Cisplatin-Mediated Upregulation of APE2 Binding to MYH9 Provokes Mitochondrial Fragmentation and Acute Kidney Injury.

Cisplatin chemotherapy is standard care for many cancers but is toxic to the kidneys. How this toxicity occurs is uncertain. In this study, we identified apurinic/apyrimidinic endonuclease 2 (APE2) as a critical molecule upregulated in the proximal tubule cells (PTC) following cisplatin-induced nuclear DNA and mitochondrial DNA damage in cisplatin-treated C57B6J mice. The APE2 transgenic mouse phenotype recapitulated the pathophysiological features of C-AKI (acute kidney injury, AKI) in the absence of cisplatin treatment. APE2 pulldown-MS analysis revealed that APE2 binds myosin heavy-Chain 9 (MYH9) protein in mitochondria after cisplatin treatment. Human MYH9-related disorder is caused by mutations in MYH9 that eventually lead to nephritis, macrothrombocytopenia, and deafness, a constellation of symptoms similar to the toxicity profile of cisplatin. Moreover, cisplatin-induced C-AKI was attenuated in APE2-knockout mice. Taken together, these findings suggest that cisplatin promotes AKI development by upregulating APE2, which leads to subsequent MYH9 dysfunction in PTC mitochondria due to an unrelated role of APE2 in DNA damage repair. This postulated mechanism and the availability of an engineered transgenic mouse model based on the mechanism of C-AKI provides an opportunity to identify novel targets for prophylactic treatment of this serious disease. SIGNIFICANCE: These results reveal and highlight an unexpected role of APE2 via its interaction with MYH9 and suggest that APE2 has the potential to prevent acute kidney injury in patients with cisplatin-treated cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/713/F1.large.jpg.

Panel 3: Genetics and Precision Medicine of Otitis Media.

Objective The objective is to perform a comprehensive review of the literature up to 2015 on the genetics and precision medicine relevant to otitis media. Data Sources PubMed database of the National Library of Medicine. Review Methods Two subpanels were formed comprising experts in the genetics and precision medicine of otitis media. Each of the panels reviewed the literature in their respective fields and wrote draft reviews. The reviews were shared with all panel members, and a merged draft was created. The entire panel met at the 18th International Symposium on Recent Advances in Otitis Media in June 2015 and discussed the review and refined the content. A final draft was made, circulated, and approved by the panel members. Conclusion Many genes relevant to otitis media have been identified in the last 4 years in advancing our knowledge regarding the predisposition of the middle ear mucosa to commensals and pathogens. Advances include mutant animal models and clinical studies. Many signaling pathways are involved in the predisposition of otitis media. Implications for Practice New knowledge on the genetic background relevant to otitis media forms a basis of novel potential interventions, including potential new ways to treat otitis media.

The roles of USH1 proteins and PDZ domain-containing USH proteins in USH2 complex integrity in cochlear hair cells.

Usher syndrome (USH) is the most common cause of inherited deaf-blindness, manifested as USH1, USH2 and USH3 clinical types. The protein products of USH2 causative and modifier genes, USH2A, ADGRV1, WHRN and PDZD7, interact to assemble a multiprotein complex at the ankle link region of the mechanosensitive stereociliary bundle in hair cells. Defects in this complex cause stereociliary bundle disorganization and hearing loss. The four USH2 proteins also interact in vitro with USH1 proteins including myosin VIIa, USH1G (SANS), CIB2 and harmonin. However, it is unclear whether the interactions between USH1 and USH2 proteins occur in vivo and whether USH1 proteins play a role in USH2 complex assembly in hair cells. In this study, we identified a novel interaction between myosin VIIa and PDZD7 by FLAG pull-down assay. We further investigated the role of the above-mentioned four USH1 proteins in the cochlear USH2 complex assembly using USH1 mutant mice. We showed that only myosin VIIa is indispensable for USH2 complex assembly at ankle links, indicating the potential transport and/or anchoring role of myosin VIIa for USH2 proteins in hair cells. However, myosin VIIa is not required for USH2 complex assembly in photoreceptors. We further showed that, while PDZ protein harmonin is not involved, its paralogous USH2 proteins, PDZD7 and whirlin, function synergistically in USH2 complex assembly in cochlear hair cells. In summary, our studies provide novel insight into the functional relationship between USH1 and USH2 proteins in the cochlea and the retina as well as the disease mechanisms underlying USH1 and USH2.

Characterization of neuronal cell death in the spiral ganglia of a mouse model of endolymphatic hydrops.

HYPOTHESIS: Spiral ganglion neurons (SGN) in the Phex male mouse, a murine model of postnatal endolymphatic hydrops (ELH) undergo progressive deterioration reminiscent of human and other animal models of ELH with features suggesting apoptosis as an important mechanism. BACKGROUND: Histologic analysis of the mutant's cochlea demonstrates ELH by postnatal Day (P) 21 and SGN loss by P90. The SGN loss seems to occur in a consistent topographic pattern beginning at the cochlear apex. METHODS: SGN were counted at P60, P90, and P120. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR, and immunohistochemical analyses of activated caspase-3, caspase-8, and caspase-9 were performed on cochlear sections obtained from mutants and controls. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay (TUNEL) was carried out on 2 mutants and 2 controls. RESULTS: Corrected SGN counts in control mice were greater in the apical turn of the cochleae at P90 and P120, respectively (p < 0.01). Increased expression of activated caspase-3, caspase-8, and caspase-9 was seen in the mutant. At later time points, activated caspase expression gradually declined in the apical turns and increased in basal turns of the cochlea. Quantitative and semiquantitative PCR analysis confirmed increased expression of caspase-3, caspase-8, and caspase-9 at P21 and P40. TUNEL staining demonstrated apoptosis at P90 in the apical and basal turns of the mutant cochleae. CONCLUSION: SGN degeneration in the Phex /Y mouse seems to mimic patterns observed in other animals with ELH. Apoptosis plays an important role in the degeneration of the SGN in the Phex male mouse.

Sh3pxd2b mice are a model for craniofacial dysmorphology and otitis media.

Craniofacial defects that occur through gene mutation during development increase vulnerability to eustachian tube dysfunction. These defects can lead to an increased incidence of otitis media. We examined the effects of a mutation in the Sh3pxd2b gene (Sh3pxd2b(nee)) on the progression of otitis media and hearing impairment at various developmental stages. We found that all mice that had the Sh3pxd2b(nee) mutation went on to develop craniofacial dysmorphologies and subsequently otitis media, by as early as 11 days of age. We found noteworthy changes in cilia and goblet cells of the middle ear mucosa in Sh3pxd2b(nee) mutant mice using scanning electronic microscopy. By measuring craniofacial dimensions, we determined for the first time in an animal model that this mouse has altered eustachian tube morphology consistent with a more horizontal position of the eustachian tube. All mutants were found to have hearing impairment. Expression of TNF-α and TLR2, which correlates with inflammation in otitis media, was up-regulated in the ears of mutant mice when examined by immunohistochemistry and semi-quantitative RT-PCR. The mouse model with a mutation in the Sh3pxd2b gene (Sh3pxd2b(nee)) mirrors craniofacial dysmorphology and otitis media in humans.

Ush1c gene expression levels in the ear and eye suggest different roles for Ush1c in neurosensory organs in a new Ush1c knockout mouse.

Usher syndrome (USH) is the most common form of deaf-blindness in humans. Molecular characterization revealed that the USH gene products form a macromolecular protein network in hair cells of the inner ear and in photoreceptor cells of the retina via binding to PDZ domains in the scaffold protein harmonin encoded by the Ush1c gene in mice and humans. Although several mouse mutants for the Ush1c gene have been described, we generated a targeted null mutation Ush1c mouse model in which the first four exons of the Ush1c gene were replaced with a reporter gene. Here, we assessed the expression pattern of the reporter gene under control of Ush1c regulatory elements and characterized the phenotype of mice defective for Ush1c. These Ush1 knockout mice are deaf but do not recapitulate vision defects before 10 months of age. Our data show LacZ expression in multiple layers of the retina but in neither outer nor inner segments of the photoreceptor layers in mice bearing the knockout construct at 1-5 months of age. The fact that Ush1c expression is much higher in the ear than in the eye suggests a different role for Ush1c in ear function than in the eye and may explain why Ush1c mutant mice do not recapitulate vision defects.

Proteomics, bioinformatics and targeted gene expression analysis reveals up-regulation of cochlin and identifies other potential biomarkers in the mouse model for deafness in Usher syndrome type 1F.

Proteins and protein networks associated with cochlear pathogenesis in the Ames waltzer (av) mouse, a model for deafness in Usher syndrome 1F (USH1F), were identified. Cochlear protein from wild-type and av mice at postnatal day 30, a time point in which cochlear pathology is well established, was analyzed by quantitative 2D gel electrophoresis followed by mass spectrometry (MS). The analytic gel resolved 2270 spots; 69 spots showed significant changes in intensity in the av cochlea compared with the control. The cochlin protein was identified in 20 peptide spots, most of which were up-regulated, while a few were down-regulated. Analysis of MS sequence data showed that, in the av cochlea, a set of full-length isoforms of cochlin was up-regulated, while isoforms missing the N-terminal FCH/LCCL domain were down-regulated. Protein interaction network analysis of all differentially expressed proteins was performed with Metacore software. That analysis revealed a number of statistically significant candidate protein networks predicted to be altered in the affected cochlea. Quantitative PCR (qPCR) analysis of select candidates from the proteomic and bioinformatic investigations showed up-regulation of Coch mRNA and those of p53, Brn3a and Nrf2, transcription factors linked to stress response and survival. Increased mRNA of Brn3a and Nrf2 has previously been associated with increased expression of cochlin in human glaucomatous trabecular meshwork. Our report strongly suggests that increased level of cochlin is an important etiologic factor leading to the degeneration of cochlear neuroepithelia in the USH1F model.

Otitis media in a mouse model for Down syndrome.

The Ts65Dn mouse shares many phenotypic characteristics of human Down syndrome. Here, we report that otitis media, characterized by effusion in the middle ear and hearing loss, was prevalent in Ts65Dn mice. Of the 53 Ts65Dn mice tested, 81.1% had high auditory-evoked brainstem response (ABR) thresholds for at least one of the stimulus frequencies (click, 8 kHz, 16 kHz and 32 kHz), in at least one ear. The ABR thresholds were variable and showed no tendency toward increase with age, from 2 to 7 months of age. Observation of pathology in mice, aged 3-4 months, revealed middle ear effusion in 11 of 15 Ts65Dn mice examined, but only in two of 11 wild-type mice. The effusion in each mouse varied substantially in volume and inflammatory cell content. The middle ear mucosae were generally thickened and goblet cells were distributed with higher density in the epithelium of the middle ear cavity of Ts65Dn mice as compared with those of wild-type controls. Bacteria of pathogenic importance to humans also were identified in the Ts65Dn mice. This is the first report of otitis media in the Ts65Dn mouse as a model characteristic of human Down syndrome.

Role for Toll-like receptor 2 in the immune response to Streptococcus pneumoniae infection in mouse otitis media.

Streptococcus pneumoniae is the most common pathogen associated with otitis media. To examine the role of Toll-like receptor 2 (TLR2) in host defense against Streptococcus pneumoniae infection in the middle ear, wild-type (WT; C57BL/6) and TLR2-deficient (TLR2(-/-)) mice were inoculated with Streptococcus pneumoniae (1 x 10(6) CFU) through the tympanic membrane. Nineteen of 37 TLR2(-/-) mice showed bacteremia and died within 3 days after the challenge, compared to only 4 of 32 WT mice that died. Of those that survived, more severe hearing loss in the TLR2(-/-) mice than in the WT mice was indicated by an elevation in auditory-evoked brain stem response thresholds at 3 or 7 days postinoculation. The histological pathology was characterized by effusion and tissue damage in the middle ear, and in the TLR2(-/-) mice, the outcome of infection became more severe at 7 days. At both 3 and 7 days postchallenge, the TLR2(-/-) mice had higher blood bacterial titers than the WT mice (P < 0.05), and typical bacteria were identified in the effusion from both ears of both mouse groups by acridine orange staining. Moreover, by 3 days postchallenge, the mRNA accumulation levels of NF-kappaB, tumor necrosis factor alpha, interleukin 1beta, MIP1alpha, Muc5ac, and Muc5b were significantly lower in the ears of TLR2(-/-) mice than in WT mice. In summary, TLR2(-/-) mice may produce relatively low levels of proinflammatory cytokines following pneumococcal challenge, thus hindering the clearance of bacteria from the middle ear and leading to sepsis and a high mortality rate. This study provides evidence that TLR2 is important in the molecular pathogenesis and host response to otitis media.

Vestibular evoked myogenic potentials in normal mice and Phex mice with spontaneous endolymphatic hydrops.

OBJECTIVE AND BACKGROUND: Vestibular evoked myogenic potentials (VEMPs) have been recorded from the neck musculature and the cervical spinal cord in humans and a limited number of laboratory animals in response to loud sound. However, the mouse VEMP has yet to be described. Evaluation of the sacculocollic pathway via VEMPs in mice can set the stage for future evaluations of mutant mice that now play an important role in research regarding human auditory and vestibular dysfunction. MATERIALS AND METHODS: Sound-evoked potentials were recorded from the neck extensor muscles and the cervical spinal cord in normal adult mice and in circling Phex(Hyp-Duk/y) mice with known vestibular abnormalities, including endolymphatic hydrops (ELH). RESULTS: Biphasic potentials were recorded from all normal animals. The mean threshold of the VEMP response in normal adult mice was 60 dB normal hearing level with a mean peak latency of 6.25 +/- 0.46 and 7.95 +/- 0.42 milliseconds for p1 and n1 peaks, respectively. At the maximum sound intensity used (100 dB normal hearing level), 4 of 5 Phex mice did not exhibit VEMP responses, and 1 showed an elevated threshold, but normal response, with regard to peak latency and amplitude. The histologic findings in all of these Phex mice were consistent with distended membranous labyrinth, displaced Reissner membrane, ganglion cell loss, and ELH. CONCLUSION: This is the first report of VEMP recordings in mice and the first report of abnormal VEMPs in a mouse model with ELH. The characteristics of these potentials such as higher response threshold in comparison to auditory brainstem response, myogenic nature of the response, and latency correlation with the cervical recording (accessory nerve nucleus) were similar to those of VEMPs in humans, guinea pigs, cats, and rats, suggesting that the mouse may be used as an animal model in the study of VEMPs. The simplicity and reliability of these recordings make the VEMP a uniquely informative test for assessing vestibular function, and these results suggest that they may be informative in mice with various mutations. However, further investigation is necessary.

Auditory brainstem responses in 10 inbred strains of mice.

The auditory brainstem response (ABR) is an evoked potential response of auditory activity in the auditory nerve and subsequent fiber tracts and nuclei within the auditory brainstem pathways. The threshold, amplitude, and latency analysis of the ABR provides information on the peripheral hearing status and the integrity of brainstem pathways. In this study, we compared the threshold, amplitude, and latency of ABRs recorded from 149 mice of 10 commonly used inbred strains (BALB/cJ, C3HeB/FeJ, C3H/HeJ, CAST/EiJ, CBA/CaJ, CBA/J, FVB/NJ, MRL/MpJ, NZB/BlNJ, and SJL/J) using clicks of different intensities. The ABR thresholds of these strains ranged from 32 to 43 dB SPL. The amplitude of both waves I and IV of ABRs, which increased monotonically with click intensity in most strains, differed significantly among different strains at each intensity tested. Moreover, the amplitude of both waves was inversely correlated with the body weight of each strain at most intensities tested. In general, the amplitude of wave IV was smaller than that of wave I resulting in the IV/I amplitude ratio of <1.0 in all strains. The peak latency of both waves I and IV decreased significantly with click intensity in each strain. However, this intensity-dependent decrease was greater for wave IV than for wave I such that the wave I-IV inter-peak latency also decreased significantly with increasing intensity. I-IV inter-peak latencies for MRL/MpJ, C3HeB/FeJ, NZB/BlNJ, and C3H/HeJ strains are longer than FVB/NJ, SJL/J, or CAST/EiJ. This work is the first step to study the genetic basis underlying strain-related differences in auditory pathway.

A comparison of vestibular and auditory phenotypes in inbred mouse strains.

The purposes of this research were to quantify gravity receptor function in inbred mouse strains and compare vestibular and auditory function for strain- and age-matched animals. Vestibular evoked potentials (VsEPs) were collected for 19 inbred strains at ages from 35 to 389 days old. On average, C57BL/6J (35 to 190 days), BALB/cByJ, C3H/HeSnJ, CBA/J, and young LP/J mice had VsEP thresholds comparable to normal. Elevated VsEP thresholds were found for elderly C57BL/6J, NOD.NONH2(kb), BUB/BnJ, A/J, DBA/2J, NOD/LtJ, A/WySnJ, MRL/MpJ, A/HeJ, CAST/Ei, SJL/J, elderly LP/J, and CE/J. These results suggest that otolithic function varies among inbred strains and several strains displayed gravity receptor deficits by 90 days old. Auditory brainstem response (ABR) thresholds were compared to VsEP thresholds for 14 age-matched strains. C57BL/6J mice (up to 190 days) showed normal VsEPs with normal to mildly elevated ABR thresholds. Four strains (BUB/BnJ, NOD/LtJ, A/J, elderly LP/J) had significant hearing loss and elevated VsEP thresholds. Four strains (DBA/2J, A/WySnJ, NOD.NONH2(kb), A/HeJ) had elevated VsEP thresholds (including absent VsEPs) with mild to moderate elevations in ABR thresholds. Three strains (MRL/MpJ, Ce/J, SJL/J) had significant vestibular loss with no concomitant hearing loss. These results suggest that functional change in one sensory system does not obligate change in the other. We hypothesize that genes responsible for early onset hearing loss may affect otolithic function, yet the time course of functional change may vary. In addition, some genetic mutations may produce primarily gravity receptor deficits. Potential genes responsible for selective gravity receptor impairment demonstrated herein remain to be identified.

A missense mutation in the previously undescribed gene Tmhs underlies deafness in hurry-scurry (hscy) mice.

Mouse deafness mutations provide valuable models of human hearing disorders and entry points into molecular pathways important to the hearing process. A newly discovered mouse mutation named hurry-scurry (hscy) causes deafness and vestibular dysfunction. Scanning electron microscopy of cochleae from 8-day-old mutants revealed disorganized hair bundles, and by 50 days of age, many hair cells are missing. To positionally clone hscy, 1,160 F(2) mice were produced from an intercross of (C57BL/6-hscy x CAST/EiJ) F(1) hybrids, and the mutation was localized to a 182-kb region of chromosome 17. A missense mutation causing a critical cysteine to phenylalanine codon change was discovered in a previously undescribed gene within this candidate interval. The gene is predicted to encode an integral membrane protein with four transmembrane helices. A synthetic peptide designed from the predicted protein was used to produce specific polyclonal antibodies, and strong immunoreactivity was observed on hair bundles of both inner and outer hair cells in cochleae of newborn +/+ controls and +/hscy heterozygotes but was absent in hscy/hscy mutants. Accordingly, the gene was given the name "tetraspan membrane protein of hair cell stereocilia," symbol Tmhs. Two related proteins (>60% amino acid identity) are encoded by genes on mouse chromosomes 5 and 6 and, together with the Tmhs-encoded protein (TMHS), comprise a distinct tetraspan subfamily. Our localization of TMHS to the apical membrane of inner ear hair cells during the period of stereocilia formation suggests a function in hair bundle morphogenesis.

A missense mutation in the mouse Col2a1 gene causes spondyloepiphyseal dysplasia congenita, hearing loss, and retinoschisis.

UNLABELLED: A missense mutation in the mouse Col2a1 gene has been discovered, resulting in a mouse phenotype with similarities to human spondyloepiphyseal dysplasia (SED) congenita. In addition, SED patients have been identified with a similar molecular mutation in human COL2A1. This mouse model offers a useful tool for molecular and biological studies of bone development and pathology. INTRODUCTION: A new mouse autosomal recessive mutation has been discovered and named spondyloepiphyseal dysplasia congenita (gene symbol sedc). MATERIALS AND METHODS: Homozygous sedc mice can be identified at birth by their small size and shortened trunk. Adults have shortened noses, dysplastic vertebrae, femora, and tibias, plus retinoschisis and hearing loss. The mutation was mapped to Chr15, and Col2a1 was identified as a candidate gene. RESULTS: Sequence analyses revealed that the affected gene is Col2a1, which has a missense mutation at exon 48 causing an amino acid change of arginine to cysteine at position 1417. Two human patients with spondyloepiphyseal dysplasia (SED) congenita have been reported with the same amino acid substitution at position 789 in the human COL2A1 gene. CONCLUSIONS: Thus, sedc/sedc mice provide a valuable model of human SED congenita with molecular and phenotypic homology. Further biochemical analyses, molecular modeling, and cell culture studies using sedc/sedc mice could provide insight into mechanisms of skeletal development dependent on Col2a1 and its role in fibril formation and cartilage template organization.