Jeong Han Lee

Interests

Teaching

Biology of the inner ear

Research

Age induced hearing lossMechanotransduction channel in hair cellsSubtypes of type1 and type2 spiral ganglion neuronsFunction of the inner/outer hair cells

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No activities entered.

Scholarly Contributions

Journals/Publications

• Kwon, H. C., Kim, S., Jin, S., Shin, S. A., Lee, J. H., Park, M. J., Kim, S. J., & Park, Y. H. (2025). Intratympanic administration of dexamethasone attenuates radiation induced damage to middle ear mucosa. Scientific reports, 15(1), 3127.
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  Radiotherapy (RTx) is a highly effective treatment for head and neck cancer that can cause concurrent damage to surrounding healthy tissues. In cases of nasopharyngeal carcinoma (NPC), the auditory apparatus is inevitably exposed to radiation fields and sustains considerable damage, resulting in dysfunction. To date, little research has been conducted on the changes induced by RTx in the middle ear and the underlying mechanisms involved. Dexamethasone (DEX) is widely used in clinical practice because of its immunosuppressive and anti-inflammatory properties. The present study investigated the effects and underlying mechanisms of DEX delivered via intratympanic administration on RTx-induced damage to the middle ear and human middle ear epithelial (HMEE) cells. Sprague-Dawley (SD) rats were exposed to fractionated RTx (6.6 Gy/day for 5 days), and middle ear samples were collected at 1 and 4 months. Rats that received RTx presented a significant increase in the thickness of the submucosal layer in the middle ear and disorganization of the ciliated epithelium in the Eustachian tube (ET) mucosa. Importantly, intratympanic administration of DEX 30 min before RTx resulted in a lower degree of damage than that in the control group. Furthermore, DEX pretreatment downregulated the expression of cell death pathway markers in HMEE cells. Our collective results potentially support the use of DEX to reduce radiation-induced damage in the middle ear and may contribute to the development of future studies.
• Chen, Y., Lee, J. H., Li, J., Park, S., Perez Flores, M. C., Peguero, B., Kersigo, J., Kang, M., Choi, J., Levine, L., Gratton, M. A., Fritzsch, B., & Yamoah, E. N. (2024). Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells. bioRxiv : the preprint server for biology.
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  Hearing loss is the most common form of sensory deficit. It occurs predominantly due to hair cell (HC) loss. Mammalian HCs are terminally differentiated by birth, making HC loss challenging to replace. Here, we show the pharmacogenetic downregulation of , a tight junction protein, generates robust supernumerary inner HCs (IHCs) in mice. The ectopic IHC shared functional and synaptic features akin to typical IHCs and were surprisingly and remarkably preserved for at least fifteen months >50% of the mouse's life cycle. , knockdown using shRNA on postnatal days (P) P2-7 yielded analogous functional ectopic IHCs that were equally durably conserved. The findings suggest that Cldn9 levels coordinate embryonic and postnatal HC differentiation, making it a viable target for altering IHC development pre- and post-terminal differentiation.
• Lee, J. H., Park, S., Perez-Flores, M. C., Chen, Y., Kang, M., Choi, J., Levine, L., Gratton, M. A., Zhao, J., Notterpek, L., & Yamoah, E. N. (2024). Demyelination and Na Channel Redistribution Underlie Auditory and Vestibular Dysfunction in PMP22-Null Mice. eNeuro, 11(2).
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  Altered expression of peripheral myelin protein 22 (PMP22) results in demyelinating peripheral neuropathy. PMP22 exhibits a highly restricted tissue distribution with marked expression in the myelinating Schwann cells of peripheral nerves. Auditory and vestibular Schwann cells and the afferent neurons also express PMP22, suggesting a unique role in hearing and balancing. Indeed, neuropathic patients diagnosed with PMP22-linked hereditary neuropathies often present with auditory and balance deficits, an understudied clinical complication. To investigate the mechanism by which abnormal expression of PMP22 may cause auditory and vestibular deficits, we studied gene-targeted -null mice. -null mice exhibit an unsteady gait, have difficulty maintaining balance, and live for only ∼3-5 weeks relative to unaffected littermates. Histological analysis of the inner ear revealed reduced auditory and vestibular afferent nerve myelination and profound Na channel redistribution without PMP22. Yet, Na current density was unaltered, in stark contrast to increased K current density. Atypical postsynaptic densities and a range of neuronal abnormalities in the organ of Corti were also identified. Analyses of auditory brainstem responses (ABRs) and vestibular sensory-evoked potential (VsEP) revealed that -null mice had auditory and vestibular hypofunction. These results demonstrate that PMP22 is required for hearing and balance, and the protein is indispensable for the formation and maintenance of myelin in the peripheral arm of the eighth nerve. Our findings indicate that myelin abnormalities and altered signal propagation in the peripheral arm of the auditory nerve are likely causes of auditory deficits in patients with PMP22-linked neuropathies.
• Lee, J. H., Perez-Flores, M. C., Park, S., Kim, H. J., Chen, Y., Kang, M., Kersigo, J., Choi, J., Thai, P. N., Woltz, R. L., Perez-Flores, D. C., Perkins, G., Sihn, C. R., Trinh, P., Zhang, X. D., Sirish, P., Dong, Y., Feng, W. W., Pessah, I. N., , Dixon, R. E., et al. (2024). The Piezo channel is a mechano-sensitive complex component in the mammalian inner ear hair cell. Nature communications, 15(1), 526.
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  The inner ear is the hub where hair cells (HCs) transduce sound, gravity, and head acceleration stimuli to the brain. Hearing and balance rely on mechanosensation, the fastest sensory signals transmitted to the brain. The mechanoelectrical transducer (MET) channel is the entryway for the sound-balance-brain interface, but the channel-complex composition is not entirely known. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as MET-complex components. The Pz channels, expressed in HC stereocilia, and cell lines are co-localized and co-assembled with MET complex partners. Mice expressing non-functional Pz1 and Pz2 at the ROSA26 locus have impaired auditory and vestibular traits that can only be explained if the Pzs are integral to the MET complex. We suggest that Pz subunits constitute part of the MET complex and that interactions with other MET complex components yield functional MET units to generate HC MET currents.
• Lee, J. H., Perez-Flores, M. C., Park, S., Kim, H. J., Chen, Y., Kang, M., Kersigo, J., Choi, J., Thai, P. N., Woltz, R., Perez-Flores, D. C., Perkins, G., Sihn, C. R., Trinh, P., Zhang, X. D., Sirish, P., Dong, Y., Feng, W. W., Pessah, I. N., , Dixon, R. E., et al. (2023). The Piezo channel is central to the mechano-sensitive channel complex in the mammalian inner ear. Research square.
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  The inner ear is the hub where hair cells transduce sound, gravity, and head acceleration stimuli carried by neural codes to the brain. Of all the senses, hearing and balance, which rely on mechanosensation, are the fastest sensory signals transmitted to the central nervous system. The mechanoelectrical transducer (MET) channel in hair cells is the entryway for the sound-balance-brain interface, but the channel's composition has eluded biologists due to its complexity. Here, we report that the mouse utilizes Piezo1 (Pz1) and Piezo2 (Pz2) isoforms as central components of the MET complex. The Pz channel subunits are expressed in hair-cell stereocilia, are co-localized and co-assembled, and are essential components of the MET complex and including integration with the transmembrane channel (Tmc1/2) protein. Mice expressing non-functional and but not functional at the ROSA26 locus under the control of hair-cell promoters, have impaired auditory and vestibular traits that can only be explained if Pz channel multimers are integral to the MET complex. We affirm that Pz protein subunits constitute MET channels and that functional interactions with components of the MET complex yield current properties resembling hair-cell MET currents. Our results demonstrate Pz is a MET channel component central to interacting with MET complex proteins. Results account for the MET channel pore and complex.
• Finno, C. J., Chen, Y., Park, S., Lee, J. H., Perez-Flores, M. C., Choi, J., & Yamoah, E. N. (2022). Cisplatin Neurotoxicity Targets Specific Subpopulations and K Channels in Tyrosine-Hydroxylase Positive Dorsal Root Ganglia Neurons. Frontiers in cellular neuroscience, 16, 853035.
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  Among the features of cisplatin chemotherapy-induced peripheral neuropathy are chronic pain and innocuous mechanical hypersensitivity. The complete etiology of the latter remains unknown. Here, we show that cisplatin targets a heterogeneous population of tyrosine hydroxylase-positive (TH) primary afferent dorsal root ganglion neurons (DRGNs) in mice, determined using single-cell transcriptome and electrophysiological analyses. TH DRGNs regulate innocuous mechanical sensation through C-low threshold mechanoreceptors. A differential assessment of wild-type and vitamin E deficient TH DRGNs revealed heterogeneity and specific functional phenotypes. The TH DRGNs comprise; fast-adapting eliciting one action potential (AP; 1-AP), moderately-adapting (≥2-APs), in responses to square-pulse current injection, and spontaneously active (SA). Cisplatin increased the input resistance and AP frequency but reduced the temporal coding feature of 1-AP and ≥2-APs neurons. By contrast, cisplatin has no measurable effect on the SA neurons. Vitamin E reduced the cisplatin-mediated increased excitability but did not improve the TH neuron temporal coding properties. Cisplatin mediates its effect by targeting outward K current, likely carried through K2P18.1 ), discovered through the differential transcriptome studies and heterologous expression. Studies show a potential new cellular target for chemotherapy-induced peripheral neuropathy and implicate the possible neuroprotective effects of vitamin E in cisplatin chemotherapy.
• Perez-Flores, M. C., Verschooten, E., Lee, J. H., Kim, H. J., Joris, P. X., & Yamoah, E. N. (2022). Intrinsic mechanical sensitivity of mammalian auditory neurons as a contributor to sound-driven neural activity. eLife, 11.
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  Mechanosensation - by which mechanical stimuli are converted into a neuronal signal - is the basis for the sensory systems of hearing, balance, and touch. Mechanosensation is unmatched in speed and its diverse range of sensitivities, reaching its highest temporal limits with the sense of hearing; however, hair cells (HCs) and the auditory nerve (AN) serve as obligatory bottlenecks for sounds to engage the brain. Like other sensory neurons, auditory neurons use the canonical pathway for neurotransmission and millisecond-duration action potentials (APs). How the auditory system utilizes the relatively slow transmission mechanisms to achieve ultrafast speed, and high audio-frequency hearing remains an enigma. Here, we address this paradox and report that the mouse, and chinchilla, AN are mechanically sensitive, and minute mechanical displacement profoundly affects its response properties. Sound-mimicking sinusoidal mechanical and electrical current stimuli affect phase-locked responses. In a phase-dependent manner, the two stimuli can also evoke suppressive responses. We propose that mechanical sensitivity interacts with synaptic responses to shape responses in the AN, including frequency tuning and temporal phase locking. Combining neurotransmission and mechanical sensation to control spike patterns gives the mammalian AN a secondary receptor role, an emerging theme in primary neuronal functions.
• Elliott, K. L., Kersigo, J., Lee, J. H., Jahan, I., Pavlinkova, G., Fritzsch, B., & Yamoah, E. N. (2021). Developmental Changes in eGFP Expression in Spiral Ganglion Neurons. Frontiers in cellular neuroscience, 15, 678113.
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  The two types of spiral ganglion neurons (SGNs), types I and II, innervate inner hair cells and outer hair cells, respectively, within the mammalian cochlea and send another process back to cochlear nuclei in the hindbrain. Studying these two neuronal types has been made easier with the identification of unique molecular markers. One of these markers, peripherin, was shown using antibodies to be present in all SGNs initially but becomes specific to type II SGNs during maturation. We used mice with fluorescently labeled peripherin (eGFP) to examine peripherin expression in SGNs during development and in aged mice. Using these mice, we confirm the initial expression of eGFP in both types I and II neurons and eventual restriction to only type II perikarya shortly after birth. However, while eGFP is uniquely expressed within type II cell bodies by P8, both types I and II peripheral and central processes continue to express eGFP for some time before becoming downregulated. Only at P30 was there selective type II eGFP expression in central but not peripheral processes. By 9 months, only the type II cell bodies and more distal central processes retain eGFP expression. Our results show that eGFP is a reliable marker for type II SGN cell bodies beyond P8; however, it is not generally a suitable marker for type II processes, except for central processes beyond P30. How the changes in eGFP expression relate to subsequent protein expression remains to be explored.
• Elliott, K. L., Kersigo, J., Lee, J. H., Yamoah, E. N., & Fritzsch, B. (2021). Sustained Loss of Affects Peripheral but Not Central Vestibular Targets. Frontiers in neurology, 12, 768456.
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  The vestibular system is vital for proper balance perception, and its dysfunction contributes significantly to fall-related injuries, especially in the elderly. Vestibular ganglion neurons innervate vestibular hair cells at the periphery and vestibular nuclei and the uvula and nodule of the cerebellum centrally. During aging, these vestibular ganglion neurons degenerate, impairing vestibular function. A complete understanding of the molecular mechanisms involved in neurosensory cell survival in the vestibular system is unknown. Brain-derived neurotrophic factor (BDNF) is specifically required for the survival of vestibular ganglion neurons, as its loss leads to early neuronal death. null mice die within 3 weeks of birth, preventing the study of the long-term effects on target cells. We use -cre to conditionally knock out , allowing mice survival to approximately 6 months of age. We show that a long-term loss of leads to a significant reduction in the number of vestibular ganglion neurons and a reduction in the number of vestibular hair cells. There was no significant decrease in the central targets lateral vestibular nucleus (LVN) or the cerebellum at 6 months. This suggests that the connectivity between central target cells and other neurons suffices to prevent their loss despite vestibular hair cell and ganglion neuron loss. Whether the central neurons would undergo eventual degeneration in the absence of remains to be determined.
• Zhang, X. D., Thai, P. N., Ren, L., Perez Flores, M. C., Ledford, H. A., Park, S., Lee, J. H., Sihn, C. R., Chang, C. W., Chen, W. C., Timofeyev, V., Zuo, J., Chan, J. W., Yamoah, E. N., & Chiamvimonvat, N. (2021). Prestin amplifies cardiac motor functions. Cell reports, 35(5), 109097.
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  Cardiac cells generate and amplify force in the context of cardiac load, yet the membranous sheath enclosing the muscle fibers-the sarcolemma-does not experience displacement. That the sarcolemma sustains beat-to-beat pressure changes without experiencing significant distortion is a muscle-contraction paradox. Here, we report that an elastic element-the motor protein prestin (Slc26a5)-serves to amplify actin-myosin force generation in mouse and human cardiac myocytes, accounting partly for the nonlinear capacitance of cardiomyocytes. The functional significance of prestin is underpinned by significant alterations of cardiac contractility in Prestin-knockout mice. Prestin was previously considered exclusive to the inner ear's outer hair cells; however, our results show that prestin serves a broader cellular motor function.
• Perkins, G., Lee, J. H., Park, S., Kang, M., Perez-Flores, M. C., Ju, S., Phillips, G., Lysakowski, A., Gratton, M. A., & Yamoah, E. N. (2020). Altered Outer Hair Cell Mitochondrial and Subsurface Cisternae Connectomics Are Candidate Mechanisms for Hearing Loss in Mice. The Journal of neuroscience : the official journal of the Society for Neuroscience, 40(44), 8556-8572.
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  Organelle crosstalk is vital for cellular functions. The propinquity of mitochondria, ER, and plasma membrane promote regulation of multiple functions, which include intracellular Ca flux, and cellular biogenesis. Although the purposes of apposing mitochondria and ER have been described, an understanding of altered organelle connectomics related to disease states is emerging. Since inner ear outer hair cell (OHC) degeneration is a common trait of age-related hearing loss, the objective of this study was to investigate whether the structural and functional coupling of mitochondria with subsurface cisternae (SSC) was affected by aging. We applied functional and structural probes to equal numbers of male and female mice with a hearing phenotype akin to human aging. We discovered the polarization of cristae and crista junctions in mitochondria tethered to the SSC in OHCs. Aging was associated with SSC stress and decoupling of mitochondria with the SSC, mitochondrial fission/fusion imbalance, a remarkable reduction in mitochondrial and cytoplasmic Ca levels, reduced K-induced Ca uptake, and marked plasticity of cristae membranes. A model of structure-based ATP production predicts profound energy stress in older OHCs. This report provides data suggesting that altered membrane organelle connectomics may result in progressive hearing loss.
• Sirish, P., Thai, P. N., Lee, J. H., Yang, J., Zhang, X. D., Ren, L., Li, N., Timofeyev, V., Lee, K. S., Nader, C. E., Rowland, D. J., Yechikov, S., Ganaga, S., Young, N., Lieu, D. K., Yamoah, E. N., Hammock, B. D., & Chiamvimonvat, N. (2020). Suppression of inflammation and fibrosis using soluble epoxide hydrolase inhibitors enhances cardiac stem cell-based therapy. Stem cells translational medicine, 9(12), 1570-1584.
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  Stem cell replacement offers a great potential for cardiac regenerative therapy. However, one of the critical barriers to stem cell therapy is a significant loss of transplanted stem cells from ischemia and inflammation in the host environment. Here, we tested the hypothesis that inhibition of the soluble epoxide hydrolase (sEH) enzyme using sEH inhibitors (sEHIs) to decrease inflammation and fibrosis in the host myocardium may increase the survival of the transplanted human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in a murine postmyocardial infarction model. A specific sEHI (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea [TPPU]) and CRISPR/Cas9 gene editing were used to test the hypothesis. TPPU results in a significant increase in the retention of transplanted cells compared with cell treatment alone. The increase in the retention of hiPSC-CMs translates into an improvement in the fractional shortening and a decrease in adverse remodeling. Mechanistically, we demonstrate a significant decrease in oxidative stress and apoptosis not only in transplanted hiPSC-CMs but also in the host environment. CRISPR/Cas9-mediated gene silencing of the sEH enzyme reduces cleaved caspase-3 in hiPSC-CMs challenged with angiotensin II, suggesting that knockdown of the sEH enzyme protects the hiPSC-CMs from undergoing apoptosis. Our findings demonstrate that suppression of inflammation and fibrosis using an sEHI represents a promising adjuvant to cardiac stem cell-based therapy. Very little is known regarding the role of this class of compounds in stem cell-based therapy. There is consequently an enormous opportunity to uncover a potentially powerful class of compounds, which may be used effectively in the clinical setting.
• Lee, J. H., Kang, M., Park, S., Perez-Flores, M. C., Zhang, X. D., Wang, W., Gratton, M. A., Chiamvimonvat, N., & Yamoah, E. N. (2019). The local translation of in dendritic projections of auditory neurons and the roles of in the transition from hidden to overt hearing loss. Aging, 11(23), 11541-11564.
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  Local and privileged expression of dendritic proteins allows segregation of distinct functions in a single neuron but may represent one of the underlying mechanisms for early and insidious presentation of sensory neuropathy. Tangible characteristics of early hearing loss (HL) are defined in correlation with nascent hidden hearing loss (HHL) in humans and animal models. Despite the plethora of causes of HL, only two prevailing mechanisms for HHL have been identified, and in both cases, common structural deficits are implicated in inner hair cell synapses, and demyelination of the auditory nerve (AN). We uncovered that Na-activated K (K) mRNA and channel proteins are distinctly and locally expressed in dendritic projections of primary ANs and genetic deletion of K channels ( and ) results in the loss of proper AN synaptic function, characterized as HHL, without structural synaptic alterations. We further demonstrate that the local functional synaptic alterations transition from HHL to increased hearing-threshold, which entails changes in global Ca homeostasis, activation of caspases 3/9, impaired regulation of inositol triphosphate receptor 1 (IPR1), and apoptosis-mediated neurodegeneration. Thus, the present study demonstrates how local synaptic dysfunction results in an apparent latent pathological phenotype (HHL) and, if undetected, can lead to overt HL. It also highlights, for the first time, that HHL can precede structural synaptic dysfunction and AN demyelination. The stepwise cellular mechanisms from HHL to canonical HL are revealed, providing a platform for intervention to prevent lasting and irreversible age-related hearing loss (ARHL).
• Lee, J. H., Park, S., Perez-Flores, M. C., Wang, W., Kim, H. J., Izu, L., Gratton, M. A., Chiamvimonvat, N., & Yamoah, E. N. (2019). Early functional alterations in membrane properties and neuronal degeneration are hallmarks of progressive hearing loss in NOD mice. Scientific reports, 9(1), 12128.
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  Presbycusis or age-related hearing loss (ARHL) is the most common sensory deficit in the human population. A substantial component of the etiology stems from pathological changes in sensory and non-sensory cells in the cochlea. Using a non-obese diabetic (NOD) mouse model, we have characterized changes in both hair cells and spiral ganglion neurons that may be relevant for early signs of age-related hearing loss (ARHL). We demonstrate that hair cell loss is preceded by, or in parallel with altered primary auditory neuron functions, and latent neurite retraction at the hair cell-auditory neuron synapse. The results were observed first in afferent inner hair cell synapse of type I neurites, followed by type II neuronal cell-body degeneration. Reduced membrane excitability and loss of postsynaptic densities were some of the inaugural events before any outward manifestation of hair bundle disarray and hair cell loss. We have identified profound alterations in type I neuronal membrane properties, including a reduction in membrane input resistance, prolonged action potential latency, and a decrease in membrane excitability. The resting membrane potential of aging type I neurons in the NOD, ARHL model, was significantly hyperpolarized, and analyses of the underlying membrane conductance showed a significant increase in K currents. We propose that attempts to alleviate some forms of ARHL should include early targeted primary latent neural degeneration for effective positive outcomes.
• Reijntjes, D. O., Lee, J. H., Park, S., Schubert, N. M., van Tuinen, M., Vijayakumar, S., Jones, T. A., Jones, S. M., Gratton, M. A., Xia, X. M., Yamoah, E. N., & Pyott, S. J. (2019). Sodium-activated potassium channels shape peripheral auditory function and activity of the primary auditory neurons in mice. Scientific reports, 9(1), 2573.
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  Potassium (K) channels shape the response properties of neurons. Although enormous progress has been made to characterize K channels in the primary auditory neurons, the molecular identities of many of these channels and their contributions to hearing in vivo remain unknown. Using a combination of RNA sequencing and single molecule fluorescent in situ hybridization, we localized expression of transcripts encoding the sodium-activated potassium channels K1.1 (SLO2.2/Slack) and K1.2 (SLO2.1/Slick) to the primary auditory neurons (spiral ganglion neurons, SGNs). To examine the contribution of these channels to function of the SGNs in vivo, we measured auditory brainstem responses in K1.1/1.2 double knockout (DKO) mice. Although auditory brainstem response (wave I) thresholds were not altered, the amplitudes of suprathreshold responses were reduced in DKO mice. This reduction in amplitude occurred despite normal numbers and molecular architecture of the SGNs and their synapses with the inner hair cells. Patch clamp electrophysiology of SGNs isolated from DKO mice displayed altered membrane properties, including reduced action potential thresholds and amplitudes. These findings show that K1 channel activity is essential for normal cochlear function and suggest that early forms of hearing loss may result from physiological changes in the activity of the primary auditory neurons.
• Park, Y. H., Moon, S. K., Lee, K. H., & Lee, J. H. (2018). Neurobiology of Hearing Loss and Ear Disease. BioMed research international, 2018, 2464251.
• Yamoah, M. A., Moshref, M., Sharma, J., Chen, W. C., Ledford, H. A., Lee, J. H., Chavez, K. S., Wang, W., López, J. E., Lieu, D. K., Sirish, P., & Zhang, X. D. (2018). Highly efficient transfection of human induced pluripotent stem cells using magnetic nanoparticles. International journal of nanomedicine, 13, 6073-6078.
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  The delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs).
• Zhang, X. D., Coulibaly, Z. A., Chen, W. C., Ledford, H. A., Lee, J. H., Sirish, P., Dai, G., Jian, Z., Chuang, F., Brust-Mascher, I., Yamoah, E. N., Chen-Izu, Y., Izu, L. T., & Chiamvimonvat, N. (2018). Coupling of SK channels, L-type Ca channels, and ryanodine receptors in cardiomyocytes. Scientific reports, 8(1), 4670.
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  Small-conductance Ca-activated K (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca microdomains necessary for SK channel activation. SK currents coupled with Ca influx via L-type Ca channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca store, suggesting that LTCCs provide the immediate Ca microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca1.2 Ca channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca signaling.
• Sirish, P., Ledford, H. A., Timofeyev, V., Thai, P. N., Ren, L., Kim, H. J., Park, S., Lee, J. H., Dai, G., Moshref, M., Sihn, C. R., Chen, W. C., Timofeyeva, M. V., Jian, Z., Shimkunas, R., Izu, L. T., Chiamvimonvat, N., Chen-Izu, Y., Yamoah, E. N., & Zhang, X. D. (2017). Action Potential Shortening and Impairment of Cardiac Function by Ablation of. Circulation. Arrhythmia and electrophysiology, 10(10).
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  Intracellular pH (pH) is critical to cardiac excitation and contraction; uncompensated changes in pH impair cardiac function and trigger arrhythmia. Several ion transporters participate in cardiac pH regulation. Our previous studies identified several isoforms of a solute carrier Slc26a6 to be highly expressed in cardiomyocytes. We show that Slc26a6 mediates electrogenic Cl/HCO exchange activities in cardiomyocytes, suggesting the potential role of Slc26a6 in regulation of not only pH, but also cardiac excitability.
• Lee, J. H., Sihn, C., Wang, W., Flores, C. M., & Yamoah, E. N. (2016). In Vitro Functional Assessment of Adult Spiral Ganglion Neurons (SGNs). Methods in molecular biology (Clifton, N.J.), 1427, 513-23.
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  Spiral ganglion neurons (SGNs) faithfully encode acoustic waves from hair cells to the cochlear nucleus (CN) using voltage-dependent ion channels. A sizable portion of our knowledge on SGN functions have been derived from pre-hearing neurons. In post-hearing SGNs, the mechanisms of how they encode the massive sound information without delay and precisely are largely unknown. Mature SGNs are housed in the central bony labyrinth of the cochlea, protected by a well-insulated myelin sheath, making it a technical feat to isolate viable neurons for rigorous functional electrophysiology. Recently, we have overcome the previous intractable hindrance in SGN functional analyses. We provide a step-by-step user-friendly protocol with practical applications, including patch-clamp recordings and imaging by using cultured SGNs.
• Sirish, P., Li, N., Timofeyev, V., Zhang, X. D., Wang, L., Yang, J., Lee, K. S., Bettaieb, A., Ma, S. M., Lee, J. H., Su, D., Lau, V. C., Myers, R. E., Lieu, D. K., López, J. E., Young, J. N., Yamoah, E. N., Haj, F., Ripplinger, C. M., , Hammock, B. D., et al. (2016). Molecular Mechanisms and New Treatment Paradigm for Atrial Fibrillation. Circulation. Arrhythmia and electrophysiology, 9(5).
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  Atrial fibrillation represents the most common arrhythmia leading to increased morbidity and mortality, yet, current treatment strategies have proven inadequate. Conventional treatment with antiarrhythmic drugs carries a high risk for proarrhythmias. The soluble epoxide hydrolase enzyme catalyzes the hydrolysis of anti-inflammatory epoxy fatty acids, including epoxyeicosatrienoic acids from arachidonic acid to the corresponding proinflammatory diols. Therefore, the goal of the study is to directly test the hypotheses that inhibition of the soluble epoxide hydrolase enzyme can result in an increase in the levels of epoxyeicosatrienoic acids, leading to the attenuation of atrial structural and electric remodeling and the prevention of atrial fibrillation.
• Zhang, X. D., Lee, J. H., Lv, P., Chen, W. C., Kim, H. J., Wei, D., Wang, W., Sihn, C. R., Doyle, K. J., Rock, J. R., Chiamvimonvat, N., & Yamoah, E. N. (2015). Etiology of distinct membrane excitability in pre- and posthearing auditory neurons relies on activity of Cl- channel TMEM16A. Proceedings of the National Academy of Sciences of the United States of America, 112(8), 2575-80.
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  The developmental rehearsal for the debut of hearing is marked by massive changes in the membrane properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Whereas the underlying mechanisms for the developing HC transition to mature stage are understood in detail, the maturation of SGNs from hyperexcitable prehearing to quiescent posthearing neurons with broad dynamic range is unknown. Here, we demonstrated using pharmacological approaches, caged-Ca(2+) photolysis, and gramicidin patch recordings that the prehearing SGN uses Ca(2+)-activated Cl(-) conductance to depolarize the resting membrane potential and to prime the neurons in a hyperexcitable state. Immunostaining of the cochlea preparation revealed the identity and expression of the Ca(2+)-activated Cl(-) channel transmembrane member 16A (TMEM16A) in SGNs. Moreover, null deletion of TMEM16A reduced the Ca(2+)-activated Cl(-) currents and action potential firing in SGNs. To determine whether Cl(-) ions and TMEM16A are involved in the transition between pre- and posthearing features of SGNs we measured the intracellular Cl(-) concentration [Cl(-)]i in SGNs. Surprisingly, [Cl(-)]i in SGNs from prehearing mice was ∼90 mM, which was significantly higher than posthearing neurons, ∼20 mM, demonstrating discernible altered roles of Cl(-) channels in the developing neuron. The switch in [Cl(-)]i stems from delayed expression of the development of intracellular Cl(-) regulating mechanisms. Because the Cl(-) channel is the only active ion-selective conductance with a reversal potential that lies within the dynamic range of SGN action potentials, developmental alteration of [Cl(-)]i, and hence the equilibrium potential for Cl(-) (ECl), transforms pre- to posthearing phenotype.
• Lv, P., Kim, H. J., Lee, J. H., Sihn, C. R., Fathabad Gharaie, S., Mousavi-Nik, A., Wang, W., Wang, H. G., Gratton, M. A., Doyle, K. J., Zhang, X. D., Chiamvimonvat, N., & Yamoah, E. N. (2014). Genetic, cellular, and functional evidence for Ca2+ inflow through Cav1.2 and Cav1.3 channels in murine spiral ganglion neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience, 34(21), 7383-93.
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  Spiral ganglion neurons (SGNs) of the eighth nerve serve as the bridge between hair cells and the cochlear nucleus. Hair cells use Cav1.3 as the primary channel for Ca(2+) inflow to mediate transmitter release. In contrast, SGNs are equipped with multiple Ca(2+) channels to mediate Ca(2+)-dependent functions. We examined directly the role of Cav1.3 channels in SGNs using Cav1.3-deficient mice (Cav1.3(-/-)). We revealed a surprising finding that SGNs functionally express the cardiac-specific Cav1.2, as well as neuronal Cav1.3 channels. We show that evoked action potentials recorded from SGNs show a significant decrease in the frequency of firing in Cav1.3(-/-) mice compared with wild-type (Cav1.3(+/+)) littermates. Although Cav1.3 is the designated L-type channel in neurons, whole-cell currents recorded in isolated SGNs from Cav1.3(-/-) mice showed a surprising remnant current with sensitivity toward the dihydropyridine (DHP) agonist and antagonist, and a depolarization shift in the voltage-dependent activation compared with that in the Cav1.3(+/+) mice. Indeed, direct measurement of the elementary properties of Ca(2+) channels, in Cav1.3(+/+) neurons, confirmed the existence of two DHP-sensitive single-channel currents, with distinct open probabilities and conductances. We demonstrate that the DHP-sensitive current in Cav1.3(-/-) mice is derived from Cav1.2 channel activity, providing for the first time, to our knowledge, functional data for the expression of Cav1.2 currents in neurons. Finally, using shRNA gene knockdown methodology, and histological analyses of SGNs from Cav1.2(+/-) and Cav1.3(+/-) mice, we were able to establish the differential roles of Cav1.2 and Cav1.3 in SGNs.
• Wang, W., Kim, H. J., Lee, J. H., Wong, V., Sihn, C. R., Lv, P., Perez Flores, M. C., Mousavi-Nik, A., Doyle, K. J., Xu, Y., & Yamoah, E. N. (2014). Functional significance of K+ channel β-subunit KCNE3 in auditory neurons. The Journal of biological chemistry, 289(24), 16802-13.
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  The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall functional relevance of the single transmembrane domain peptide on the pore-forming K(+) channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3(-/-) mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K(+) currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.
• Kang, T. H., Hong, B. N., Jung, S. Y., Lee, J. H., So, H. S., Park, R., & You, Y. O. (2013). Curculigo orchioides protects cisplatin-induced cell damage. The American journal of Chinese medicine, 41(2), 425-41.
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  Cisplatin is commonly used as a chemotherapeutic agent against many human cancers. However, it generates reactive oxygen species (ROS) and has serious dose-limiting side effects, including ototoxicity. The roots of Curculigo orchioides (C. orchioides) have been used to treat auditory diseases such as tinnitus and hearing loss in Chinese traditional medicine. In the present study, we investigated the protective effects of an ethanol extract obtained from C. orchioides rhizome (COR) on cisplatin-induced cell damage in auditory cells (HEI-OC1). COR (2.5-25 μg/ml) inhibited cisplatin-induced HEI-OC1 cell damage in a dose-dependent manner. To investigate the protective mechanism of COR on cisplatin cytotoxicity in HEI-OC1 cells, we measured the effects of COR on ROS generation and lipid peroxidation in cisplatin-treated cells as well as its scavenging activities against superoxide radicals, hydroxyl radicals, hydrogen peroxide, and DPPH radicals. COR (1-25 μg/ml) had scavenging activities against superoxide radicals, hydroxyl radicals, hydrogen peroxide, and DPPH radicals, as well as reduced lipid peroxidation. In in vivo experiments, COR was shown to reduce cochlear and peripheral auditory function impairments through cisplatin-induced auditory damage in mice. These results indicate that COR protects from cisplatin-induced auditory damage by inhibiting lipid peroxidation and scavenging activities against free radicals.
• Kim, H. J., Gratton, M. A., Lee, J. H., Perez Flores, M. C., Wang, W., Doyle, K. J., Beermann, F., Crognale, M. A., & Yamoah, E. N. (2013). Precise toxigenic ablation of intermediate cells abolishes the "battery" of the cochlear duct. The Journal of neuroscience : the official journal of the Society for Neuroscience, 33(36), 14601-6.
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  The extracellular potential of excitable and nonexcitable cells with respect to ground is ∼0 mV. One of the known exceptions in mammals is the cochlear duct, where the potential is ∼80-100 mV, called the endocochlear potential (EP). The EP serves as the "battery" for transduction of sound, contributing toward the sensitivity of the auditory system. The stria vascularis (StV) of the cochlear duct is the station where the EP is generated, but the cell-specific roles in the StV are ill defined. Using the intermediate cell (IC)-specific tyrosinase promoter, under the control of diphtheria toxin (DT), we eliminated and/or halted differentiation of neural crest melanocytes after migration to the StV. The ensuing adult transgenic mice are profoundly deaf. Additionally, the EP was abolished. Expression of melanocyte early marker and Kir4.1 in ICs precedes the onset of pigment synthesis. Activation of DT leads to loss of ICs. Finally, in accord with the distinct embryology of retinal pigmented cells, transgenic mice with toxigenic ablation of neural crest-derived melanocytes have intact visual responses. We assert that the tyrosinase promoter is the distinct target for genetic manipulation of IC-specific genes.
• Lee, J. H., Park, C., Kim, S. J., Kim, H. J., Oh, G. S., Shen, A., So, H. S., & Park, R. (2013). Different uptake of gentamicin through TRPV1 and TRPV4 channels determines cochlear hair cell vulnerability. Experimental & molecular medicine, 45(3), e12.
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  Hair cells at the base of the cochlea appear to be more susceptible to damage by the aminoglycoside gentamicin than those at the apex. However, the mechanism of base-to-apex gradient ototoxicity by gentamicin remains to be elucidated. We report here that gentamicin caused rodent cochlear hair cell damages in a time- and dose-dependent manner. Hair cells at the basal turn were more vulnerable to gentamicin than those at the apical turn. Gentamicin-conjugated Texas Red (GTTR) uptake was predominant in basal turn hair cells in neonatal rats. Transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4) expression was confirmed in the cuticular plate, stereocilia and hair cell body of inner hair cells and outer hair cells. The involvement of TRPV1 and TRPV4 in gentamicin trafficking of hair cells was confirmed by exogenous calcium treatment and TRPV inhibitors, including gadolinium and ruthenium red, which resulted in markedly inhibited GTTR uptake and gentamicin-induced hair cell damage in rodent and zebrafish ototoxic model systems. These results indicate that the cytotoxic vulnerability of cochlear hair cells in the basal turn to gentamicin may depend on effective uptake of the drug, which was, in part, mediated by the TRPV1 and TRPV4 proteins.
• Park, S., Lee, J. H., Cho, H. J., Lee, K. Y., Kim, M. O., Yun, B. W., & Ryoo, Z. (2013). tmie Is required for gentamicin uptake by the hair cells of mice. Comparative medicine, 63(2), 136-42.
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  The circling (cir/cir) mouse is a spontaneous model of deafness due to deletion of a 40-kb genomic region that includes the transmembrane inner ear (tmie) gene. In addition to being deaf, cir/cir mice exhibit abnormal behaviors including circling and hyperactivity. Here we investigated differences between 3-d-old (that is, before hair-cell degeneration) cir/cir and phenotypically normal (+/cir) mice and the reason underlying the degeneration of the inner ear structure of cir/cir mice. To this end, we used gentamicin, gentamicin-Texas red conjugate, and FM1-43 to investigate mechanotransducer channel activity in the hair cells of cir/cir mice; these compounds are presumed to enter hair cells through the mechanotransducer channel. Although the structure of the inner ear of +/cir mice was equivalent to that of cir/cir mice, the hair cells of cir/cir mice (unlike +/cir) did not take up gentamicin, gentamicin-Texas red conjugate, or FM1-43. These findings suggest that hair cells in cir/cir mice demonstrate abnormal maturation and mechanotransduction. In addition, our current results indicate that tmie is required for maturation and maintenance of hair cells.
• Kim, S. J., Lee, J. H., Kim, B. S., So, H. S., Park, R., Myung, N. Y., Um, J. Y., & Hong, S. H. (2012). (-)-Epigallocatechin-3-gallate protects against NO-induced ototoxicity through the regulation of caspase- 1, caspase-3, and NF-κB activation. PloS one, 7(9), e43967.
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  Excessive nitric oxide (NO) production is toxic to the cochlea and induces hearing loss. However, the mechanism through which NO induces ototoxicity has not been completely understood. The aim of this study was to gain further insight into the mechanism mediating NO-induced toxicity in auditory HEI-OC1 cells and in ex vivo analysis. We also elucidated whether and how epigallocatechin-3-gallate (EGCG), the main component of green tea polyphenols, regulates NO-induced auditory cell damage. To investigate NO-mediated ototoxicity, S-nitroso-N-acetylpenicillamine (SNAP) was used as an NO donor. SNAP was cytotoxic, generating reactive oxygen species, releasing cytochrome c, and activating caspase-3 in auditory cells. NO-induced ototoxicity also mediated the nuclear factor (NF)-κB/caspase-1 pathway. Furthermore, SNAP destroyed the orderly arrangement of the 3 outer rows of hair cells in the basal, middle, and apical turns of the organ of Corti from the cochlea of Sprague-Dawley rats at postnatal day 2. However, EGCG counteracted this ototoxicity by suppressing the activation of caspase-3/NF-κB and preventing the destruction of hair cell arrays in the organ of Corti. These findings may lead to the development of a model for pharmacological mechanism of EGCG and potential therapies against ototoxicity.
• Jeong, H. J., Choi, Y., Kim, M. H., Kang, I. C., Lee, J. H., Park, C., Park, R., & Kim, H. M. (2011). Rosmarinic acid, active component of Dansam-Eum attenuates ototoxicity of cochlear hair cells through blockage of caspase-1 activity. PloS one, 6(4), e18815.
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  Cisplatin causes auditory impairment due to the apoptosis of auditory hair cells. There is no strategy to regulate ototoxicity by cisplatin thus far. Dansam-Eum (DSE) has been used for treating the central nerve system injury including hearing loss in Korea. However, disease-related scientific investigation by DSE has not been elucidated. Here, we demonstrated that DSE and its component rosmarinic acid (RA) were shown to inhibit apoptosis of the primary organ of Corti explants as well as the auditory cells. Administration of DSE and RA reduced the thresholds of the auditory brainstem response in cisplatin-injected mice. A molecular docking simulation and a kinetic assay show that RA controls the activity of caspase-1 by interaction with the active site of caspase-1. Pretreatment of RA inhibited caspase-1 downstream signal pathway, such as the activation of caspase-3 and 9, release of cytochrome c, translocation of apoptosis-inducing factor, up-regulation of Bax, down-regulation of Bcl-2, generation of reactive oxygen species, and activation of nuclear factor-κB. Anticancer activity by cisplatin was not affected by treatment with RA in SNU668, A549, HCT116, and HeLa cells but not B16F10 cells. These findings show that blocking a critical step by RA in apoptosis may be useful strategy to prevent harmful side effects of ototoxicity in patients with having to undergo chemotherapy.
• Kim, H. J., Oh, G. S., Lee, J. H., Lyu, A. R., Ji, H. M., Lee, S. H., Song, J., Park, S. J., You, Y. O., Sul, J. D., Park, C., Chung, S. Y., Moon, S. K., Lim, D. J., So, H. S., & Park, R. (2011). Cisplatin ototoxicity involves cytokines and STAT6 signaling network. Cell research, 21(6), 944-56.
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  We herein investigated the role of the STAT signaling cascade in the production of pro-inflammatory cytokines and cisplatin ototoxicity. A significant hearing impairment caused by cisplatin injection was observed in Balb/c (wild type, WT) and STAT4(-/-), but not in STAT6(-/-) mice. Moreover, the expression levels of the protein and mRNA of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were markedly increased in the serum and cochlea of WT and STAT4(-/-), but not STAT6(-/-) mice. Organotypic culture revealed that the shape of stereocilia bundles and arrays of sensory hair cell layers in the organ of Corti from STAT6(-/-) mice were intact after treatment with cisplatin, whereas those from WT and STAT4(-/-) mice were highly distorted and disarrayed after the treatment. Cisplatin induced the phosphorylation of STAT6 in HEI-OC1 auditory cells, and the knockdown of STAT6 by STAT6-specific siRNA significantly protected HEI-OC1 auditory cells from cisplatin-induced cell death and inhibited pro-inflammatory cytokine production. We further demonstrated that IL-4 and IL-13 induced by cisplatin modulated the phosphorylation of STAT6 by binding with IL-4 receptor alpha and IL-13Rα1. These findings suggest that STAT6 signaling plays a pivotal role in cisplatin-mediated pro-inflammatory cytokine production and ototoxicity.
• Kim, S. J., Myung, N. Y., Shin, B. G., Lee, J. H., So, H. S., Park, R. K., Um, J. Y., & Hong, S. H. (2011). Protective effect of a Chrysanthemum indicum containing formulation in cadmium-induced ototoxicity. The American journal of Chinese medicine, 39(3), 587-600.
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  Chungshinchongyitang (CSCYT) is an herbal drug formula containing Chrysanthemum indicum and 13 other herbs used for treating auditory diseases. Irreversible hearing loss is a characteristic effect of a number of heavy metals. Cadmium (Cd(2+)) is an environmental contaminant that causes a variety of adverse effects. In the present study, we investigate the protective effects of CSCYT against Cd(2+) induced ototoxicity in vitro and ex vivo. The findings of this study show that CSCYT prevents the destruction of hair cell arrays induced by Cd(2+) in the rat organ of Corti primary explants. CSCYT inhibited cell death, release of cytochrome c and generation of reactive oxygen species induced by Cd(2+) in HEI-OC1 auditory cell line. In addition, we also demonstrated that CSCYT exerted its effect by modulating of apoptosis via the caspase-3 activation and extracellular signal-regulated kinase activation. These results are expected to improve the understanding of the pharmacological mechanism of CSCYT and aid in the development of potential therapeutic strategies against ototoxicity.
• Kim, H. J., Lee, J. H., Kim, S. J., Oh, G. S., Moon, H. D., Kwon, K. B., Park, C., Park, B. H., Lee, H. K., Chung, S. Y., Park, R., & So, H. S. (2010). Roles of NADPH oxidases in cisplatin-induced reactive oxygen species generation and ototoxicity. The Journal of neuroscience : the official journal of the Society for Neuroscience, 30(11), 3933-46.
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  In our previous study, we clearly demonstrated the roles of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1beta (IL-1beta), and IL-6, and subsequent reactive oxygen species (ROS) generation on the pathogenesis of cisplatin ototoxicity in vitro and in vivo. ROS generation in cisplatin-treated HEI-OC1 auditory cells was also correlated with changing mitochondrial membrane potential. However, the roles of NADPH oxidase in cisplatin-induced ROS generation and ototoxicity have not been fully elucidated. Herein, immunohistochemical studies demonstrated that treatment of cisplatin induced the expression of NADPH oxidase isoforms NOX-1 and NOX-4 in HEI-OC1 auditory cells. Expression of mRNA for NOX-1, NOX-4, NOXO1, NOXA1, p47(phox), and p67(phox) was also increased. Inhibition of NADPH oxidase with diphenyleniodonium chloride or apocynin abolished ROS production and the subsequent apoptotic cell death in cisplatin-treated cells. Furthermore, suppression of NOX1 and NOX4 expression by small interfering RNA transfection markedly abolished the cytotoxicity and ROS generation by cisplatin. Together, our data suggest that ROS generated, in part, through the activation of NADPH oxidase plays an essential role in cisplatin ototoxicity.
• Kim, H. S., Cho, K. R., Lee, J. H., Kim, Y. H., Lim, S. H., Lee, K. M., Cheong, S. H., Kim, Y. J., Shin, C. M., & Lee, J. Y. (2010). Prevention of pain during injection of microemulsion propofol: application of lidocaine mixture and the optimal dose of lidocaine. Korean journal of anesthesiology, 59(5), 310-3.
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  Similar to lipid emulsion propofol, microemulsion propofol also causes a high incidence of pain during intravenous injection. Various methods have been used to minimize the incidence and severity of pain on injection of lipid emulsion propofol. In this study, we investigated the effect of a lidocaine mixture on pain induced by microemulsion propofol injection, and sought to determine the optimal dose of lidocaine that could reduce pain on injecting a propofol-lidocaine mixture.
• Shin, M. J., Lee, J. H., Yu, D. H., Kim, H. J., Bae, K. B., Yuh, H. S., Kim, M. O., Hyun, B. H., Lee, S., Park, R., & Ryoo, Z. Y. (2010). Spatiotemporal expression of tmie in the inner ear of rats during postnatal development. Comparative medicine, 60(4), 288-94.
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  The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.
• Kim, S. J., Park, C., Han, A. L., Youn, M. J., Lee, J. H., Kim, Y., Kim, E. S., Kim, H. J., Kim, J. K., Lee, H. K., Chung, S. Y., So, H., & Park, R. (2009). Ebselen attenuates cisplatin-induced ROS generation through Nrf2 activation in auditory cells. Hearing research, 251(1-2), 70-82.
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  Ebselen, an organoselenium compound that acts as a glutathione peroxidase mimetic, has been demonstrated to possess antioxidant and anti-inflammatory activities. However, the molecular mechanism underlying this effect is not fully understood in auditory cells. The purpose of the present study is to investigate the protective effect of ebselen against cisplatin-induced toxicity in HEI-OC1 auditory cells, organotypic cultures of cochlear explants from two-day postnatal rats (P(2)) and adult Balb/C mice. Pretreatment with ebselen ameliorated apoptotic death induced by cisplatin in HEI-OC1 cells and organotypic cultures of Corti's organ. Ebselen pretreatment also significantly suppressed cisplatin-induced increases in intracellular reactive oxygen species (ROS), intracellular reactive nitrogen species (RNS) and lipid peroxidation levels. Ebselen dose-dependently increased the expression level of an antioxidant response element (ARE)-luciferase reporter in HEI-OC1 cells through the translocation of Nrf2 into the nucleus. Furthermore, we found that pretreatment with ebselen significantly restored Nrf2 function, whereas it ameliorated the cytotoxicity of cisplatin in cells transfectants with either a pcDNA3.1 (control) or a DN-Nrf2 (dominant-negative) plasmid. We also observed that Nrf2 activation by ebselen increased the expression of phase II antioxidant genes, including heme oxygenase (HO-1), NAD(P)H:quinine oxidoreductase, and gamma-glutamylcysteine synthetase (gamma-GCS). Treatment with ebselen resulted in an increased expression of HO-1 and intranuclear Nrf2 in hair cells of organotypic cultured cochlea. After intraperitoneal injection with cisplatin, auditory brainstem responses (ABRs) threshold was measured on 8th day in Balb/C mice. ABR threshold shift was marked occurred in mice injected with cisplatin (16 mg/kg, n=5; Click and 8-kHz stimuli, p
• Kim, S. J., Shin, B. G., Choi, I. Y., Kim, D. H., Kim, M. C., Myung, N. Y., Moon, P. D., Lee, J. H., An, H. J., Kim, N. H., Lee, J. Y., So, H. S., Park, R. K., Jeong, H. J., Um, J. Y., Kim, H. M., & Hong, S. H. (2009). Hwanggunchungyitang prevents cadmium-induced ototoxicity through suppression of the activation of caspase-9 and extracellular signal-related kinase in auditory HEI-OC1 cells. Biological & pharmaceutical bulletin, 32(2), 213-9.
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  Hwanggunchungyitang (HGCYT) is a newly designed herbal drug formula for the purpose of treating auditory diseases. A number of heavy metals have been associated with toxic effects to the peripheral or central auditory system. Cadmium (Cd(2+)) is a heavy metal and a potent carcinogen implicated in tumor development through occupational and environmental exposure. However, the auditory effect of Cd(2+) is not poorly understood. The purpose of the present study was to investigate whether HGCYT prevent the ototoxic effects induced by Cd(2+) in auditory cell line, HEI-OC1. HGCYT inhibited the cell death, reactive oxygen species generation (ROS), activation of caspase-9, and extracellular signal-related kinase (ERK) induced by Cd(2+). In addition, we observed that cochlear hair cells in middle turn were damaged by Cd(2+). However, HGCYT prevented the destruction of hair cell arrays of the rat primary organ of Corti explants in the presence of Cd(2+). These results support the notion that ROS are involved in Cd(2+) ototoxicity and suggest HGCYT therapeutic usefulness, against Cd(2+)-induced activation of caspase-9 and ERK.
• Park, D. S., So, H. S., Lee, J. H., Park, H. Y., Lee, Y. J., Cho, J. H., Yoon, K. H., Park, C., Yun, K., & Park, R. (2009). Simvastatin treatment induces morphology alterations and apoptosis in murine cochlear neuronal cells. Acta oto-laryngologica, 129(2), 166-74.
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  Simvastatin presented neurodegenerative morphological changes and cell death via its specific inhibition of mevalonate pathway induced apoptosis in cultured cochlear neuronal cells. These findings might contribute to understanding the auditory neurobiological effects of HMG-CoA reductase inhibitors.
• Kim, H. J., So, H. S., Lee, J. H., Park, C., Lee, J. B., Youn, M. J., Kim, S. J., Yang, S. H., Lee, K. M., Kwon, K. B., Park, B. H., & Park, R. (2008). Role of proinflammatory cytokines in cisplatin-induced vestibular hair cell damage. Head & neck, 30(11), 1445-56.
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  Cisplatin causes the impairment of inner ear functions, including hearing and balance, through the involvement of a number of mechanisms. However, no laboratory studies have been performed on involvement of inflammation-related events in cisplatin-mediated vestibular dysfunction.
• Kim, S. J., Jeong, H. J., Myung, N. Y., Kim, M. C., Lee, J. H., So, H. S., Park, R. K., Kim, H. M., Um, J. Y., & Hong, S. H. (2008). The protective mechanism of antioxidants in cadmium-induced ototoxicity in vitro and in vivo. Environmental health perspectives, 116(7), 854-62.
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  Several heavy metals have been shown to have toxic effects on the peripheral and central auditory system. Cadmium (Cd2+) is an environmental contaminant showing a variety of adverse effects. Given the current rate of release into the environment, the amount of Cd2+ present in the human body and the incidence of Cd2+-related diseases are expected to increase.
• Kim, Y., So, H. S., Kim, S. J., Youn, M. J., Lee, J. H., Kim, N. S., Lee, J. H., Woo, W. H., Lee, D. W., Cho, K. H., Moon, B. S., & Park, R. (2008). Antiinflammatory effect of Daesiho, a Korean traditional prescription for cerebral infarct patients. Phytotherapy research : PTR, 22(6), 829-35.
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  Daesiho, a prescription composed of eight herbal mixtures, has been widely used in the treatment of cerebral infarct in Oriental medicine. However, the mechanisms by which the formula affects the production of pro-inflammatory cytokines in cerebral infarct patients remains unknown. The levels of secretory protein pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were significantly increased in both lipopolysaccharide (LPS) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from cerebral infarct patients and LPS-stimulated THP-1 differentiated macrophage-like cells (THP-1/M). However, pretreatment with Daesiho significantly inhibited the secretion of pro-inflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, in stimulated PBMCs and THP-1/M cells. In addition, Daesiho significantly suppressed mRNA expression of pro-inflammatory cytokines. Therefore, these data indicate that Daesiho may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression of the production of pro-inflammatory cytokines via inhibition of mRNA expression.
• Shin, M. J., Lee, J. H., Yu, D. H., Kim, B. S., Kim, H. J., Kim, S. H., Kim, M. O., Park, C., Hyun, B. H., Lee, S., So, H. S., Park, R., & Ryoo, Z. Y. (2008). Ectopic expression of tmie transgene induces various recovery levels of behavior and hearing ability in the circling mouse. Biochemical and biophysical research communications, 374(1), 17-21.
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  The circling (cir/cir) mouse is one of the murine models for human non-syndromic deafness DFNB6. The mice have abnormal circling behavior, suggesting a balanced disorder and profound deafness. The causative gene was transmembrane inner ear (tmie) gene of which the mutation is a 40-kb genomic deletion including tmie gene itself. In this study, tmie-overexpression trasngenic mice were established. Individuals with germline transmission have been mated with circling homozygous mutant mice (cir/cir) in order to produce the transgenic mutant mice (cir/cir-tg) as a gene therapy. After the genotyping, phenotypic analyses were performed so that the insertion of the new gene might compensate for the diseases such as hearing loss, circling behavior, or swimming inability. Some individuals exhibited complete recovery in their behavior and hearing but the others did not show any amelioration in behavior or hearing. Individual mice had very different levels of tmie transgene expression in the cochlea. These results clearly indicate that tmie protein plays an important role when the appropriate expression level of tmie was expressed in the inner ear. The protein levels were variable in each individual and these are thought to induce the differences in disease amelioration levels.
• Youn, M. J., So, H. S., Cho, H. J., Kim, H. J., Kim, Y., Lee, J. H., Sohn, J. S., Kim, Y. K., Chung, S. Y., & Park, R. (2008). Berberine, a natural product, combined with cisplatin enhanced apoptosis through a mitochondria/caspase-mediated pathway in HeLa cells. Biological & pharmaceutical bulletin, 31(5), 789-95.
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  Berberine, a main component of Coptidis Rhizoma, has been extensively studied and is known to exhibit multiple pharmacologic activities. In this study, we investigated whether the combination of berberine and cisplatin exhibited significant cytotoxicity in HeLa cells. Apoptosis was evaluated based on DNA fragmentation and cytofluorometrically with the annexin-V/propidium iodide labeling method. Combined treatment with berberine and cisplatin acted in concert to induce loss of mitochondrial membrane potential (Delta Psi m), release of cytochrome-c from mitochondria, and decreased expression of antiapoptotic Bcl-2, Bcl-x/L, resulting in activation of caspases and apoptosis. Further study showed that cell death induced by the combined treatment was associated with increased reactive oxygen species generation and lipid peroxidation. Moreover, we discovered that the combined treatment-induced apoptosis was mediated by the activation of the caspase cascade. These results indicated that the potential of cytotoxicity mediated through the mitochondria-caspase pathway is primarily involved in the combined treatment-induced apoptosis.
• So, H., Kim, H., Lee, J. H., Park, C., Kim, Y., Kim, E., Kim, J. K., Yun, K. J., Lee, K. M., Lee, H. Y., Moon, S. K., Lim, D. J., & Park, R. (2007). Cisplatin cytotoxicity of auditory cells requires secretions of proinflammatory cytokines via activation of ERK and NF-kappaB. Journal of the Association for Research in Otolaryngology : JARO, 8(3), 338-55.
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  The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, through the activation of ERK and NF-kappaB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-alpha. Immunohistochemical studies revealed that TNF-alpha expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kappaB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-alpha, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.
• Kim, H. J., So, H. S., Lee, J. H., Lee, J. H., Park, C., Park, S. Y., Kim, Y. H., Youn, M. J., Kim, S. J., Chung, S. Y., Lee, K. M., & Park, R. (2006). Heme oxygenase-1 attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation. Free radical biology & medicine, 40(10), 1810-9.
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  Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme catabolism, is known to modulate various cellular functions, including cytokine production, cell proliferation, and apoptosis, in stress-related conditions. However, the role of HO-1 in the auditory system remains elusive. Herein, we demonstrate that pharmacologic induction of HO-1 along with catalytic activation significantly suppressed apoptosis of HEI-OC1 cells induced by cisplatin. Studies of ectopic expression of pcDNA3-HO-1 and siRNA of HO-1 further revealed the protective role of HO-1 against cisplatin in HEI-OC1 cells. Among the catabolic metabolites of HO-1, both carbon monoxide (CO) and bilirubin were directly involved in the protective role of HO-1 against cisplatin through inhibition of reactive oxygen species generation. Furthermore, pharmacological induction of HO-1 completely prevented the destruction of outer hair cell arrays by cisplatin through a CO-dependent mechanism in organotrophic culture of the rat primary organ of Corti explants. These results suggest that HO-1 may serve as a safeguard of auditory sensory hair cells against a variety of challenges of oxidative stress, including noise trauma, presbycusis, and ototoxic drugs, respectively.