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Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus

c-Met is expressed in lateral line neuromasts. (A) Neuromast of a Brn3c:mGFP transgenic zebrafish with clearly labeled hair cell boundaries. (B) Anti-c-Met labeling (red punctae) is present throughout the neuromast. (C) Merged image shows c-Met is present near the hair cell membrane and in surrounding cells. (D–F) Brn3c:mGFP larvae incubated with secondary antibody only show no c-Met labeling. (G) *AB adult liver tissue labeled with DAPI (blue) and anti-c-Met (red) demonstrates robust, punctate c-Met expression. Scale bar in (C) represents 5 μm and applies to images (A–F). Scale bar in (G) represents 5 μm.
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Figure 1: c-Met is expressed in lateral line neuromasts. (A) Neuromast of a Brn3c:mGFP transgenic zebrafish with clearly labeled hair cell boundaries. (B) Anti-c-Met labeling (red punctae) is present throughout the neuromast. (C) Merged image shows c-Met is present near the hair cell membrane and in surrounding cells. (D–F) Brn3c:mGFP larvae incubated with secondary antibody only show no c-Met labeling. (G) *AB adult liver tissue labeled with DAPI (blue) and anti-c-Met (red) demonstrates robust, punctate c-Met expression. Scale bar in (C) represents 5 μm and applies to images (A–F). Scale bar in (G) represents 5 μm.

Mentions: We immunohistochemically processed 5 dpf Brn3c:mGFP larvae using anti-c-Met to determine if c-Met expression is present in anterior lateral line neuromasts. In these fish, GFP localizes to the hair cell membrane, allowing for easy delineation of cell boundaries (Figure 1A). c-Met expression was widespread in the skin epithelium, including in neuromasts (red punctae in Figure 1B). No red punctae were present in a secondary antibody only control that was not incubated in anti-c-Met (Figures 1D–F). c-Met labeling is present both in hair cells and in other cell types in the neuromast (Figure 1C). To verify the validity of observed c-Met expression in hair cells, adult *AB liver tissue was immunohistochemically processed for anti-c-Met and DAPI (blue). c-Met expression in the adult liver is widespread and punctate, similar to the neuromast expression profile (Figure 1G). These results indicate that our c-Met labeling is reliable and that anterior lateral line neuromasts should be receptive to c-Met modulation.


Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

c-Met is expressed in lateral line neuromasts. (A) Neuromast of a Brn3c:mGFP transgenic zebrafish with clearly labeled hair cell boundaries. (B) Anti-c-Met labeling (red punctae) is present throughout the neuromast. (C) Merged image shows c-Met is present near the hair cell membrane and in surrounding cells. (D–F) Brn3c:mGFP larvae incubated with secondary antibody only show no c-Met labeling. (G) *AB adult liver tissue labeled with DAPI (blue) and anti-c-Met (red) demonstrates robust, punctate c-Met expression. Scale bar in (C) represents 5 μm and applies to images (A–F). Scale bar in (G) represents 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309183&req=5

Figure 1: c-Met is expressed in lateral line neuromasts. (A) Neuromast of a Brn3c:mGFP transgenic zebrafish with clearly labeled hair cell boundaries. (B) Anti-c-Met labeling (red punctae) is present throughout the neuromast. (C) Merged image shows c-Met is present near the hair cell membrane and in surrounding cells. (D–F) Brn3c:mGFP larvae incubated with secondary antibody only show no c-Met labeling. (G) *AB adult liver tissue labeled with DAPI (blue) and anti-c-Met (red) demonstrates robust, punctate c-Met expression. Scale bar in (C) represents 5 μm and applies to images (A–F). Scale bar in (G) represents 5 μm.
Mentions: We immunohistochemically processed 5 dpf Brn3c:mGFP larvae using anti-c-Met to determine if c-Met expression is present in anterior lateral line neuromasts. In these fish, GFP localizes to the hair cell membrane, allowing for easy delineation of cell boundaries (Figure 1A). c-Met expression was widespread in the skin epithelium, including in neuromasts (red punctae in Figure 1B). No red punctae were present in a secondary antibody only control that was not incubated in anti-c-Met (Figures 1D–F). c-Met labeling is present both in hair cells and in other cell types in the neuromast (Figure 1C). To verify the validity of observed c-Met expression in hair cells, adult *AB liver tissue was immunohistochemically processed for anti-c-Met and DAPI (blue). c-Met expression in the adult liver is widespread and punctate, similar to the neuromast expression profile (Figure 1G). These results indicate that our c-Met labeling is reliable and that anterior lateral line neuromasts should be receptive to c-Met modulation.

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus