Unraveling the molecular landscape of lead-induced cochlear synaptopathy: a quantitative proteomics analysis.

Introduction: Exposure to heavy metal lead can cause serious health effects such as developmental neurotoxicity in infants, cognitive impairment in children, and cardiovascular and nephrotoxic effects in adults. Hearing loss is one of the toxic effects induced by exposure to lead. Previous studies demonstrated that exposure to lead causes oxidative stress in the cochlea and disrupts ribbon synapses in the inner hair cells. Methods: This study investigated the underlying mechanism by evaluating the changes in the abundance of cochlear synaptosomal proteins that accompany lead-induced cochlear synaptopathy and hearing loss in mice. Young-adult CBA/J mice were given lead acetate in drinking water for 28 days. Results: Lead exposure significantly increased the hearing thresholds, particularly at the higher frequencies in both male and female mice, but it did not affect the activity of outer hair cells or induce hair cell loss. However, lead exposure decreased wave-I amplitude, suggesting lead-induced cochlear synaptopathy. In agreement, colocalization of pre- and post-synaptic markers indicated that lead exposure decreased the number of paired synapses in the basal turn of the cochlea. Proteomics analysis indicated that lead exposure increased the abundance of 352 synaptic proteins and decreased the abundance of 394 synaptic proteins in the cochlea. Bioinformatics analysis indicated that proteins that change in abundance are highly enriched in the synaptic vesicle cycle pathway. Discussion: Together, these results suggest that outer hair cells are not the primary target in lead-induced ototoxicity, that lead-induced cochlear synaptopathy is more pronounced in the basal turn of the cochlea, and that synaptic vesicle cycle signaling potentially plays a critical role in lead-induced cochlear synaptopathy.

Quantitative profiling of cochlear synaptosomal proteins in cisplatin-induced synaptic dysfunction.

The disruption of ribbon synapses in the cochlea impairs the transmission of auditory signals from the cochlear sensory receptor cells to the auditory cortex. Although cisplatin-induced loss of ribbon synapses is well-documented, and studies have reported nitration of cochlear proteins after cisplatin treatment, yet the underlying mechanism of cochlear synaptopathy is not fully understood. This study tests the hypothesis that cisplatin treatment alters the abundance of cochlear synaptosomal proteins, and selective targeting of nitrative stress prevents the associated synaptic dysfunction. Auditory brainstem responses of mice treated with cisplatin showed a reduction in amplitude and an increase in latency of wave I, indicating cisplatin-induced synaptic dysfunction. The mass spectrometry analysis of cochlear synaptosomal proteins identified 102 proteins that decreased in abundance and 249 that increased in abundance after cisplatin treatment. Pathway analysis suggested that the dysregulated proteins were involved in calcium binding, calcium ion regulation, synapses, and endocytosis pathways. Inhibition of nitrative stress by co-treatment with MnTBAP, a peroxynitrite scavenger, attenuated cisplatin-induced changes in the abundance of 27 proteins. Furthermore, MnTBAP co-treatment prevented the cisplatin-induced decrease in the amplitude and increase in the latency of wave I. Together, these findings suggest a potential role of oxidative/nitrative stress in cisplatin-induced cochlear synaptic dysfunction.

Lead exposure induces nitrative stress and disrupts ribbon synapses in the cochlea.

Environmental exposure to heavy metal lead, a public health hazard in many post-industrial cities, causes hearing impairment upon long-term exposure. Lead-induced cochlear and vestibular dysfunction is well-documented in animal models. Although short-term exposure to lead at concentrations relevant to environmental settings does not cause significant shifts in hearing thresholds in adults, moderate- to low-level lead exposures induce neuronal damage and synaptic dysfunction. We reported that lead exposure induces oxidative stress in the mouse cochlea. However, lead-induced nitrative stress and potential damage to cochlear ribbon synapses are yet to be fully understood. Therefore, this study has evaluated cochlear synaptopathy and nitrative stress in young-adult mice exposed to 2 mM lead acetate for 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated that this exposure significantly increased the blood lead levels. Assessment of hair cell loss by immunohistochemistry analysis and outer hair cell (OHC) activity by recording distortion product otoacoustic emissions (DPOAEs) indicated that the structure and function of the hair cells were not affected by lead exposure. However, this exposure significantly decreased the expression of C-terminal-binding protein-2 (CtBP2) and GluA2, pre- and post-synaptic protein markers in the inner hair cell synapses, particularly in the basal turn of the organ of Corti, suggesting lead-induced disruption of ribbon synapses. In addition, lead exposure significantly increased the nitrotyrosine levels in spiral ganglion cells, suggesting lead-induced nitrative stress in the cochlea. Collectively, these findings suggest that lead exposure even at levels that do not affect the OHCs induces cochlear nitrative stress and causes cochlear synaptopathy.

Nitrative Stress and Auditory Dysfunction.

Nitrative stress is increasingly recognized as a critical mediator of apoptotic cell death in many pathological conditions. The accumulation of nitric oxide along with superoxide radicals leads to the generation of peroxynitrite that can eventually result in the nitration of susceptible proteins. Nitrotyrosine is widely used as a biomarker of nitrative stress and indicates oxidative damage to proteins. Ototoxic insults, such as exposure to noise and ototoxic drugs, enhance the generation of 3-nitrotyrosine in different cell types in the cochlea. Nitrated proteins can disrupt critical signaling pathways and eventually lead to apoptosis and loss of sensory receptor cells in the cochlea. Accumulating evidence shows that selective targeting of nitrative stress attenuates cellular damage. Anti-nitrative compounds, such as peroxynitrite decomposition catalysts and inducible nitric oxide synthase inhibitors, prevent nitrative stress-mediated auditory damage. However, the role of nitrative stress in acquired hearing loss and its potential significance as a promising interventional target is yet to be fully characterized. This review provides an overview of nitrative stress mechanisms, the induction of nitrative stress in the auditory tissue after ototoxic insults, and the therapeutic value of targeting nitrative stress for mitigating auditory dysfunction.

Lmo4 Deficiency Enhances Susceptibility to Cisplatin-Induced Cochlear Apoptosis and Hearing Loss.

Cisplatin, a potent chemotherapeutic drug, induces ototoxicity, which limits its clinical utility. Cisplatin-induced oxidative stress plays a causal role in cochlear apoptosis while the consequent nitrative stress leads to the nitration of LIM domain only 4 (LMO4), a transcriptional regulator, and decreases its cochlear expression levels. Here, we show a direct link between cochlear LMO4 and cisplatin-induced hearing loss by employing a Lmo4 conditional knockout mouse model (Lmo4lox/lox; Gfi1Cre/+). Hair cell-specific deletion of Lmo4 did not alter cochlear morphology or affect hearing thresholds and otoacoustic emissions, in the absence of apoptotic stimuli. Cisplatin treatment significantly elevated the auditory brainstem response thresholds of conditional knockouts, across all frequencies. Moreover, deletion of Lmo4 compromised the activation of STAT3, a downstream target that regulates anti-apoptotic machinery. Immunostaining indicated that the expression of phosphorylated STAT3 was significantly decreased while the expression of activated caspase 3 was significantly increased in Lmo4 deficient hair cells, post-cisplatin treatment. These findings suggest an otoprotective role of LMO4 as cisplatin-induced decrease in cochlear LMO4 could compromise the LMO4/STAT3 cellular defense mechanism to induce ototoxicity.

Cisplatin-induced hair cell loss in zebrafish neuromasts is accompanied by protein nitration and Lmo4 degradation.

Generation of reactive oxygen species, a critical factor in cisplatin-induced ototoxicity, leads to the formation of peroxynitrite, which in turn results in the nitration of susceptible proteins. Previous studies indicated that LMO4, a transcriptional regulator, is the most abundantly nitrated cochlear protein after cisplatin treatment and that LMO4 nitration facilitates ototoxicity in rodents. However, the role of this mechanism in regulating cisplatin-induced hair cell loss in non-mammalian models is unknown. As the mechanosensory hair cells in the neuromasts of zebrafish share many features with mammalian inner ear and is a good model for studying ototoxicity, we hypothesized that cisplatin treatment induces protein nitration and Lmo4 degradation in zebrafish hair cells, thereby facilitating hair cell loss. Immunostaining with anti-parvalbumin revealed a significant decrease in the number of hair cells in the neuromast of cisplatin treated larvae. In addition, cisplatin treatment induced a significant decrease in the expression of Lmo4 protein and a significant increase in nitrotyrosine levels, in the hair cells. The cisplatin-induced changes in Lmo4 and nitrotyrosine levels strongly correlated with hair cell loss, implying a potential link. Furthermore, a significant increase in the expression of activated Caspase-3 in zebrafish hair cells, post cisplatin treatment, suggested that cisplatin-induced decrease in Lmo4 levels is accompanied by apoptosis. These findings suggest that nitrative stress and Lmo4 degradation are important factors in cisplatin-induced hair cell loss in zebrafish neuromasts and that zebrafish could be used as a model to screen the otoprotective efficacy of compounds that inhibit protein nitration.

Environmental Exposures and Hearing Loss.

Pollutants that contaminate the natural or built environment adversely affect the health of living organisms. Although exposure to many of them could be avoided or minimized by careful preventive measures, it is impossible to totally avoid exposure to all pollutants. Ototraumatic agents, such as noise, chemicals, and heavy metals, are pervasive pollutants, mostly produced by human activity, and are critical factors in inducing acquired hearing loss. More importantly, exposure to these pollutants often occurs concurrently and, therefore, the synergistic interactions potentiate auditory dysfunction in susceptible individuals. Epidemiological studies have provided compelling data on the incidence of auditory dysfunction after exposure to a number of ototraumatic agents in the environment, while animal studies have offered crucial insights for understanding the underlying molecular mechanisms. Together, they provide a framework for developing effective interventional approaches for mitigating the adverse impacts of environmental or occupational exposure to ototraumatic agents. This article provides a brief overview of the common pollutants that cause hearing loss.

Tinnitus and Self-Perceived Hearing Handicap in Firefighters: A Cross-Sectional Study.

Firefighters are susceptible to auditory dysfunction due to long-term exposure to noise from sirens, air horns, equipment, and tools used in forcible entry, ventilation, and extrication. In addition, they are exposed to ototoxic chemicals, particularly, during overhaul operations. Studies indicate that 40% of firefighters have hearing loss in the noise-sensitive frequencies of 4 and 6 kHz. Noise-induced hearing loss (NIHL) is often accompanied by tinnitus, which is characterized by ringing noise in the ears. The presence of phantom sounds can adversely affect the performance of firefighters. However, there has been limited research conducted on the prevalence of tinnitus in firefighters. We enrolled firefighters from Michigan, with at least 5 years of continuous service. The hearing handicap inventory for adults (HHIA) was used to determine the difficulty in hearing perceived by the firefighters and the tinnitus functional index (TFI) was used to determine the severity of tinnitus. Self-perceived hearing handicap was reported by 36% of the participants, while tinnitus was reported by 48% of the participants. The TFI survey indicated that 31% perceived tinnitus as a problem. More importantly, self-perceived hearing handicap was significantly associated with the incidence of tinnitus in firefighters, suggesting a potential link between occupational exposure to ototraumatic agents and tinnitus in firefighters.

Inhibition of protein nitration prevents cisplatin-induced inactivation of STAT3 and promotes anti-apoptotic signaling in organ of Corti cells.

JAK/STAT pathway is one among the several oxidative stress-responsive signaling pathways that play a critical role in facilitating cisplatin-induced ototoxicity. Cisplatin treatment decreases the levels of cochlear LMO4, which acts as a scaffold for IL6-GP130 protein complex. Cisplatin-induced nitration and degradation of LMO4 could destabilize this protein complex, which in turn could compromise the downstream STAT3-mediated cellular defense mechanism. Here, we investigated the link between cisplatin-induced nitrative stress and STAT3-mediated apoptosis by using organ of Corti cell cultures. SRI110, a peroxynitrite decomposition catalyst that prevented cisplatin-induced decrease in LMO4 levels and ototoxicity, was used to inhibit nitrative stress. Immunoblotting and immunostaining indicated that cisplatin treatment decreased the expression levels, phosphorylation, and nuclear localization of STAT3 in UB/OC1 cells. Inhibition of nitration by SRI110 co-treatment prevented cisplatin-induced inactivation of STAT3 and promoted its nuclear localization. SRI110 co-treatment reversed the cisplatin-induced changes in the expression levels of Bcl2l1, Ccnd1, Jak2, Jak3, and Src and significantly attenuated the changes in the expression levels of Cdkn1a, Egfr, Fas, Il6st, Jak1, Stat3, and Tyk2. Collectively, these results suggest that the inhibition of cisplatin-induced nitration prevents the inactivation of STAT3, which in turn enables the transcription of anti-apoptotic genes and thereby helps to mitigate cisplatin-induced toxicity.

Chronic lead exposure induces cochlear oxidative stress and potentiates noise-induced hearing loss.

Acquired hearing loss is caused by complex interactions of multiple environmental risk factors, such as elevated levels of lead and noise, which are prevalent in urban communities. This study delineates the mechanism underlying lead-induced auditory dysfunction and its potential interaction with noise exposure. Young-adult C57BL/6 mice were exposed to: 1) control conditions; 2) 2 mM lead acetate in drinking water for 28 days; 3) 90 dB broadband noise 2 h/day for two weeks; and 4) both lead and noise. Blood lead levels were measured by inductively coupled plasma mass spectrometry analysis (ICP-MS) lead-induced cochlear oxidative stress signaling was assessed using targeted gene arrays, and the hearing thresholds were assessed by recording auditory brainstem responses. Chronic lead exposure downregulated cochlear Sod1, Gpx1, and Gstk1, which encode critical antioxidant enzymes, and upregulated ApoE, Hspa1a, Ercc2, Prnp, Ccl5, and Sqstm1, which are indicative of cellular apoptosis. Isolated exposure to lead or noise induced 8-12 dB and 11-25 dB shifts in hearing thresholds, respectively. Combined exposure induced 18-30 dB shifts, which was significantly higher than that observed with isolated exposures. This study suggests that chronic exposure to lead induces cochlear oxidative stress and potentiates noise-induced hearing impairment, possibly through parallel pathways.

CRISPR/Cas9-mediated knockout of Lim-domain only four retards organ of Corti cell growth.

Lim-domain only 4 (LMO4) plays a critical role in mediating the ototoxic side-effects of cisplatin, a highly effective anti-cancer drug. However, the signaling mechanism by which cochlear LMO4 mediates otopathology is yet to be fully understood. Knockout cell culture models are useful tools for investigating the functional roles of novel genes and delineating associated signaling pathways. Therefore, LMO4 knockout organ of Corti cells were generated by using the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system. Successful knockout of LMO4 in UB/OC1 cells was verified by the absence of LMO4 protein bands in immunoblots. Though the Knockout of LMO4 retarded the growth rate and the migratory potential of the cells it did not inhibit their long-term viability as the LMO4 knockout UB/OC1 cells were able to survive, proliferate, and form colonies. In addition, the knockout of LMO4 did not alter the expression of myosin VIIa, a biomarker of hair cells, suggesting that the knockout cells retain important characteristic features of cochlear sensory receptor cells. Thus, the findings of this study indicate that CRISPR/Cas9 system is a simple and versatile method for knocking out genes of interest in organ of Corti cells and that LMO4 knockout UB/OC1 cells are viable experimental models for studying the functional role of LMO4 in ototoxicity.

Targeting nitrative stress for attenuating cisplatin-induced downregulation of cochlear LIM domain only 4 and ototoxicity.

Cisplatin-induced ototoxicity remains a primary dose-limiting adverse effect of this highly effective anticancer drug. The clinical utility of cisplatin could be enhanced if the signaling pathways that regulate the toxic side-effects are delineated. In previous studies, we reported cisplatin-induced nitration of cochlear proteins and provided the first evidence for nitration and downregulation of cochlear LIM domain only 4 (LMO4) in cisplatin ototoxicity. Here, we extend these findings to define the critical role of nitrative stress in cisplatin-induced downregulation of LMO4 and its consequent ototoxic effects in UBOC1 cell cultures derived from sensory epithelial cells of the inner ear and in CBA/J mice. Cisplatin treatment increased the levels of nitrotyrosine and active caspase 3 in UBOC1 cells, which was detected by immunocytochemical and flow cytometry analysis, respectively. The cisplatin-induced nitrative stress and apoptosis were attenuated by co-treatment with SRI110, a peroxynitrite decomposition catalyst (PNDC), which also attenuated the cisplatin-induced downregulation of LMO4 in a dose-dependent manner. Furthermore, transient overexpression of LMO4 in UBOC1 cells prevented cisplatin-induced cytotoxicity while repression of LMO4 exacerbated cisplatin-induced cell death, indicating a direct link between LMO4 protein levels and cisplatin ototoxicity. Finally, auditory brainstem responses (ABR) recorded from CBA/J mice indicated that co-treatment with SRI110 mitigated cisplatin-induced hearing loss. Together, these results suggest that cisplatin-induced nitrative stress leads to a decrease in the levels of LMO4, downregulation of LMO4 is a critical determinant in cisplatin-induced ototoxicity, and targeting peroxynitrite could be a promising strategy for mitigating cisplatin-induced hearing loss.

Quantitative PCR analysis and protein distribution of drug transporter genes in the rat cochlea.

Membrane transporters can be major determinants in the targeting and effectiveness of pharmaceutical agents. A large number of biologically important membrane transporters have been identified and localized to a variety of tissues, organs and cell types. However, little is known about the expression of key membrane transporters in the inner ear, a promising site for targeted therapeutics, as well as a region vulnerable to adverse drug reactions and environmental factors. In this study, we examined the levels of endogenous membrane transporters in rat cochlea by targeted PCR array analysis of 84 transporter genes, followed by validation and localization in tissues by immunohistochemistry. Our studies indicate that several members of the SLC, VDAC and ABC membrane transporter families show high levels of expression, both at the RNA and protein levels in the rat cochlea. Identification and characterization of these membrane transporters in the inner ear have clinical implications for both therapeutic and cytotoxic mechanisms that may aid in the preservation of auditory function.

Cisplatin-induced apoptosis in auditory, renal, and neuronal cells is associated with nitration and downregulation of LMO4.

Cytotoxic effects of cisplatin occur primarily through apoptosis. Though several pro- and anti-apoptotic signaling molecules have been identified to play an important role in mediating the ototoxic, nephrotoxic, and neurotoxic side-effects of cisplatin, the underlying mechanism is yet to be fully characterized. We reported that nitration of LIM domain only 4 (LMO4), a transcriptional regulator, facilitates cochlear apoptosis in cisplatin-induced ototoxicity. However, its role in cisplatin-mediated nephrotoxicity and neurotoxicity is poorly understood. Therefore, HK2, and SH-SY5Y cells were employed along with UBOC1 cells, to investigate the perturbations of LMO4 in cisplatin-induced cytotoxicity, in renal, neuronal, and auditory cells, respectively. Cisplatin induced an increase in the expression of active caspase-3, indicating cellular apoptosis, and increased the nitration of proteins, 24 h post-treatment. Immunostaining with anti-nitrotyrosine and anti-LMO4 indicated that nitrotyrosine co-localized with LMO4 protein in cisplatin treated cells. Immunoblotting with anti-LMO4 indicated that cisplatin induced a decrease in LMO4 protein levels. However, a corresponding decrease in LMO4 gene levels was not observed. Inhibition of protein nitration with SRI110, a peroxynitrite decomposition catalyst, attenuated cisplatin-induced downregulation of LMO4. More importantly, overexpression of LMO4 mitigated the cytotoxic effects of cisplatin in UBOC1 cells while a dose-dependent decrease in LMO4 protein strongly correlated with cell viability in UBOC1, HK2, and SH-SY5Y cells. Collectively, these findings suggested a potential role of LMO4 in facilitating the cytotoxic effects of cisplatin in auditory, renal, and neuronal cells.

Downstream targets of Lmo4 are modulated by cisplatin in the inner ear of Wistar rats.

Lmo4, a transcriptional regulator, appears to be a key player in mediating the cochlear pathology in cisplatin ototoxicity, as it controls cellular responses by modulating the formation of transcriptional complexes. We provided the first evidence of in vivo nitration of Lmo4 in cisplatin ototoxicity. Our data suggested that nitration of Lmo4 and associated decrease in its cochlear expression has the potential to play a pivotal role in cisplatin ototoxicity. However, the Lmo4 interactomes that signal the downstream events in the cochlea are poorly understood. Therefore, custom-made gene arrays were employed to evaluate the modulation of known binding partners or targets of Lmo4, in Wistar rats treated with 16 mg/kg cisplatin. RT-PCR analysis, 3 days post cisplatin treatment, indicated that cisplatin induced up/down regulation of multiple cochlear genes associated with Lmo4 signaling. The cochlear expression of Esr1 was significantly up-regulated by cisplatin treatment, while the expression of Stat3 was down-regulated. Co-treatment with Trolox, an otoprotective antioxidant, attenuated the cisplatin-induced modulation of 5 genes in the cochlea. Consistent with the changes observed at the gene level, immunoblots with anti-Stat3 indicated that cisplatin-induced decrease in cochlear protein levels were attenuated by Trolox co-treatment. These results suggest that cisplatin-induced decreases in the cochlear Lmo4 upon nitration, and associated modulation in the cochlear expression of its binding partners Esr1 and Jak1, probably facilitates the repression of Stat3, a downstream target of Lmo4 implicated in drug mediated apoptosis. Collectively, these findings provide insights on Lmo4 downstream events and indicate a potential role of Jak/Stat transcriptional machinery in relaying the Lmo4 protein signaling in cisplatin-induced ototoxicity.

Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

Cisplatin inhibits hippocampal cell proliferation and alters the expression of apoptotic genes.

The hippocampus, which is critical for memory and spatial navigation, contains a proliferating stem cell niche that is especially vulnerable to antineoplastic drugs such as cisplatin. Although the damaging effects of cisplatin have recently been recognized, the molecular mechanisms underlying its toxic effects on this vital region are largely unknown. Using a focused apoptosis gene array, we analyzed the early cisplatin-induced changes in gene expression in the hippocampus of adult Sprague-Dawley rats and compared the results to those from the inferior colliculus, a non-mitotic auditory region resistant to cisplatin-induced cell death. Two days after a 12 mg/kg dose of cisplatin, significant increases were observed in five proapoptotic genes: Bik, Bid, Bok, Trp53p2, and Card6 and a significant decrease in one antiapoptotic gene Bcl2a1. In contrast, Nol3, an antiapoptotic gene, showed a significant increase in expression. The cisplatin-induced increase in Bid mRNA and decrease in Bcl2a1 mRNA were accompanied by a corresponding increase and decrease of their respective proteins in the hippocampus. In contrast, the cisplatin-induced changes in Bcl2a1, Bid, Bik, and Bok gene expression in the inferior colliculus were strikingly different from those in the hippocampus consistent with the greater susceptibility of the hippocampus to cisplatin toxicity. Cisplatin also significantly reduced immunolabeling of the cell proliferation marker Ki67 in the subgranular zone of the hippocampus 2 days post-treatment. These results indicate that cisplatin-induced hippocampal cell death is mediated by increased expression of proapoptotic and decreased antiapoptotic genes and proteins that likely inhibit hippocampal cell proliferation.

Is S-nitrosylation of cochlear proteins a critical factor in cisplatin-induced ototoxicity?

S-nitrosylation is a redox-sensitive protein modification, which is a highly specific, but reversible mechanism that regulates several signal transduction cascades. Oxidative stress plays a causal role in the ototoxic effects of an anti-neoplastic drug, cisplatin. Despite emerging evidence implicating nitroxidative stress as a critical factor in cisplatin toxicity, the significance of the cochlear protein S-nitrosylation in cisplatin ototoxicity is yet to be understood. In the present study, a 16-mg/kg dose of cisplatin, induced a significant shift in the amplitudes of distortion product otoacoustic emissions, a measure of outer hair cell activity, in Wistar rats, 3 days post-treatment. These ototoxic effects were accompanied by significant increases in the S-nitrosylation of at least three cochlear proteins. Biological significance of these S-nitrosylated proteins was indicated by their immunolocalization in organ of Corti, stria vascularis, and spiral ganglions, which are known cochlear targets of cisplatin toxicity. In addition, co-treatment with Trolox, an inhibitor of peroxynitrite, attenuated cisplatin-induced S-nitrosylation of cochlear proteins and prevented the associated hearing loss. The cisplatin-induced S-nitrosylation of inner ear proteins, their sensitive cochlear localization, and their potential association with cisplatin-induced hearing loss suggests that S-nitrosylation of cochlear proteins might play a crucial role in mediating cisplatin ototoxicity.

Cisplatin-induced ototoxicity is mediated by nitroxidative modification of cochlear proteins characterized by nitration of Lmo4.

Tyrosine nitration is an important sequel of cellular signaling induced by reactive oxygen species. Cisplatin is an anti-neoplastic agent that damages the inner ear through reactive oxygen species and by the formation of DNA adducts. This study reveals a correlation between cisplatin-mediated hearing loss and nitroxidative modification of cochlear proteins and is the first to report nitration of Lmo4. Cisplatin induced a dose-dependent increase in hearing loss in Wistar rats. A 10-15-dB decrease in distortion product amplitude and massive loss of outer hair cells at the basal turn of the cochlea was observed 3 days post-treatment after a 16 mg/kg dose. Cisplatin induced nitration of cellular proteins within the organ of Corti, spiral ganglion, and stria vascularis, which are known targets of cisplatin ototoxicity. Nitration of a 76-kDa cochlear protein correlated with cisplatin dose. The nitrated protein was identified as Lmo4 (LIM domain only 4) by MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass spectrometry and confirmed by reciprocal immunoprecipitation and immunoblotting. Co-localization of nitrotyrosine and Lmo4 was particularly high in outer hair cell nuclei after cisplatin treatment. Cochlear levels of Lmo4 were decreased in rats treated with cisplatin. In vitro studies supported the repression of Lmo4 in nitroxidative conditions and the induction of apoptosis upon repression of Lmo4. Inhibition of cochlear protein nitration prevented cisplatin-induced hearing loss. As Lmo4 is a transcriptional regulator that controls the choice between cell survival and cell death, these results support the hypothesis that nitration of Lmo4 influences cisplatin-induced ototoxicity.

Noise induced changes in the expression of p38/MAPK signaling proteins in the sensory epithelium of the inner ear.

Noise exposure is a major cause of hearing loss. Classical methods of studying protein involvement have provided a basis for understanding signaling pathways that mediate hearing loss and damage repair but do not lend themselves to studying large networks of proteins that are likely to increase or decrease during noise trauma. To address this issue, antibody microarrays were used to quantify the very early changes in protein expression in three distinct regions of the chinchilla cochlea 2h after exposure to a 0.5-8 kHz band of noise for 2h at 112 dB SPL. The noise exposure caused significant functional impairment 2h post-exposure which only partially recovered. Distortion product otoacoustic emissions were abolished 2h after the exposure, but at 4 weeks post-exposure, otoacoustic emissions were present, but still greatly depressed. Cochleograms obtained 4 weeks post-exposure demonstrated significant loss of outer hair cells in the basal 60% of the cochlea corresponding to frequencies in the noise spectrum. A comparative analysis of the very early (2h post-exposure) noise-induced proteomic changes indicated that the sensory epithelium, lateral wall and modiolus differ in their biological response to noise. Bioinformatic analysis of the cochlear protein profile using "The Database for Annotation, Visualization and Integrated Discovery 2008" (DAVID - http://david.abcc. ncifcrf.gov) revealed the initiation of the cell death process in sensory epithelium and modiolus. An increase in Fas and phosphorylation of FAK and p38/MAPK in the sensory epithelium suggest that noise-induced stress signals at the cell membrane are transmitted to the nucleus by Fas and focal adhesion signaling through the p38/MAPK signaling pathway. Up-regulation of downstream nuclear proteins E2F3 and WSTF in immunoblots and microarrays along with their immunolocalization in the outer hair cells supported the pivotal role of p38/MAPK signaling in the mechanism underlying noise-induced hearing loss.