Maria Cristina Perez Flores

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• 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.
• 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.
• 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.
• Perez-Flores, M. C., Lee, J. H., Park, S., Zhang, X. D., Sihn, C. R., Ledford, H. A., Wang, W., Kim, H. J., Timofeyev, V., Yarov-Yarovoy, V., Chiamvimonvat, N., Rabbitt, R. D., & Yamoah, E. N. (2020). Cooperativity of K7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells. Science advances, 6(15), eaba1104.
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  The mammalian cochlea relies on active electromotility of outer hair cells (OHCs) to resolve sound frequencies. OHCs use ionic channels and somatic electromotility to achieve the process. It is unclear, though, how the kinetics of voltage-gated ionic channels operate to overcome extrinsic viscous drag on OHCs at high frequency. Here, we report ultrafast electromechanical gating of clustered K7.4 in OHCs. Increases in kinetics and sensitivity resulting from cooperativity among clustered-K7.4 were revealed, using optogenetics strategies. Upon clustering, the half-activation voltage shifted negative, and the speed of activation increased relative to solitary channels. Clustering also rendered K7.4 channels mechanically sensitive, confirmed in consolidated K7.4 channels at the base of OHCs. K7.4 clusters provide OHCs with ultrafast electromechanical channel gating, varying in magnitude and speed along the cochlea axis. Ultrafast K7.4 gating provides OHCs with a feedback mechanism that enables the cochlea to overcome viscous drag and resolve sounds at auditory frequencies.
• 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.
• Finno, C. J., Peterson, J., Kang, M., Park, S., Bordbari, M. H., Durbin-Johnson, B., Settles, M., Perez-Flores, M. C., Lee, J. H., & Yamoah, E. N. (2019). Single-Cell RNA-seq Reveals Profound Alterations in Mechanosensitive Dorsal Root Ganglion Neurons with Vitamin E Deficiency. iScience, 21, 720-735.
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  Ninety percent of Americans consume less than the estimated average requirements of dietary vitamin E (vitE). Severe vitE deficiency due to genetic mutations in the tocopherol transfer protein (TTPA) in humans results in ataxia with vitE deficiency (AVED), with proprioceptive deficits and somatosensory degeneration arising from dorsal root ganglia neurons (DRGNs). Single-cell RNA-sequencing of DRGNs was performed in Ttpa mice, an established model of AVED. In stark contrast to expected changes in proprioceptive neurons, Ttpa DRGNs showed marked upregulation of voltage-gated Ca and K channels in mechanosensitive, tyrosine-hydroxylase positive (TH+) DRGNs. The ensuing significant conductance changes resulted in reduced excitability in mechanosensitive Ttpa DRGNs. A highly supplemented vitE diet (600 mg dl-α-tocopheryl acetate/kg diet) prevented the cellular and molecular alterations and improved mechanosensation. VitE deficiency profoundly alters the molecular signature and functional properties of mechanosensitive TH+ DRGN, representing an intriguing shift of the prevailing paradigm from proprioception to mechanical sensation.
• 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.
• Yu, Y., Hu, B., Bao, J., Mulvany, J., Bielefeld, E., Harrison, R. T., Neton, S. A., Thirumala, P., Chen, Y., Lei, D., Qiu, Z., Zheng, Q., Ren, J., Perez-Flores, M. C., Yamoah, E. N., & Salehi, P. (2018). Otoprotective Effects of Stephania tetrandra S. Moore Herb Isolate against Acoustic Trauma. Journal of the Association for Research in Otolaryngology : JARO, 19(6), 653-668.
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  Noise is the most common occupational and environmental hazard, and noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit. Although therapeutics that target the free-radical pathway have shown promise, none of these compounds is currently approved against NIHL by the United States Food and Drug Administration. The present study has demonstrated that tetrandrine (TET), a traditional Chinese medicinal alkaloid and the main chemical isolate of the Stephania tetrandra S. Moore herb, significantly attenuated NIHL in CBA/CaJ mice. TET is known to exert antihypertensive and antiarrhythmic effects through the blocking of calcium channels. Whole-cell patch-clamp recording from adult spiral ganglion neurons showed that TET blocked the transient Ca current in a dose-dependent manner and the half-blocking concentration was 0.6 + 0.1 μM. Consistent with previous findings that modulations of calcium-based signaling pathways have both prophylactic and therapeutic effects against neural trauma, NIHL was significantly diminished by TET administration. Importantly, TET has a long-lasting protective effect after noise exposure (48 weeks) in comparison to 2 weeks after noise exposure. The otoprotective effects of TET were achieved mainly by preventing outer hair cell damage and synapse loss between inner hair cells and spiral ganglion neurons. Thus, our data indicate that TET has great potential in the prevention and treatment of NIHL.
• 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.
• 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.