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.

The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell type-specific target genes.

The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3' RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.

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.

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.

Reanalysis of BRCA1/2 negative high risk ovarian cancer patients reveals novel germline risk loci and insights into missing heritability.

While up to 25% of ovarian cancer (OVCA) cases are thought to be due to inherited factors, the majority of genetic risk remains unexplained. To address this gap, we sought to identify previously undescribed OVCA risk variants through the whole exome sequencing (WES) and candidate gene analysis of 48 women with ovarian cancer and selected for high risk of genetic inheritance, yet negative for any known pathogenic variants in either BRCA1 or BRCA2. In silico SNP analysis was employed to identify suspect variants followed by validation using Sanger DNA sequencing. We identified five pathogenic variants in our sample, four of which are in two genes featured on current multi-gene panels; (RAD51D, ATM). In addition, we found a pathogenic FANCM variant (R1931*) which has been recently implicated in familial breast cancer risk. Numerous rare and predicted to be damaging variants of unknown significance were detected in genes on current commercial testing panels, most prominently in ATM (n = 6) and PALB2 (n = 5). The BRCA2 variant p.K3326*, resulting in a 93 amino acid truncation, was overrepresented in our sample (odds ratio = 4.95, p = 0.01) and coexisted in the germline of these women with other deleterious variants, suggesting a possible role as a modifier of genetic penetrance. Furthermore, we detected loss of function variants in non-panel genes involved in OVCA relevant pathways; DNA repair and cell cycle control, including CHEK1, TP53I3, REC8, HMMR, RAD52, RAD1, POLK, POLQ, and MCM4. In summary, our study implicates novel risk loci as well as highlights the clinical utility for retesting BRCA1/2 negative OVCA patients by genomic sequencing and analysis of genes in relevant pathways.

Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens.

Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB). No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

Folate and colorectal cancer in rodents: a model of DNA repair deficiency.

Fortification of grains has resulted in a positive public health outcome vis-a-vis reduced incidence of neural tube defects. Whether folate has a correspondingly beneficial effect on other disease outcomes is less clear. A role for dietary folate in the prevention of colorectal cancer has been established through epidemiological data. Experimental data aiming to further elucidate this relationship has been somewhat equivocal. Studies report that folate depletion increases DNA damage, mutagenesis, and chromosomal instability, all suggesting inhibited DNA repair. While these data connecting folate depletion and inhibition of DNA repair are convincing, we also present data demonstrating that genetic inhibition of DNA repair is protective in the development of preneoplastic colon lesions, both when folate is depleted and when it is not. The purpose of this paper is to (1) give an overview of the data demonstrating a DNA repair defect in response to folate depletion, and (2) critically compare and contrast the experimental designs utilized in folate/colorectal cancer research and the corresponding impact on tissue folate status and critical colorectal cancer endpoints. Our analysis suggests that there is still an important need for a comprehensive evaluation of the impact of differential dietary prescriptions on blood and tissue folate status.

Epigenetic and functional analysis of IGFBP3 and IGFBPrP1 in cellular immortalization.

Carcinogenic transformation of a cell requires bypassing senescence and becoming immortalized. A cellular senescence-like phenotype can be induced in immortal Li-Fraumeni syndrome (LFS) cells by treating them with the DNA methyltransferase inhibitor 5-aza-deoxycytidine. Our microarray-based expression profiling studies of spontaneously immortalized LFS cell lines identified genes that may provide the growth advantage required for the cells to become immortal. Several members of the IGFBP superfamily of genes fit the profile of genes involved in immortalization: silenced during immortalization and reactivated by 5-aza-deoxycytidine. Overexpression of IGFBP3 or IGFBPrP1 in the immortal LFS cell lines suppressed cell growth and inhibited colony formation. Both genes have the expression pattern of an epigenetically regulated gene and contain CpG islands suitable for methylation-dependent silencing. Analysis of how IGFBPs regulate immortalization will lead to a better understanding of this process and may lead to novel methods for the prevention and treatment of cancer.