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Ototoxicity refers to damage of sensory hair cells and functional hearing impairment following aminoglycosides exposure. Previously, we have determined that ferulic acid (FA) protected hair cells against serial concentrations of neomycin-induced ototoxic damage. The aim of the present study is to assess the mechanism and effects of FA on neomycin-induced hair cells loss and impact on mechanosensory-mediated behaviors alteration using transgenic zebrafish (pvalb3b: TagGFP). We first identified the optimal protective condition as pre/co-treatment method in early fish development. Pretreatment of the larvae with FA significantly protected against neomycin-induced hair cells loss through preventing neomycin passed through the cytoplasm of hair cells, and subsequently decreased reactive oxygen species production and TUNEL signals in 4 day post-fertilization (dpf) transgenic zebrafish larvae. Moreover, preservation of functional hair cells correlated directly with rescue of the altered swimming behavior, indicates FA pretreatment protects against neomycin ototoxic damage in 7-dpf transgenic zebrafish larvae. Together, our findings unravel the otoprotective role of FA as an effective agent against neomycin-induced ototoxic effects and offering the theoretical foundation for discovering novel candidates for hearing protection.

We previously reported that Schwann cells undergo apoptosis after serum withdrawal. Insulin-like growth factor-I, via phosphatidylinositol-3 kinase, inhibits caspase activation and rescues Schwann cells from serum withdrawal-induced apoptosis. In this study, we examined the role of c-jun N-terminal protein kinase (JNK) in Schwann cell apoptosis induced by serum withdrawal. Activation of both JNK1 and JNK2 was detected 1 h after serum withdrawal with the maximal level detected at 2 h. A dominant negative JNK mutant, JNK (APF), blocked JNK activation induced by serum withdrawal and Schwann cell apoptosis, suggesting JNK activation participates in Schwann cell apoptosis. Serum withdrawal-induced JNK activity was caspase dependent and inhibited by a caspase 3 inhibitor, Ac-DEVD-CHO. Because insulin-like growth factor-I and Bcl-XL are both Schwann cell survival factors, we tested their effects on JNK activation during apoptosis. Insulin-like growth factor-I treatment decreased both JNK1 and JNK2 activity induced by serum withdrawal. LY294002, a phosphatidylinositol-3 kinase inhibitor, blocked insulin-like growth factor-I inhibition on JNK activation, suggesting that phosphatidylinositol-3 kinase mediates the effects of insulin-like growth factor-I. Overexpression of Bcl-XL also resulted in less Schwann cell death and inhibition of JNK activation after serum withdrawal. Collectively, these results suggest JNK activation is involved in Schwann cell apoptosis induced by serum withdrawal. Insulinlike growth factor-I and Bcl family proteins rescue Schwann cells, at least in part, by inhibition of JNK activity.

The insulin-like growth factors (IGFs) are trophic factors whose growth-promoting actions are mediated via the IGF-I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF-I and IGF-I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF-I and IGFBP-5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF-I positive. In vitro, IGF-I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF-I receptor and IGFBP-5. IGF-I treatment increased the intensity of IGFBP-5 without affecting gene expression. Des(1-3)IGF-I, an IGF-I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP-5 with SC conditioned media revealed IGF-I protection of IGFBP-5 from proteolysis, implying the presence of an IGFBP-5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF-I and SC express the IGF-I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF-I bioavailability.

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin-like growth factor-I (IGF-I) prevents apoptosis in neurons in vitro. To investigate whether IGF-I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum-containing media until confluency. When cells were switched to serum-free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF-I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF-I had normal nuclear morphology. The phosphatidylinositol 3-kinase (PI 3-K) inhibitor LY294002 blocked IGF-I–mediated protection. Caspase-3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF-I rescues SC from apoptosis via PI 3-K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999

Insulin-like growth factor-I (IGF-I) promotes the proliferation and differentiation of Schwann cells (SC). We use SC/dorsal root ganglion neuron (DRG) cocultures to examine the effects of IGF-I on the interaction between axons and SC. As SC extend processes toward the axon in the presence of IGF-I, these processes attach to and ensheath axons. Continued IGF-I exposure leads to enhanced P0 expression and long-term myelination. No myelination occurs in the absence of IGF-I. These data imply that IGF-I is critical not only for SC attachment and ensheathment of axons but also for long-term myelination.

Caveolae are non-clathrin-coated invaginations of the plasma membrane, which are present in most cell types. An integral component of caveolae is the caveolin family of related proteins, which not only forms the structural framework of caveolae, but also likely subserves its functional roles, including regulation of signal transduction and cellular transport, in particular, cholesterol trafficking. Although caveolae have been identified ultrastructurally in the peripheral nervous system (PNS), caveolin expression has not previously been studied. To date, three caveolin genes have been reported. Here, we show for the first time that caveolin-1 is expressed by Schwann cells (SC) as well as several SC-derived cell lines. Caveolin-1 is enriched in the buoyant, detergent-insoluble membranes of rat sciatic nerve (SN) and SC, a hallmark of the caveolar compartment. Caveolin-1 exists as both soluble and insoluble forms in rat SN and SC, and localizes to SC cytoplasm and abaxonal myelin. SC caveolin-1 decreases after axotomy, when SC revert to a premyelinating phenotype. We speculate that caveolin-1 may regulate signal transduction and/or cholesterol transport in myelinating SC.

Schwann cells (SCs) are the myelin producing cells of the peripheral nervous system. During development, SCs cease proliferation and differentiate into either a myelin-forming or non-myelin forming mature phenotype. We are interested in the role of insulin-like growth factor-I (IGF-I) in SC development. We have shown previously SCs proliferate in response to IGF-I in vitro. In the current study, we investigated the role of IGF-I in SC differentiation. SC differentiation was determined by morphological criteria and expression of myelin proteins. Addition of 1 mM 8-bromo cyclic AMP (cAMP) or growth on Matrigel matrix decreased proliferation and induced differentiation of SCs. IGF-I enhanced both cAMP and Matrigel matrix-induced SC differentiation, as assessed by both morphological criteria and myelin gene expression. Cultured SCs also express IGF binding protein-5 (IGFBP-5), which can modulate the actions of IGF-I. We examined the expression of IGFBP-5 during SC differentiation. Both cAMP and Matrigel matrix treatment enhanced IGFBP-5 protein expression and cAMP increased IGFBP-5 gene expression five fold. These findings suggest IGF-I potentiates SC differentiation. The concomitant up-regulation of IGFBP-5 may play a role in targeting IGF-I to SCs and thus increase local IGF-I bioavailability.