Effects of peroxisome proliferator activated receptors (PPAR)-γ and -α agonists on cochlear protection from oxidative stress

<div><p>Various insults cause ototoxicity in mammals by increasing oxidative stress leading to apoptosis of auditory hair cells (HCs). The thiazolidinediones (TZDs; e.g., pioglitazone) and fibrate (e.g., fenofibrate) drugs are used for the treatment of diabetes and dyslipidemia. These agents target the peroxisome proliferator-activated receptors, PPARγ and PPARα, which are transcription factors that influence glucose and lipid metabolism, inflammation, and organ protection. In this study, we explored the effects of pioglitazone and other PPAR agonists to prevent gentamicin-induced oxidative stress and apoptosis in mouse organ of Corti (OC) explants. Western blots showed high levels of PPARγ and PPARα proteins in mouse OC lysates. Immunofluorescence assays indicated that PPARγ and PPARα proteins are present in auditory HCs and other cell types in the mouse cochlea. Gentamicin treatment induced production of reactive oxygen species (ROS), lipid peroxidation, caspase activation, PARP-1 cleavage, and HC apoptosis in cultured OCs. Pioglitazone mediated its anti-apoptotic effects by opposing the increase in ROS induced by gentamicin, which inhibited the subsequent formation of 4-hydroxy-2-nonenal (4-HNE) and activation of pro-apoptotic mediators. Pioglitazone mediated its effects by upregulating genes that control ROS production and detoxification pathways leading to restoration of the reduced:oxidized glutathione ratio. Structurally diverse PPAR agonists were protective of HCs. Pioglitazone (PPARγ-specific), tesaglitazar (PPARγ/α-specific), and fenofibric acid (PPARα-specific) all provided >90% protection from gentamicin toxicity by regulation of overlapping subsets of genes controlling ROS detoxification. This study revealed that PPARs play important roles in the cochlea, and that PPAR-targeting drugs possess therapeutic potential as treatment for hearing loss.</p></div

Pioglitazone (PIO) inhibited the production of reactive oxygen species (ROS) and the lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE).

<p>(A-D) Mouse OCs were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188596#pone.0188596.g002" target="_blank">Fig 2</a> 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).</p

Effects of structurally diverse PPAR agonists on redox gene expression in mouse organ of Corti (OC) explants.

<p>Pioglitazone (PIO), muraglitazar (MURA) and tesaglitazar (TESA) significantly induced expression of of superoxide dismutase (<i>Sod1</i>), glutathione peroxidase (<i>Gpx1</i>), catalase (<i>Cat</i>), and uncoupling protein 2 (<i>Ucp2</i>), 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.</p

Expression and localization of PPARγ and PPARα proteins in mouse cochlea.

<p>(A) Fluorescence micrographs of adult mouse cochlear sections stained for PPARα and PPARγ. Negative controls (<i>panels 1 and 2</i>), red immunostaining for PPARα (<i>panels 3 and 5</i>) and for PPARγ (<i>panels 4 and 6</i>). Scale bar: 50 μm. DAPI: nuclear stain (blue). (B) Western blots of protein extracts show (<i>left</i>) PPARα and (<i>right</i>) 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.</p

Structurally diverse PPAR agonists prevented gentamicin (GM)-induced hair cell death in mouse organ of Corti (OC) explants.

<p>(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 (<i>from top to bottom in each column</i>): 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: (<i>left</i>) tesaglitazar (2 or 10 μM), (<i>middle</i>) muraglitazar (2 or 10 μM), and (<i>right</i>) 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.</p

Pioglitazone (PIO) restored the redox balance in mouse OCs after exposure to gentamicin (GM).

<p>Mouse OCs were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188596#pone.0188596.g002" target="_blank">Fig 2</a> 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 <i>Sod1</i>, <i>Gpx1</i>, or <i>Cat</i>, but upregulated <i>Ucp2</i> expression. Pioglitazone upregulated the expression of all four genes, including <i>Ucp2</i> 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.</p

Pioglitazone (PIO) prevented gentamicin (GM)-induced activation of pro-apoptotic caspases in mouse organ of Corti.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188596#pone.0188596.g002" target="_blank">Fig 2</a>, 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.</p

Pioglitazone prevented gentamicin (GM)-induced hair cell death in mouse organ of Corti (OC) explants.

<p>(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 (<i>top to bottom</i>): 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.</p