Fig 1.
Expression and localization of PPARγ and PPARα proteins in mouse cochlea.
(A) Fluorescence micrographs of adult mouse cochlear sections stained for PPARα and PPARγ. Negative controls (panels 1 and 2), red immunostaining for PPARα (panels 3 and 5) and for PPARγ (panels 4 and 6). Scale bar: 50 μm. DAPI: nuclear stain (blue). (B) Western blots of protein extracts show (left) PPARα and (right) PPARγ levels in the organ of Corti (OC) from 5-day old neonatal mice. ß-actin was used as a loading control. OC, organ of Corti; Br, mouse brain; L, mouse liver. kDa, kilodalton, TM, tectorial membrane; DAPI, 4',6-diamidino-2-phenylindol.
Fig 2.
Pioglitazone prevented gentamicin (GM)-induced hair cell death in mouse organ of Corti (OC) explants.
(A) Representative fluorescence micrographs of the basal turn of OCs show auditory hair cells detected with Alexa Fluor 488-phalloidin (green). OC were incubated in the following conditions (top to bottom): medium alone for 48 h; medium 24 h, then GM (50 μM) for 24 h; pioglitazone (10 μM) alone for 48 h; and pioglitazone at 2 or 10 μM for 48 h, with GM (50 μM) added for the last 24 h. Scale bar: 50 μm. (B) Quantitative analysis of hair survival. N = 20 explants per group; ns (not significant), *p<0.05 and ****p<0.0001 compared to GM treatment alone. Data are the mean number of surviving hair cells ± SD. OHC, outer hair cell; IHC, inner hair cell.
Fig 3.
Structurally diverse PPAR agonists prevented gentamicin (GM)-induced hair cell death in mouse organ of Corti (OC) explants.
(A) Representative fluorescence micrographs of the basal turn of OCs show auditory hair cells stained with Alexa Fluor 488-phalloidin (green) and counted under a fluorescence microscopy. OCs were incubated in the following conditions (from top to bottom in each column): medium alone (Control) for 48 h, medium 24 h, then PPAR agonist (two concentrations) for 48 h, with GM (50 μM) added for the last 24 h; the PPAR agonists were: (left) tesaglitazar (2 or 10 μM), (middle) muraglitazar (2 or 10 μM), and (right) fenofibric acid (25 or 150 μM). Scale bar: 50 μm. (B) Quantitative analysis of hair cell survival. N = 20 explants per group. **p<0.01 and ****p<0.0001, compared to GM treatment alone. Data are the mean number of surviving hair cells ± SD. OHC = outer hair cell; IHC = inner hair cell.
Fig 4.
Pioglitazone (PIO) prevented gentamicin (GM)-induced activation of pro-apoptotic caspases in mouse organ of Corti.
(A) Representative fluorescence micrographs of the basal turn of OCs show auditory hair cells detected with rhodamine-phalloidin (red), and activated caspases detected with Caspatag (green). Mouse OCs were treated as described in Fig 2, without (control, CTRL) or with GM +/- PIO. Scale bar: 50 μm (B) Quantification of the fluorescent signal in panel A. N = 10 explants per group; ****p <0.0001. (C) Representative Western blot shows the cleavage product of caspase 3 in lysates from OCs treated without (CTR) or with GM (50 μM) or GM + PIO (10 μM); ß-actin is the loading control. (D) Quantification of Western blots for the caspase 3 cleavage product in Panel C. The values shown indicate the mean expression levels normalized to ß-actin. N = 3; (E) Representative Western blot shows full-length PARP-1 and its cleaved 24kDa fragment in lysates from hair cells treated without (CTR) or with GM (50 μM) or GM with PIO (10 μM). (F) Quantification of Western blot signals represented by ratio of signals cleaved/full-length PARP-1 normalized to ß-actin. N = 3; **p<0.01.
Fig 5.
Pioglitazone (PIO) inhibited the production of reactive oxygen species (ROS) and the lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE).
(A-D) Mouse OCs were treated as described in Fig 2 with gentamicin (GM) +/- PIO. Representative micrographs from the basal turn of the organ of Corti were stained with Alexa Fluor 488-phalloidin (green) and either (A) the ROS indicator, CellRox (red) or (C) the 4-HNE antibody (red). Scale bar: 50 μm. (B, D) Quantification of signal intensities. GM strongly induced ROS production and lipid peroxidation. Both effects were nearly completely prevented by PIO. Values are the mean ± SD (N = 10 explants per group; ****p <0.0001).
Fig 6.
Pioglitazone (PIO) restored the redox balance in mouse OCs after exposure to gentamicin (GM).
Mouse OCs were treated as described in Fig 2 with GM +/- 10 μM PIO. (A) GM caused depletion of endogenous antioxidants, as reflected by a 75% reduction in the ratio of reduced:oxidized glutathione (GSH/GSSG). (B) GM alone had no effect on the relative expression of Sod1, Gpx1, or Cat, but upregulated Ucp2 expression. Pioglitazone upregulated the expression of all four genes, including Ucp2 expression, which correlates with significant improvement in the cellular redox state as reflected in panel A. Results are the mean fold-change in transcript levels ± SD. N = 3; *p<0.05; **p<0.01, compared to untreated OCs.
Fig 7.
Effects of structurally diverse PPAR agonists on redox gene expression in mouse organ of Corti (OC) explants.
Pioglitazone (PIO), muraglitazar (MURA) and tesaglitazar (TESA) significantly induced expression of of superoxide dismutase (Sod1), glutathione peroxidase (Gpx1), catalase (Cat), and uncoupling protein 2 (Ucp2), while fenofibric acid (FFA) repressed their expression. Results are the mean fold-change in transcript levels ± SD. N = 3; ns (not significant); *p<0.05; **p<0.01; ***p<0.001, compared to untreated OCs.