Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
[Preprint]. 2024 Jul 12:2024.05.30.596537.
doi: 10.1101/2024.05.30.596537.

Multiple mechanisms of aminoglycoside ototoxicity are distinguished by subcellular localization of action

Patricia Wu  1   2 , Francisco Barros Becker  1   3 , Roberto Ogelman  1   2 , Esra D Camci  1   3 , Tor H Linbo  2   3 , Julian A Simon  4 , Edwin W Rubel  1 , David W Raible  1   2   3
Affiliations

Multiple mechanisms of aminoglycoside ototoxicity are distinguished by subcellular localization of action

Patricia Wu et al. bioRxiv. .

Update in

Abstract

Mechanosensory hair cells of the inner ears and lateral line of vertebrates display heightened vulnerability to environmental insult, with damage resulting in hearing and balance disorders. An important example is hair cell loss due to exposure to toxic agents including therapeutic drugs such as the aminoglycoside antibiotics such as neomycin and gentamicin and antineoplastic agents. We describe two distinct cellular pathways for aminoglycoside-induced hair cell death in zebrafish lateral line hair cells. Neomycin exposure results in death from acute exposure with most cells dying within 1 hour of exposure. By contrast, exposure to gentamicin results primarily in delayed hair cell death, taking up to 24 hours for maximal effect. Washout experiments demonstrate that delayed death does not require continuous exposure, demonstrating two mechanisms where downstream responses differ in their timing. Acute damage is associated with mitochondrial calcium fluxes and can be alleviated by the mitochondrially-targeted antioxidant mitoTEMPO, while delayed death is independent of these factors. Conversely delayed death is associated with lysosomal accumulation and is reduced by altering endolysosomal function, while acute death is not sensitive to lysosomal manipulations. These experiments reveal the complexity of responses of hair cells to closely related compounds, suggesting that intervention focusing on early events rather than specific death pathways may be a successful therapeutic strategy.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
AGs differ in relative hair cell toxicity dependent on time of exposure and length of incubation. (A-C) Fish were treated with 200 μM neomycin (Neo) or gentamicin (Gent) (red bar) for time indicated, or rinsed into fresh medium (blue bar). A) Hair cells were exposed for 1hr acute treatment with AGs. Hair cells are effectively killed by Neo, but are spared by Gent. n=9-11 fish, 4 NMs/fish. Unpaired t test with Welch's correction, **** P value <0.0001 B) Chronic 24hrs treatment with Neo or Gent results in hair cell loss. n=9-11 fish, 4 NMs/fish. Unpaired t test with Welch's correction, **** P value <0.0001 C) Delayed death occurs after 1hr treatment with Neo or Gent, rinsing, and then incubation for 23hrs in fresh medium (1+23hrs). n=9-11 fish, 4 NMs/fish. Unpaired t test with Welch's correction, ** P value = 0.0073 D) Dose-dependent loss of hair cells after treatment with neomycin for 1hr, 24hrs or 1+23hrs. Differences between treatments were highly significant (2-way ANOVA, Tukey’s multiple comparison, p<0.0005). n=9-11 fish, 4 NMs/fish for each condition. E) Dose-dependent loss of hair cells after treatment with gentamicin for 1hr, 24hrs or 1+23hrs. Differences between treatments were highly significant (2-way ANOVA, Tukey’s multiple comparison, p<0.0001). n=9-11 fish, 4 NMs/fish for each condition. Error bars represent Standard Deviation.
Figure 2.
Figure 2.
The rate of delayed hair cell loss is dependent on initial gentamicin concentration. Fish were treated with doses of gentamicin (25, 50, 100, 200 μM) for 1hr (red bar), then rinsed and incubated in fresh medium (blue bar). Loss of hair cells was assessed at 5hrs, 11hrs, 17hrs and 23hrs after the 1hr incubation period. Increasing initial dose results in more rapid delayed hair cell loss. Differences between 25 μM, 50 μM and 200 μM treatments were significant (Tukey’s multiple comparison, p<0.0001). There was no significant difference between 100 μM and 200 μm treatments. n=9-11 fish, 4 NMs/fish for each treatment. Error bars represent Standard Deviation.
Figure 3.
Figure 3.
Different mitochondrial Ca2+ responses during acute or delayed hair cell death. A-C) Fish were incubated in AG (red bar) for time indicated, or rinsed into fresh medium (blue bar). Fluorescence changes above baseline (F/F0) from mitoGCaMP in response to AG addition were monitored at 30 sec intervals by spinning disk microscopy over the interval indicated (black bar). Individual traces represent responses of individual cells. Traces are aligned to time of cell fragmentation. A) Changes in mitoGCaMP signal in cells undergoing acute death in response to 100 μM neomycin. Hair cells were imaged during the first hour of neomycin exposure. Increases in mitochondrial Ca2+ were observed in 16/16 dying cells. B) Changes in mitoGCaMP signal in cells undergoing delayed death after exposure to 100 μM G418. Cells were exposed to G418 for 1hr followed by rinse and incubation in fresh embryo medium (EM) for 1.5hrs, and then imaged over an additional 2hr period. Increases in mitochondrial Ca2+ were observed in 2/11 dying cells. C) Changes in mitoGCaMP signal in cells undergoing acute death in response to 400 μM G418. Hair cells were imaged during the first hour of G418 exposure. Increases in mitochondrial Ca2+ were observed in 10/14 dying cells. D) Maximum mitoGCaMP signal compared to baseline for dying cells after neomycin or G418 exposure. ***Dunn’s multiple comparison test p<0.0005. Error bars represent Standard Deviation.
Figure 4.
Figure 4.
The mitochondrially-targeted antioxidant mitoTEMPO protects against acute neomycin damage from neomycin but not delayed damage from G418. 50 μM mitoTEMPO was added for 30min before AG (purple bar), co-treated with neomycin for 1hr or co-treated with G418 for 1hr (red bar), rinsed, and incubated with mitoTEMPO alone for 23hrs (purple bar). A) mitoTEMPO partially protected against damage from both 100 μM and 200 μM neomycin treatment. **** Two-way ANOVA, Sidak’s multiple comparison, p<0.0001. B) mitoTEMPO offered no protection against damage from either 100 μM or 200 μM G418 treatment. ns Two-way ANOVA, Sidak’s multiple comparison, p = 0.98. n=9-11 fish, 4 NMs/fish for each treatment group. Error bars represent Standard Deviation.
Figure 5.
Figure 5.
Neomycin and G418 differentially accumulate in Rab7+ vesicles. A) G418-TR (magenta) accumulation in Rab7+ vesicles (green) in neuromast from Tg(myosin6b:EGFP-Rab7a) transgenic line. A’) G418-TR signal is mainly in vesicles. B) Neo-TR (magenta) accumulation in Rab7+ vesicles (green) in neuromast from Tg(myosin6b:EGFP-Rab7a) transgenic line. B’) Neo-TR signal is found in vesicles and cytoplasm. C) Ratio of G418-TR and Neo-TR found in Rab7+ vesicles compared to cytoplasm. ** Unpaired T test, p<0.01. Error bars represent Standard Deviation.
Figure 6.
Figure 6.
GPN treatment reduces the number of vesicles accumulating G418. A) Image of neuromast treated with G418-TR. A’) Mask of segmented hair cells. A’’) Mask of segmented vesicles. B) Image of neuromast treated with G418-TR and GPN. B’) Mask of segmented hair cells. B’’) Mask of segmented vesicles. C) Number of vesicles per hair cell, mean for each neuromast analyzed. ****Mann-Whitney test, p<0.0001. D) Vesicle volume per hair cell, mean for each neuromast. ns, Mann-Whitney test. E) Vesicle fluorescence per hair cell, mean for each neuromast analyzed. ns, Mann-Whitney test. Error bars represent Standard Deviation.
Figure 7.
Figure 7.
GPN treatment protects against delayed gentamicin damage but not acute neomycin damage. 250 μM GPN was added for 30min before AG (purple bar), co-treated with neomycin for 1hr or co-treated with G418 for 1hr (red bar), rinsed, and incubated with GPN alone for 23hrs (purple bar). A) GPN treatment offers robust protection against delayed death from all concentrations of gentamicin (Two-way ANOVA, Sidak’s multiple comparison, p<0.0001). B) GPN does not protect against acute death from neomycin at any concentration. n=9-11 fish, 4 NMs/fish for each treatment group. Error bars represent Standard Deviation.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Alharazneh A, Luk L, Huth M, Monfared A, Steyger PS, Cheng AG, Ricci AJ. (2011). Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity. PLoS One. 6:e22347. - PMC - PubMed
    1. Angeli S, Lin X, Liu XZ. (2012) Genetics of hearing and deafness. Anat Rec., 295:1812–29. - PMC - PubMed
    1. Atakpa P, van Marrewijk LM, Apta-Smith M, Chakraborty S, Taylor CW. (2019). GPN does not release lysosomal Ca2+ but evokes Ca2+ release from the ER by increasing the cytosolic pH independently of cathepsin C. J Cell Sci. 32:jcs223883. - PMC - PubMed
    1. Au S, Weiner ND, Schacht J. (1987) Aminoglycoside antibiotics preferentially increase permeability in phosphoinositide-containing membranes: a study with carboxyfluorescein in liposomes. Biochim Biophys Acta. 902:80–6. - PubMed
    1. Ballabio A, Bonifacino JS. (2020) Lysosomes as dynamic regulators of cell and organismal homeostasis. Nat Rev Mol Cell Biol. 21:101–118. - PubMed

Publication types

LinkOut - more resources