A small molecule mitigates hearing loss in a mouse model of Usher syndrome III.

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein.

Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation. SIGNIFICANCE STATEMENT: Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. This approach illuminates the role of clarin-1 and the molecular mechanism linked to the CLRN1(N48K) mutation in sensory hair cells of the inner ear. Additionally, the investigation provided an in vivo model to guide future drug discovery to rescue the hCLRN1(N48K) in hair cells.

Noddy, a mouse harboring a missense mutation in protocadherin-15, reveals the impact of disrupting a critical interaction site between tip-link cadherins in inner ear hair cells.

In hair cells of the inner ear, sound or head movement increases tension in fine filaments termed tip links, which in turn convey force to mechanosensitive ion channels to open them. Tip links are formed by a tetramer of two cadherin proteins: protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which have 11 and 27 extracellular cadherin (EC) repeats, respectively. Mutations in either protein cause inner ear disorders in mice and humans. We showed recently that these two cadherins bind tip-to-tip in a "handshake" mode that involves the EC1 and EC2 repeats of both proteins. However, a paucity of appropriate animal models has slowed our understanding both of the interaction and of how mutations of residues within the predicted interface compromise tip link integrity. Here, we present noddy, a new mouse model for hereditary deafness. Identified in a forward genetic screen, noddy homozygotes lack inner ear function. Mapping and sequencing showed that noddy mutant mice harbor an isoleucine-to-asparagine (I108N) mutation in the EC1 repeat of PCDH15. Residue I108 interacts with CDH23 EC2 in the handshake and its mutation impairs the interaction in vitro. The noddy mutation allowed us to determine the consequences of blocking the handshake in vivo: tip link formation and bundle morphology are disrupted, and mechanotransduction channels fail to remain open at rest. These results offer new insights into the interaction between PCDH15 and CDH23 and help explain the etiology of human deafness linked to mutations in the tip-link interface.

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.

The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene.

Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America.

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.

Modeling and Preventing Progressive Hearing Loss in Usher Syndrome III.

Usher syndrome type III (USH3) characterized by progressive loss of vision and hearing is caused by mutations in the clarin-1 gene (CLRN1). Clrn1 knockout (KO) mice develop hair cell defects by postnatal day 2 (P2) and are deaf by P21-P25. Early onset profound hearing loss in KO mice and lack of information about the cochlear cell type that requires Clrn1 expression pose challenges to therapeutic investigation. We generated KO mice harboring a transgene, TgAC1, consisting of Clrn1-UTR (Clrn1 cDNA including its 5' and 3' UTR) under the control of regulatory elements (Atoh1 3' enhancer/ß-globin basal promoter) to direct expression of Clrn1 in hair cells during development and down regulate it postnatally. The KO-TgAC1 mice displayed delayed onset progressive hearing loss associated with deterioration of the hair bundle structure, leading to the hypothesis that hair cell expression of Clrn1 is essential for postnatal preservation of hair cell structure and hearing. Consistent with that hypothesis, perinatal transfection of hair cells in KO-TgAC1 mice with a single injection of AAV-Clrn1-UTR vector showed correlative preservation of the hair bundle structure and hearing through adult life. Further, the efficacy of AAV-Clrn1 vector was significantly attenuated, revealing the potential importance of UTR in gene therapy.

Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection--a technical report based on preliminary findings.

Laser Capture Microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. We first tested this method with sections of adult mouse inner ears and subsequently applied it to human inner ear sections. The morphology of the various cell types within the inner ear is well preserved in formalin fixed paraffin embedded (FFPE) sections, making it easier to identify cell types and their boundaries. Recovery of good quality RNA from FFPE sections can be challenging, however, recent studies in cancer research demonstrated that it is possible to carry out gene expression analysis of FFPE material. Thus, a method developed using mouse FFPE tissue can be applied to human archival temporal bones. This is important because the majority of human temporal bone banks have specimens preserved in formalin and a technique for retrospective analysis of human archival ear tissue is needed. We used mouse FFPE inner ear sections to procure distinct populations of cells from the various functional domains (organ of Corti, spiral ganglion, etc.) by LCM. RNA was extracted from captured cells, amplified, and assessed for quality. Expression of selected genes was tested by RT-PCR. In addition to housekeeping genes, we were able to detect cell type specific markers, such as Myosin 7a, p27(kip1) and neurofilament gene transcripts that confirmed the likely composition of cells in the sample. We also tested the method described above on FFPE sections from human crista ampullaris. These sections were approximately a year old. Populations of cells from the epithelium and stroma were collected and analyzed independently for gene expression. The method described here has potential use in many areas of hearing research. For example, following exposure to noise, ototoxic drugs or age, it would be highly desirable to analyze gene expression profiles of selected populations of cells within the organ of Corti or spiral ganglion cells rather than a mixed population of cells from whole inner ear tissue. Also, this method can be applied for analysis of human archival ear tissue.

Identification and characterization of mouse cochlear stem cells.

Genetic, noise- and drug-induced loss of hair cells in the mouse and human cochlea leads to permanent hearing loss due to lack of regeneration of hair cells, which may be due to reduced numbers or loss of the regenerative ability of stem cells in the adult cochlea. We hypothesized that the mouse neonate cochlea harbors stem cells capable of differentiating into hair cells. Cells from the primary neonate cochlear culture began to proliferate and formed floating spheres after 14 days in vitro (DIV). By comparison, spheres from the primary culture of the cortex were observed after 7 DIV. Cochlear sphere cells could be passaged and the new spheres were observed after 7 DIV. Cochlear sphere cells were capable of differentiating into astrocytes and oligodendrocytes, but not neurons under the conditions tested. Cochlear sphere cells expressed Sox2 and Myo7a, but failed to show markers that are expressed exclusively in mature cochlear tissue, while cells from cortex spheres expressed Sox2 and Otx2, but not Myo7a. Our results show that cochleae from neonatal mice harbor cells capable of forming spheres and cells from these spheres appear to be better endowed to become hair cells.

Continuous infusion of oxalate by minipumps induces calcium oxalate nephrocalcinosis.

It is hypothesized that oxalate plays an active role in calcium oxalate (CaOx) nephrocalcinosis and oxalate driven nephrolithiasis by interacting with the kidney. We developed an adjustable, nonprecursor, continuous infusion model of hyperoxaluria and CaOx nephrocalcinosis to investigate this hypothesis. Minipumps containing PBS or KOx (60-360 micromol/day; n = 5-7/dose) were implanted subcutaneously in male Sprague-Dawley rats on D0 and D6. Rats were killed on D13. Oxalate excretion and CaOx crystalluria were monitored by 20+4 h urine collections. Localization and content of intrarenal crystals were determined on frozen sections using polarization and microFTIR. Oxalate excretion was significantly elevated in all KOx rats (P < or = 0.005). CaOx crystalluria was most persistent in the 240-360 micromol/day KOx rats, but even 60 micromol/day KOx rats showed sporadic crystalluria. One hundred percent of KOx rats had CaOx nephrocalcinosis as confirmed by microFTIR. Most crystals were localized to the lumens of the corticomedullary collecting ducts. A few crystals are localized just under the papillar urothelium. The minipump model is the first model of hyperoxaluria to provide continuous infusion of oxalate. It permits control of the levels of hyperoxaluria, crystalluria and CaOx nephrocalcinosis. The level of sustained hyperoxaluria and CaOx nephrocalcinosis induced by treatment with 360 micromol/day KOx for 13D models the conditions frequently observed in jejunoileal bypass patients. Adjustments in the length of treatment and level of hyperoxaluria may allow this model to also be used to study the oxalate driven CaOx-nephrolithiasis common in patients with hyperoxaluria due to other causes.

Decreased renal expression of the putative calcium oxalate inhibitor Tamm-Horsfall protein in the ethylene glycol rat model of calcium oxalate urolithiasis.

PURPOSE: Tamm-Horsfall protein is believed to inhibit calcium oxalate crystallization, aggregation or adhesion to the renal epithelium. We determined whether ethylene glycol induced urolithiasis changes the expression of renal and urinary Tamm-Horsfall protein. For comparison the expression of another calcium oxalate inhibitor, osteopontin, was also analyzed. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol plus an AIN-76 diet (Dyets, Bethlehem Pennsylvania) (ethylene glycol group) or standard rat chow and water (control group) for up to 8 weeks (6 per group for 8 weeks and 3 per group for 3 days to 6 weeks). Kidneys and urine (8 weeks only) were harvested and analyzed by Northern and Western blot analysis, and immunohistochemistry. RESULTS: Tamm-Horsfall protein message and protein (membrane bound form) were decreased, while those of osteopontin were increased in the kidneys of rats treated with ethylene glycol for 8 weeks. As judged by immunochemistry Tamm-Horsfall protein and osteopontin were consistently present in a few tubules in rats in the ethylene glycol and control groups, respectively. In urine expression of the free form of Tamm-Horsfall protein (approximately 75 kDa.) was decreased but detectable in ethylene glycol treated rats. Although readily detected in tissue, osteopontin was not detected in the urine of control or ethylene glycol treated rats. In the time course experiment Tamm-Horsfall protein did not decrease until 4 weeks, when calcium oxalate crystals were detectable in the kidneys of treated rats. In contrast, osteopontin was increased, although inconsistently, beginning at 3 days. CONCLUSIONS: Unlike other calcium oxalate inhibitors, such as osteopontin, renal message and protein for Tamm-Horsfall protein was decreased in ethylene glycol treated rats. Tamm-Horsfall protein expression did not decrease until aggregates of crystals had been deposited in the kidneys, while osteopontin expression began to increase almost immediately. Comparisons of the data on kidneys and urine obtained by RNA or protein blot analysis and immunochemistry underscore the need to examine tissue and urine by multiple techniques to obtain the most accurate assessment of how protein expression is changed by a given treatment.

Microarray analysis of changes in renal phenotype in the ethylene glycol rat model of urolithiasis: potential and pitfalls.

OBJECTIVES: To investigate, in an initial study, the use of microarray analysis (MA) to develop an information base for urolithiasis. MA enables the screening of thousands of genes simultaneously making it the technique of choice for situations where the results are known, but the underlying mechanisms are not. Little is known about the pathological changes occurring in the kidney during urolithiasis and this has severely hampered efforts to develop effective therapeutics. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol for 2, 4 or 8 weeks; after death the kidneys were processed for RNA isolation and MA, conducted using a rat-based chip (one kidney/chip) and the results confirmed by reverse transcription-polymerase chain reaction (RT-PCR, 21 probe sets; control, four rats; treated, five rats). Targets were defined as different by the software if the fold change (FC) was >or= 2, and sorted into functional categories using a data-mining tool. The repeatability of MA was investigated by subjecting the 4-week samples to MA in two independent runs. RESULTS: The results for targets with a FC of >or= 2 were plotted (y = 1.01x - 0.75; r(2) 0.84). Comparing the results obtained by RT-PCR and MA showed a good qualitative correlation for those targets having a FC of >or= 5 as determined by MA. Changes in the expression of genes associated with tubule function and regulation, oxidative damage, and inflammation were the most common in the functional categories. Changes in the expression of tubule-specific markers indicated that there was damage to the proximal (gamma-adducin, organic anion and cation transporters, sodium-hydrogen exchange protein-isoform 3) and distal tubules (gamma-adducin, kallikrein) at 2 and 4 weeks. Increased expression of mitochondrial uncoupling protein indicated that there were changes to the mitochondria and oxidative stress at 2 and 4 weeks. CONCLUSION: This study shows the power of MA as an exploratory technique, and changes in the expression of several physiologically important genes whose expression has not previously been reported to be affected by hyperoxaluria or calcium oxalate crystalluria.

Minipump induced hyperoxaluria and crystal deposition in rats: a model for calcium oxalate urolithiasis.

PURPOSE: Unraveling the mechanisms leading to clinically active calcium oxalate (CaOx) stone disease and the development of effective medical therapies to treat it have been hampered by the lack of appropriate animal models. To address this problem we developed a model of hyperoxaluria and calcium oxalate crystal deposition by implanting osmotic minipumps subcutaneously into male rats, that is minipump induced hyperoxaluria and crystal deposition in rats. MATERIALS AND METHODS: Male Harlan-Sprague Dawley rats (225 to 290 gm) were implanted subcutaneously with 1-week 2 ml osmotic minipumps containing 1.5 M potassium oxalate (360 microM KOx/24 hours, [KOx-trt], 11) or phosphate buffered saline (PBS-trt, 9) on days 1 and 7. The 24-hour urine collections were performed on days 0, 4, 7, 11 and 14. Data were analyzed by ANOVA and Tukey's HSD. Urinary crystals were analyzed by light microscopy. Kidneys were harvested on day 14 and processed for light and polarizing microscopy, and RNA analysis.RESULTS Mean overall creatinine excretion +/- SEM (PBS-trt 107 +/- 7 and KOx-trt 123 +/- 6 microM/24 hours, p >0.07) and day 14 serum creatinine (PBS-trt 83 +/- 4 and KOx-trt 83 +/- 5 microM, p >or=0.9) were similar in the 2 treatment groups. Overall urinary volume (PBS-trt 11.3 +/- 0.8 and KOx-trt 18.0 +/- 1.5 ml/24 hours, p