Piplartine attenuates aminoglycoside-induced TRPV1 activity and protects from hearing loss in mice

Abstract

Hearing loss is a major health concern in our society, affecting more than 400 million people worldwide. Among the causes, aminoglycoside therapy can result in permanent hearing loss in 40% to 60% of patients receiving treatment, and despite these high numbers, no drug for preventing or treating this type of hearing loss has yet been approved by the US Food and Drug Administration. We have previously conducted high-throughput screenings of bioactive compounds, using zebrafish as our discovery platform, and identified piplartine as a potential therapeutic molecule. In the present study, we expanded this work and characterized piplartine's physicochemical and therapeutic properties. We showed that piplartine had a wide therapeutic window and neither induced nephrotoxicity in vivo in zebrafish nor interfered with aminoglycoside antibacterial activity. In addition, a fluorescence-based assay demonstrated that piplartine did not inhibit cytochrome C activity in microsomes. Coadministration of piplartine protected from kanamycin-induced hair cell loss in zebrafish and protected hearing function, outer hair cells, and presynaptic ribbons in a mouse model of kanamycin ototoxicity. Last, we investigated piplartine's mechanism of action by phospho-omics, immunoblotting, immunohistochemistry, and molecular dynamics experiments. We found an up-regulation of AKT1 signaling in the cochleas of mice cotreated with piplartine. Piplartine treatment normalized kanamycin-induced up-regulation of TRPV1 expression and modulated the gating properties of this receptor. Because aminoglycoside entrance to the inner ear is, in part, mediated by TRPV1, these results suggested that by regulating TRPV1 expression, piplartine blocked aminoglycoside's entrance, thereby preventing the long-term deleterious effects of aminoglycoside accumulation in the inner ear compartment.

Fig. 1.. PL properties.

(A) Percentage of protection of neuromast HCs. Zebrafish were cotreated with PL (8 μM to 8 pM) and kanamycin (400 μM) and immunostained for otoferlin; 100% represents vehicle-treated fish and 0% kanamycin-treated fish. PL-only: #1 = 25 μM, #2 = 1 μM, and #3 = 0.1 μM. At least three neuromasts per fish were inspected (five or six fish). (B to E) Representative images of zebrafish green fluorescent protein (GFP)–positive neuromast HCs immunostained for otoferlin (red). Zebrafish were treated with DMSO (control) (B), kanamycin alone at 400 μM (KM) (C), and KM + PL at 120 pM (D) or 15 nM (E). Scale bar, 5 μm. (F) Toxicity assay in HEI-OC1 cells. PL, 1 mM to 512 nM. Results are expressed as the percentage of survival from control (three independent experiments). PL’s chemical structure is shown with the colchicine motif in red. (G to I) Representative images of α-tubulin immunostaining (gray) in HEI-OC1 cells from three independent experiments. Vehicle control (DMSO) (G), PL at 25 μM (H), or colchicine at 10 μM (I). Scale bar, 10 μm. (J) Proximal tubule function in zebrafish (6 to 7 dpf) measured as in (26), in the presence/absence of kanamycin and PL (PL1 = 8 μM, PL2 = 150 nM, and PL3 = 150 pM). Inset indicates the standard curve. Circles represent independent values. OD, optical density. (K) Cytochrome activity was assessed by the amount of fluorophore generated in the absence or presence of different PL concentrations. Ket indicates the negative control. Results are presented as the average fluorophore concentration. Means ± SEM. Brown-Forsythe and Welch analysis of variance (ANOVA) followed by Dunnett's T3 posttest for multiple comparisons. *P < 0.05 and **P < 0.01 versus KM400 (J) or DMSO (K).

Fig. 2.. PL protects from AGIHL in mice.

(A) ABR thresholds at 4- to 64-kHz frequencies measured before (baseline; gray) and 4 weeks after treatment. Seven- to 8-week-old mice treated with vehicle (black), kanamycin (KM600; red), kanamycin + PL (KM600 + PL; blue), or PL (orange). (B) ABR threshold shifts. Means ± SEM. Circles represent individual values. (C) Representative ABR traces at the 22.6-kHz frequency for the four treatments. (D and E) DPOAEs at 4- to 22.6-kHz frequencies measured before (D) and 4 weeks after treatment (E).Threshold averages ± SEM. Two-way [(A), (D), and (E)] and one-way ANOVA (B) followed by Dunnett's posttest for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001 control versus KM600 [(A), (D), and (E); red], control versus KM600 + PL [(A), (D), and (E); blue], and KM600 versus KM600 + PL (B). Number of animals per group is indicated in parentheses.

Fig. 3.. PL protects OHCs from AG toxicity.

(A to D) Representative micrographs of the organ of Corti at five frequency regions. Control (vehicle) (A), kanamycin (KM600) (B), kanamycin + PL (KM600 + PL40) (C), and PL-only (PL40) (D). Gray scale: myosin-7A (myo-7A) immunostaining alone. Color images: myo-7A (green) and phalloidin (phal) (red). Red asterisks denote missing OHCs. Scale bars, 10 μm. (E) OHC quantification (number of animals: control vehicle = 3, KM600 = 3 or 4, KM600 + PL40 = 3, and PL40 = 3) . Means ± SEM. Circles represent individual values. Two-way ANOVA followed by Dunnett's posttest for multiple comparisons. ***P < 0.001 versus corresponding control.

Fig. 4.. PL protects presynaptic ribbons from AG-induced toxicity.

Representative micrographs of the organ of Corti at five frequency regions. (A) Control (vehicle), (B) kanamycin (KM600), (C) kanamycin + PL (KM600 + PL40), and (D) PL-only (PL40). Ctbp2 (green) and myo-7A (magenta) immunostaining. Scale bars, 10 μm. (E) Presynaptic ribbon quantification (number of animals: control vehicle = 3, KM600 = 6 or 7, KM600 + PL40 = 3 to 5, and PL40 = 3 or 4). Means ± SEM. Circles represent individual values. Two-way ANOVA followed by Dunnett's posttest for multiple comparisons. ***P < 0.001, **P < 0.01, and *P < 0.05 versus corresponding control.

Fig. 5.. PL signaling after kanamycin treatment.

(A) Illustration of PamGene platform workflow. ATP, adenosine 5′-triphosphate; ADP, adenosine 5′-diphosphate. (B) Heatmap of phosphorylation activity of the reporter peptides on the STK kinome panel. Unbiased clustering of control (CTL), kanamycin-only (KM), kanamycin + PL (KM + PL), and PL-only (PL). Each row represents a peptide. (C) Box and whisker plots of log₂ fold activity changes in reporter peptides “mapped” as putative targets of AKT1 and ERK kinase families (23 and 22 peptides, respectively). An average of triplicates was used for each peptide per condition (four animals were used per treatment condition). (D) Top: Representative immunoblots for anti–AKT1-S473 (pAKT). Numbers on the left indicate molecular weights. Bottom: Quantification of at least three independent experiments. Membranes were reprobed with anti-tAKT (total-AKT). (E) Top: Representative immunoblots for anti–ERK1/2-T202/Y204 (pERK). Membranes were reprobed with anti-tERK1/2 (total-ERK1/2). Bottom: Immunoblot quantification of at least three independent experiments. Three animals were used per treatment for AKT and ERK immunoblots. Statistical analysis: Two-way ANOVA followed by Dunnett's posttest for multiple comparisons (C) and Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 posttest for multiple comparisons (D). ***P < 0.001 and **P < 0.01 versus kanamycin.

Fig. 6.. PL affects AKT1 phosphorylation.

(A to L) Representative micrograph images of cochlea cross sections. [(A) to (D)] Organ of Corti. OHCs and IHCs are delineated with a dashed white line in (A) and (B). PCs, pillar cells. [(E) to (H)] SV. [(I) to (L)] SGNs. Scale bars, 10 μm [(A) to (D) and (I) to (L)] and 20 μm [(E) to (H)]. Samples stained for pAKT1 (red), Sox2/4′,6-diamidino-2-phenylindole (DAPI) (blue), and Tuj1 (I to L) (green). Micrographs were counterstained with phalloidin [(A) to (H), green; (I) to (L), magenta]. (M) Quantification is presented as the ratio of fluorescence for each region relative to the total fluorescence. Means ± SEM. Circles represent values from different animals. Three independent samples were analyzed. One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *P < 0.05 versus kanamycin.

Fig. 7.. PL treatment results in TRPV1 down-regulation.

(A to P) Representative micrograph images of cochlea cross sections. (A to D) Low magnification. (E to H) Organ of Corti. OHCs and IHCs are delineated with a dashed white line in (E) and (G). [(I) to (L)] SV. [(M) to (P)] SGNs. Scale bars, 50 μm [(A) to (D)] and 10 μm [(E) to (P)]. Samples were stained for TRPV1 (red), Sox2/DAPI (blue), Tuj1 (green), and phalloidin [green (A) to (H) or magenta (M) to (P)]. (Q) Quantification is presented as the percentage of fluorescence for each region of interest relative to the total fluorescence. Means ± SEM. Circles represent values from different animals. Three independent tissue samples were analyzed (three animals per condition). One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *P < 0.05 versus kanamycin.

Fig. 8.. PL regulates AG uptake through TRPV1 interaction.

(A and B) Structural model depicting the selectivity filter (A) and S6 gate (B) regions of TRPV1 bound to PL (left), CP (center), or unligated (Apo) (right). Images were retrieved from a representative frame of the MD trajectories. TRPV1’s chains are presented in different colors. Purple measurements are the distances in angstroms from each carbon alpha from residue M644. (C) Structural model of TRPV1 illustrating the receptor upon binding to PL (left) compared with binding to CP (right). (D) Representative immunoblot of KPT2 cells stably expressing TRPV1 (KPT2-Trpv1), incubated with medium (control), CP + PL (1 μM for CP + PL1 or 0.1 μM for CP + PL2), CP only, or PL only (1 μM; PL2). Numbers indicate pAKT/tAKT ratios for that particular experiment. (E) Quantification of pAKT/tAKT ratios from three independent immunoblots. One-way ANOVA followed by Dunnett's posttest for multiple comparisons. *P < 0.05 versus CP. (F to I) Representative images of KPT2-Trpv1 cells incubated with medium-only (F), CP (G), or CP + PL [0.1 μM for (H) and 1 μM for (I)] and stained for pAKT1 (red) and phalloidin (green). (J to M) Representative images of KPT2- Trpv1 cells incubated with GTTR alone (red) (J), GTTR + CP (K), GTTR + CP + PL (1 μM) (L), and GTTR + capsazepine (GTTR + CPZ) (M) counterstained with phalloidin (green). Numbers in (J) to (M) are the percentages of GTTR-positive cells. Scale bars, 20 μm.