Inactivation of NADPH oxidase organizer 1 Results in Severe Imbalance

Abstract

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.

Subcellular localization and function of alternatively spliced Noxo1 isoforms

“…We do not know the relevant oxidase systems expressed in these cells nor could we compare Noxo1 isoform functions in these lines, because of their low transfection efficiency. Noxo1 is also a physiologically relevant partner of Nox3, since lesions in either murine gene result in the same distinctive inner ear phenotype, defective otoconia formation and loss of balance [23,24]. We observed the following rank order of Nox3 activities supported by Noxo1 isoforms in the HEK293 cell model: Noxo1β > Noxo1γ > Noxo1α, similar to that observed with Nox1 (data not shown).…”

“…These structural and functional similarities between the Nox and phox regulators may explain why Noxo1 and Noxa1 can function in partially compensating for p47 phox and p67 phox in supporting Nox2 activity [8,[13][14][15]. Noxo1, Noxa1, and Rac1 also support Nox3 activity, which appears to function critically in the inner ear [17,[20][21][22]; mice with lesions in either the NOX3 or NOXO1 gene have defects in sensing gravity resulting from impaired otoconia formation [23,24]. Nox3 activity can be supported by p47 phox and p67 phox , although mice deficient in p47 phox or chronic granulomatous disease patients deficient in either protein show no deficits in balance.…”

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…Mutant mice that show an absence of otoconia exhibit a "head slant" phenotype characterized by defective gravity sensing. Several of these mouse strains showed mutations in either Nox3 [183] or its regulatory subunit NOXO1 [184].…”

Biological Roles for the NOX Family NADPH Oxidases

“…Homologs of p47 phox and p67 phox , named NOX organizing (NOXO1) and activating (NOXA1) proteins to reflect their presumed function, have been identified (29 -31), isoforms of NOXO1 described (32,33), and each implicated in the activity of NOX1-3. Of special note, dependence of NOX3 on NOXA1 exhibits species specificity; whereas murine NOX3 requires both NOXO1 and NOXA1 for maximal activity, optimal activity of human NOX3 depends on NOXO1 alone (30,34). Human and murine NOXO1 are 67% identical at the amino acid level, with most differences at the C terminus.…”