Loss of Slc4a1b Chloride/Bicarbonate Exchanger Function Protects Mechanosensory Hair Cells from Aminoglycoside Damage in the Zebrafish Mutant <em>persephone</em>

<div><p>Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The <em>persephone</em> mutation confers resistance to aminoglycosides. 5 dpf homozygous <em>persephone</em> mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in <em>persephone</em> maps to the chloride/bicarbonate exchanger <em>slc4a1b</em> and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both <em>persephone</em> mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. <em>persephone</em> mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglcyoside antibiotics.</p> </div

<i>persephone</i> hair cells have normal morphology and show only modest ultrastructural changes when treated with neomycin.

<p>Zebrafish (5 dpf) treated with or without neomycin were euthanized, tails were removed for genotyping, and heads were fixed for transmission electron microscopy. Images of transverse sections through neuromasts are shown. (N: hair cell nuclei, m:mitochondria, sc: support cell, k: kinocilia. Top panels A–C and bottom panels G–I show hair cells from wildtype siblings. Top panels D–F and bottom panels J–L show hair cells from <i>persephone</i> homozygotes. Panels were either mock-treated or exposed to 50 µM or 200 µM neomycin. Hair cells of mock-treated wildtype siblings and <i>persephone</i> mutants show organized stereocilia, a large central nucleus, and normal mitochondrial morphology. There are no readily apparent differences between the wildtype siblings and <i>persephone</i> mutants. Hair cells of <i>persephone mutants</i> treated with 50 µM neomycin show either mild mitochondrial swelling (E,K) or are indistinguishable from mock-treated wildtype siblings. In comparison wildtype siblings (B,H) show swollen mitochondria and fused stereocilia. Most neuromasts from wildtype siblings treated with 200 µM neomycin lack hair cells, and remaining hair cells exhibit severe damage including condensed nuclei and severely swollen mitochondria or signs of cytolysis (C,I). Hair cells of <i>persephone</i> mutants treated with 200 µM neomycin show minimal damage. Stereocilia are typically oraganized and intact; some but not all hair cells show modestly swollen mitochondria (F,L). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002971#pgen-1002971-t001" target="_blank">Table 1</a> for quantification of neomycin-induced damage across genotypes. Top scale bar = 5 µm; Bottom scale bar = 1 µm.</p

Protection of hair cells by zSlc4a1b inhibitors and substrates.

<p>(A) Treatment with the Slc4 inhibitor DIDS protects hair cells from exposure to neomycin. 5 dpf zebrafish were pretreated with DIDS for 1 hr; then co-treated with DIDS and neomycin for 1 hr. Treatment with DIDS resulted in modest but significant reduction in hair cell death. (n≥10 larvae per group, 3 neuromasts per larvae. Error bars: S.D.; ANOVA p value indicating significance of DIDS to protection: <0.001) (B) Treatment with the Slc4 inhibitor SITS protects hair cells from neomycin toxicity. SITS protects hair cells in a concentration-dependent manner against hair cell loss caused by exposure to 200 µM neomycin. (n≥10 fish per group, 3 neuromasts per fish. Error bars: S.D.; ANOVA p value indicating significance of SITS to protection: <0.0001) (C) Increased bicarbonate in the embryo media protects hair cells from exposure to neomycin. 5 dpf zebrafish were exposed for 1 hr to embryo media with a 10 fold range of bicarbonate concentrations (0.714 mM to 7 mM). Neomycin was then added at the indicated concentrations for 30 min. Larvae were rinsed three times in fresh media, and allowed to recover for 1 hr. Bicarbonate protects hair cells in a concentration-dependent manner. (n≥10 fish per group, 3 neuromasts per fish. Error bars: S.D.; ANOVA p value indicating significance of bicarbonate to protection: <0.001). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002971#pgen.1002971.s002" target="_blank">Figure S2</a>. (D) <i>persephone</i> mutants show dramatically increased bicarbonate-mediated hair cell protection relative to heterozygous and homozygous wildtype siblings. Progeny of an in-cross of <i>persephone</i> heterozygotes (5 dpf) were exposed for 1 hr to embryo media with bicarbonate added at the indicated concentrations, and then treated an additional 1 hr with or without 500 µM neomycin. Fish were allowed to recover for 1 hr, then were assayed individually for hair cell survival prior to euthanasia and genotyping by dCAPS. Whereas 10× [bicarbonate] only modestly protected wildtype and heterozygous siblings (solid blue line), <i>persephone</i> mutants (solid orange line) were dramatically protected. (n≥10 fish per group, 3 neuromasts per fish. Error bars: S.D.; ANOVA p value indicating significance of genotype to protection: <0.0001).</p

Phenotype of the <i>persephone</i> mutant.

<p>(A) Hair cell protection in homozygous <i>persephone</i> mutants. 5 dpf zebrafish (progeny of a heterozygous incross) were treated with or without 200 µM neomycin for 30 min, and then rinsed. After 1 hr recovery in fresh embryo media, hair cells were labeled with the vital dye DASPEI. Left panels are differential interference contrast (DIC) images and right panels are corresponding fluorescent images. Top, wildtype sibling (+/+), mock-treated, Middle, wildtype sibling (+/+), treated with 200 µM neomycin, and Bottom, <i>persephone</i> homozygote treated with 200 µM neomycin. Red arrows indicate examples of neuromasts present in the untreated wildtype and treated <i>persephone</i> larvae. Mutant larvae show dramatic retention of hair cells relative to their siblings. (B) Parvalbumin antibody staining of hair cells in representative fish from an in-cross of <i>persephone</i> heterozygotes. Wildtype siblings show loss of hair cells when treated with 200 µM neomycin. Homozygous <i>persephone</i> mutants show a dramatic retention of parvalbumin-stained hair cells following neomycin treatment, consistent with DASPEI hair cell staining results. (C) Protection observed in <i>persephone</i> is not due to a delay in neomycin-induced hair cell death. <i>persephone</i> mutants (blue line) exposed to neomycin for 1 hr, rinsed and maintained for 6 or 24 hr in fresh EM, and then assessed for hair cell death, do not show significantly greater hair cell death than those assayed after 1 hr. Hair cell death is not delayed in <i>persephone</i>. Wildtype siblings treated in parallel (green line) are shown for comparison. (n = 10 fish, 10 neuromasts per fish; Error bars: S.D.; p value<0.001).</p

Protection of hair cells in the <i>persephone</i> mutant.

<p>(A) Neomycin dose response curve for progeny from a <i>persephone</i> incross. Homozygous <i>persephone</i> mutants (orange line) show dramatic protection at all tested neomycin concentrations (50, 100, 200 and 400 µM) as compared to wildtype (green line) and heterozygous siblings (blue line) that show no protection. (n≥10 fish per group, 10 neuromasts per fish. Error bars: S.D.; ANOVA p value<0.0001) (B) <i>persephone</i> protects hair cells from loss induced by the aminoglycoside kanamycin. Larvae were maintained in embryo media with kanamycin (0, 50, and 200 µM) for 24 hr prior to assaying hair cell death. Mutants (orange line) are protected as compared to the combined wildtype and heterozygous siblings (teal line). (n≥10 fish per group, 10 neuromasts per fish. Error bars: S.D.; ANOVA p value<0.0001) (C) <i>persephone</i> protects hair cells from loss induced by the aminoglycoside gentamicin. Larvae were maintained in embryo media with gentamicin (0, 50, and 200 µM) for 6 hr prior to assaying hair cell death. Mutants (orange line) are protected as compared to the combined wildtype and heterozygous siblings (teal line). (n≥10 fish per group, 10 neuromasts per fish. Error bars: S.D.; ANOVA p value<0.0001) (D) <i>persephone</i> protects against the hair cell toxin cisplatin. Larvae were exposed for 24 hr to cisplatin (50, 100 or 200 µM) and assayed for hair cell survival by hair cell counts prior to genotyping. Homozygous <i>persephone</i> mutants (orange line) show modest but significant protection compared to wildtype siblings (teal line) (n≥10 fish per group, 3 neuromasts per fish. Error bars: S.D.; ANOVA p value<0.0001.).</p

Ultrastructural damage is strongly reduced in <i>persephone</i> homozygotes versus wild-type siblings after neomycin exposure.

<p>n = number of fish in each category.</p><p>Damage scale:</p><p>A = All hair cells of the neuromast appear normal.</p><p>B = Slight mitochondrial swelling (modest within range of mock-treated controls).</p><p>C = One or more hair cells exhibit some mitochondrial swelling. One or more hair cells also exhibit some nuclear condensation.</p><p>D = Many hair cells show severe damage, including distended mitochondria, highly pycnotic nuclei, or condensation of the entire cell.</p><p>E = Hair cells are absent, or all hair cells show severe damage, including severely distended mitochondria, highly pycnotic nuclei, or condensation of the entire cell.</p

Mapping the <i>persephone</i> mutant.

<p>(A) Histograms showing the distribution of hair cell labeling in wildtype fish. 40 individual wildtype fish were mock-treated (light green bars) or treated with 200 µM neomycin for 30 min (dark green bars). Mock-treated wildtype larvae show a distribution centered near 85%. The distribution is dramatically reduced in larvae exposed to neomycin (mean centered near 10%). (B) Histogram showing distribution of hair cell retention in progeny from an in-cross of <i>persephone</i> heterozygotes. Individual larvae were assayed for hair cell staining, and then genotyped (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002971#s4" target="_blank">Materials and Methods</a>). Orange bars show binned hair cell staining of homozygous <i>persephone</i> mutants; blue bars show binned hair cell staining of wildtype siblings (both <i>pers/+</i> and <i>+/+</i>). The distribution of hair cell retention in <i>persephone</i> homozygotes is dramatically shifted with a mean broadly centered around 80%. In contrast, heterozygous and homozygous wildtype siblings do not show protection from neomycin exposure; their mean distribution is centered around 5%. (C) Schematic diagram of the region of zebrafish chromosome 12 that cosegregates with the <i>persephone</i> mutation. Genetic markers used for fine mapping are indicated with the number and % of recombinants observed. Positions on the heat shock (HS) genetic map and the physical map are from the Zv9 genomic assembly. (D) Sequencing chromatogram of a <i>persephone</i> heterozygote showing the mutated codon and the two preceding codons. The C→T mutation in <i>persephone</i> converts the wildtype Ser to Phe at amino acid 298 in the mutant allele. (E) Alignment of zslc4a1b homologs shows evolutionary conservation of Ser 298 mutated in the <i>persephone</i> mutant. The indicated serine residue is invariant across a diverse range of taxa; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002971#pgen.1002971.s001" target="_blank">Figure S1</a>.</p

FM1-43 uptake—used as a proxy for mechanotransduction activity—is reduced in <i>persephone</i> mutants.

<p>Free swimming 5dpf progeny of an incross of <i>persephone</i> heterozygotes were individually exposed to FM1-43 for 1 min and rinsed by basket transfer. Neuromasts of individual fish were immediately imaged and total fluorescence signal from FM1-43 was quantified as described for GTTR quantification in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002971#s4" target="_blank">Materials and Methods</a>. After imaging, fish were individually genotyped by dCAPS. (n≥10 larvae per group, 3 neuromasts per larvae. Error bars: S.D., p value: 0.003 for difference in means between wildtype siblings and <i>persephone</i> larvae. +/+ and <i>pers</i>/+ larvae are not significantly different (p value 0.15). Uptake in <i>persephone</i> is significantly higher than in the loss of mechanotransduction mutant sputnik (p value 0.02.).</p

Mutant Slc4a1b is mislocalized and fails to transport chloride.

<p>(A) Confocal images of zebrafish embryos transiently expressing wildtype Slc4a1b–GFP. mRNA was injected into one cell stage embryos. Cells along the anterior dorsal aspect of embryos were imaged at 48 hpf. Wildtype protein is localized to the plasma membrane. B) Confocal images of zebrafish embryos expressing mutant Slc4a1b(S298F)-GFP. Slc4(S298F)-GFP is present in intracellular vesicles, with complete loss of plasma membrane localization. (C) Chloride influx into <i>Xenopus laevis</i> oocytes expressing either zebrafish Slc4a1b (zSlc4a1b) or the closest human homolog, SLC4A1 (hSlc4a1). Zebrafish Slc4a1b transports radiolabeled chloride across oocyte membranes as efficiently as its nearest human homolog. This activity is blocked by DIDS (200 µM), an inhibitor of Slc4 family-mediated anion exchangers. (D) Comparison of chloride influx into <i>Xenopus laevis</i> oocytes expressing wildtype or mutant (S298F) zSlc4a1b, or wildtype zSlc4a1b-GFP. zSlc4a1(S298F), the <i>persephone</i> gene product, exhibits complete loss of chloride influx activity. Notably, GFP-tagged wildtype slc4a1b exhibits no significant reduction of chloride transport activity. (n indicated above each sample; Error bars = SEM).</p

Conditioned media depleted of NM23-H1 no longer supports pluripotent stem cell growth.

<p><b>a</b>) Conditioned media from either murine (MEFs) or human (HS27) embryonic fibroblast cells was immuno-depleted of NM23-H1. Western blot confirmed that the depleted media no longer contains NM23-H1. <b>b</b>) NM23-H1 was captured by affinity chromatography, eluted and its concentration quantified by the optical density at 280nm. Human conditioned media contains 5-times more NM23-H1 than murine conditioned media. Human ES cells on Matrigel were cultured with: <b>c, d</b>) NM23-H1-depleted media, <b>e, f</b>) bFGF plus complete conditioned media. NM23-H1-depleted conditioned media induced differentiation (<b>c, d</b>).</p