Portal de Periódicos da CAPES

Auditory Pathology in a Transgenic mtTFB1 Mouse Model of Mitochondrial Deafness

2015; Elsevier BV; Volume: 185; Issue: 12 Linguagem: Inglês

10.1016/j.ajpath.2015.08.014

1525-2191

Sharen E. McKay, Wayne Yan, Jessica Nouws, Maximilian J. Thormann, Nuno Raimundo, Abdul Khan, Joseph Santos‐Sacchi, Lei Song, Gerald S. Shadel,

RNA modifications and cancer

The A1555G mutation in the 12S rRNA gene of human mitochondrial DNA causes maternally inherited, nonsyndromic deafness, an extreme case of tissue-specific mitochondrial pathology. A transgenic mouse strain that robustly overexpresses the mitochondrial 12S ribosomal RNA methyltransferase TFB1M (Tg-mtTFB1 mice) exhibits progressive hearing loss that we proposed models aspects of A1555G-related pathology in humans. Although our previous studies of Tg-mtTFB1 mice implicated apoptosis in the spiral ganglion and stria vascularis because of mitochondrial reactive oxygen species–mediated activation of AMP kinase (AMPK) and the nuclear transcription factor E2F1, detailed auditory pathology was not delineated. Herein, we show that Tg-mtTFB1 mice have reduced endocochlear potential, indicative of significant stria vascularis dysfunction, but without obvious signs of strial atrophy. We also observed decreased auditory brainstem response peak 1 amplitude and prolonged wave I latency, consistent with apoptosis of spiral ganglion neurons. Although no major loss of hair cells was observed, there was a mild impairment of voltage-dependent electromotility of outer hair cells. On the basis of these results, we propose that these events conspire to produce the progressive hearing loss phenotype in Tg-mtTFB1 mice. Finally, genetically reducing AMPK α1 rescues hearing loss in Tg-mtTFB1 mice, confirming that aberrant up-regulation of AMPK signaling promotes the observed auditory pathology. The relevance of these findings to human A1555G patients and the potential therapeutic value of reducing AMPK activity are discussed. The A1555G mutation in the 12S rRNA gene of human mitochondrial DNA causes maternally inherited, nonsyndromic deafness, an extreme case of tissue-specific mitochondrial pathology. A transgenic mouse strain that robustly overexpresses the mitochondrial 12S ribosomal RNA methyltransferase TFB1M (Tg-mtTFB1 mice) exhibits progressive hearing loss that we proposed models aspects of A1555G-related pathology in humans. Although our previous studies of Tg-mtTFB1 mice implicated apoptosis in the spiral ganglion and stria vascularis because of mitochondrial reactive oxygen species–mediated activation of AMP kinase (AMPK) and the nuclear transcription factor E2F1, detailed auditory pathology was not delineated. Herein, we show that Tg-mtTFB1 mice have reduced endocochlear potential, indicative of significant stria vascularis dysfunction, but without obvious signs of strial atrophy. We also observed decreased auditory brainstem response peak 1 amplitude and prolonged wave I latency, consistent with apoptosis of spiral ganglion neurons. Although no major loss of hair cells was observed, there was a mild impairment of voltage-dependent electromotility of outer hair cells. On the basis of these results, we propose that these events conspire to produce the progressive hearing loss phenotype in Tg-mtTFB1 mice. Finally, genetically reducing AMPK α1 rescues hearing loss in Tg-mtTFB1 mice, confirming that aberrant up-regulation of AMPK signaling promotes the observed auditory pathology. The relevance of these findings to human A1555G patients and the potential therapeutic value of reducing AMPK activity are discussed. Mitochondria are essential organelles that produce ATP via the process of oxidative phosphorylation, but are multifunctional, playing additional key roles in metabolism, as well as other cellular processes like apoptosis, inflammation, and signal transduction.1West A.P. Shadel G.S. Ghosh S. Mitochondria in innate immune responses.Nat Rev Immunol. 2011; 11: 389-402Crossref PubMed Scopus (868) Google Scholar, 2Kasahara A. Scorrano L. Mitochondria: from cell death executioners to regulators of cell differentiation.Trends Cell Biol. 2014; 24: 761-770Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 3Chandel N.S. Mitochondria as signaling organelles.BMC Biol. 2014; 12: 34Crossref PubMed Scopus (320) Google Scholar, 4Hill S. Van Remmen H. Mitochondrial stress signaling in longevity: a new role for mitochondrial function in aging.Redox Biol. 2014; 2: 936-944Crossref PubMed Scopus (89) Google Scholar Human mitochondria possess a 16,565-bp circular genome that is maternally inherited and present at hundreds to thousands of copies per cell in most tissues.5Shadel G.S. Clayton D.A. Mitochondrial DNA maintenance in vertebrates.Annu Rev Biochem. 1997; 66: 409-435Crossref PubMed Scopus (814) Google Scholar In mammals, mitochondrial DNA (mtDNA) encodes 13 oxidative phosphorylation complex subunits and the two rRNAs and 22 tRNAs needed for translation of these by dedicated mitochondrial ribosomes.6Shutt T. Shadel G. A compendium of human mitochondrial gene expression machinery with links to disease.Environ Mol Mutagen. 2010; 51: 360-379PubMed Google Scholar All other components of the estimated 1200-member mitochondrial proteome, including the remaining 70 to 75 oxidative phosphorylation subunits and all of the factors needed for mtDNA replication and expression, are encoded by nuclear genes and imported into the organelle.7Bestwick M.L. Shadel G.S. Accessorizing the human mitochondrial transcription machinery.Trends Biochem Sci. 2013; 38: 283-291Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Thus, signaling pathways between mitochondria and the nucleus are required to coordinate the biogenesis, composition, and activity of mitochondria and to trigger homeostatic nuclear gene expression responses to mitochondrial dysfunction. These responses can be beneficial or harmful, depending on the precise cellular context and, to date, remain poorly understood. Mitochondrial dysfunction causes human diseases, with an estimated occurrence of 1 in 5000 to 10,000 live births.8Wallace D.C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.Annu Rev Genet. 2005; 39: 359-407Crossref PubMed Scopus (2530) Google Scholar, 9DiMauro S. Schon E. Mitochondrial respiratory-chain diseases.N Engl J Med. 2003; 348: 2656-2668Crossref PubMed Scopus (1293) Google Scholar, 10Vafai S.B. Mootha V.K. Mitochondrial disorders as windows into an ancient organelle.Nature. 2012; 491: 374-383Crossref PubMed Scopus (500) Google Scholar These can be inherited maternally, because of mutations in mtDNA, or in a Mendelian manner, because of mutations in nuclear genes encoding mitochondrial components. Because mtDNA is present in multiple copies per cell, and different organs vary in their energy requirements, mitochondrial diseases are complicated and heterogeneous, characterized by cell- and tissue-specific responses and pathology.2Kasahara A. Scorrano L. Mitochondria: from cell death executioners to regulators of cell differentiation.Trends Cell Biol. 2014; 24: 761-770Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 8Wallace D.C. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.Annu Rev Genet. 2005; 39: 359-407Crossref PubMed Scopus (2530) Google Scholar, 9DiMauro S. Schon E. Mitochondrial respiratory-chain diseases.N Engl J Med. 2003; 348: 2656-2668Crossref PubMed Scopus (1293) Google Scholar, 10Vafai S.B. Mootha V.K. Mitochondrial disorders as windows into an ancient organelle.Nature. 2012; 491: 374-383Crossref PubMed Scopus (500) Google Scholar An extreme example of tissue specificity is the A1555G mtDNA mutation that causes maternally inherited deafness. This mutation causes a progressive, nonsyndromic hearing loss that can vary from mild to profound and has a variable age of onset.11Guan M.X. Fischel-Ghodsian N. Attardi G. Nuclear background determines biochemical phenotype in the deafness-associated mitochondrial 12S rRNA mutation.Hum Mol Genet. 2001; 10: 573-580Crossref PubMed Scopus (163) Google Scholar, 12Prezant T.R. Shohat M. Jaber L. Pressman S. Fischel-Ghodsian N. Biochemical characterization of a pedigree with mitochondrially inherited deafness.Am J Med Genet. 1992; 44: 465-472Crossref PubMed Scopus (27) Google Scholar A1555G carriers can also be predisposed to aminoglycoside-induced deafness.13Prezant T.R. Agapian J.V. Bohlman M.C. Bu X. Oztas S. Qiu W.Q. Arnos K.S. Cortopassi G.A. Jaber L. Rotter J.I. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness.Nat Genet. 1993; 4: 289-294Crossref PubMed Scopus (995) Google Scholar Hearing loss induced by A1555G is incompletely penetrant, which has been attributed, at least in part, to multiple nuclear and mtDNA modifying loci.11Guan M.X. Fischel-Ghodsian N. Attardi G. Nuclear background determines biochemical phenotype in the deafness-associated mitochondrial 12S rRNA mutation.Hum Mol Genet. 2001; 10: 573-580Crossref PubMed Scopus (163) Google Scholar, 14Zhao H. Li R. Wang Q. Yan Q. Deng J.-H. Han D. Bai Y. Young W.-Y. Guan M.-X. Maternally inherited aminoglycoside-induced and nonsyndromic deafness is associated with the novel C1494T mutation in the mitochondrial 12S rRNA gene in a large Chinese family.Am J Hum Genet. 2004; 74: 139-152Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 15Dai P. Liu X. Han D. Qian Y. Huang D. Yuan H. Li W. Yu F. Zhang R. Lin H. He Y. Yu Y. Sun Q. Qin H. Li R. Zhang X. Kang D. Cao J. Young W.Y. Guan M.X. Extremely low penetrance of deafness associated with the mitochondrial 12S rRNA mutation in 16 Chinese families: implication for early detection and prevention of deafness.Biochem Biophys Res Commun. 2006; 340: 194-199Crossref PubMed Scopus (52) Google Scholar One such nuclear modifier is the TFB1M gene, which encodes an RNA methyltransferase that post-transcriptionally modifies the mtDNA-encoded 12S rRNA in mitochondrial ribosomes.16Bykhovskaya Y. Mengesha E. Wang D. Yang H. Estivill X. Shohat M. Fischel-Ghodsian N. Human mitochondrial transcription factor B1 as a modifier gene for hearing loss associated with the mitochondrial A1555G mutation.Mol Genet Metab. 2004; 82: 27-32Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar The A1555G mutation is located near a highly conserved stem loop in the 12S rRNA that contains adjacent adenines that are dimethylated by TFB1M. We showed previously that patient-derived A1555G cybrid cell lines exhibit increased stem-loop dimethylation and/or increased amounts of 12S rRNA methylated at this site.17Cotney J. McKay S.E. Shadel G.S. Elucidation of separate, but collaborative functions of the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 in mitochondrial biogenesis reveals new insight into maternally inherited deafness.Hum Mol Genet. 2009; 18: 2670-2682Crossref PubMed Scopus (69) Google Scholar, 18Raimundo N. Song L. Shutt T.E. McKay S.E. Cotney J. Guan M.-X. Gilliland T.C. Hohuan D. Santos-Sacchi J. Shadel G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell. 2012; 148: 716-726Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar These cells also exhibited enhanced apoptotic susceptibility in culture17Cotney J. McKay S.E. Shadel G.S. Elucidation of separate, but collaborative functions of the rRNA methyltransferase-related human mitochondrial transcription factors B1 and B2 in mitochondrial biogenesis reveals new insight into maternally inherited deafness.Hum Mol Genet. 2009; 18: 2670-2682Crossref PubMed Scopus (69) Google Scholar, 18Raimundo N. Song L. Shutt T.E. McKay S.E. Cotney J. Guan M.-X. Gilliland T.C. Hohuan D. Santos-Sacchi J. Shadel G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell. 2012; 148: 716-726Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar because of mitochondrial reactive oxygen species (ROS)–dependent activation of 5′-AMP–activated protein kinase (AMPK) that unmasks the pro-apoptotic function of the nuclear transcription factor E2F1. These phenotypes are recapitulated in cell lines that overexpress the TFB1M methyltransferase in the absence of the A1555G mutation. TFB1M binds to the mitochondrial RNA polymerase (POLRMT) in the context of mitochondrial ribosomes to promote proper 12S rRNA methylation and ribosome biogenesis.19Surovtseva Y.V. Shadel G.S. Transcription-independent role for human mitochondrial RNA polymerase in mitochondrial ribosome biogenesis.Nucleic Acids Res. 2013; 41: 2479-2488Crossref PubMed Scopus (28) Google Scholar Thus, disruption of these interactions in the mature ribosome or during ribosome assembly may cause unique perturbations in mitochondrial homeostasis and increased ROS production that promote deafness. How this unique mitochondrial perturbation leads to tissue-specific pathology remains unknown. Consistent with the above pathogenic mechanism, we showed that transgenic mice that globally overexpress TFB1M (Tg-mtTFB1) exhibit premature hearing loss in an E2F1-dependent manner.18Raimundo N. Song L. Shutt T.E. McKay S.E. Cotney J. Guan M.-X. Gilliland T.C. Hohuan D. Santos-Sacchi J. Shadel G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell. 2012; 148: 716-726Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar These mice have the increased 12S rRNA methylation signature and AMPK up-regulation in multiple tissues, as well as increased caspase 3 staining and E2F1 up-regulation in two tissues in the inner ear, the stria vascularis and spiral ganglion neurons. This suggested that deafness in Tg-mtTFB1 mice is induced by a pro-apoptotic, mROS-AMPK-E2F1 pathway similar to the one we delineated in the A1555G patient cybrids, making Tg-mtTFB1 mice an indirect, yet potentially useful, animal model for maternally inherited deafness caused by the A1555G mutation.18Raimundo N. Song L. Shutt T.E. McKay S.E. Cotney J. Guan M.-X. Gilliland T.C. Hohuan D. Santos-Sacchi J. Shadel G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell. 2012; 148: 716-726Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Herein, we have characterized the mechanism of hearing loss in Tg-mtTFB1 mice and addressed directly the involvement of AMPK signaling in the pathogenic response in vivo. The original mixed C57BL/6J × SJL/J transgenic Tg-mtTFB1 mice18Raimundo N. Song L. Shutt T.E. McKay S.E. Cotney J. Guan M.-X. Gilliland T.C. Hohuan D. Santos-Sacchi J. Shadel G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell. 2012; 148: 716-726Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar were extensively back-crossed to the C57BL/6J genetic background. F8 and F9 backcrossed animals were bred to generate the Tg-mtTFB1 and wild-type (WT) littermates used in electrophysiological and histological studies. F8 Tg-mtTFB1 mice were bred to C57BL/6J heterozygous knockouts of AMPKα1+/− (Prkaa1+/−) obtained from Dr. Benoit Viollet (INSERM, Paris, France),20Jørgensen S.B. Wojtaszewski J.F.P. Viollet B. Andreelli F. Birk J.B. Hellsten Y. Schjerling P. Vaulont S. Neufer P.D. Richter E.A. Pilegaard H. Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle.FASEB J. 2005; 19: 1146-1148Crossref PubMed Scopus (236) Google Scholar, 21Faubert B. Boily G. Izreig S. Griss T. Samborska B. Dong Z. Dupuy F. Chambers C. Fuerth B.J. Viollet B. Mamer O.A. Avizonis D. DeBerardinis R.J. Siegel P.M. Jones R.G. AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo.Cell Metab. 2013; 17: 113-124Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar and the resulting AMPKα1+/−/Tg-mtTFB1 mice were bred to AMPKα1+/+ for studies of AMPK knockdown. Animals were anesthetized with either 48 mg/kg pentobarbital or 480 mg/kg chloral hydrate (i.p.), and all recordings were conducted in a sound-attenuating chamber (Industrial Acoustics Corp., Bronx, NY). A customized TDT3 system (Tucker-Davis Technologies, Inc., Alachua, FL) was used for auditory brainstem response (ABR) recordings. Subdermal needle electrodes (Rochester Electro-Medical, Inc., Lutz, FL) were positioned at the vertex (active, noninverting), the infra-auricular mastoid region (reference, inverting), and the neck region (ground). Differentially recorded scalp potentials were bandpass filtered between 0.05 and 3 kHz over a 15-millisecond (ms) epoch. A total of 400 trials were averaged for each waveform for each stimulus condition. Symmetrically shaped tone bursts were 3 ms long (1 ms raised cosine on/off ramps and 1 ms plateau). All acoustic stimuli were delivered free field via a speaker (Tucker Davis Technologies, Inc., Part FF1 2021) positioned 10 cm from the vertex. Stimulus levels were calibrated using a 0.5-in condenser microphone (model 4016; ACO Pacific, Belmont, CA) positioned at the approximate location of the animal's head during recording sessions and are reported in decibels sound pressure level (dB SPL; referenced to 20 μPa). Stimuli of alternating polarity were delivered at a rate of approximately 20 per second. Tone burst responses were collected in half octave steps ranging from 32 to 2.0 kHz. The effects of level were determined by decreasing stimulus intensity in 5-dB steps. A maximum stimulus level of 90 dB SPL was used first to avoid overstimulation. If the thresholds exceed 90 dB SPL, gain was adjusted to 40 dB to deliver a maximum stimulus level of 110 dB SPL. ABR thresholds were determined visually by noting the response waveforms exceeding a 1:1 signal to noise ratio. A two-way analysis of variance was used to determine the overall effect of genotype on ABR thresholds at different frequencies. The uncorrected Fisher's least significant difference test was used to make post hoc comparisons for determining statistical significance at each frequency between WT and Tg-mtTFB1 mice. Latencies of the initial four ABR peaks were measured from animals aged 3 to 6 months or 9 to 12 months by setting time markers at maxima of the peaks and measuring the time from onset of stimulus to peaks. Measurements were made at 8 and 11 kHz on traces with visible peaks. Latencies of peak I and central conduction time (peak I to IV) were used in the analysis. Amplitudes of peak I were assessed by taking the mean of the ΔV of the upward and downward slopes of peak I. Tg-mtTFB1 and WT animals, aged 9 to 12 months, were anesthetized with sodium pentobarbital (48 mg/kg body weight as initial dose and supplement as needed at 24 mg/kg). Animals were then placed onto a stereotaxic mouse head holder (MA-6N; Narishige, Tokyo, Japan) mounted onto a ball-and-socket stage and a magnetic base (M-RN-56; Newport Corp.). The round window was exposed through a ventral approach by opening the bulla of the temporal bone. A sharp electrode (10 to 15 MΩ, 1B150F-4; World Precision Instruments, Sarasota, FL) with 3 mol/L KCl pipette solution was mounted onto a micromanipulator with a pulse motor driving unit (PF5-1; Narishige). Electrodes were first placed at the round window, with visualization under a surgical microscope. An Axon 200A patch clamp amplifier was used for current clamp recording with an Axon Digidata 1321A and jClamp software version 22.8.4 (Scisoft, Inc., Ridgefield, CT). When the electrode was inserted into the scala tympani, voltage was balanced to 0 mV and then the electrodes were advanced through the basilar membrane into the scala media to measure the endocochlear potential (EP). Pipettes were then withdrawn back to the scala tympani or advanced through the scala vestibuli for confirmation of EP. Data analyses were performed offline. Whole cell patch clamp recordings were made from single isolated outer hair cells (OHCs) from the organ of Corti of Tg-mtTFB1 and WT mice. The temporal bones were excised, and the cochleae were dissected free. Enzyme treatment (1 mg/mL dispase I, 10 to 12 minutes) preceded gentle trituration, and isolated OHCs were placed in a glass-bottom recording chamber. An E600-FN microscope (Nikon, Tokyo, Japan) with a 40× water immersion objective was used to observe cells during voltage clamp. Experiments were performed at room temperature. The base high chloride ionic blocking solution contained (in mmol/L) the following: NaCl, 100; TEA-Cl, 20; CsCl, 20; CoCl2, 2; MgCl2, 1; CaCl2, 1; and HEPES, 10. Base intracellular solutions contained (in mmol/L) the following: CsCl, 140; MgCl2, 2; HEPES, 10; and EGTA, 10. An Axon 200B amplifier was used for the whole-cell patch-clamping recording. Nonlinear capacitance (NLC) was measured using a continuous, high-resolution (2.56 ms sampling), two-sine stimulus protocol (10-mV peak at both 390.6 and 781.2 Hz) superimposed onto the voltage ramp range from −200 to 200 mV.22Santos-Sacchi J. Kakehata S. Takahashi S. Effects of membrane potential on the voltage dependence of motility-related charge in outer hair cells of the guinea-pig.J Physiol. 1998; 510: 225-235Crossref PubMed Scopus (115) Google Scholar, 23Santos-Sacchi J. Determination of cell capacitance using the exact empirical solution of partial differential Y/partial differential Cm and its phase angle.Biophys J. 2004; 87: 714-727Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Capacitance data were fit to the first derivative of a two-state Boltzmann function.Cm=QmaxzekTb(1+b)2+Clin(1) whereb=exp(−ze(Vm−VpkCm)kT).(2) Qmax is the maximum nonlinear charge moved, Vh is voltage at peak capacitance or equivalently, at half maximum charge transfer, Vm is membrane potential, z is valence, Clin is linear membrane capacitance, e is electron charge, k is Boltzmann constant, and T is absolute temperature. The temporal bones of 9- to 12-month-old WT (n = 5) were dissected, and cochlea removed and immersion fixed for 24 to 48 hours in 4% paraformaldehyde in phosphate-buffered saline (PBS). Bony capsules were partially dissected to enable fluid penetration, and the entire structure was then transferred to a blocking solution containing 10% normal goat serum and 0.1% Tween 20 in PBS. Mouse monoclonal Myo7a antibody (Developmental Hybridoma Studies Bank, University of Iowa, Iowa City, IA) was applied in blocking buffer at a 1:500 dilution overnight at 4°C and detected using an Alexa 546 donkey anti-mouse secondary antibody (Jackson Immunological, West Grove, PA). The bony capsule was then carefully removed, and the organ of Corti was detached from the modiolus as a single strip. Spiral ligaments were then dissected away to allow good exposure of the organ of Corti. The organ of Corti was then dissected and mounted. The apical region ranged from 57% to 64% from the base. This region has been described by Müller et al24Müller M. von Hünerbein K. Hoidis S. Smolders J.W.T. A physiological place-frequency map of the cochlea in the CBA/J mouse.Hear Res. 2005; 202: 63-73Crossref PubMed Scopus (295) Google Scholar as corresponding to frequencies between 13.5 and 16 kHz, which encompasses the range of frequencies showing elevated ABR thresholds in the Tg-mtTFB1 mice. Myo7a-labeled inner hair cells (IHCs) and OHCs in this region were visualized with an IX-71 inverted fluorescence microscope (Olympus, Center Valley, PA), and images were collected and analyzed with a Spot camera (Diagnostic Instruments, Inc, Sterling Heights, MI). The number of cells was normalized and expressed as hair cells per 100 μm. Temporal bones were dissected and fixed in 4% paraformaldehyde in PBS at 4°C for 24 to 48 hours, followed by decalcification in 10% EDTA in PBS for 96 hours. The cochleae were bisected in the midmodiolar plane, and the two halves were embedded in paraffin, divided into sections, and stained with hematoxylin and eosin (Yale Pathology Developmental Histology Service, New Haven, CT). The width and thickness of intact, cross-sectioned striae were measured at multiple points at apical-basal locations25Santi P.A. Muchow D.C. Morphometry of the chinchilla organ of Corti and stria vascularis.J Histochem Cytochem. 1979; 27: 1539-1542Crossref PubMed Scopus (15) Google Scholar, 26Schmitz H.M. Johnson S.B. Santi P.A. Kanamycin-furosemide ototoxicity in the mouse cochlea: a 3-dimensional analysis.Otolaryngol Head Neck Surg. 2014; 150: 666-672Crossref PubMed Scopus (15) Google Scholar in each animal using an Olympus microscope and Spot camera software version 5.1 for analysis. Strial width was measured as the length of a curved line between the two end points of the stria in cross section, one near the insertion of Reissner's membrane and the other near the spiral ligament. Thickness was measured by a straight line drawn through the midpoint of the section. We previously characterized hearing loss in Tg-mtTFB1 mice as elevated ABR thresholds that progressed with age, accompanied by increased caspase 3 staining in the stria vascularis and spiral ganglia and reduced numbers of spiral ganglion neurons.18Raimundo N. Song L. Shutt T.E. McKay S.E. Cotney J. Guan M.-X. Gilliland T.C. Hohuan D. Santos-Sacchi J. Shadel G.S. Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.Cell. 2012; 148: 716-726Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Because defects in the stria vascularis, a three-layered epithelial organ lining the scala media and responsible for maintaining the requisite elevated extracellular potassium necessary for hair cell function, can also result in hearing loss characterized by increased ABR thresholds, we measured the EP to assess strial function directly. After confirming that ABR thresholds were prematurely elevated in a cohort of Tg-mtTFB1 mice now extensively backcrossed to the C57BL/6J background (Figure 1A), we recorded EP in a group of WT and Tg-mtTFB1 animals aged 9 to 12 months. We observed a 40% reduction in EP in Tg-mtTFB1 mice (meanTg-mtTFB1 = 60 ± 10 mV) compared with WT controls, which was near 100 mV (meanWT = 100.2 ± 2.1 mV) (Figure 1B). Hearing loss accompanied by strial dysfunction in aging humans is often accompanied by atrophy of the epithelium. To assess atrophy, the width and thickness of the stria were measured at multiple sites in cochlear cross sections of nine Tg-mtTFB1 and six WT animals. The widths, indicators of the cochlear spiral location of the stria, ranged from 131 to 387 μm in WT and from 98 to 358 μm in Tg-mtTFB1 animals. The thickness of the stria ranged from 13 to 22 μm in the WT and from 14 to 28 μm in the Tg-mtTFB1 animals. There was no significant correlation in either genotype between the width of the stria and the thickness (WT, r2 = −0.0049; Tg-mtTFB1, r2 = 0.0077). Because there was no difference in the thickness as a function of apical-basal location, we combined all of the thickness measurements and compared the mean values by a t-test. There was no significant difference between WT and Tg-mtTFB1 mice (Figure 1C). These data suggest that the strial dysfunction in Tg-mtTFB1 mice is either distinct from presbycusis or we are sampling at a stage of progressive hearing loss that precedes gross strial atrophy. To more precisely determine the source of auditory dysfunction, we analyzed details of the ABRs of WT and Tg-mtTFB1 mice with hearing loss. Mouse ABRs have five characteristic peaks, the first of which represents activity in nerve VIII. Changes in the latency or amplitude of peak I reflect dysfunctional cochlear and/or nerve VIII processing. By using two-way analysis of variance to assess the latency to peak I, we observed significant increases in 9- to 12-month-old Tg-mtTFB1 mice at both 8 kHz [F(1,109) = 34, P < 0.001] (Figure 2A) and 11 kHz [F(1,127) = 28, P < 0.001] (Figure 2B). When the same analysis was performed on 3- to 6-month-old animals, when only moderate ABR threshold shifts are observed in Tg-mtTFB1, the latencies were not significantly affected at 8 kHz [F(1,128) = 0.23] (Figure 2C) or 11 kHz [F(1,152) = 0.04] (Figure 2D). Thus, increased latency to peak I is only observed in older Tg-mtTFB1 animals with hearing loss. In contrast, central conduction time (elapsed time between peak I and peak IV), which reflects synaptic and nerve conduction timing after cochlear/nerve VIII processing, was unchanged in animals with severe hearing loss [F(1,104) = 1.2 and F(1,105) = 1.0] (Figure 2, E and F), respectively. These data suggest that hearing loss in Tg-mtTFB1 mice is induced by progressive pathological changes in the cochlea/nerve VIII, not central auditory system defects (ie, brainstem). To determine whether hair cells are directly affected, we dissected the organ of Corti from cochleae of 9-month-old WT and Tg-mtTFB1 mice and labeled IHCs and OHCs with an antibody for Myo7a. Despite considerable ABR threshold changes at frequencies from 4 to 16 kHz (Figure 1A), the hair cells in the apex of the cochlea, which detect frequencies in the range of hearing loss in Tg-mtTFB1 mice,24Müller M. von Hünerbein K. Hoidis S. Smolders J.W.T. A physiological place-frequency map of the cochlea in the CBA/J mouse.Hear Res. 2005; 202: 63-73Crossref PubMed Scopus (295) Google Scholar are largely intact, as evidenced by quantifying Myo7a-labeled IHCs and OHCs in the most apical 2.5 to 3.0 mm of the organ of Corti (Figure 3A). Although there might be a downward trend in the number of hair cells in Tg-mtTFB1 mice, a two-way analysis of variance shows that the counts do not differ significantly from WT [F(1,10) = 0.90]. Next, to determine whether OHC function was altered, NLC, the electrical correlate of electromotility, was measured in OHCs isolated from WT and Tg-mtTFB1 mice between 9 and 12 months of age. NLC was measured by whole-cell patch clamp using symmetric chloride solutions (140 mmol/L Cl− intracellular and extracellular). OHCs from Tg-mtTFB1 mice exhibited a positive shift of nearly 20 mV in the voltage at peak capacitance (Vh; meanWT = −94 ± 2.6 mV; meanTg-mtTFB1 = −76 ± 5 mV) (Figure 3B), suggesting that TFB1M overexpression in the cochlea induces a long-term change in OHC voltage responsiveness. To determine whet