Histology and immunocytochemical localization of glial fibrillary acidic protein in the inner ear of Scincella tsinlingensis / Histología y localización inmunocitoquímica de la proteína ácida fibrilar glial en el oído interno de Scincella tsinlingensis

SUMMARY: The microstructure of inner ear in Scincella tsinlingensis was observed by light microscopy and the expression of glial fibrillary acidic protein (GFAP) in membranous labyrinth among the juvenile age group, subadult age group and adult age group were also detected by methods of immunohistochemistry. The inner ear in S. tsinlingensis resembled those in other Scincid lizards in their anatomy and histology. Large and elongate cochlear duct was slightly bowed or arched laterally. There was no hint of limbic modifications and the limbic lip was absent in cochlear recess. The basilar papilla elongated anteroventrally possessed specialized tectorial sallets. GFAP staining was significantly distributed in supporting cells of the sensory epithelia of cochlear duct, while the utricular macula and canal ampullae showed immunopositive for the GFAP antibody, with weaker staining in the saccular macula. The membranous inner ear of three different age groups revealed the similar pattern of GFAP expression, which suggested that the distribution of supporting cells were independent of age in S. tsinlingensis.

RESUMEN: La microestructura del oído interno en Scincella tsinlingensis fue analizada mediante microscopía óptica y por otra parte, fue cuantificada la expresión de la proteína ácida fibrilar glial (GFAP) en el laberinto membranoso, entre los grupos de edad juvenil, subadulto y adulto, utilizándose métodos inmunohistoquímicos. El oído interno de S. tsinlingensis se asemejaba al de otros lagartos Scincid tanto en su anatomía como en su histología. El conducto coclear mayor estaba ligeramente arqueado o arqueado lateralmente. No había indicios de modificaciones límbicas y no se evidenció el labio en el receso coclear. La papila basilar alargada anteroventralmente poseía sallets tectoriales especializados. La tinción de GFAP se distribuyó significativamente en las células del epitelio sensorial del conducto coclear, mientras que la mácula utricular y la ampolla del canal mostraron inmunopositividad para el anticuerpo GFAP, con una tinción más débil en la mácula sacular. El oído interno membranoso de los tres grupos de edad diferentes reveló un patrón similar de expresión de GFAP, lo que sugiere que la distribución de las células de soporte son independiente de la edad en S. tsinlingensis.

Enrichment of damaging missense variants in genes related with axonal guidance signalling in sporadic Meniere's disease.

INTRODUCTION: Meniere's disease (MD) is a rare inner ear disorder with a significant genetic contribution defined by a core phenotype: episodic vertigo, sensorineural hearing loss and tinnitus. It has been mostly described in sporadic cases, familial cases being around 10% of the observed individuals. It is associated with an accumulation of endolymph in the inner ear, but the molecular underpinnings remain largely unknown. The main molecular pathways showing higher differentially expressed genes in the supporting cells of the inner ear are related to cochlea-vestibular innervation, cell adhesion and leucocyte extravasation. In this study, our objective is to find a burden of rare variants in genes that interact with the main signalling pathways in supporting cells of the inner ear in patients with sporadic MD. METHODS: We designed a targeted-sequencing panel including genes related with the main molecular pathways in supporting cells and sequenced 860 Spanish patients with sporadic MD. Variants with minor allele frequencies <0.1 in the gene panel were compared with three independent reference datasets. Variants were classified as loss of function, missense and synonymous. Missense variants with a combined annotation-dependent depletion score of >20 were classified as damaging missense variants. RESULTS: We have observed a significant burden of damaging missense variants in few key genes, including the NTN4 gene, associated with axon guidance signalling pathways in patients with sporadic MD. We have also identified active subnetworks having an enrichment of rare variants in sporadic MD. CONCLUSION: The burden of missense variants in the NTN4 gene suggests that axonal guidance signalling could be a novel pathway involved in sporadic MD.

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

Localization of Glucose Transporter 10 to Hair Cells' Cuticular Plate in the Mouse Inner Ear.

This study aimed to investigate the localization pattern of glucose transporters (Gluts) in mouse cochlea. Genome-wide gene expression analysis using CodeLink™ bioarrays indicated that Glut1 and Glut10 were highly expressed (~10-fold) in mouse cochlea compared with the other members of glucose transporters (Glut2-6, Glut8, and Glut9). Semiquantitative RT-PCR and western blotting confirmed that Glut10 expression in mouse cochlea was high throughout the embryogenesis and postnatal development. Immunofluorescent staining showed that Glut10 protein was localized in the cuticular plate of the outer and inner cochlear hair cells and in the ampullary crest of the vestibular system. Based on these results, it was supposed that Glut10 may contribute to glucose transport from the endolymph to the hair cells across the cuticular plate.

Arsenic level in toenails is associated with hearing loss in humans.

Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12-55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz [odds ratio (OR) = 4.27; 95% confidence interval (CI): 1.51, 12.05], 8 kHz (OR = 3.91; 95% CI: 1.47, 10.38) and 12 kHz (OR = 4.15; 95% CI: 1.55, 11.09) by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails (r = 0.8113, p = 0.0014) in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans.

Endolymph composition: paradigm or inevitability?

This review is focused on the unusual composition of the endolymph of the inner ear and its function in mechanoelectrical transduction. The role of K(+) and Ca(2+) in excitatory influx, the very low Na(+), Ca(2+) and Mg(2+) concentrations of endolymph, stereocilia structure of hair cells and some proteins involved in mechanosensory signal transduction with emphasis on auditory receptors are presented and analyzed in more details. An alternative hypothetical model of ciliary structure and endolymph with a 'normal' composition is discussed. It is concluded that the unique endolymph cation content is more than an energy saving mechanism that avoids disturbing circulatory vibrations to achieve a much better mechanosensory resolution. It is the only possible way to fulfil the requirements for a precise ciliary mechanoelectrical transduction in conditions where pressure events with quite diverse amplitudes and duration are transformed into adequate hair cell membrane depolarizations, which are regulated by a sensitive Ca(2+)-dependent feedback tuning.

A gene network regulated by FGF signalling during ear development.

During development cell commitment is regulated by inductive signals that are tightly controlled in time and space. In response, cells activate specific programmes, but the transcriptional circuits that maintain cell identity in a changing signalling environment are often poorly understood. Specification of inner ear progenitors is initiated by FGF signalling. Here, we establish the genetic hierarchy downstream of FGF by systematic analysis of many ear factors combined with a network inference approach. We show that FGF rapidly activates a small circuit of transcription factors forming positive feedback loops to stabilise otic progenitor identity. Our predictive network suggests that subsequently, transcriptional repressors ensure the transition of progenitors to mature otic cells, while simultaneously repressing alternative fates. Thus, we reveal the regulatory logic that initiates ear formation and highlight the hierarchical organisation of the otic gene network.

Spatiotemporal expression patterns of clusterin in the mouse inner ear.

Clusterin (CLU) is an extracellular chaperone protein that is implicated in diverse physiological and pathophysiological cellular processes. CLU expression is upregulated in response to cellular stress and under certain conditions, such as neurodegenerative disease and cancer. CLU primarily functions as a chaperone that exerts cytoprotective effects by removing cellular debris and misfolded proteins and also acts as a signaling molecule that regulates pro-survival pathways. Deafness is caused by genetic factors and various extrinsic insults, including ototoxic drugs, exposure to loud sounds and aging. Considering its cytoprotectivity, CLU may also mediate cellular defense mechanisms against hearing loss due to cellular stresses. To understand the function of CLU in the inner ear, we analyze CLU expression patterns in the mouse inner ear during development and in the adult stage. Results of quantitative real-time polymerase chain reaction analysis showed that Clu mRNA levels in the inner ear were increased during embryogenesis and were constantly expressed in the adult. Detailed spatial expression patterns of Clu both in the mRNA and protein levels were analyzed throughout various developmental stages via in situ hybridization and immunofluorescence staining. Clu expression was found in specific domains of developing inner ear starting from the otocyst stage, mainly adjacent to the prosensory domain of the cochlear epithelium. In the mature inner ear, Clu expression was observed in Deiter's cells and pillar cells of the organ of Corti, outer sulcus and in basal cells of the stria vascularis in the cochlea. These specific spatiotemporal expression patterns suggest the possible roles of CLU in inner ear development and in maintaining proper hearing function.

Innate immune defense in the inner ear - mucines are expressed by the human endolymphatic sac.

The human endolymphatic sac has been shown recently to have immunological capacities and has thus been proposed as the main entity protecting the inner ear from pathogen invasion, equivalent to mucosa-associated lymphoid tissue (MALT). Although the sac expresses molecules of the innate immune system, the potential expression of members of the important mucin family has not been detailed. Thus, this paper explores endolymphatic sac expression of a number of mucins and mucin precursors. Twelve fresh tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery. The expression of Mucin 1, 2, 5B/AC and 16, as well as the core structure elements (mucin precursors) T-antigen, Tn-antigen and Sialyl-Tn-antigen was investigated by immunohistochemistry. The endolymphatic sac epithelium expressed MUC1 (both apically towards the endolymphatic sac (ES) lumen and basally towards the capillary network), MUC 16 and Tn-antigen. There was no labeling after incubation with antibodies against T-antigen, sialyl-Tn-antigen, MUC2 and MUC5B/AC. We conclude that the human endolymphatic sac epithelium expresses a number of mucin molecules, which supports the hypothesis of the sac as the primary immunological tissue structure of the inner ear, equivalent to MALT in other organs. The mucins may also play a role in the formation and continuous homeostasis of the inner ear fluids, as well as the pathogenesis of Meniere's disease.

BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of Opsanus tau.

We report on a new xyloside conjugated to BODIPY, BX and its utility to prime fluorescent glycosaminoglycans (BX-GAGs) within the inner ear in vivo. When BX is administered directly into the endolymphatic space of the oyster toadfish (Opsanus tau) inner ear, fluorescent BX-GAGs are primed and become visible in the sensory epithelia of the semicircular canals, utricle, and saccule. Confocal and 2-photon microscopy of vestibular organs fixed 4 h following BX treatment, reveal BX-GAGs constituting glycocalyces that envelop hair cell kinocilium, nerve fibers, and capillaries. In the presence of GAG-specific enzymes, the BX-GAG signals are diminished, suggesting that chondroitin sulfates are the primary GAGs primed by BX. Results are consistent with similar click-xylosides in CHO cell lines, where the xyloside enters the Golgi and preferentially initiates chondroitin sulfate B production. Introduction of BX produces a temporary block of hair cell mechanoelectrical transduction (MET) currents in the crista, reduction in background discharge rate of afferent neurons, and a reduction in sensitivity to physiological stimulation. A six-degree-of-freedom pharmacokinetic mathematical model has been applied to interpret the time course and spatial distribution of BX and BX-GAGs. Results demonstrate a new optical approach to study GAG biology in the inner ear, for tracking synthesis and localization in real time.

RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays.

The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs.

A comprehensive catalogue of the coding and non-coding transcripts of the human inner ear.

The mammalian inner ear consists of the cochlea and the vestibular labyrinth (utricle, saccule, and semicircular canals), which participate in both hearing and balance. Proper development and life-long function of these structures involves a highly complex coordinated system of spatial and temporal gene expression. The characterization of the inner ear transcriptome is likely important for the functional study of auditory and vestibular components, yet, primarily due to tissue unavailability, detailed expression catalogues of the human inner ear remain largely incomplete. We report here, for the first time, comprehensive transcriptome characterization of the adult human cochlea, ampulla, saccule and utricle of the vestibule obtained from patients without hearing abnormalities. Using RNA-Seq, we measured the expression of >50,000 predicted genes corresponding to approximately 200,000 transcripts, in the adult inner ear and compared it to 32 other human tissues. First, we identified genes preferentially expressed in the inner ear, and unique either to the vestibule or cochlea. Next, we examined expression levels of specific groups of potentially interesting RNAs, such as genes implicated in hearing loss, long non-coding RNAs, pseudogenes and transcripts subject to nonsense mediated decay (NMD). We uncover the spatial specificity of expression of these RNAs in the hearing/balance system, and reveal evidence of tissue specific NMD. Lastly, we investigated the non-syndromic deafness loci to which no gene has been mapped, and narrow the list of potential candidates for each locus. These data represent the first high-resolution transcriptome catalogue of the adult human inner ear. A comprehensive identification of coding and non-coding RNAs in the inner ear will enable pathways of auditory and vestibular function to be further defined in the study of hearing and balance. Expression data are freely accessible at https://www.tgen.org/home/research/research-divisions/neurogenomics/supplementary-data/inner-ear-transcriptome.aspx.

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Inner ear morphological correlates of ultrasonic hearing in frogs.

Three species of anuran amphibians (Odorrana tormota, Odorrana livida and Huia cavitympanum) have recently been found to detect ultrasounds. We employed immunohistochemistry and confocal microscopy to examine several morphometrics of the inner ear of these ultrasonically sensitive species. We compared morphological data collected from the ultrasound-detecting species with data from Rana pipiens, a frog with a typical anuran upper cut-off frequency of ∼3 kHz. In addition, we examined the ears of two species of Lao torrent frogs, Odorrana chloronota and Amolops daorum, that live in an acoustic environment approximating those of ultrasonically sensitive frogs. Our results suggest that the three ultrasound-detecting species have converged on small-scale functional modifications of the basilar papilla (BP), the high-frequency hearing organ in the frog inner ear. These modifications include: 1. reduced BP chamber volume, 2. reduced tectorial membrane mass, 3. reduced hair bundle length, and 4. reduced hair cell soma length. While none of these factors on its own could account for the US sensitivity of the inner ears of these species, the combination of these factors appears to extend their hearing bandwidth, and facilitate high-frequency/ultrasound detection. These modifications are also seen in the ears of O. chloronota, suggesting that this species is a candidate for high-frequency hearing sensitivity. These data form the foundation for future functional work probing the physiological bases of ultrasound detection by a non-mammalian ear.

Altered expression of middle and inner ear cytokines in mouse otitis media.

OBJECTIVES/HYPOTHESIS: The inner ear is at risk for sensorineural hearing loss in both acute and chronic otitis media (OM), but the mechanisms underlying sensorineural hearing loss are unknown. Previous gene expression array studies have shown that cytokine genes might be upregulated in the cochleas of mice with acute and chronic OM. This finding implies that the inner ear could manifest a direct inflammatory response to OM that may cause sensorineural damage. Therefore, to better understand inner ear cytokine gene expression during OM, quantitative real-time polymerase chain reaction and immunohistochemistry were used in mouse models to evaluate middle and inner ear inflammatory and remodeling cytokines. STUDY DESIGN: Basic science experiment. METHODS: An acute OM model was created in Balb/c mice by a transtympanic injection of Streptococcus pneumoniae in one ear; the other ear was used as a control. C3H/HeJ mice were screened for unilateral chronic OM, with the noninfected ear serving as a control. RESULTS: Both acute and chronic OM caused both the middle ear and inner tissues in these two mouse models to overexpress numerous cytokine genes related to tissue remodeling (tumor necrosis factor-α, bone morphogenetic proteins, fibroblast growth factors) and angiogenesis (vascular endothelial growth factor), as well as inflammatory cell proliferation (interleukin [IL]-1α,ß, IL-2, IL-6). Immunohistochemistry confirmed that both the middle ear and inner ear tissues expressed these cytokines. CONCLUSIONS: Cochlear tissues are capable of expressing cytokine mRNA that contributes to the inflammation and remodeling that occur in association with middle ear disease. This provides a potential molecular basis for the transient and permanent sensorineural hearing loss often reported with acute and chronic OM.

OTO-104: a sustained-release dexamethasone hydrogel for the treatment of otic disorders.

HYPOTHESIS: To investigate whether OTO-104, a poloxamer-based hydrogel containing micronized dexamethasone for intratympanic delivery, can provide long-lasting inner ear exposure and be well tolerated. METHODS: OTO-104 was administered intratympanically to guinea pigs and sheep, and its pharmacokinetic and toxicity profiles were examined. RESULTS: After a single intratympanic injection of OTO-104 (from 0.6% to 20%, w/w), significant and prolonged exposure to dexamethasone in the inner ear was observed. Increasing the concentration of OTO-104 resulted in higher perilymph drug levels as well as a more prolonged duration of exposure. At the highest dose, therapeutic perilymph levels of dexamethasone could be sustained over 3 months in guinea pigs and more than 1 month in sheep. A toxicologic evaluation was conducted, including assessments of middle and inner ear function and physiology, as well as appraisal of local and systemic toxicity. A small and transient shift in hearing threshold was observed, most probably conductive in nature. No significant histologic changes in middle or inner ear tissues were noted. Although macroscopically mild erythema/inflammation was documented in a subset of guinea pigs treated with 20% OTO-104, the nature and the severity of these changes were not different between the poloxamer vehicle, saline, and 20% OTO-104 groups. No evidence of acute dermal toxicity, delayed hypersensitivity, or systemic adverse effects was found. CONCLUSION: OTO-104 is a novel proprietary therapeutic delivery system that can achieve prolonged, sustained release of dexamethasone within the inner ear fluids. The administration of this clinical candidate formulation via intratympanic injection is expected to be well tolerated both locally and systemically.

Mammalian Otolin: a multimeric glycoprotein specific to the inner ear that interacts with otoconial matrix protein Otoconin-90 and Cerebellin-1.

BACKGROUND: The mammalian otoconial membrane is a dense extracellular matrix containing bio-mineralized otoconia. This structure provides the mechanical stimulus necessary for hair cells of the vestibular maculae to respond to linear accelerations and gravity. In teleosts, Otolin is required for the proper anchoring of otolith crystals to the sensory maculae. Otoconia detachment and subsequent entrapment in the semicircular canals can result in benign paroxysmal positional vertigo (BPPV), a common form of vertigo for which the molecular basis is unknown. Several cDNAs encoding protein components of the mammalian otoconia and otoconial membrane have recently been identified, and mutations in these genes result in abnormal otoconia formation and balance deficits. PRINCIPAL FINDINGS: Here we describe the cloning and characterization of mammalian Otolin, a protein constituent of otoconia and the otoconial membrane. Otolin is a secreted glycoprotein of ∼70 kDa, with a C-terminal globular domain that is homologous to the immune complement C1q, and contains extensive posttranslational modifications including hydroxylated prolines and glycosylated lysines. Like all C1q/TNF family members, Otolin multimerizes into higher order oligomeric complexes. The expression of otolin mRNA is restricted to the inner ear, and immunohistochemical analysis identified Otolin protein in support cells of the vestibular maculae and semi-circular canal cristae. Additionally, Otolin forms protein complexes with Cerebellin-1 and Otoconin-90, two protein constituents of the otoconia, when expressed in vitro. Otolin was also found in subsets of support cells and non-sensory cells of the cochlea, suggesting that Otolin is also a component of the tectorial membrane. CONCLUSION: Given the importance of Otolin in lower organisms, the molecular cloning and biochemical characterization of the mammalian Otolin protein may lead to a better understanding of otoconial development and vestibular dysfunction.

A method for MS(E) differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue.

Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography-mass spectrometry (LC-MS) method for tissue sections embedded with celloidin. Q-TOF (Quadrupole-time of flight mass spectrometry) MS(E) (mass spectrometry where E represents collision energy) and Identity(E)™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins. Formalin-fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin de-cross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 µm sections of mouse inner ear samples, and FFCE 20 µm human inner ear and human otic capsule bone sections. Three of the five-tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified Liquid Tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Modification of loop 1 affects the nucleotide binding properties of Myo1c, the adaptation motor in the inner ear.

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c(1IQ), as well as chimeras of Myo1c(1IQ) with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca(2+) sensitivity of Myo1c(1IQ). Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c(1IQ)-tonic) or replacement with a single glycine (Myo1c(1IQ)-G) accelerated the release of ADP from A.M 2-3-fold in Ca(2+), whereas substitution with loop 1 from phasic muscle myosin II (Myo1c(1IQ)-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single alpha-helix, or SAH, domain showed that Myo1c(SAH)-tonic translocated actin in vitro twice as fast as Myo1c(SAH)-WT and 3-fold faster than Myo1c(SAH)-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras.