Sirtuin 1 and Autophagy Attenuate Cisplatin-Induced Hair Cell Death in the Mouse Cochlea and Zebrafish Lateral Line.
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ABSTRACT:
SUBMITTER: Pang J
PROVIDER: S-EPMC6339946 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Here we present spatio-temporal localization of Kremen1, a transmembrane receptor, in the mammalian cochlea, and investigate its role in the formation of sensory organs in mammal and fish model organisms. We show that Kremen1 is expressed in prosensory cells during cochlear development and in supporting cells of the adult mouse cochlea. Based on this expression pattern, we investigated whether Kremen1 functions to modulate cell fate decisions in the prosensory domain of the developing cochlea. We used gain and loss-of-function experiments to show that Kremen1 is sufficient to bias cells towards supporting cell fate, and is implicated in suppression of hair cell formation. In addition to our findings in the mouse cochlea, we examined the effects of over expression and loss of Kremen1 in the zebrafish lateral line. In agreement with our mouse data, we show that over expression of Kremen1 has a negative effect on the number of mechanosensory cells that form in the zebrafish neuromasts, and that fish lacking Kremen1 protein develop more hair cells per neuromast compared to wild type fish. Collectively, these data support an inhibitory role for Kremen1 in hair cell fate specification.
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Project description:Aminoglycoside antibiotics cause death of sensory hair cells. Research over the past decade has identified several key players in the intracellular cascade. However, the role of the extracellular environment in aminoglycoside ototoxicity has received comparatively little attention. The present study uses the zebrafish lateral line to demonstrate that extracellular calcium and magnesium ions modulate hair cell death from neomycin and gentamicin in vivo, with high levels of either divalent cation providing significant protection. Imaging experiments with fluorescently-tagged gentamicin show that drug uptake is reduced under high calcium conditions. Treating fish with the hair cell transduction blocker amiloride also reduces aminoglycoside uptake, preventing the toxicity, and experiments with variable calcium and amiloride concentrations suggest complementary effects between the two protectants. Elevated magnesium, in contrast, does not appear to significantly attenuate drug uptake, suggesting that the two divalent cations may protect hair cells from aminoglycoside damage through different mechanisms. These results provide additional evidence for calcium- and transduction-dependent aminoglycoside uptake. Divalent cations provided differential protection from neomycin and gentamicin, with high cation concentrations almost completely protecting hair cells from neomycin and acute gentamicin toxicity, but offering reduced protection from continuous (6 h) gentamicin exposure. These experiments lend further support to the hypothesis that aminoglycoside toxicity occurs via multiple pathways in a both a drug and time course-specific manner.
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Project description:Exposure to ototoxic drugs is a significant cause of hearing loss that affects about 30 thousand children with potentially serious physical, social and psychological dysfunctions every year. Cisplatin (CP) and aminoglycosides are effective antineoplastic or bactericidal drugs, and their application has a high probability of ototoxicity which results from the death of hair cells (HCs). Here, we describe the therapeutic effect of the flavonoid compound naringin (Nar) against ototoxic effects of cisplatin and aminoglycosides include gentamicin (GM) and neomycin (Neo) in zebrafish HCs. Animals incubated with Nar (100-400 μmol/L) were protected against the pernicious effects of CP (150-250 μmol/L), GM (50-150 μmol/L) and Neo (50-150 μmol/L). We also provide evidence for the potential mechanism of Nar against ototoxicity, including antioxidation, anti-apoptosis, promoting proliferation and hair cell regeneration. We found that mRNA levels of the apoptotic- and pyroptosis-related genes are regulated by Nar both in vivo and in vitro. Finally, by proving that Nar does not affect the anti-tumour efficacy of CP and antibacterial activity of aminoglycosides in vitro, we highlight its value in clinical application. In conclusion, these results unravel a novel therapeutic role for Nar as an otoprotective drug against the adverse effects of CP and aminoglycosides.
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Project description:We report a series of experiments investigating the kinetics of hair cell loss in lateral line neuromasts of zebrafish larvae following exposure to aminoglycoside antibiotics. Comparisons of the rate of hair cell loss and the differential effects of acute versus chronic exposure to gentamicin and neomycin revealed markedly different results. Neomycin induced rapid and dramatic concentration-dependent hair cell loss that is essentially complete within 90 min, regardless of concentration or exposure time. Gentamicin-induced loss of half of the hair cells within 90 min and substantial additional loss, which was prolonged and cumulative over exposure times up to at least 24h. Small molecules and genetic mutations that inhibit neomycin-induced hair cell loss were ineffective against prolonged gentamicin exposure supporting the hypothesis that these two drugs are revealing at least two cellular pathways. The mechanosensory channel blocker amiloride blocked both neomycin and gentamicin-induced hair cell death acutely and chronically indicating that these aminoglycosides share a common entry route. Further tests with additional aminoglycosides revealed a spectrum of differential responses to acute and chronic exposure. The distinctions between the times of action of these aminoglycosides indicate that these drugs induce multiple cell death pathways.
| S-EPMC3167481 | biostudies-literature
Project description:Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ?AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure.
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Project description:Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.
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Project description:Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/?-catenin signaling following hair cell death. We propose that Wnt/?-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.
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Project description:The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration.
| S-EPMC3281009 | biostudies-literature
Project description:Key pointsWe investigated hair-cell regeneration in the zebrafish lateral line following the application of the ototoxic compound copper. In early-larval zebrafish (<10 days post-fertilisation), regenerated hair cells drive action potentials (APs) in the afferent neurons 24 h post-copper treatment (24 hpt). Full regeneration of the early-larval neuromasts, the organs containing the hair cells, requires ∼48 h due to the progressive addition of hair cells and synaptic refinement. In older larval zebrafish, regenerated hair cells are active and drive afferent APs by 48 hpt, which is comparable to larvae, but the functional recovery of their neuromasts requires >120 hpt. Afferent terminals within the regenerating neuromast appear to initially contact supporting cells, and their complete ablation prevents the timely reappearance of supporting cells and hair cells. We conclude that the regeneration of zebrafish neuromasts is slower after the initial developmental stages, and that the afferent input plays a key role in driving this process.AbstractHair cells are mechanosensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. Different from mammals, the hair cells of lower vertebrates, including those present in the neuromasts of the zebrafish lateral line, regenerate following environmental or chemical insults. Here we investigate the time course of regeneration of hair cells in vivo using electrophysiology, two-photon imaging and immunostaining applied to wild-type and genetically encoded fluorescent indicator zebrafish lines. Functional hair cells drive spontaneous action potentials in the posterior lateral line afferent fibres, the frequency of which progressively increases over the first 10 days post-fertilisation (dpf). Higher firing-rate fibres are only observed from ∼6 dpf. Following copper treatment, newly formed hair cells become functional and are able to drive APs in the afferent fibres within 48 h in both early-larval (≤8 dpf) and late-larval (12-17 dpf) zebrafish. However, the complete functional regeneration of the entire neuromast is delayed in late-larval compared to early-larval zebrafish. We propose that while individual regenerating hair cells can rapidly become active, the acquisition of fully functional neuromasts progresses faster at early-larval stages, a time when hair cells are still under development. At both ages, the afferent terminals in the regenerating neuromast appear to make initial contact with supporting cells. The ablation of the lateral line afferent neurons prevents the timely regeneration of supporting cells and hair cells. These findings indicate that the afferent system is likely to facilitate or promote the neuromast regeneration process.
| S-EPMC7612129 | biostudies-literature
Project description:In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.
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