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1.
Front Cell Dev Biol ; 11: 1247324, 2023.
Article in English | MEDLINE | ID: mdl-37900280

ABSTRACT

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.

2.
Nat Commun ; 13(1): 5818, 2022 10 07.
Article in English | MEDLINE | ID: mdl-36207323

ABSTRACT

Human papillomavirus (HPV)-associated cervical cancer is a leading cause of cancer deaths in women. Here we present an integrated multi-omic analysis of 643 cervical squamous cell carcinomas (CSCC, the most common histological variant of cervical cancer), representing patient populations from the USA, Europe and Sub-Saharan Africa and identify two CSCC subtypes (C1 and C2) with differing prognosis. C1 and C2 tumours can be driven by either of the two most common HPV types in cervical cancer (16 and 18) and while HPV16 and HPV18 are overrepresented among C1 and C2 tumours respectively, the prognostic difference between groups is not due to HPV type. C2 tumours, which comprise approximately 20% of CSCCs across these cohorts, display distinct genomic alterations, including loss or mutation of the STK11 tumour suppressor gene, increased expression of several immune checkpoint genes and differences in the tumour immune microenvironment that may explain the shorter survival associated with this group. In conclusion, we identify two therapy-relevant CSCC subtypes that share the same defining characteristics across three geographically diverse cohorts.


Subject(s)
Carcinoma, Squamous Cell , Papillomavirus Infections , Uterine Cervical Neoplasms , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Female , Human papillomavirus 16/genetics , Humans , Papillomaviridae/genetics , Papillomavirus Infections/complications , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Prognosis , Tumor Microenvironment , Uterine Cervical Neoplasms/pathology
3.
iScience ; 24(5): 102486, 2021 May 21.
Article in English | MEDLINE | ID: mdl-34027326

ABSTRACT

Across their lives, biological sensors maintain near-constant functional outputs despite countless exogenous and endogenous perturbations. This sensory homeostasis is the product of multiple dynamic equilibria, the breakdown of which contributes to age-related decline. The mechanisms of homeostatic maintenance, however, are still poorly understood. The ears of vertebrates and insects are characterized by exquisite sensitivities but also by marked functional vulnerabilities. Being under the permanent load of thermal and acoustic noise, auditory transducer channels exemplify the homeostatic challenge. We show that (1) NompC-dependent mechanotransducers in the ear of the fruit fly Drosophila melanogaster undergo continual replacement with estimated turnover times of 9.1 hr; (2) a de novo synthesis of NompC can restore transducer function in the adult ears of congenitally hearing-impaired flies; (3) key components of the auditory transduction chain, including NompC, are under activity-dependent transcriptional control, likely forming a transducer-operated mechanosensory gain control system that extends beyond hearing organs.

4.
JCI Insight ; 6(7)2021 04 08.
Article in English | MEDLINE | ID: mdl-33735112

ABSTRACT

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.


Subject(s)
Aminoglycosides/adverse effects , Cochlea/drug effects , Ototoxicity/prevention & control , Animals , Cochlea/cytology , Drug Evaluation, Preclinical/methods , Embryo, Nonmammalian/drug effects , Female , Gentamicins/adverse effects , Gentamicins/pharmacology , Hair Cells, Auditory/drug effects , Male , Mechanotransduction, Cellular/drug effects , Mice, Inbred Strains , Microbial Sensitivity Tests , Microphthalmia-Associated Transcription Factor/genetics , Neomycin/adverse effects , Organ Culture Techniques , Ototoxicity/etiology , Protective Agents/administration & dosage , Protective Agents/pharmacology , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics
5.
Front Cell Neurosci ; 13: 416, 2019.
Article in English | MEDLINE | ID: mdl-31572129

ABSTRACT

Aminoglycoside antibiotics are widely prescribed to treat a variety of serious bacterial infections. They are extremely useful clinical tools, but have adverse side effects such as oto- and nephrotoxicity. Once inside a cell they are thought to cause mitochondrial dysfunction, subsequently leading to apoptotic cell death due to an increase in reactive oxygen species (ROS) production. Here we present evidence of a direct effect of gentamicin (the most commonly prescribed aminoglycoside) on the respiratory activities of isolated rat liver and kidney mitochondria. We show that gentamicin stimulates state 4 and inhibits state 3u respiratory rates, thereby reducing the respiratory control ratio (RCR) whilst simultaneously causing a collapse of the mitochondrial membrane potential (MtMP). We propose that gentamicin behaves as an uncoupler of the electron transport chain (ETC) - a hypothesis supported by our evidence that it reduces the production of mitochondrial ROS (MtROS). We also show that gentamicin collapses the MtMP in the sensory hair cells (HCs) of organotypic mouse cochlear cultures.

6.
JCI Insight ; 4(15)2019 08 08.
Article in English | MEDLINE | ID: mdl-31391343

ABSTRACT

Aminoglycoside (AG) antibiotics are widely used to prevent life-threatening infections, and cisplatin is used in the treatment of various cancers, but both are ototoxic and result in loss of sensory hair cells from the inner ear. ORC-13661 is a new drug that was derived from PROTO-1, a compound first identified as protective in a large-scale screen utilizing hair cells in the lateral line organs of zebrafish larvae. Here, we demonstrate, in zebrafish larvae and in mouse cochlear cultures, that ORC-13661 provides robust protection of hair cells against both ototoxins, the AGs and cisplatin. ORC-13661 also prevents both hearing loss in a dose-dependent manner in rats treated with amikacin and the loading of neomycin-Texas Red into lateral line hair cells. In addition, patch-clamp recordings in mouse cochlear cultures reveal that ORC-13661 is a high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in outer hair cells, suggesting that it may reduce the toxicity of AGs by directly competing for entry at the level of the MET channel and of cisplatin by a MET-dependent mechanism. ORC-13661 is therefore a promising and versatile protectant that reversibly blocks the hair cell MET channel and operates across multiple species and toxins.


Subject(s)
Anti-Bacterial Agents/toxicity , Antineoplastic Agents/toxicity , Hair Cells, Auditory/drug effects , Ototoxicity/prevention & control , Protective Agents/pharmacology , Thiophenes/pharmacology , Urea/analogs & derivatives , Amikacin/toxicity , Aminoglycosides/toxicity , Animals , Cell Culture Techniques , Cells, Cultured , Cisplatin/toxicity , Disease Models, Animal , Dose-Response Relationship, Drug , Hair Cells, Auditory/metabolism , Humans , Intravital Microscopy , Ion Channels/antagonists & inhibitors , Ion Channels/metabolism , Male , Mechanotransduction, Cellular/drug effects , Mice , Ototoxicity/etiology , Patch-Clamp Techniques , Protective Agents/therapeutic use , Rats , Thiophenes/therapeutic use , Time-Lapse Imaging , Urea/pharmacology , Urea/therapeutic use , Zebrafish
7.
J Med Chem ; 62(11): 5312-5329, 2019 06 13.
Article in English | MEDLINE | ID: mdl-31083995

ABSTRACT

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell's mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions.


Subject(s)
Aminoglycosides/adverse effects , Carvedilol/chemical synthesis , Carvedilol/pharmacology , Drug Design , Hair Cells, Auditory/cytology , Hair Cells, Auditory/drug effects , Mechanotransduction, Cellular/drug effects , Animals , Carvedilol/chemistry , Chemistry Techniques, Synthetic , Cytoprotection/drug effects , Dose-Response Relationship, Drug , Electrophysiological Phenomena/drug effects , Mice , Zebrafish
8.
Cell Death Dis ; 9(9): 922, 2018 09 11.
Article in English | MEDLINE | ID: mdl-30206231

ABSTRACT

In view of the prevalence of sensorineural hearing defects in an ageing population, the development of protocols to generate cochlear hair cells and their associated sensory neurons as tools to further our understanding of inner ear development are highly desirable. We report herein a robust protocol for the generation of both vestibular and cochlear hair cells from human pluripotent stem cells which represents an advance over currently available methods that have been reported to generate vestibular hair cells only. Generating otic organoids from human pluripotent stem cells using a three-dimensional culture system, we show formation of both types of sensory hair cells bearing stereociliary bundles with active mechano-sensory ion channels. These cells share many morphological characteristics with their in vivo counterparts during embryonic development of the cochlear and vestibular organs and moreover demonstrate electrophysiological activity detected through single-cell patch clamping. Collectively these data represent an advance in our ability to generate cells of an otic lineage and will be useful for building models of the sensory regions of the cochlea and vestibule.


Subject(s)
Ear, Inner/cytology , Hair Cells, Auditory, Inner/cytology , Hair Cells, Vestibular/cytology , Organogenesis/physiology , Pluripotent Stem Cells/cytology , Cell Culture Techniques/methods , Cell Differentiation/physiology , Cells, Cultured , Hearing Loss, Sensorineural/pathology , Hearing Loss, Sensorineural/therapy , Humans
9.
JCI Insight ; 2(24)2017 12 21.
Article in English | MEDLINE | ID: mdl-29263311

ABSTRACT

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red-conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem.


Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Hair Cells, Auditory/drug effects , Neuroprotective Agents/pharmacology , Aminoglycosides/antagonists & inhibitors , Animals , Cell Death/drug effects , Cochlea/drug effects , Drug Evaluation, Preclinical/methods , Female , Gentamicins/antagonists & inhibitors , Gentamicins/pharmacology , Ion Channels/drug effects , Male , Mice , Tissue Culture Techniques , Zebrafish
10.
Front Cell Neurosci ; 11: 262, 2017.
Article in English | MEDLINE | ID: mdl-28928635

ABSTRACT

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell's stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 µM) or berbamine (≥1.55 µM) protect zebrafish hair cells in vivo from neomycin (6.25 µM, 1 h). Protection of zebrafish hair cells against gentamicin (10 µM, 6 h) was provided by ≥25 µM dTC or ≥12.5 µM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 µM, 48 h) by 20 µM berbamine or 25 µM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 µM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 µM (dTC) and 2.8 µM (berbamine) in the presence of 1.3 mM Ca2+ at -104 mV. Berbamine, but not dTC, also blocks the hair cell's basolateral K+ current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants.

11.
J Neurosci ; 34(16): 5505-14, 2014 Apr 16.
Article in English | MEDLINE | ID: mdl-24741041

ABSTRACT

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca(2+) chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current-voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.


Subject(s)
Cell Membrane Permeability/physiology , Hair Cells, Auditory/physiology , Ion Channels/physiology , Mechanotransduction, Cellular/physiology , Animals , Animals, Newborn , Cadherin Related Proteins , Cadherins/genetics , Cell Membrane Permeability/genetics , Chelating Agents/pharmacology , Dihydrostreptomycin Sulfate/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Embryo, Mammalian , Female , Hair Cells, Auditory/cytology , Hair Cells, Auditory/drug effects , In Vitro Techniques , Ion Channels/drug effects , Male , Mechanotransduction, Cellular/drug effects , Mechanotransduction, Cellular/genetics , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myosin VIIa , Myosins/genetics , Organ of Corti/cytology , Protein Precursors/genetics
12.
Curr Biol ; 23(1): R22-3, 2013 Jan 07.
Article in English | MEDLINE | ID: mdl-23305665

ABSTRACT

Two new studies in the fruit fly Drosophila demonstrate unexpected molecular, and mechanistic, overlaps between the different senses. In the centre stand two long-established families of sensory proteins--rhodopsins and TRP channels.


Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Light Signal Transduction , Mechanotransduction, Cellular , Sensory Rhodopsins/physiology , Transient Receptor Potential Channels/physiology , Animals , Drosophila Proteins/metabolism , Evoked Potentials, Visual , Light , Microscopy, Atomic Force , Photoreceptor Cells, Invertebrate/physiology , Sensory Rhodopsins/metabolism , Transient Receptor Potential Channels/metabolism
13.
Open Biol ; 2(5): 120068, 2012 May.
Article in English | MEDLINE | ID: mdl-22724068

ABSTRACT

Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.


Subject(s)
Hair Cells, Auditory/physiology , Mechanotransduction, Cellular/physiology , Nerve Tissue Proteins/physiology , Sensory Receptor Cells/physiology , TRPC Cation Channels/physiology , Action Potentials/drug effects , Animals , Cell Size , Cells, Cultured/drug effects , Cells, Cultured/physiology , Evoked Potentials, Auditory, Brain Stem , Ganglia, Spinal/cytology , Hair Cells, Auditory/classification , Hair Cells, Auditory/drug effects , Hair Cells, Auditory, Outer/drug effects , Hair Cells, Auditory, Outer/physiology , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/physiopathology , Hypesthesia/genetics , Hypesthesia/physiopathology , Imidazoles/pharmacology , Ion Transport/drug effects , Ion Transport/physiology , Mechanotransduction, Cellular/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Primary Cell Culture , Sensory Receptor Cells/classification , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/ultrastructure , TRPC Cation Channels/biosynthesis , TRPC Cation Channels/deficiency , TRPC Cation Channels/genetics , TRPC6 Cation Channel , Vestibular Diseases/genetics , Vestibular Diseases/physiopathology
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