Guilty as CHARGED: p53's expanding role in disease.

Unrestrained p53 activity during development, as occurs upon loss of the p53 negative regulators Mdm2 or Mdmx, causes early embryonic lethality. Surprisingly, co-expression of wild-type p53 and a transcriptionally-dead variant of p53, with mutations in both transactivation domains (p53(L25Q,W26S,F53Q,F54S)), also causes lethality, but later in gestation and in association with a host of very specific phenotypes reminiscent of a syndrome known as CHARGE. Molecular analyses revealed that wild-type p53 is inappropriately activated in p53(5,26,53,54/)(+) embryos, triggering cell-cycle arrest or apoptosis during development to cause CHARGE phenotypes. In addition, CHARGE syndrome is typically caused by mutations in the CHD7 chromatin remodeler, and we have shown that activated p53 contributes to phenotypes caused by CHD7-deficiency. Together, these studies provide new insight into CHARGE syndrome and expand our understanding of the role of p53 in diseases other than cancer.

Anti-apoptotic gene Bcl2 is required for stapes development and hearing.

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2⁻/⁻ mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2⁻/⁻ mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2⁻/⁻Bim⁻/⁻ mice and partially rescued in Bcl2⁻/⁻Bim⁺/⁻ mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2⁻/⁻ defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.

Monaural conductive hearing loss alters the expression of the GluA3 AMPA and glycine receptor α1 subunits in bushy and fusiform cells of the cochlear nucleus.

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABA(A) receptor subunits ß2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABA(A) ß2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

Early age conductive hearing loss causes audiogenic seizure and hyperacusis behavior.

Recent clinical reports found a high incidence of recurrent otitis media in children suffering hyperacusis, a marked intolerance to an otherwise ordinary environmental sound. However, it is unclear whether the conductive hearing loss caused by otitis media in early age will affect sound tolerance later in life. Thus, we have tested the effects of tympanic membrane (TM) damage at an early age on sound perception development in rats. Two weeks after the TM perforation, more than 80% of the rats showed audiogenic seizure (AGS) when exposed to loud sound (120 dB SPL white noise, < 1 min). The susceptibility of AGS lasted at least sixteen weeks after the TM damage, even the hearing loss recovered. The TM damaged rats also showed significantly enhanced acoustic startle responses compared to the rats without TM damage. These results suggest that early age conductive hearing loss may cause an impaired sound tolerance during development. In addition, the AGS can be suppressed by the treatment of vigabatrin, acute injections (250 mg/kg) or oral intakes (60 mg/kg/day for 7 days), an antiepileptic drug that inhibits the catabolism of GABA. c-Fos staining showed a strong staining in the inferior colliculus (IC) in the TM damaged rats, not in the control rats, after exposed to loud sound, indicating a hyper-excitability in the IC during AGS. These results indicate that early age conductive hearing loss can impair sound tolerance by reducing GABA inhibition in the IC, which may be related to hyperacusis seen in children with otitis media.

Reversible conductive hearing loss: restored activity in the central auditory system.

The effect of a reversible, unilateral hearing loss on 2-deoxyglucose (2-DG) uptake in the central auditory system was studied using young gerbils. All animals had a unilateral conductive hearing loss (CHL), induced by atresia, on postnatal day 21 (P21). One week later, on P28, animals had their atresia repaired (CHL/R), or not repaired (CHL/NR), and CHL/NR animals entered the 2-DG experiments. CHL/R animals were allowed a 1-week period of restored binaural hearing experience prior to entering 2-DG experiments on P35. Animals in each group were injected with 2-DG and exposed to ambient sounds for 45 min prior to sacrifice. Uptake of 2-DG was measured in the anteroventral cochlear nucleus (AVCN), the medial superior olive (MSO), and the inferior colliculus (IC) on both sides of the brain. In CHL/NR animals, there were significant differences in uptake between the AVCN, MSO, and IC ipsilateral versus contralateral to the manipulated ear, indicating an imbalance in ascending afferent activity. In CHL/R animals, there were no significant differences, suggesting that 1 week after CHL repair, the appearance of balanced afferent activity had been restored.

TAP deficiency syndrome: chronic rhinosinusitis and conductive hearing loss.

Nose-ear-throat manifestations of immunodeficiency disorders represent a diagnostic challenge for clinicians as these diseases often constitute the initial sign for connective disorders or autoimmune disease. The history of chronic rhinosinusitis and conductive hearing loss is often non specific. Therefore attention to an HLA class I deficiency must be considered if the disease has not been diagnosed on routine examination. One of the syndromes is due to a defective TAP complex, the peptide transporter complex associated with antigen presentation. Herein, we report two sisters with TAP-deficiency. The treatment of choice for TAP-deficient patients is conservative.

Consequences of unilateral hearing loss: time dependent regulation of protein synthesis in auditory brainstem nuclei.

Conductive hearing impairment results in marked changes in neuronal activity in the central auditory system, particularly in young animals [Tucci, D.L., Cant, N.B., Durham, D., 1999. Conductive hearing loss results in a decrease in central auditory system activity in the young gerbil. Laryngoscope 109, 1359-1371]. To better understand the effects of conductive hearing loss (CHL) on cellular metabolism, incorporation of (3)H-leucine was used as a measure of protein synthesis in immature postnatal day 21 gerbils subjected to either unilateral CHL by malleus removal or profound sensorineural hearing loss by cochlear ablation. (3)H-leucine uptake was measured after survival times of 6 or 48h. Protein synthesis values were standardized to measurements from the abducens nucleus and compared with measurements from sham animals at similar age/survival times. Protein synthesis in the medial superior olive (MSO) was found to be significantly down-regulated (bilaterally) after CHL in animals surviving 48h. However, 6h after CHL manipulation, protein synthesis is up-regulated in MSO (bilaterally) and in the ipsilateral medial nucleus of the trapezoid body.

Mechanisms of hearing loss resulting from middle-ear fluid.

Fluid in the middle ear, a defining feature of otitis media with effusion (OME), is commonly associated with a 20- to 30-dB conductive hearing loss. The effects and relative importance of various mechanisms leading to conductive hearing loss were investigated in a human temporal bone preparation. Umbo velocity in response to ear-canal sound was measured with a laser vibrometer while saline and silicone fluids of viscosity 5-12,000 cSt were introduced into the middle ear to contact part or all of the tympanic membrane (TM) and fill part or all of the middle ear. At low frequencies, reductions in umbo velocity (deltaVU) of up to 25 dB depended on the percentage of the original middle-ear air space that remained air-filled, which suggests that the primary mechanism in hearing loss at low frequencies is a reduction of the admittance of the middle-ear air space due to displacement of air with fluid. At higher frequencies, deltaVU (of up to 35 dB) depended on the percentage of the TM contacted by fluid, which suggests that the primary mechanism at high frequencies is an increase in tympanic membrane mass by entrained fluid. The viscosity of the fluid had no significant effect on umbo velocity. deltaVU for the fluid-filled middle ear matched hearing losses reported in patients whose middle ear was believed to be completely filled with fluid. The difference between deltaVU for a partly-filled middle ear and hearing losses reported in patients whose middle ear was believed to be incompletely fluid-filled is consistent with the reported effect of middle-ear underpressure (commonly seen in OME) on umbo velocity. Small amounts of air in the middle ear are sufficient to facilitate umbo motion at low frequencies.

The neonate has a temporary conductive hearing loss due to fluid in the middle ear.

Postnatal functional changes in the activity of the ear and auditory pathway in neonatal guinea pigs [from day of birth (postnatal day, PND = 0), PNDs 1-4, 7 and then weekly up to 7 weeks] were studied as a model of maturation of hearing in human neonates. On the day of birth there were signs of a conductive hearing loss: negative middle ear pressure, auditory nerve brainstem evoked response (ABR) threshold elevation, ABR wave 1 latency prolongation and low amplitude otoacoustic emissions. The conductive hearing loss is probably a result of the (amniotic) fluid found in the neonatal middle-ear cavity. Over the next PNDs, this conductive hearing loss was resolved. In order to confirm this neonatal conductive hearing loss and its resolution, saline was instilled into the middle ear of guinea pigs. This induced signs of a conductive hearing loss similar to those seen in the neonatal guinea pigs which disappeared with clearance of this fluid. Therefore it may be concluded that most of the changes in auditory function seen over the first PNDs are due to absorption of amniotic fluid from the middle-ear cavity.

Early unilateral auditory deprivation increases 2-deoxyglucose uptake in contralateral auditory cortex of juvenile Mongolian gerbils.

The effects of early onset, unilateral conductive hearing loss on tone-induced 2-deoxyglucose (2-DG) uptake in the auditory cortex of juvenile Mongolian gerbils (Meriones unguiculatus) were studied. Atresia of the left ear canal was induced at postnatal day 9 (P9) to achieve reversible auditory deprivation prior to onset of hearing (around P12). Atresia either persisted (ATR, n=4) or the canal was opened 15 min before the 2-DG experiments (RE, n=4) at P27. Control animals were either non-deprived (CON, n=4), or their left ears were plugged acutely (PAX, n=4). In PAX, 2-DG uptake in primary auditory cortex (AI) and anterior auditory field (AAF) was lower in right than in left AI and AAF. In contrast, in ATR and RE, uptake was significantly higher on the right side contralateral to the atresia. Hence, atresia during early development leads to plastic changes resulting in an interhemispheric imbalance of functional metabolism in favor of the auditory cortex contralateral to the manipulated ear. Distances between tone-induced 2-DG labeling in AI and AAF were increased in PAX, but smaller in ATR in the right compared to the left hemisphere, suggesting effects of atresia also on spatial relations in cortical tonotopic maps.

Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia.

Haploinsufficiency for human EYA1, a homologue of the Drosophila melanogaster gene eyes absent (eya), results in the dominantly inherited disorders branchio-oto-renal (BOR) syndrome and branchio-oto (BO) syndrome, which are characterized by craniofacial abnormalities and hearing loss with (BOR) or without (BO) kidney defects. To understand the developmental pathogenesis of organs affected in these syndromes, we inactivated the gene Eya1 in mice. Eya1 heterozygotes show renal abnormalities and a conductive hearing loss similar to BOR syndrome, whereas Eya1 homozygotes lack ears and kidneys due to defective inductive tissue interactions and apoptotic regression of the organ primordia. Inner ear development in Eya1 homozygotes arrests at the otic vesicle stage and all components of the inner ear and specific cranial sensory ganglia fail to form. In the kidney, Eya1 homozygosity results in an absence of ureteric bud outgrowth and a subsequent failure of metanephric induction. Gdnf expression, which is required to direct ureteric bud outgrowth via activation of the c-ret Rtk (refs 5, 6, 7, 8), is not detected in Eya1-/- metanephric mesenchyme. In Eya1-/- ear and kidney development, Six but not Pax expression is Eya1 dependent, similar to a genetic pathway elucidated in the Drosophila eye imaginal disc. Our results indicate that Eya1 controls critical early inductive signalling events involved in ear and kidney formation and integrate Eya1 into the genetic regulatory cascade controlling kidney formation upstream of Gdnf. In addition, our results suggest that an evolutionarily conserved Pax-Eya-Six regulatory hierarchy is used in mammalian ear and kidney development.

Conductive hearing loss results in a decrease in central auditory system activity in the young gerbil.

OBJECTIVES/HYPOTHESIS: The impact of childhood conductive HL (CHL) on development of auditory function has long been debated. The present study was conducted to define and compare the consequences of CHL and cochlear ablation (CA) in young and adult animals, using 2-deoxyglucose (2-DG) uptake as a measure of metabolic activity. It was hypothesized that, for both ages, CHL would result in a decrease in activity in the major ascending central auditory system pathway of the manipulated ear, but that this decrease would be significantly less than that observed with CA. STUDY DESIGN: Sham-controlled study of metabolic effects of CHL during sound stimulation. METHODS: Gerbils (aged 21 days or adult), underwent malleus removal, CA, or a sham procedure. Young animals survived either 48 hours or 3 weeks; adults survived 3 weeks. Each age/survival CHL group contained eight animals; otherwise, each group (CA and sham) contained five animals, for a total number of 54. At the appropriate survival time, animals were given an intracardiac injection of 14C-2-DG, and sacrificed under anesthesia after 45 minutes of exposure to normal laboratory sounds. Tissue sections were prepared for exposure to x-ray film for optical density measurements, and alternate sections stained for identification of nuclei. Measurements from auditory nuclei of experimental animals were corrected against an unaffected control area (abducens nucleus) and compared with measurements taken from animals in the sham group. Auditory evoked potential thresholds to both air- and bone-conducted stimuli were obtained in a second group of neonatal and adult animals. RESULTS: Both CHL and CA resulted in a marked decrease in 2-DG uptake in the major ascending projection of the manipulated ear, in both the neonatal and adult animals. In young animals, effects of CHL and CA were similar. Effects of CHL in adult animals were less marked and significantly different from either effects of CHL in young animals or effects of CA in adult animals. HL following malleus removal only was purely conductive and ranged from 38 to 55 dB across frequency. CONCLUSIONS: Results suggest that, particularly in young animals, a unilateral CHL may have profound effects on metabolic activity in the central auditory system.

Vestibular and hearing loss in genetic and metabolic disorders.

Hearing loss affects about 4% of people under 45 years of age and comprises a broad spectrum of clinical presentations (congenital or late-onset, conductive or sensorineural, and syndromic or nonsyndromic). Approximately 30% of genetically determined deafness is reported to occur in syndromic form and 70% in nonsyndromic form. This review highlights recent advances in the molecular and genetic basis of hearing loss, which will help in understanding the biology of normal and abnormal hearing.

Regulation of D-aspartate release and uptake in adult brain stem auditory nuclei after unilateral middle ear ossicle removal and cochlear ablation.

In young adult guinea pigs, the effects of unilateral ossicle removal and cochlear ablation were determined on transmitter release from glutamatergic presynaptic endings and glutamate inactivation via uptake. (i) D-[3H]Aspartate release and uptake were measured in subdivisions of the cochlear nucleus (CN) and in nuclei of the superior olive (SOC) and auditory midbrain (MB) up to 145 days after placing the lesions. Activities were compared to those from age-matched unlesioned controls. Fiber degeneration was visualized histologically. (ii) In the ipsilateral CN, changes in release and uptake were governed by the type of lesion. Ossicle removal produced sparse pruning of fibers only after 112 days and decreased release and uptake at 145 days, consistent with regulatory weakening of excitatory glutamatergic transmission. Cochlear ablation deafferented the CN, producing deficient release and uptake at 2 days and abundant fiber degeneration at 7 days. Subsequently, the residual release and uptake increased in magnitude, consistent with strengthening of excitatory glutamatergic transmission. (iii) In the contralateral CN, after either lesion, changes in release and uptake usually matched those in the ipsilateral CN. Thus, the auditory pathway associated with the lesioned ear probably provided cues for the regulation of synaptic strength in the contralateral CN. (iv) Both lesions increased release in the SOC and MB, and uptake in the SOC, consistent with strengthening of excitatory glutamatergic transmission. Sparse fiber degeneration, suggesting axonal pruning, appeared in the SOC and MB after cochlear ablation. (v) The strengthening of excitatory glutamatergic transmission may facilitate and maintain symptoms such as loudness recruitment and tinnitus which often accompany hearing loss.

Postnatal morphologic changes and glial fibrillary acidic protein immunoreactivity in the anteroventral cochlear nucleus of the acoustically-deprived gerbil.

This study investigated the morphological changes and glial fibrillary acidic protein immunoreactivity (GFAP-IR) in the anteroventral cochlear nucleus (AVCN) of acoustically-deprived gerbils during postnatal development. The mongolian gerbil, Meriones unguiculatus, had been acoustically deprived on the right side or left side by a surgical ligation of the external auditory canal at postnatal day 12-14. No discernible microcysts were located in the ipsilateral AVCN at one, three, six and nine months after monaural ligation. Also, no discernible microcysts were located in the contralateral AVCN at one and three months after monaural ligation. Numerous microcysts were located in the contralateral AVCN at six months after monaural ligation and were slightly reduced in number at nine months after monaural ligation. Some of the microcysts closely apposed to and connected with the blood vessels through a leakage route or channel. A foamy region was found in the superficial granule cell cap of the AVCN. The foamy region became evident in the ipsilateral AVCN at three months after monaural ligation. However, the foamy region became evident in the contralateral AVCN at three and nine months after monaural ligation. Vacuoles were mainly found in the neuronal cells at the junction of the superficial and deep layers in the AVCN. These vacuoles were found in the contralateral AVCN at one, three, six, and nine months after monaural ligation. However, vacuoles were found in the ipsilateral AVCN only at three months after monaural ligation. Morphological changes of the myelin sheath were found to be more severe in the contralateral AVCN than in the ipsilateral. GFAP-IR was located in the superficial layer of the contralateral AVCN at three and nine months after monaural ligation. However, GFAP-IR was found in the superficial and deep layers of the ipsilateral AVCN at three and nine months after monaural ligation. GFAP-IR was also found in the superficial layers of the ipsilateral AVCN at six months after monoaural ligation. Microcysts are presumably derived from the detachment of the myelin sheath from the retracted axons, protrusion of the myelin sheath, and disruption of the myelin sheath. The major conclusions were that (1) microcysts were greatly reduced following acoustical ligation during postnatal development, and (2) blood vessels and GFAP-immunoreactive astrocytes may be involved in the depletion of microcysts for maintaining the homeostasis of the microenvironment in the cochlear nuclei.