Cristian Setz

Simvastatin protects auditory hair cells from gentamicin-induced toxicity and activates Akt signaling in vitro

BackgroundInhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, known as statins, are commonly used as cholesterol-lowering drugs. During the past decade, evidence has emerged that statins also have neuroprotective effects. Research in the retina has shown that simvastatin, a commonly used statin, increases Akt phosphorylation in vivo, indicating that the PI3K/Akt pathway contributes to the protective effects achieved. While research about neuroprotective effects have been conducted in several systems, the effects of statins on the inner ear are largely unknown.ResultsWe evaluated whether the 3-hydroxy-3-methylglutaryl-coenzyme A reductase is present within the rat cochlea and whether simvastatin is able to protect auditory hair cells from gentamicin-induced apoptotic cell death in a in vitro mouse model. Furthermore, we evaluated whether simvastatin increases Akt phosphorylation in the organ of Corti. We detected 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in organ of Corti, spiral ganglion, and stria vascularis by reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, we observed a dose-dependent and significant reduction of hair cell loss in organs of Corti treated with simvastatin in addition to gentamicin, as compared to samples treated with gentamicin alone. The protective effect of simvastatin was reversed by addition of mevalonate, a downstream metabolite blocked by simvastatin, demonstrating the specificity of protection. Finally, Western blotting showed an increase in organ of Corti Akt phosphorylation after simvastatin treatment in vitro.ConclusionThese results suggest a neuroprotective effect of statins in the inner ear, mediated by reduced 3-hydroxy-3-methylglutaryl-coenzyme A reductase metabolism and Akt activation.

All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.

Inhibition of mTOR by Rapamycin Results in Auditory Hair Cell Damage and Decreased Spiral Ganglion Neuron Outgrowth and Neurite Formation In Vitro.

Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR) by blocking the mTOR complex 1 (mTORC1). mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp.) are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzed in vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation.

Metformin Protects Auditory Hair Cells from Gentamicin-Induced Toxicity in vitro

Metformin is a commonly used antidiabetic drug. It has been shown that this drug activates the AMP-activated protein kinase, which inhibits downstream the mammalian target of rapamycin. In addition, several studies indicate that metformin reduces intracellular reactive oxygen species. Our data, using an in vitro rat model, indicate that metformin is able to protect auditory hair cells (HCs) from gentamicin-induced apoptotic cell death. Moreover, metformin has no toxic effect on spiral ganglion neuronal survival or outgrowth in vitro. These results suggest a protective effect of metformin on auditory HC survival in gentamicin-induced HC loss in vitro.

Role of Somatostatin Receptor-2 in Gentamicin-Induced Auditory Hair Cell Loss in the Mammalian Inner Ear

Hair cells and spiral ganglion neurons of the mammalian auditory system do not regenerate, and their loss leads to irreversible hearing loss. Aminoglycosides induce auditory hair cell death in vitro, and evidence suggests that phosphatidylinositol-3-kinase/Akt signaling opposes gentamicin toxicity via its downstream target, the protein kinase Akt. We previously demonstrated that somatostatin—a peptide with hormone/neurotransmitter properties—can protect hair cells from gentamicin-induced hair cell death in vitro, and that somatostatin receptors are expressed in the mammalian inner ear. However, it remains unknown how this protective effect is mediated. In the present study, we show a highly significant protective effect of octreotide (a drug that mimics and is more potent than somatostatin) on gentamicin-induced hair cell death, and increased Akt phosphorylation in octreotide-treated organ of Corti explants in vitro. Moreover, we demonstrate that somatostatin receptor-1 knockout mice overexpress somatostatin receptor-2 in the organ of Corti, and are less susceptible to gentamicin-induced hair cell loss than wild-type or somatostatin-1/somatostatin-2 double-knockout mice. Finally, we show that octreotide affects auditory hair cells, enhances spiral ganglion neurite number, and decreases spiral ganglion neurite length.

Simvastatin Results in a Dose-Dependent Toxic Effect on Spiral Ganglion Neurons in an In Vitro Organotypic Culture Assay

Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme necessary for the production of mevalonate. They are widely used as cholesterol-lowering drugs. However, conflicting data about the effect of statins on neuronal cells has been published. To explore the effect of simvastatin on spiral ganglion neurons (SGNs), SG explants of 5-day-old rats were treated with increasing concentrations of simvastatin. In addition, SG explants were treated with mevalonate and with the combination of simvastatin and mevalonate. SGN number, length of the neurites, area of nonneuronal supporting cells, and neuronal survival were analyzed. Simvastatin treatment results in a significant dose-dependent decrease of SG neurite number, length of neurites, area of supporting cells, and SG neuronal survival compared to control. Interestingly, treatment with mevalonate in addition to simvastatin increased SG neuronal survival compared to simvastatin treatment only. However, treatment with mevalonate in addition to simvastatin did not influence SG neurite number, length of neurites, and area of supporting cells compared to simvastatin treatment only. Our results suggest a neurotoxic effect of simvastatin on SGNs in vitro. Neurotoxicity seems to be at least partially mediated by the mevalonate pathway. Therefore, caution is warranted to use simvastatin as a potential otoprotective drug.

T-cadherin in the mammalian cochlea

Cadherins are a superfamily of transmembrane glycoproteins, which mediate calcium‐dependent intercellular adhesions. T‐cadherin is an atypical member of the cadherin family in regard to its structure; it acts as a signalling receptor rather than an adhesion molecule. In this study we examine the role of T‐cadherin in the mammalian cochlea.

Expression of Advanced Glycation End-Product Receptors in the Cochlea

Advanced glycation end products (AGE) have recently been implicated in aging changes within different tissues of the body. The role of AGEs and their receptors in the mammalian inner ear is largely unknown. In this study we analyzed for the expression of two AGE receptors, namely RAGE and Ddost (AGE‐R1).

TARGETING THE SOMATOSTATIN RECEPTORS AS A THERAPEUTIC APPROACH FOR THE PRESERVATION AND PROTECTION OF THE MAMMALIAN COCHLEA FROM EXCITOTOXICITY

The neuropeptide somatostatin (SST) is an important modulator of neurotransmission in the central nervous system (CNS) and binds to G-protein-coupled receptors (SSTR1-5) on target cells. Little is known about the expression and function of the somatostatinergic system in the mammalian cochlea. We analyzed the expression of SSTR1-SSTR5 in the immature mammalian cochlea. The peak in the expression of SSTR1 and SSTR2 at mRNA and protein level is around the onset of hearing to airborne sound, at postnatal day (P)14. This suggests their involvement in the maturation of the mammalian cochlea. We demonstrated that all five receptors are expressed in the inner hair cells (IHC) and outer hear cells (OHC) as well as in defined supporting cells of the organ of Corti (OC) in the adult mouse cochlea. A similar expression of the SSTRs in the IHC and OHC was found in cultivated P6 mouse OC explants as well as in neuroepithelial cell culture. In order to learn more about the regulation of SSTRs, we used mice with either a deletion of SSTR1, SSTR2 or SSTR1/SSTR2 double knock out (DKO). In DKO mice, SSTR5 was up-regulated and SSTR3 and SSTR4 were down regulated. These findings provide evidence of a compensatory regulation in the mammalian cochlea as a consequence of a receptor subtype deletion. In addition, we observed reduced levels of phospho-Akt and total-Akt in SSTR1 KO and DKO mice as compared to wild type (WT) mice. Akt is likely to be involved in hair cell survival. Most importantly, we found improved hair cell survival in somatostatin and octreotide treated OC explants that had been exposed to gentamicin compared to those explants exposed to gentamicin alone. These findings propose that the somatostatinergic system within the cochlea may have neuroprotective properties.