Inactivation of NADPH oxidase organizer 1 results in severe imbalance.

Otoconia are biominerals of the vestibular system that are indispensable for the perception of gravity. Despite their importance, the process of otoconia genesis is largely unknown. Reactive oxygen species (ROS) have been recognized for their toxic effects in antimicrobial host defense as well as in aging and carcinogenesis. Enzymes evolved for ROS production belong to the recently discovered NADPH oxidase (Nox) enzyme family . Here we show that the inactivation of a regulatory subunit, NADPH oxidase organizer 1 (Noxo1), resulted in the severe balance deficit seen in the spontaneous mutant "head slant" (hslt) mice whose phenotype was rescued by Noxo1 transgenes. Wild-type Noxo1 was expressed in the vestibular and cochlear epithelia and was required for ROS production by an oxidase complex. In contrast, the hslt mutation of Noxo1 was biochemically inactive and led to an arrest of otoconia genesis, characterized by a complete lack of calcium carbonate mineralization and an accumulation of otoconial protein, otoconin-90/95 (OC-90/95). These results suggest that ROS generated by a Noxo1-dependent vestibular oxidase are critical for otoconia formation and may be required for interactions among otoconial components. Noxo1 mutants implicate a constructive developmental role for ROS, in contrast to their previously described toxic effects.

Mixing model systems: using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development.

Human vestibular dysfunction is an increasing clinical problem. Degeneration or displacement of otoconia is a significant etiology of age-related balance disorders and Benign Positional Vertigo (BPV). In addition, commonly used antibiotics, such as aminoglycoside antibiotics, can lead to disruption of otoconial structure and function. Despite such clinical significance, relatively little information has been compiled about the development and maintenance of otoconia in humans. Recent studies in model organisms and other mammalian organ systems have revealed some of the proteins and processes required for the normal biomineralization of otoconia and otoliths in the inner ear of vertebrates. Orchestration of extracellular biomineralization requires bringing together ionic and proteinaceous components in time and space. Coordination of these events requires the normal formation of the otocyst and sensory maculae, specific secretion and localization of extracellular matrix proteins, as well as tight regulation of the endolymph ionic environment. Disruption of any of these processes can lead to the formation of abnormally shaped, or ectopic, otoconia, or otoconial agenesis. We propose that normal generation of otoconia requires a complex temporal and spatial control of developmental and biochemical events. In this review, we suggest a new hypothetical model for normal otoconial and otolith formation based on matrix vesicle mineralization in bone which we believe to be supported by information from existing mutants, morphants, and biochemical studies.

Effect of Otoconial Proteins Fetuin A, Osteopontin, and Otoconin 90 on the Nucleation and Growth of Calcite.

We investigated the roles of three proteins associated with the formation of otoconia including fetuin A, osteopontin (OPN), and otoconin 90 (OC90). In situ atomic force microscopy (AFM) studies of the effects of these proteins on the growth of atomic steps on calcite surfaces were performed to obtain insight into their effects on the growth kinetics. We also used scanning electron microscopy to examine the effects of these proteins on crystal morphology. All three proteins were found to be potent inhibitors of calcite growth, although fetuin A promoted growth at concentrations below about 40 nM and only became an inhibitor at higher concentrations. We then used in situ optical microscopy to observe calcite nucleation on films of these proteins adsorbed onto mica surfaces. By measuring the calcite nucleation rate as a function of supersaturation, the value of the interfacial energy that controls the free energy barrier to heterogeneous nucleation was determined for each protein. OPN and OC90 films led to significantly reduced interfacial energies as compared to the value for homogeneous calcite nucleation in bulk solution. The value for fetuin A was equal to that for bulk solution within experimental error. Zeta potential measurements showed all of the proteins possessed negative surface charge and varied in magnitude according to sequence fetuin A > OC90 > OPN. In addition, the interfacial energies exhibited an inverse scaling with the zeta potential. In analogy to previous measurements on polysaccharide films, this scaling indicates the differences between the proteins arise from the effect of protein surface charge on the solution-substrate interfacial energy.

The cochlear F-box protein OCP1 associates with OCP2 and connexin 26.

OCP1 and OCP2 are the most abundant proteins in the organ of Corti. Their distributions map identically to the epithelial gap-junction system, which unites the supporting cell population. Sequence data imply that OCP1 and OCP2 are subunits of an SCF E3 ubiquitin ligase. Consistent with that hypothesis, electrophoretic mobility-shift assays and pull-down assays with immobilized OCP1 demonstrate the formation of an OCP1-OCP2 complex. Sedimentation equilibrium data indicate that the complex is heterodimeric. The coincidence of the OCP1-OCP2 distribution and the epithelial gap-junction system suggests that one or more connexin isoforms may be targets of an SCF(OCP1) complex. Significantly, immobilized OCP1 binds (35)S-labeled connexin 26 (Cx26) produced by in vitro transcription-translation. Moreover, Cx26 can be co-immunoprecipitated from extracts of the organ of Corti by immobilized anti-OCP1, implying that OCP1 and Cx26 may associate in vivo. Given that lesions in the Cx26 gene (GJB2) are the most common cause of hereditary deafness, the OCP1-Cx26 interaction has substantial biomedical relevance.

Molecular mechanisms underlying ectopic otoconia-like particles in the endolymphatic sac of embryonic mice.

Otoconin-90, the principal otoconial matrix protein, provided a tool to investigate the molecular mechanism of otoconial morphogenesis. The endolymphatic sac of the embryonic chick and guinea pig contain otoconia. Here, we show that the embryonic mouse transiently expresses ectopic otoconia in the endolymphatic sac. Massive precipitate of otoconin-90-positive material is detectable in the lumen of the endolymphatic sac between embryonic day 14.5 and 17.5 with frequent accretion into more heavily staining otoconia-like particles. Otoconin-90 was also localized at the surface and the interior of epithelial cells lining the endolymphatic sac as well as incorporated into free floating cells. In contrast, in situ hybridization failed to detect mRNA in the endolymphatic duct and sac, even though the adjacent nonsensory vestibular structures are heavily stained. Because of ample expression of otoconin-90 protein in the absence of the corresponding mRNA, we conclude that the luminal otoconin-90 is imported via longitudinal flow from the vestibular compartments, where both mRNA and protein are strongly expressed. Because of absence of mRNA, the expression of the corresponding protein by the epithelia lining the endolymphatic sac can only be explained by a resorptive process, as previously proposed on the basis of the movement of luminal macromolecules. The data do not support the previous hypothesis that the transient expression of otoconia-like particles of the endolymphatic sac represents a vestigial phenomenon from the amphibian stage, since amphibia express ample mRNA encoding otoconin-22 in the endolymphatic sac system.

Toward an understanding of cochlear homeostasis: the impact of location and the role of OCP1 and OCP2.

The central role of the supporting cell population, or epithelial support complex (ESC), in cochlear homeostasis has gained general acceptance. That the details of this role may vary markedly with location, however, remains poorly appreciated. For example, the K+ recirculation pathway may well be dictated by position along the cochlear axis: a perilymphatic route near the apex and a transcellular one near the base. The ESC expresses very high levels of OCP1 and OCP2, now known to be components of a novel, organ of Corti (OC)-specific SCF ubiquitin ligase (SCF(OCP1)). In the SCF(OCP1) cnmplex, OCP1 presumably binds selected protein targets, positioning them for ubiquitination. The recent demonstration that recombinant OCP1 interacts non-covalently with Cx26 suggests that the connexins may be target proteins for SCF(OCP1). Although ubiquitination has classically been viewed as a signal for subsequent destruction by the 26S proteasome, the energy-limited state of the OC prompts consideration of alternative fates, e.g. reversible internalization. The ESC also expresses several components of the Wingless/Wnt signaling pathway. Significantly, two of the gap-junction proteins expressed in the OC, Cx43 and Cx30, are known targets of the Wnt pathway. On the basis of these observations, a working hypothesis is proposed wherein the Wnt pathway activates connexin expression, while OCP1 regulates its degradation.

In vitro calcite crystal morphology is modulated by otoconial proteins otolin-1 and otoconin-90.

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.

Immunogold TEM of otoconin 90 and otolin - relevance to mineralization of otoconia, and pathogenesis of benign positional vertigo.

Implementation of the deep-etch technique enabled unprecedented definition of substructural elements of otoconia, including the fibrillar meshwork of the inner core with its globular attachments. Subsequently the effects of the principal soluble otoconial protein, otoconin 90, upon calcite crystal growth in vitro were determined, including an increased rate of nucleation, inhibition of growth kinetics and significant morphologic changes. The logical next step, ultrastructural localization of otoconin 90, by means of immunogold TEM in young mature mice, demonstrated a high density of gold particles in the inner core in spite of a relatively low level of mineralization. Here gold particles are typically arranged in oval patterns implying that otoconin 90 is attached to a scaffold consisting of the hexagonal fibrillar meshwork, characteristic of otolin. The level of mineralization is much higher in the outer cortex where mineralized fiber bundles are arranged parallel to the surface. Following decalcification, gold particles, as well as matrix fibrils, presumed to consist of a linear structural phenotype of otolin, are aligned in identical direction, suggesting that they serve as scaffold to guide mineralization mediated by otoconin 90. In the faceted tips, the level of mineralization is highest, even though the density of gold particles is relatively low, conceivably due to the displacement by the dense mineral phase. TEM shows that individual crystallites assemble into iso-oriented columns. Columns are arranged in parallel lamellae which convert into mineralized blocks for hierarchical assembly into the complex otoconial mosaic. Another set of experiments based on immunogold TEM in young mice demonstrates that the fibrils interconnecting otoconia consist of the short chain collagen otolin. By two years of age the superficial layer of mouse otoconia (corresponding to mid-life human) has become demineralized resulting in weakening or loss of anchoring of the fibrils interconnecting otoconia. Consequently, otoconia detached from each other may be released into the endolymphatic space by minor mechanical disturbances. In humans, benign positional vertigo (BPV) is believed to result from translocation of otoconia from the endolymphatic space into the semi-circular canals rendering their receptors susceptible to stimulation by gravity causing severe attacks of vertigo. The combinations of these observations in humans, together with the presented animal experiments, provide a tentative pathogenetic basis of the early stage of BPV.

Significance of tertiary conformation of otoconial matrix proteins - clinical implications.

CONCLUSION: Co-option of the enzyme secretory phospholipase A2 (sPLA2) and adoption of tertiary conformation are essential factors in the multifunctionality of otoconin 90 (OC90) and homologous modulators. OBJECTIVE: To present results of in vitro studies of recombinant otoconial proteins for the understanding of current concepts of biomolecular mechanisms controlling otoconial mineralization. METHODS: In vitro characterization of recombinant otoconial proteins with respect to crystal growth parameters and solution state behavior. Evaluation by HR-SEM, micro-Raman, circular dichroism, in combination with molecular modeling of individual domains and whole OC90. RESULTS: Polymorph selection: recombinant otoconin 22 (rOC22) in vitro selects calcite rather than aragonite, expression of which requires association with an insoluble scaffold most likely provided by Otolin. Alternate folding of rOC22 results in formation of vaterite, the polymorph of primitive fish otoconia and of diseased human otoconia (e.g. Potter's syndrome). Molecular models of OC90 exhibit a surface of uniform negative electrostatic potential, enabling localized supersaturation. We propose that OC90 interacts with Otolin in formation of iso-oriented columns of nano-crystallites, which should ultimately result in assembly of the complex mosaic of native otoconia.

In vitro effects of recombinant otoconin 90 upon calcite crystal growth. Significance of tertiary structure.

Otoconia are biomineral particles of microscopic size essential for perception of gravity and maintenance of balance. Millions of older Americans are affected in their mobility, quality of life and in their health by progressive demineralization of otoconia. Currently, no effective means to prevent or counteract this process are available. Because of prohibitive anatomical and biological constraints, otoconial research is lagging far behind other systems such as bone and teeth. We have overcome these obstacles by generating otoconial matrix proteins by recombinant techniques. In the present study, we evaluated the effects of recombinant Otoconin 90 (OC90), the principal soluble matrix protein upon calcite crystal growth patterns in vitro. Our findings highlight multiple effects, including facilitation of nucleation, and inhibition of crystal growth in a concentration-dependent manner. Moreover, OC90 induces morphologic changes characteristic of native otoconia. OC90 is considerably less acidic than the prototypical invertebrate CaCO(3) -associated protein, but is nevertheless an effective modulator of calcite crystal growth. Based on homology modeling of the sPLA2-like domains of OC90, we propose that the lower density of acidic residues of the primary sequence is compensated by formation of major anionic surface clusters upon folding into tertiary conformation.

Microscale analysis of proteins in inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and organ of Corti.

Here we describe preparatory techniques adapted for the study of proteins of inner ear tissues and fluids that have allowed us to apply state-of-the-art analytical techniques in spite of the minute size and anatomical complexities of this organ. Illustrative examples address unresolved issues of functional and clinical significance. First, we demonstrate how quick-freezing and freeze drying prevents artifacts that arise from sampling endolymphatic sac (ES) content in the liquid state. This set the stage for the generation of the first protein profile of the ES. Identification of crucial proteins will help elucidate mechanisms of endolymph volume regulation and pathogenesis of Meniere's disease. Second, we show how a unique situation allowed identification of otoconial proteins by mass spectrometric analysis without prior separation and we discuss possible roles for these minor otoconins in otoconial development and prevention of degenerative diseases that affect balance. Finally, we demonstrate techniques for the precise dissection of organ of Corti and its substructures, while preserving their near normal chemical state. We extended an earlier study in which we identified a novel calcium-binding protein by IEF, oncomodulin, localized in the outer hair cells and show here the applicability of prefractionation for the screening of calcium-binding proteins of organ of Corti. These studies demonstrate how advanced preparatory and analytical techniques can be applied to studies of the inner ear.

Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1.

Otoconia are biominerals within the utricle and saccule of the inner ear that are critical for the perception of gravity and linear acceleration. The classical mouse mutant tilted (tlt) and a new allele, mergulhador (mlh), are recessive mutations that affect balance by impairing otoconial morphogenesis without causing collateral deafness. The mechanisms governing otoconial biosynthesis are not known. Here we show that tlt and mlh are mutant alleles of a novel gene (Otopetrin 1, Otop1), encoding a multi-transmembrane domain protein that is expressed in the macula of the developing otocyst. Both mutants carry single point mutations leading to non-conservative amino acid substitutions that affect two putative transmembrane (TM) domains (tlt, Ala(151)-->Glu in TM3; mlh, Leu(408)-->Gln in TM8). Otop1 and Otop1-like paralogues, Otop2 and Otop3, define a new gene family with homology to the C. elegans and D. melanoganster DUF270 genes.

Otopetrin 1 is required for otolith formation in the zebrafish Danio rerio.

Orientation with respect to gravity is essential for the survival of complex organisms. The gravity receptor is one of the phylogenetically oldest sensory systems, and special adaptations that enhance sensitivity to gravity are highly conserved. The fish inner ear contains three large extracellular biomineral particles, otoliths, which have evolved to transduce the force of gravity into neuronal signals. Mammalian ears contain thousands of small particles called otoconia that serve a similar function. Loss or displacement of these structures can be lethal for fish and is responsible for benign paroxysmal positional vertigo (BPPV) in humans. The distinct morphologies of otoconial particles and otoliths suggest divergent developmental mechanisms. Mutations in a novel gene Otopetrin 1 (Otop1), encoding multi-transmembrane domain protein, result in nonsyndromic otoconial agenesis and a severe balance disorder in mice. Here we show that the zebrafish, Danio rerio, contains a highly conserved gene, otop1, that is essential for otolith formation. Morpholino-mediated knockdown of zebrafish Otop1 leads to otolith agenesis without affecting the sensory epithelium or other structures within the inner ear. Despite lack of otoliths in early development, otolith formation partially recovers in some fish after 2 days. However, the otoliths are malformed, misplaced, lack an organic matrix, and often consist of inorganic calcite crystals. These studies demonstrate that Otop1 has an essential and conserved role in the timing of formation and the size and shape of the developing otolith.