Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

Myosin light chain kinase regulates hearing in mice by influencing the F-actin cytoskeleton of outer hair cells and cochleae.

Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chains to facilitate its interaction with actin filaments and produce contractile activity. The outer hair cells (OHCs) in the ear contain large amounts of actin and a variety myosins. The stereociliary and somatic motility of OHCs are closely related to hearing. It appears likely that MLCK may play an important role in acoustic trans-duction. In this study, we analyzed, both in vivo and in vitro, the OHCs of mice bearing a specific deletion of the MLCK gene and the OHCs of control mice. The phenotype was assessed by auditory function [acoustic brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs)], inner ear morphology and histology. MLCK-deficient mice aged 6-7 months showed impaired hearing, a 5- to 10-dB sound pressure level (SPL) increase in the ABR thresholds, when responding to clicks and tones of different frequencies (8 and 16 kHz) (P<0.05). The DPOAE amplitudes of 3-month-old MLCK-deficient mice decreased significantly (>10 dB SPL) at low frequencies (4, 5 and 6 kHz). The OHCs in the MLCK-deficient mice increased with abnormal stereocilia. The staining of F-actin and the phosphorylation of the regulatory light chain in MLCK-deficient OHCs was weak. Our results indicate that MLCK may regulate the structure and the motility of stereocilia through F-actin polymerization.

Inhibition of Myo6 gene expression by co­expression of a mutant of transcription factor POU4F3 (BRN­3C) in hair cells.

An eight­base pair (bp) deletion in the Pou4f3 gene in hair cells is associated with DFNA15, a hereditary form of hearing loss. To explore the pathological mechanisms underlying the development of DFNA15, the effect of the mutation in Pou4f3 on the activity of the myosin VI (Myo6) promoter, was investigated. The upstream regulatory sequence of Myo6 (2625 bp), consisting of an 1899 bp upstream sequence and a 727 bp intron 1 sequence, was amplified using polymerase chain reaction and subcloned into the pGL3­Basic vector expressing firefly luciferase. For verification of inserted fragments, plasmids were subjected to restriction analysis and then sequenced. HEK293T human embryonic kidney cells were transiently transfected with renilla luciferase­thymidine kinase vectors expressing Renilla luciferase and the Myo6 promoter­driven firefly luciferase expressing vectors along with pIRES2­enhanced green fluorescent protein (EGFP)­Pou4f3 (expressing wild­type Pou4f3) or pIRES2­EGFP­Pou4f3 (expressing the truncation mutant of Pou4f3). The relative luciferase activities were measured to determine the activity of the Myo6 promoter. The Myo6 promoter activity was not affected by co­expression of wild­type Pou4f3, as indicated by the comparable relative luciferase activities in the presence of the pIRES2­EGFP­Pou4f3 and the empty control vectors. However, co­expression of mutated Pou4f3 significantly inhibited the activity of the Myo6 promoter to almost half of that of the control (P<0.001). The data suggests that mutated Pou4f3 has a negative role in the promoter activity of Myo6, and by extension, the expression of myosin VI, and this may be an underlying mechanism of DFNA15 hearing loss.

[Study on conditional myosin light chain kinase gene knockout mice resulting in hearing loss].

OBJECTIVE: To investigate the function of myosin light chain kinase (MLCK) in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing. METHODS: Cross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response (ABR) to evaluate mice hearing function at different frequencies. RESULTS: Mlck knockout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones (8 kHz, 16 kHz, 32 kHz), and the rise was over 20 dB at high-frequency(32 kHz). Further analyses of waveforms showed that wave-I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone (16 kHz) were less than control group(P < 0.05) on average, but the ratio of wave I/II and I/III were not difference (P > 0.05). CONCLUSIONS: Mlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high-frequencies.