Yves Brand

Ras/p38 and PI3K/Akt but not Mek/Erk signaling mediate BDNF-induced neurite formation on neonatal cochlear spiral ganglion explants.

Neurotrophins participate in regulating the survival, differentiation, and target innervation of many neurons, mediated by high-affinity Trk and low-affinity p75 receptors. In the cochlea, spiral ganglion (SG) neuron survival is strongly dependent upon neurotrophic input, including brain-derived neurotrophic factor (BDNF), which increases the number of neurite outgrowth in neonatal rat SG in vitro. Less is known about signal transduction pathways linking the activation of neurotrophin receptors to SG neuron nuclei. In particular, the p38 and cJUN Kinase (JNK), mitogen-activated protein kinase (MAPK) pathways, which participate in JNK signaling in other neurons, have not been studied. We found that inhibition of Ras, p38, phosphatidyl inositol 3 kinase (PI3K) or Akt signaling reduced or eliminated BDNF mediated increase in number of neurite outgrowth, while inhibition of Mek/Erk had no influence. Inhibition of Rac/cdc42, which lies upstream of JNK, modestly enhanced BDNF induced formation of neurites. Western blotting implicated p38 and Akt signaling, but not Mek/Erk. The results suggest that the Ras/p38 and PI3K/Akt are the primary pathways by which BDNF promotes its effects. Activation of Rac/cdc42/JNK signaling by BDNF may reduce the formation of neurites. This is in contrast to our previous results on NT-3, in which Mek/Erk signaling was the primary mediator of SG neurite outgrowth in vitro. Our data on BDNF agree with prior results from others that have implicated PI3K/Akt involvement in mediating the effects of BDNF on SG neurons in vitro, including neuronal survival and neurite extension. However, the identification of p38 and JNK involvement is entirely novel. The results suggest that neurotrophins can exert opposing effects on SG neurons, the balance of competing signals influencing the generation of neurites. This competition could provide a potential mechanism for the control of neurite number during development.

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.

Somatostatin and Gentamicin-Induced Auditory Hair Cell Loss

Hair cells of the mammalian auditory system do not regenerate, and therefore their loss leads to irreversible hearing loss. Aminoglycosides, among other substances, can irreversibly damage hair cells. Somatostatin, a peptide with hormone/neurotransmitter properties, has neuroprotective effects by binding to its receptor. In this study, we tested whether somatostatin can protect hair cells from gentamicin‐induced damage in vitro.

Somatostatin receptor types 1 and 2 in the developing Mammalian cochlea

The neuropeptide somatostatin (SST) exerts several important physiological actions in the adult central nervous system through interactions with membrane-bound receptors. Transient expression of SST and its receptors has been described in several brain areas during early ontogeny. It is therefore believed that SST may play a role in neural maturation. The present study provides the first evidence for the developmental expression of SST receptors in the mammalian cochlea, emphasizing their possible roles in cochlear maturation. In the developing mouse cochlea, cells immunoreactive to somatostatin receptor 1 (SSTR1) and somatostatin receptor 2 (SSTR2) were located in the embryonic cochlear duct on Kolliker’s organ as early as embryonic day (E) 14 (E14). At E17, the expression of both receptors was high and already located at the hair cells and supporting cells along the length of the cochlear duct, which have become arranged into the characteristic pattern for the organ of Corti (OC) at this stage. At birth, SSTR1- and SSTR2-containing cells were only localized in the OC. In general, immunoreactivity for both receptors increased in the mouse cochlea from postnatal day (P) 0 (P0) to P10; the majority of immunostained cells were inner hair cells, outer hair cells, and supporting cells. Finally, a peak in the mRNA and protein expression of both receptors is present near the time when they respond to physiological hearing (i.e., hearing of airborne sound) at P14. At P21, SSTR1 and SSTR2 levels decrease dramatically. A similar developmental pattern was observed for SSTR1 and SSTR2 mRNA, suggesting that the expression of the SSTR1 and SSTR2 genes is controlled at the transcriptional level throughout development. In addition, we observed reduced levels of phospho-Akt and total Akt in SSTR1 knockout and SSTR1/SSTR2 double-knockout mice compared with wild-type mice. We know from previous studies that Akt is involved in hair cell survival. Taken together, the dynamic nature of SSTR1 and SSTR2 expression at a time of major developmental changes in the cochlea suggests that SSTR1 and SSTR2 (and possibly other members of this family) are involved in the maturation of the mammalian cochlea.

Stress and Survival Pathways in the Mammalian Cochlea

Studies conducted over the last few years demonstrated that signaling pathways that operate in the organs of Corti (OC) play a central role in survival and death of hair cells. An important goal of molecular otology is to characterize these signaling pathways in normal inner ears and inner ears exposed to a variety of different forms of stress, such as ototoxic substances and noise overexposure. In this study, we used high-performance reverse protein microarray technology and phospho-specific antibodies to examine the activation status of defined molecules involved in cellular signaling. We demonstrate that reverse protein microarrays based on the highly sensitive planar-waveguide technology provide an effective and high-throughput means to assess the activation state of key molecules involved in apoptotic and prosurvival signaling in microdissected OC explants over time. In this study, we show that gentamicin and a specific NF-ĸB inhibitor increase the ratio of phospho-c-Jun/c-Jun in OC explants of postnatal rats soon after exposure to these drugs. In addition, we found a decrease in the phospho-Akt/Akt ratio in OC explants early after NF-ĸB inhibition. Finally, we observed an early and consistent decrease in the phospho-p38/p38 ratio in OC explants exposed to the NF-ĸB inhibitor and only a transient decrease in this ratio in OC examples after gentamicin exposure.

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.

Neural Cell Adhesion Molecule L1 Modulates Type I But Not Type II Inner Ear Spiral Ganglion Neurite Outgrowth in an In Vitro Alternate Choice Assay

L1, a neural cell adhesion molecule of the immunoglobulin superfamily, is widely expressed in the nervous system and important in axonal outgrowth, guidance, synapse formation, and signaling. Gene deletion studies emphasize the significance of L1 during development of the central nervous system and L1 is crucial for the topographic targeting of retinal axons. In contrast to the brain and retina, the role of L1 in the inner ear is largely unknown. While previous studies have localized L1 in the developing inner ear of the chicken and mouse, its function during the innervation of the cochlea still remains largely unclear. We therefore investigated the functional role of L1 in the mammalian inner ear. Our aim was to determine whether or not L1 can modulate type I and/or type II spiral ganglion neuron outgrowth using an in vitro alternate choice assay. We found that L1, presented in stripe micropatterns, provide directional cues to neonatal rodent type I but not type II inner ear spiral ganglion neurites. The results suggest that L1 may play a role in axonal pathfinding of type I spiral ganglion dendrites toward their inner hair cell targets but not of type II toward the outer hair cells.

Neural Cell Adhesion Molecule NrCAM Is Expressed in the Mammalian Inner Ear and Modulates Spiral Ganglion Neurite Outgrowth in an In Vitro Alternate Choice Assay

Neuron-glial-related cell adhesion molecule (NrCAM) is a neuronal cell adhesion molecule involved in neuron–neuron and neuron–glial adhesion as well as directional signaling during axonal cone growth. NrCAM has been shown to be involved in several cellular processes in the central and peripheral nervous systems, including neurite outgrowth, axonal pathfinding and myelination, fasciculation of nerve fibers, and cell migration. This includes sensory systems such as the eye and olfactory system. However, there are no reports on the expression/function of NrCAM in the auditory system. The aim of the present study was to elucidate the occurrence of NrCAM in the mammalian cochlea and its role in innervation of the auditory end organ. Our work indicates that NrCAM is highly expressed in the developing mammalian cochlea (position consistent with innervation). Moreover, we found that NrCAM, presented in stripe micropatterns, provide directional cues to neonatal rat inner ear spiral ganglion neurites in vitro. Our results are consistent with a role for NrCAM in the pathfinding of spiral ganglion dendrites toward their hair cell targets in the sensory epithelium.