Dopamine in the auditory brainstem and midbrain: co-localization with amino acid neurotransmitters and gene expression following cochlear trauma.

Dopamine (DA) modulates the effects of amino acid neurotransmitters (AANs), including GABA and glutamate, in motor, visual, olfactory, and reward systems (Hnasko et al., 2010; Stuber et al., 2010; Hnasko and Edwards, 2012). The results suggest that DA may play a similar modulatory role in the auditory pathways. Previous studies have shown that deafness results in decreased GABA release, changes in excitatory neurotransmitter levels, and increased spontaneous neuronal activity within brainstem regions related to auditory function. Modulation of the expression and localization of tyrosine hydroxylase (TH; the rate limiting enzyme in the production of DA) in the IC following cochlear trauma has been previously reported (Tong et al., 2005). In the current study the possibility of co-localization of TH with AANs was examined. Changes in the gene expression of TH were compared with changes in the gene expression of markers for AANs in the cochlear nucleus (CN) and inferior colliculus (IC) to determine whether those deafness related changes occur concurrently. The results indicate that bilateral cochlear ablation significantly reduced TH gene expression in the CN after 2 months while in the IC the reduction in TH was observed at both 3 days and 2 months following ablation. Furthermore, in the CN, glycine transporter 2 (GLYT2) and the GABA transporter (GABAtp) were also significantly reduced only after 2 months. However, in the IC, DA receptor 1 (DRDA1), vesicular glutamate transporters 2 and 3 (VGLUT2, VGLUT3), GABAtp and GAD67 were reduced in expression both at the 3 days and 2 months time points. A close relationship between the distribution of TH and several of the AANs was determined in both the CN and the IC. In addition, GLYT2 and VGLUT3 each co-localized with TH within IC somata and dendrites. Therefore, the results of the current study suggest that DA is spatially well positioned to influence the effects of AANs on auditory neurons.

Induction of heat shock proteins by hyperthermia and noise overstimulation in hsf1 -/- mice.

Diverse cellular and environmental stresses can activate the heat shock response, an evolutionarily conserved mechanism to protect proteins from denaturation. Stressors activate heat shock transcription factor 1 (HSF1), which binds to heat shock elements in the genes for heat shock proteins, leading to rapid induction of these important molecular chaperones. Both heat and noise stress are known to activate the heat shock response in the cochlea and protect it from subsequent noise trauma. However, the contribution of HSF1 to induction of heat shock proteins following noise trauma has not been investigated at the molecular level. We evaluated the role of HSF1 in the cochlea following noise stress by examining induction of heat shock proteins in Hsf1 ( +/- ) control and Hsf1 ( -/- ) mice. Heat stress rapidly induced expression of Hsp25, Hsp47, Hsp70.1, Hsp70.3, Hsp84, Hsp86, and Hsp110 in the cochleae of wild-type and Hsf1 ( +/- ) mice, but not in Hsf1 ( -/- ) mice, confirming the essential role of HSF1 in mediating the heat shock response. Exposure to broadband noise (2-20 kHz) at 106 dB SPL for 2 h produced partial hearing loss. Maximal induction of heat shock proteins occurred 4 h after the noise. In comparison to heat stress, noise stress resulted in lower induced levels of Hsp25, Hsp70.1, Hsp70.3, Hsp86, and Hsp110 in Hsf1 ( +/- ) mice. Induction of these heat shock proteins was attenuated, but not completely eliminated, in Hsf1 ( -/- ) mice. These same noise exposure conditions induced genes for several immediate early transcription factors and maximum induction occurred earlier than for heat shock proteins. Thus, additional signaling pathways and transcriptional regulators that are activated by noise probably contribute to induction of heat shock proteins in the cochlea.

Isolation of epithelial cells in the developing primary lip and palate.

Failure of the primary lip and palate to fuse leads to clefts of the lip, a birth defect with an incidence of 1 for every 500 in some races. Epithelial cells lining the facial processes of the primary lip and palate, the lateral and medial nasal processes (LNP and MNP), must first make contact to go through a series of highly regulated and coordinated sequence of events to form the normal midface. As yet, many of the basic mechanisms underlying the fusion events of the epithelial-lined surfaces are not known. This is due in part to the difficulty associated with the isolation of the epithelial cells for further study and analysis. The objective of this study was to test the use of laser capture microdissection to collect clean populations of primary lip and palate epithelial cells destined to fuse. Fusing and nonfusing epithelial cell populations of the MNP and LNP were isolated by laser capture microdissection and assayed for gene expression of Bmp-4, Tgfß-2, and their type 1 receptors, Alk-3 and Alk-5, respectively. Transcripts of Bmp-4/Alk-3 and Tgfß-2/Alk-5 were restricted to the epithelial seam of the fusion site, and the epithelium of the prefusion site, in patterns previously reported. Data indicated our ability to isolate clean populations of epithelial and mesenchymal cells around the primary palate fusion site, allowing precise analysis of tissue and site-specific gene expression at high resolution. This study provides the basis of further analysis of the potential molecular players of MNP and LNP fusion and nonfusion of epithelial cells.

Mature middle and inner ears express Chd7 and exhibit distinctive pathologies in a mouse model of CHARGE syndrome.

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears.

Notch signaling and Atoh1 expression during hair cell regeneration in the mouse utricle.

The mammalian vestibular epithelium has a limited capacity for spontaneous hair cell regeneration. The mechanism underlying the regeneration is not well understood. Because the Notch signaling pathway mediates the formation of the sensory epithelial mosaic patterning during ear development, it may also play a role in hair cell regeneration in the mature mammalian vestibular epithelium after a lesion. To investigate the process of spontaneous regeneration in the vestibular epithelium vis-à-vis changes in Notch signaling, we induced a unilateral lesion by infusing streptomycin into the mouse posterior semicircular canal, and examined Notch signaling molecules and their mRNA expression levels by immunohistochemistry and quantitative real-time polymerase chain reaction (qRTPCR), respectively. We detected Jagged1 in supporting cells in both normal and lesioned utricles. Atoh1, a marker for early developing hair cells, was absent in the intact mature tissue, but re-appeared after the lesion. Many cells were either positive for both Atoh1 and myosin VIIa, or for one of them. qRTPCR data showed a post trauma decrease of Hes5 and an increase in Atoh1. Atoh1 up-regulation may either be a result of Hes5 down-regulation or mediated by another signaling pathway.

Deafness and permanently reduced potassium channel gene expression and function in hypothyroid Pit1dw mutants.

The absence of thyroid hormone (TH) during late gestation and early infancy can cause irreparable deafness in both humans and rodents. A variety of rodent models have been used in an effort to identify the underlying molecular mechanism. Here, we characterize a mouse model of secondary hypothyroidism, pituitary transcription factor 1 (Pit1(dw)), which has profound, congenital deafness that is rescued by oral TH replacement. These mutants have tectorial membrane abnormalities, including a prominent Hensen's stripe, elevated beta-tectorin composition, and disrupted striated-sheet matrix. They lack distortion product otoacoustic emissions and cochlear microphonic responses, and exhibit reduced endocochlear potentials, suggesting defects in outer hair cell function and potassium recycling. Auditory system and hair cell physiology, histology, and anatomy studies reveal novel defects of hormone deficiency related to deafness: (1) permanently impaired expression of KCNJ10 in the stria vascularis of Pit1(dw) mice, which likely contributes to the reduced endocochlear potential, (2) significant outer hair cell loss in the mutants, which may result from cellular stress induced by the lower KCNQ4 expression and current levels in Pit1(dw) mutant outer hair cells, and (3) sensory and strial cell deterioration, which may have implications for thyroid hormone dysregulation in age-related hearing impairment. In summary, we suggest that these defects in outer hair cell and strial cell function are important contributors to the hearing impairment in Pit1(dw) mice.

Age-related changes in cochlear gene expression in normal and shaker 2 mice.

The vertebrate cochlea is a complex organ optimized for sound transduction. Auditory hair cells, with their precisely arranged stereocilia bundles, transduce sound waves to electrical signals that are transmitted to the brain. Mutations in the unconventional myosin XV cause deafness in both human DFNB3 families and in shaker 2 (sh2) mice as a result of defects in stereocilia. In these mutant mice, hair cells have relatively normal spatial organization of stereocilia bundles but lack the graded, stair-step organization. We used sh2 mice as an experimental model to investigate the molecular consequences of the sh2 mutation in the Myo15 gene. Gene expression profiling with Affymetrix GeneChips in deaf homozygous (sh2/sh2) mice at 3 weeks and 3 months of age, and in age-matched, normal-hearing heterozygotes (+/sh2) identified only a few genes whose expression was affected by genotype, but a large number with age-associated changes in expression in both normal mice and sh2/sh2 homozygotes. Microarray data analyzed using Robust Multiarray Average identified Aim1, Dbi, and Tm4sf3 as genes with increased expression in sh2/sh2 homozygotes. These increases were confirmed by quantitative reverse transcription-polymerase chain reaction. Genes exhibiting altered expression with age encoded collagens and proteins involved in collagen maturation, extracellular matrix, and bone mineralization. These results identified potential cellular pathways associated with myosin XV defects, and age-associated molecular events that are likely to be involved in maturation of the cochlea and auditory function.

The fate of outer hair cells after acoustic or ototoxic insults.

In epithelial sheets, clearance of dead cells may occur by one of several routes, including extrusion into the lumen, phagocytic clearance by invading lymphocytes, or phagocytosis by neighboring cells. The fate of dead cochlear outer hair cells is unclear. We investigated the fate of the "corpses" of dead outer hair cells in guinea pigs and mice following drug or noise exposure. We examined whole mounts and plastic sections of normal and lesioned organ of Corti for the presence of prestin, a protein unique to outer hair cells. Supporting cells, which are devoid of prestin in the normal ear, contained clumps of prestin in areas of hair cell loss. The data show that cochlear supporting cells surround the corpses and/or debris of degenerated outer hair cells, and suggest that outer hair cell remains are phagocytosed by supporting cells within the epithelium.

Gene and protein expression of transforming growth factor beta 2 gene during murine primary palatogenesis.

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these facial primordia leads to the common human craniofacial birth defects, clefts of the lip with or without clefts of the palate. Members of the transforming growth factor beta (Tgfbeta) superfamily have been shown to play critical roles during craniofacial development. Specifically, the role of Tgfbeta-3 in mediating the fusion of the embryonic secondary palatal shelves is well documented. In a screen for genes expressed during fusion of the murine midfacial processes, Tgfbeta2 was identified as a gene differentially expressed during fusion of the lateral and medial nasal processes. The objective of our study was to analyze the spatial and temporal expression of Tgfbeta2 during critical stages of midfacial morphogenesis at both the transcript and protein levels. We also compared the pattern of expression of Tgfbeta2 with that of Bmp4, a gene shown previously to be involved in mediating the fusion process in the midface. Our results showed Tgfbeta2 expression in a very restrictive area of the epithelial layer along the borders of the midfacial primordia, in a pattern very similar to that of Bmp4. The highly restrictive and spatial and temporal pattern of expression of Tgfbeta2 implicates its role in mediating the fusion of the midfacial processes, possibly through interacting with Bmp4 in the regulation of apoptosis and/or epithelial-mesenchymal transformation. A greater understanding of the role of this gene will clarify how the normal midface grows and the mechanisms behind cleft development.

Noise overstimulation induces immediate early genes in the rat cochlea.

In mammals, exposure to intense noise produces a permanent hearing loss called permanent threshold shift (PTS), whereas a moderate noise produces only a temporary threshold shift (TTS). Little is known about the molecular responses to such high intensity noise exposures. In this study we used gene arrays to examine the early response to acoustic overstimulation in the rat cochlea. We compared cochlear RNA from noise-exposed rats with RNA from unexposed controls. The intense PTS noise induced several immediate early genes encoding both transcription factors (c-FOS, EGR1, NUR77/TR3) and cytokines (PC3/BTG2, LIF and IP10). In contrast, the TTS noise down-regulated the gene for growth hormone. The response of these genes to different noise intensities was examined by quantitative RT-PCR 2.5 h after the 90-min noise exposure. For most genes, the extent of induction correlates with the intensity of the noise exposure. Three proteins (EGR1, NUR77/TR3, and IP10) were detected in many regions of the unexposed cochlea. After exposure to 120 dB noise, these proteins were present at higher levels or showed extended expression in additional regions of the cochlea. LIF was undetectable in the cochlea of unexposed rats, but could be seen in the organ of Corti and spiral ganglion neurons following noise. NUR77/TR3 was a nuclear protein before noise, but following noise translocated to the cytoplasm. These studies provide new insights into the molecular response to noise overstimulation in the mammalian cochlea.

Identification and characterization of choline transporter-like protein 2, an inner ear glycoprotein of 68 and 72 kDa that is the target of antibody-induced hearing loss.

The Kresge Hearing Research Institute-3 (KHRI-3) antibody binds to a guinea pig inner ear supporting cell antigen (IESCA) and causes hearing loss. To gain insight into the mechanism of antibody-induced hearing loss, we used antibody immunoaffinity purification to isolate the IESCA, which was then sequenced by mass spectroscopy, revealing 10 guinea pig peptides identical to sequences in human choline transporter-like protein 2 (CTL2). Full-length CTL2 cDNA sequenced from guinea pig inner ear has 85.9% identity with the human cDNA. Consistent with its expression on the surface of supporting cells in the inner ear, CTL2 contains 10 predicted membrane-spanning regions with multiple N-glycosylation sites. The 68 and 72 kDa molecular forms of inner ear CTL2 are distinguished by sialic acid modification of the carbohydrate. The KHRI-3 antibody binds to an N-linked carbohydrate on CTL2 and presumably damages the organ of Corti by blocking the transporter function of this molecule. CTL2 mRNA and protein are abundantly expressed in human inner ear. Sera from patients with autoimmune hearing loss bind to guinea pig inner ear with the same pattern as CTL2 antibodies. Thus, CTL2 is a possible target of autoimmune hearing loss in humans.

Characterization of the human UBE3B gene: structure, expression, evolution, and alternative splicing.

E3 ubiquitin ligases target proteins for degradation by adding ubiquitin residues. We characterized full-length cDNAs for human and mouse UBE3B, a novel HECT-domain E3 ligase, and analyzed the structure of human UBE3B on chromosome 12q24.1. Alternative splicing of exon 20 of UBE3B generated two major transcripts. The 5.7-kb mRNA lacked exon 20 and encoded a full-length protein ligase, variant 1 (UBE3B_v1). A second transcript contained a 97-bp insertion encoded by exon 20 that introduced an in-frame stop codon. The predicted protein (UBE3B_v2) would lack the HECT domain and would be nonfunctional, since the HECT domain constitutes the active site for ubiquitin transfer. No alternative splicing was observed in this region of mouse UBE3B. Elimination of the HECT domain by alternative splicing has not been reported in any genes encoding HECT domain ligases and may represent a novel mechanism in regulating intracellular levels of functional HECT-domain ligases.

Gene expression profiles of the rat cochlea, cochlear nucleus, and inferior colliculus.

High-throughput DNA microarray technology allows for the assessment of large numbers of genes and can reveal gene expression in a specific region, differential gene expression between regions, as well as changes in gene expression under changing experimental conditions or with a particular disease. The present study used a gene array to profile normal gene expression in the rat whole cochlea, two subregions of the cochlea (modiolar and sensorineural epithelium), and the cochlear nucleus and inferior colliculus of the auditory brainstem. The hippocampus was also assessed as a well-characterized reference tissue. Approximately 40% of the 588 genes on the array showed expression over background. When the criterion for a signal threshold was set conservatively at twice background, the number of genes above the signal threshold ranged from approximately 20% in the cochlea to 30% in the inferior colliculus. While much of the gene expression pattern was expected based on the literature, gene profiles also revealed expression of genes that had not been reported previously. Many genes were expressed in all regions while others were differentially expressed (defined as greater than a twofold difference in expression between regions). A greater number of differentially expressed genes were found when comparing peripheral (cochlear) and central nervous system regions than when comparing the central auditory regions and the hippocampus. Several families of insulin-like growth factor binding proteins, matrix metalloproteinases, and tissue inhibitor of metalloproteinases were among the genes expressed at much higher levels in the cochlea compared with the central nervous system regions.

Differential Gene Expression Following Noise Trauma in Birds and Mammals.

Acoustic overstimulation has very different outcomes in birds and mammals. When noise exposure kills hair cells in birds, these cells can regenerate and hearing will recover. In mammals, however, the hair cell loss, and resulting hearing loss, is permanent. Changes in gene expression form the basis for important biological processes, including repair, regeneration, and plasticity. We are therefore using a battery of molecular approaches to identify and compare changes in gene expression following noise trauma in birds and mammals. Both differential display and subtractive hybridisation were used to identify genes whose expression increased in the chick basilar papilla immediately following exposure to an octave band noise (118 dB, centre frequency 1.5 kHz) for 4-6 hr. Among those upregulated genes were two involved in actin signalling: the CDC42 gene encoding a Rho GTPase, and WDR1, which encodes a protein involved in actin dynamics. A third gene, UBE3B, encodes an E3 ubiquitin ligase involved in protein turnover. A fourth gene encodes a cystein-rich secreted protein that may interact with calcium channels. To examine the mammalian response, gene microarrays on nylon membranes (Clontech Atlas Gene Arrays) were used to examine global changes in gene expression 30 minutes after TTS (110 dB broadband noise 50% duty cycle) or PTS (125 dB, 100% duty cycle) noise overstimulation (each for 90 minutes) in the rat cochlea. Several genes, including classic immediate early response genes such as c-fos, EGR1/NGFI-A, and NGFI-B, were upregulated at this early time point following the PTS exposure, but were not upregulated following the TTS exposure.