IR was made as described in methods section. Age= years;* = hearing and vestibular function normal; MD: Meniere's disease; SJS: Sjogren's syndrome, mild hearing loss; RA: Rheumatoid arthritis; BHL: bilateral hearing loss; DS: Down syndrome; BPPV: Benign paroxysmal vertigo.

18FemaleDS***
216MaleNormal***
317MaleNormal***
455FemaleMD
559FemaleNormal***
660FemaleRA*
763FemaleOtotoxicity
865FemaleOtosclerosis
965FemaleSJS
1068FemaleMD***
1275FemaleBPPV
1176FemaleBHL, dizziness
1378MaleBHL**
1480MalePresbycusis
1583FemaleMD
1692FemaleMD

Temporal bone removal and tissue processing: methods for human tissue processing have been described in detail (Lopez et al., 2005, 2007; Ishiyama et al., 2009, 2010). In brief, at autopsy the whole brain, including the brainstem and blood vessels, was removed from the cranial cavity. The eighth cranial nerve and vascular bundle were sectioned outside the internal auditory canal. The temporal bones were then removed using a bone plug cutter. The bones were then immediately immersed for 16 hr in cold 4% paraformaldehyde in 0.11 M sodium phosphate buffer (PBS), pH 7.4. Thereafter, the fixative was removed by washing with PBS (10 min × 3). The temporal bones were placed under a dissecting microscope (Nikon SMZ1500), and using forceps, the muscle, connective tissue, and bone surrounding the membranous labyrinth were carefully removed as previously described (Lopez et al., 2005). Individual endorgans (i.e., crista, macula, and cochlea) were then removed and immersed in 30% sucrose in PBS for 7 days. Before sectioning, each cochlea was embedded in Tissue-Tek and placed on Teflon embedding molds (Polysciences) and properly oriented under the dissecting microscope to obtain longitudinal mid-modiolar sections. Twenty-micron thick serial sections were obtained using a Microm-H cryostat (Microm-HN505E). The cryosections were mounted on Superfrost plus glass slides (Fisher Scientific, Pittsburgh, PA) and stored at −80°C until their use.

Immunofluorescence

Tissue sections were incubated at room temperature for 60 min with a blocking solution containing 1% bovine serum albumin (BSA) fraction-V (Sigma, St. Louis, MO) and 0.5% Triton X-100 (Sigma) in PBS. At the end of the incubation, the blocking solution was removed and the primary polyclonal antibody against Tom20 was applied. The primary antibody was incubated for 16 hr at 4°C in a humid chamber. Alexa 594 anti-rabbit secondary antibody (1:1,000, Molecular Probes, Carlsbad, CA) was applied for 2 hr at room temperature in the dark. Then, sections were washed with PBS (3 × 10 min) and covered with Vectashield mounting media (Vector Labs, Burlingame, CA) containing DAPI to visualize all cell nuclei.

Affinity purified rabbit polyclonal antibodies against Tom20 (1:500, Santa Cruz, CA) were used. Tom20 antibody (FL-145) cat # sc-11415 (Santa Cruz, CA), is a rabbit polyclonal antibody raised against aminoacids 1-145 representing full length Tom20 of human origin. This antibody is recommended for detection of Tom20 from mouse, rat, and human. It recognizes a band of 20 kDa by Western blot in whole cell lysates.

Immunofluorescence Staining in the Mouse Inner Ear

Mice were handled and cared for in accordance with the Animal Welfare Act and in strict compliance with the National Institute of Health Guidelines. CBA/J mice (6 weeks of age; n = 4, male 12-weeks-old) were over anesthetized with halothane and then decapitated. Methods for mouse tissue processing have been described in detail (Lopez et al., 2009). Twelve-micron thick cryostat sections were obtained (Microm HM505E) from the temporal bones and immunofluorescence staining was performed as described above for the human inner ear, and according to Lopez et al. (2009).

Controls

Cryostat sections from rat and mouse cerebellum were incubated with the antibodies described above. These sections were subjected to the same immunocytochemistry protocol. As negative control, the primary antibody was omitted and the immunoreaction was performed in the human cochlea sections as described above.

Archival Celloidin Specimens

The methodology for celloidin removal and antigen retrieval steps has been described (O'Malley et al., 2009) and adapted to our laboratory. Celloidin sections were placed in a glass Petri dish and immersed in 100% acetone for 2 × 15 min. Thereafter, sections were sequentially immersed in a mixture of sodium-ethoxide-100% ethanol (1:3) for 10 min; 100% ethanol, 50% ethanol, and distilled water (5 min each) and then immersed in hydrogen peroxide 3% in methanol (10 min). Slides were then washed with double distilled water before the antigen retrieval step. Slides were placed horizontally in a glass Petri dish that contained antigen retrieval solution (Vector Antigen Unmasking Solution, Vector Labs, Burlingame CA diluted 1:200 with distilled water). Sections were heated in the microwave oven using intermittent heating of two 2-min cycles with an interval of 1 min between the heating cycles. The Petri dishes were allowed to cool for 15 min and washed with PBS for 10 min.

Immunocytochemistry

Sections were incubated for 1 hr with a blocking solution containing 5% normal goat serum/1% BSA fraction-V (Sigma, St. Louis, MO) and 0.5% Triton X-100 (Sigma) in PBS. Incubation with primary antibodies against Tom20 was performed for 48 hr at 4°C in a humid chamber. The sections were washed with PBS (15 min × 3), and then incubated for 1 hr with biotinylated secondary antibody, goat anti-rabbit polyclonal IgG (1:1,000, Vector Labs, Burlingame, CA), and then washed with PBS (5 min × 3). Next, 1-hr incubation was performed with Vectastain Elite ABC reagent (Vector Labs) followed by PBS washes (15 min × 3). Immunoperoxidase staining was performed using Immpact DAB solution (Vector Labs). The reaction was stopped with distilled water washes (5 min × 3) after 1–2 min. Slides were mounted with Vectamount AQ aqueous mounting media (Vector Labs) and glass cover slips.

Microscopic Observation and Documentation

Immunoreacted tissue sections were viewed and imaged with an Olympus BX51 fluorescent microscope (Olympus America, NY) equipped with an Olympus DP70 digital camera. To provide unbiased comparisons of the immunoreactive signal between each specimen, all images were captured using strictly the same camera settings. Images were acquired using MicroSuite™ Five software (Olympus America). All images were prepared using the Adobe Photoshop software program run in a Dell Precision 380 computer. Confocal images were acquired using a Carl Zeiss Laser Scanning Microscope model LSM 510 Meta Zeiss (Jena, Germany).

Quantification of Immunostained Areas

Quantitative immunocytochemistry for Tom20 in the cochlea was performed as described by Ishiyama et al. (2010). To minimize bias in the analysis, the observer was “blinded” to the identity of the tissue samples to be analyzed. A second person not blinded to the sample identity, coded each sample. Observations were made at the apical, medial, and basal portions of the spiral ganglia, organ of Corti and lateral wall (at low magnification 200×). Image acquisition and quantitative analyses were made using micrographs acquired at 400×. The area immunostained was quantified using the computer image analysis software ImageJ (http://rsb.info.nih.gov/ij/index.html, version v1.47a) with the protocol described by Ishiyama et al. (2010) and Calzada et al. (2012). Tom20 immunoreactive area was measured at the apical, middle, and basal region of each cochlea section to determine whether there were regional variations in its expression. The immunostained area measured in each region included the SGNs and their surrounding neuropil.

Statistical Analysis

For each specimen, mean values of the immunoreacted area were averaged and subjected to one-way repeated measures analysis of variance. Comparisons were made between the three cochlea regions as follow: base versus medial, base versus apical, and medial versus apical. Values were considered statistically significant at P ≤ 0.05. The Sigma Stat 3.1 software program (Sigma Stat, Ashburn, VA) was used for statistical analysis. Because of the small number of specimens, no comparisons of Tom20-IR were made between different diseases, age, or gender.

RESULTS

Tom20 Immunolocalization in the Normal Cochlea (Frozen Formalin-Fixed Sections)

Tom20 IR was localized within cells of the stria vascularis and the spiral ligament (Fig. 1a). In the organ of Corti, Tom20-IR was present in supporting cells (Deiters cells, inner and outer pillar cells), inner and outer hair cells (Fig. 1b). Fig. 1b′ outlined the location of the different cells of the organ of Corti. Tom20-IR was also present in the SGNs (Fig. 1c). Consistent with its localization in mitochondria, Tom20-IR appeared as granular dense accumulations within the cytoplasm of cells in the spiral ligament, stria vascularis, organ of Corti and SGNs.

Figure 1.

Confocal images of Tom20 IR in the normal human inner ear. (a) Stria vascularis, (b) Organ of Corti, (1b′) outlined the locations of the different cells of the organ of Corti. (c) SGNs. * indicate the non-immunoreactive cell nucleus. sl: spiral ligament; sv: stria vascularis; ihc: inner hair cells; ohc: outer hair cells; tc: tunnel of Corti; ipc: inner pillar cells; outer pillar cells (opc); Dc; Deiters cells. Bar is 50 μm.

Tom20-IR in Celloidin Embedded Sections

Tom20-IR was also investigated in celloidin-embedded temporal bone specimens from patients with normal auditory function (n = 3), patients diagnosed with Meniere's disease (n = 4), and other otologic and non-otologic diseases (n = 9) (Table 1). In the normal cochlea (female 59-year-old), Tom20-IR localized to the stria vascularis and the spiral ligament (Fig. 2a), the organ of Corti (Fig. 2a1), and SGNs (Fig. 2a2). In one specimen diagnosed with Meniere's disease (female, 55-year-old), Tom20-IR in the spiral ligament (Fig. 2b), organ of Corti (Fig. 2b1), and SGNs (Fig. 2b1) was similar to the normal specimen. In contrast in an 82-year-old patient diagnosed with Meniere's disease there was a marked decrease in Tom20-IR in the stria vascularis (Fig. 2c) and organ of Corti (Fig. 2c1) but not in SGNs (Fig. 2c2). A similar decrease in Tom20-IR in the organ of Corti was observed in an 80-year-old patient diagnosed with presbycusis (age-related sensorineural hearing loss).

Figure 2.

Distribution of Tom20 in celloidin embedded temporal bone sections. (a, a1, and a2) Tom20-IR in the normal cochlea (55-year-old female). Tom20-IR was localized in the stria vascularis, organ of Corti and neurons of the spiral ganglia, respectively. b, b1, and b2 show Tom20-IR in the stria vascularis organ of Corti and SGNs, and of a Meniere's disease specimen (59-year-old, female). c, c1, and c2 show Tom20-IR in the stria vascularis organ of Corti and SGN, and of a Meniere's disease specimen (83-year-old, female). * indicate the non-immunoreactive cell nucleus. Ihc: inner hair cells, ohc: outer hair cells, tc: tunnel of Corti, sl: spiral ligament, sv: stria vascularis Magnification bar is 150 μm in all figures.

Tom20-IR comparisons between the basal and medial region, basal and apical portion, and medial versus the apical regions of the spiral ganglia showed statistically significant differences in seven patients (Table 1). The basal and medial region showed more intense Tom20 staining than the apical region. In contrast, nine patients showed no statistically significant differences in Tom20-IR in SGNs between the three regions. Given the diversity of specimens used (age, sex, normal, and disease) and small sample size, no Tom20-IR comparisons were made between specimens.

Tom20-IR in the Mouse Cochlea

Similar to the human cochlea, Tom20-IR was observed in supporting cells, and inner and outer hair cells of the organ of Corti of the mouse (Fig. 3a). There was no discernible regional variation of Tom20-IR pattern from the basal to the apical portion of the mouse cochlea. Figure 3b shows a high magnification of the organ of Corti; the cytoplasm of inner hair cells shows strong punctated IR. Tom20-IR also localized in Dieters cells located underneath the outer hair cells. The inset in Fig. 3b outlines the location of the different cells types of the organ of Corti. SGNs were also immunoreactive to Tom20 (Fig. 3c). Negative controls (incubation with normal serum or no primary antibody) did not demonstrate IR within the cochlear tissues. In a cross-section of the human cochlea immunostained with all the reagents except for the Tom20 antibody, no specific IR was observed (Fig 3d).

Figure 3.

Tom20-IR in the mouse cochlea. (a) Tom20-IR was present in the spiral ligament, the stria vascularis or organ of Corti. (b) shows high magnification view of the organ of Corti. (c) SGNs were also immunoreactive. (d) Shows a cross-section of the human cochlea in the SGNs area immunostained with all the reagents except for the Tom20 antibody, no specific reaction was observed. Ihc: inner hair cells, ohc: outer hair cells, tc: tunnel of Corti, sl: spiral ligament, sv: tria vascularis. Magnification bar in (a) is, 200 μm, (b) 100, (c) 150 μm, and (d) 200 μm.

DISCUSSION

Using formalin fixed sections from temporal bones obtained at autopsy and archival temporal bone celloidin-embedded sections, we determined the immunolocalization of Tom20 in the human inner ear. Tom20-IR was ubiquitously distributed in the human cochlea. Specifically, Tom20 was found in cells of the stria vascularis, spiral ligament, inner and outer hair cells, and supporting cells in the organ of Corti, and neurons of the spiral ganglia. Tom20-IR was qualitatively decreased in the cochlea of old age and Meniere's disease specimens when compared with normal cochlea sections.

In this study, we used celloidin embedded sections from our temporal bones collected for several years, or human inner ear tissue obtained postmortem. Frozen formalin-fixed sections obtained from the mouse cochlea showed a similar immunoreactive pattern. Once the localization of Tom20 is shown, it will be possible to design specific in vitro assays to investigate its function in the inner ear of animal models from which it may be possible to isolate viable mitochondria that can be used to test, for example, changes in membrane potential.

There were no regional changes in Tom20-IR in SGNs from the basal to the apical regions in Meniere's specimens. In contrast, a study of super oxide dismutase-2 (SOD2) expression in the cochlea of mammals (Ying and Balaban, 2009), showed that SOD2 is expressed highly in SGNs in the apex and the base. An ALS-linked mutation of SOD-1 showed that mitochondrial specific pathways are damaged, including reduced protein import and decreased complex I activity (Li et al., 2010). The alteration of SOD-1 changed the expression of Tom22 and Tom40.

The decreased Tom20-IR in the stria vascularis in Meniere's disease specimens suggests that there may be strial dysfunction or loss of mitochondria in the strial cells. Alternatively, the diminished immunostaining may be due to strial and organ of Corti degeneration. No changes of Tom20-IR were detected in the SGNs of Meniere's disease specimens when compared with normal specimens of a similar age; however, from a 83- and 92-year-old Meniere's disease specimens there was a pronounced decrease in Tom20-IR, suggesting that the loss of Tom20 maybe due to loss of cells from aging and/or disease rather than loss of function of Tom20. It remains to be determined whether or not an alteration in Tom20 expression results in auditory dysfunction.

We performed our study mainly in older individuals that in general show a decline in auditory function, due to a decrease in the number of hair cells, and SGNs among other changes. In this respect, atrophy of the stria vascularis has been documented in patients with Meniere's disease (Ishiyama et al., 2007), aging, ototoxicity, and noise exposure.

Disease and aging can decrease activities of the mitochondrial transport chain complexes, and increase the release of ROS from mitochondria, suggesting that the mitochondrial loss of function may result from the increased oxidative damage to proteins, lipids, and DNA of this organelle (Figueiredo et al., 2008). Although the most common pathological correlate of Meniere's disease is endolymphatic hydrops, the cause remains unclear and the hydrops may be an epiphenomenon (Merchant et al., 2005). Recent theories, including genetics, autoimmune disorders, perturbation in fluid dynamics and oxidative stress have been implicated in the development of endolymphatic hydrops (Semaan et al., 2005; Calabrese et al., 2010).

Oxidative stress in a physiological setting occurs when there is an excessive bioavailability of ROS, which is the net result of an imbalance between production and destruction of ROS (with the latter being influenced by antioxidant defenses). Oxidative stress has been shown to be a component of many neurodegenerative diseases, and it has been implicated in other conditions such as asthma, cancer, and autoimmune disease (Khansari et al., 2009). A recent study by Calabrese et al. (2010), detected reduced glutathione and thioredoxin in lymphocytes from Meniere's disease patients. The authors noted that in specimens from Meniere's disease, the induction of HSP70 is maintained in response to counteracting the intracellular pro-oxidant status generated by decreased content of GHS and the expression of thioredoxyn. Persistent oxidative stress will affect mitochondrial activity that in turn will affect adequate intracellular metabolic energy crucial for the transport of ions and water. Cytochrome oxidase, one of the most important respiratory enzymes, is decreased in a guinea pig model of endolymphatic hydrops (Hsu, 1992).

Changes in cytochrome C IR with age have been detected in the human temporal bone (Keithley et al., 2001). The ubiquitous presence of Tom20 in the inner ear and decreased Tom20-IR in temporal bones from older patients with Meniere's disease, support the idea that a mitochondrial dysfunction may be a contributor of hearing loss in Meniere's disease. To date no studies on Tom-20 normal expression and experimental paradigms have been performed in the inner ear of any animal model. Our results suggest that Tom20 can be used as a marker for mitochondria and could indicate to some extent the functional status of these energy producing organelles in the inner ear. The localization of Tom20 in both mouse and human inner ear could allow the design of physiological and molecular biological experiments to test the normal function of these transporters in the inner ear.

In conclusion, Tom20 is widely expressed in the human cochlea. There was a consistent decrease of Tom20-IR in the organ of Corti and stria vascularis in subjects with older age and Meniere's disease. There was also diminished Tom20 expression in the stria and the organ of Corti in presbycusis. Other important markers for inner ear function can be detected using human inner ear sections obtained from microdissected temporal bone specimens or archived celloidin-embedded human inner ear sections.

Acknowledgements

The authors acknowledge Dr Larry Hoffman from the Department of Head and Neck Surgery at UCLA for the use of the laser confocal microscope.

Ancillary