Heather Jensen-Smith

MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival

MicroRNAs (miRNAs) post‐transcriptionally repress complementary target gene expression and can contribute to cell differentiation. The coordinate expression of miRNA‐183 family members (miR‐183, miR‐96, and miR‐182) has been demonstrated in sensory cells of the mouse inner ear and other vertebrate sensory organs. To further examine hair cell miRNA expression in the mouse inner ear, we have analyzed miR‐183 family expression in wild type animals and various mutants with defects in neurosensory development. miR‐183 family member expression follows neurosensory cell specification, exhibits longitudinal (basal‐apical) gradients in maturating cochlear hair cells, and is maintained in sensory neurons and most hair cells into adulthood. Depletion of hair cell miRNAs resulting from Dicer1 conditional knockout (CKO) in Atoh1‐Cre transgenic mice leads to more disparate basal‐apical gene expression profiles and eventual hair cell loss. Results suggest that hair cell miRNAs subdue cochlear gradient gene expression and are required for hair cell maintenance and survival. Developmental Dynamics 240:808–819, 2011.

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.

Differential synthesis of β-tubulin isotypes in gerbil nasal epithelia

Abstract. Compartmentalization of β-tubulin isotypes within cells according to function was examined in gerbil olfactory and respiratory epithelia by using specific antibodies to four β-tubulin isotypes (βI, βII, βIII, and βIV). Isotype synthesis was cell-type-specific, but the localization of the isotypes was not compartmentalized. All four isotypes were found in the cilia, dendrites, somata, and axons of olfactory neurons. Only two isotypes (βI and βIV) were present in the cilia of nasal respiratory epithelial cells. The βIV isotype, thought to be an essential component of cilia, was present in olfactory neurons and respiratory epithelial cells, which are ciliated, but was not found in basal cells (the stem cells of olfactory sensory neurons, which have no cilia). Olfactory neurons therefore do not synthesize βIV-tubulin until they mature, when functioning cilia are also elaborated. The failure to observe compartmentalization of β-tubulin isotypes in olfactory neurons sheds new light on potential functions of the β-tubulin isotypes.

Long-distance three-color neuronal tracing in fixed tissue using NeuroVue dyes.

Dissecting development of neuronal connections is critical for understanding neuronal function in both normal and diseased states. Charting the development of the multitude of connections is a monumental task, since a given neuron typically receives hundreds of convergent inputs from other neurons and provides divergent outputs for hundreds of other neurons. Although progress is being made utilizing various mutants and/or genetic constructs expressing fluorescent proteins like GFP, substantial work remains before a database documenting the development and final location of the neuronal pathways in an adult animal is completed. The vast majority of developing neurons cannot be specifically labeled with antibodies and making specific GFP-expressing constructs to tag each of them is an overwhelming task. Fortunately, fluorescent lipophilic dyes have emerged as very useful tools to systematically compare changes in neuronal networks between wild-type and mutant mice. These dyes diffuse laterally along nerve cell membranes in fixed preparations, allowing tracing of the position of a given neuron within the neuronal network in murine mutants fixed at various stages of development. Until recently, however, most evaluations have been limited to one, or at most, two color analyses. We have previously reported three color neuronal profiling using the novel lipophilic dyes NeuroVue (NV) Green, Red and Maroon (Fritzsch et al., Brain. Res. Bull. 66: 249–258, 2005). Unfortunately such three color experiments have been limited by the fact that NV Green and its brighter successor, NV Emerald, both exhibit substantially decreased signal intensities when times greater than 48 hours at 37°C are required to achieve neuronal profile filling (unpublished observations). Here we describe a standardized test system developed to allow comparison of candidate dyes and its use to evaluate a series of 488 nm-excited green-emitting lipophilic dyes. The best of these, NV Jade, has spectral properties well matched to NV Red and NV Maroon, better solubility in DMF than DiO or DiA, improved thermostability compared with NV Emerald, and the ability to fill neuronal profiles at rates of 1 mm per day for periods of at least 5 days. Use of NV Jade in combination with NV Red and NV Maroon substantially improves the efficiency of connectional analysis in complex mutants and transgenic models where limited numbers of specimens are available.

The roles of conserved and nonconserved cysteinyl residues in the oligomerization and function of mammalian prestin

The creation of several prestin knockout and knockin mouse lines has demonstrated the importance of the intrinsic outer hair cell membrane protein prestin to mammalian hearing. However, the structure of prestin remains largely unknown, with even its major features in dispute. Several studies have suggested that prestin forms homo-oligomers that may be stabilized by disulfide bonds. Our phylogenetic analysis of prestin sequences across chordate classes suggested that the cysteinyl residues could be divided into three groups, depending on the extent of their conservation between prestin orthologs and paralogs or homologs. An alanine scan functional analysis was performed of all nine cysteinyl positions in mammalian prestin. Prestin function was assayed by measurement of prestin-associated nonlinear capacitance. Of the nine cysteine-alanine substitution mutations, all were properly membrane targeted and all demonstrated nonlinear capacitance. Four mutations (C124A, C192A, C260A, and C415A), all in nonconserved cysteinyl residues, significantly differed in their nonlinear capacitance properties compared with wild-type prestin. In the two most severely disrupted mutations, substitution of the polar residue seryl for cysteinyl restored normal function in one (C415S) but not the other (C124S). We assessed the relationship of prestin oligomerization to cysteine position using fluorescence resonance energy transfer. With one exception, cysteine-alanine substitutions did not significantly alter prestin-prestin interactions. The exception was C415A, one of the two nonconserved cysteinyl residues whose mutation to alanine caused the most disruption in function. We suggest that no disulfide bond is essential for prestin function. However, C415 likely participates by hydrogen bonding in both nonlinear capacitance and oligomerization.

Localization of βV tubulin in the cochlea and cultured cells with a novel monoclonal antibody

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of alpha and beta subunits; both alpha and beta exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the beta(I), beta(II), beta(III), beta(IV) and beta(V) isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the beta(I), beta(II), beta(III), and beta(IV) isotypes; however, little is known about the beta(V) isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for beta(V). The antibody was designed to be specific for the C-terminal sequence EEEINE, which is unique to rodent and chicken beta(V). The antibody was found to bind specifically to the C-terminal peptide EEEINE, and does not cross-react with the carboxy-termini of either alpha-tubulin or the other beta-tubulin isotypes. However, the antibody also binds to the peptide EEEVNE, but not to the peptide EEEIDG, corresponding respectively to the C-terminal peptides of bovine and human beta(V). Immunofluorescence analysis indicates that beta(V) is found in microtubules of both the interphase network and the mitotic spindle. In gerbils, beta(V) also occurs in the cochlea where it is found largely in the specialized cells that are unique in containing bundled microtubules with 15 protofilaments.

A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages

Our laboratory has developed a flow cytometric assay to quantify alveolar macrophage (Mcapital EF, Cyrillic) phagocytosis of bacteria within a live animal. Mcapital EF, Cyrillics collected by bronchoalveolar lavage from rats infected transtracheally with Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry to quantify their bacterial uptake.

The Morphological Specializations and Electromotility of the Mammalian Outer Hair Cell

More than 20 years have elapsed since the discovery of induced and electrically driven length changes in isolated outer hair cells (OHCs) (Brownell 1984; Brownell et al. 1985). At the time, the phenomenon was embraced as the potential solution to two problems: the paradoxical nature of the OHC and the origin of the frequencyand level-specific mechanical energy input to the cochlea that we call the cochlear amplifier. Today, while we know much more about the physiology and electromechanical activity of the OHC, we are no closer to understanding its role in cochlear transduction. Indeed, as we shall see, there are good reasons to believe that its role in hearing is something far distant from that originally envisaged. To understand the paradox, recall that OHCs are an unusual hair cell in many respects. As we describe here, the OHC is highly specialized and has characteristics possessed by no other vertebrate hair cell. Foremost among these is the electromotility of the OHC, its ability to extend and contract at acoustic frequencies in response to membrane potential change. OHCs are clearly the most vulnerable cell type in the cochlea. Major hearing pathologies such as aminoglycoside ototoxicity, noise trauma, and aging strike primarily at OHCs. And yet, while OHCs are four times as numerous as inner hair cells (IHCs), they receive one-twentieth of the afferent innervation. The consequence of OHC loss appears to be loss of the cochlear amplifier, that is, a profound loss of sensitivity and tuning that is altogether incongruous when compared to the sparse and diffuse innervation of OHCs. What could OHCs contribute that was so important, if not afferent activity? Arguing for the need for mechanical amplification in the vibration of the basilar membrane (BM), investigators seized on the newly discovered electromechanical activity of OHCs as the solution to both problems. For a mechanism such as OHC electromotility to be considered as an explanation of the cochlear amplifier, it must fulfill at least the following criteria: (1) it

Homo‐ and Hetero‐Oligomerization in the Slc26a Protein Family

The motor‐protein prestin is thought to be a homo‐oligomer, but the oligomerization motifs are unknown. We used the acceptor‐photobleach variant of fluorescence‐lifetime based FRET analysis to demonstrate that homo‐oligomerization is not only common to mammalian and non‐mammalian prestins, but occurs in widely divergent members of the Slc26a family proteins. We therefore tested the hypothesis that oligomerization is conserved across the Slc26a family by measuring FRET between different Slc26a family molecules. Our results show that hetero‐oligomerization is common between family members, which suggest that common oligomerization motifs exist.