Richard Hallworth

Pathology of the olfactory epithelium: Smoking and ethanol exposure

Objective: To investigate the effects of tobacco smoke on the olfactory epithelium. Cigarette smoking has been associated with hyposmia; however, the pathophysiology is poorly understood. The sense of smell is mediated by olfactory sensory neurons (OSNs) exposed to the nasal airway, rendering them vulnerable to environmental injury and death. As a consequence, a baseline level of apoptotic OSN death has been demonstrated even in the absence of obvious disease. Dead OSNs are replaced by the mitosis and maturation of progenitors to maintain sufficient numbers of neurons into adult life. Disruption of this balance has been suggested as a common cause for clinical smell loss. This current study will evaluate the effects of tobacco smoke on the olfactory mucosa, with emphasis on changes in the degree of OSN apoptosis.

Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.

Modulation of outer hair cell compliance and force by agents that affect hearing

Force generated by outer hair cells is thought to be an essential source of mechanical input to the normal cochlea. Many disease processes in the inner ear act via outer hair cells. It is therefore plausible that such disease processes modulate outer hair cell force generation. The force generated by an isolated, electrically stimulated outer hair cell against a load may be represented by an intrinsic motor and a passive axial stiffness in series. Thus modulation of outer hair cell force generation may occur either by action on the motor or indirectly by an action on cell stiffness. In this study, the effects of agents that affect hearing on outer hair cell stiffness and force generation have been examined. Overstimulation and hypoosmotic challenge caused cells to decrease in length and increase in stiffness. The force generated by a constant voltage stimulus increased consequent to the stiffness increase. Hyperosmotic challenge elicited a stiffness decrease and a force decrease. In contrast, salicylate caused a decrease in force without stiffness change. The results suggest that outer hair cell force generation in vivo may be modulated in at least two ways.

Evolutionary insights into the unique electromotility motor of mammalian outer hair cells

SUMMARY Prestin (SLC26A5) is the molecular motor responsible for cochlear amplification by mammalian cochlea outer hair cells and has the unique combined properties of energy‐independent motility, voltage sensitivity, and speed of cellular shape change. The ion transporter capability, typical of SLC26A members, was exchanged for electromotility function and is a newly derived feature of the therian cochlea. A putative minimal essential motif for the electromotility motor (meEM) was identified through the amalgamation of comparative genomic, evolution, and structural diversification approaches. Comparisons were done among nonmammalian vertebrates, eutherian mammalian species, and the opossum and platypus. The opossum and platypus SLC26A5 proteins were comparable to the eutherian consensus sequence. Suggested from the point‐accepted mutation analysis, the meEM motif spans all the transmembrane segments and represented residues 66–503. Within the eutherian clade, the meEM was highly conserved with a substitution frequency of only 39/7497 (0.5%) residues, compared with 5.7% in SLC26A4 and 12.8% in SLC26A6 genes. Clade‐specific substitutions were not observed and there was no sequence correlation with low or high hearing frequency specialists. We were able to identify that within the highly conserved meEM motif two regions, which are unique to all therian species, appear to be the most derived features in the SLC26A5 peptide.

Differential expression of β tubulin isotypes in the adult gerbil cochlea

Tubulin, the principal component of microtubules, exists as two polypeptides, termed alpha and beta. Seven isotypes of beta tubulin are known to exist in mammals. The distributions of four beta tubulin isotypes, beta(I), beta(II), beta(III), and beta(IV), have been examined in the adult cochlea by indirect immunofluorescence using isotype-specific antibodies. In the organ of Corti, outer hair cells contained only beta(I) and beta(IV), while inner hair cells contained only beta(I) and beta(II). Inner and outer pillar cells contained beta(II) and beta(IV), but Deiters cells contained those isotypes plus beta(I). Fine fibers in the inner spiral bundle, tunnel crossing fibers, and outer spiral fibers, probably efferent in character, contained beta(I), beta(II), and beta(III), but not beta(IV). In the spiral ganglion, the somas and axons of neurons contained all four isotypes, and the myelination of ganglion cells also contained beta(I). Fibers of the intraganglionic spiral bundle contained beta(I), beta(II), and beta(III). No antibody labeled the dendritic processes of spiral ganglion neurons. The differences in isotype distribution in organ of Corti and neurons described here are consistent with and support the multi-tubulin hypothesis, which states that tubulin isotypes are expressed specifically in different cell types and may therefore have different functions.

Cell type-specific reduction of β tubulin isotypes synthesized in the developing gerbil organ of Corti

There are seven isotypic forms of the microtubule protein β tubulin in mammals, but not all isotypes are synthesized in every cell type. In the adult organ of Corti, each of the five major cell types synthesizes a different subset of isotypes. Inner hair cells synthesize only βI and βII tubulin, while outer hair cells make βI and βIV tubulin. Only βII and βIV tubulin are found in inner and outer pillar cells, while βI, βII, and βIV tubulin are present in Deiters cells, and βI, βII and βIII tubulin are found in organ of Corti dendrites. During post-natal organ of Corti development in the gerbil, microtubules are elaborated in an orderly temporal sequence beginning with hair cells, followed by pillar cells and Deiters cells. Using β tubulin isotype-specific antibodies, we show that, in the gerbil cochlea, the same three isotypes are present in each cell type at birth, and that a cell type-specific reduction in the isotypes synthesized occurs in hair cells and pillar cells at an unusually late stage in development. No β tubulin isotypes were detected in mature afferent dendrites, but we show that this is because few microtubules are present in mature dendrites. In addition, we show that primary cilia in inner hair cells, a feature of early development, persist much later than previously reported. The findings represent the first description of developmental cell type-specific reductions in tubulin isotypes in any system.

Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells.

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.

Prestin in HEK cells is an obligate tetramer

The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiometry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

Metabolic imaging using two-photon excited nadh intensity and fluorescence lifetime imaging

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Tubulin expression in the developing and adult gerbil organ of Corti.

In the late stages of inner ear development, the relatively undifferentiated cells of Kollickers organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to beta-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.