Systematic analysis of mitochondrial genes associated with hearing loss in the Japanese population: dHPLC reveals a new candidate mutation.

BACKGROUND: Variants of mitochondrial DNA (mtDNA) have been evaluated for their association with hearing loss. Although ethnic background affects the spectrum of mtDNA variants, systematic mutational analysis of mtDNA in Japanese patients with hearing loss has not been reported. METHODS: Using denaturing high-performance liquid chromatography combined with direct sequencing and cloning-sequencing, Japanese patients with prelingual (N = 54) or postlingual (N = 80) sensorineural hearing loss not having pathogenic mutations of m.1555A > G and m.3243A > G nor GJB2 were subjected to mutational analysis of mtDNA genes (12S rRNA, tRNALeu(UUR), tRNASer(UCN), tRNALys, tRNAHis, tRNASer(AGY), and tRNAGlu). RESULTS: We discovered 15 variants in 12S rRNA and one homoplasmic m.7501A > G variant in tRNASer(UCN); no variants were detected in the other genes. Two criteria, namely the low frequency in the controls and the high conservation among animals, selected the m.904C > T and the m.1105T > C variants in 12S rRNA as candidate pathogenic mutations. Alterations in the secondary structures of the two variant transcripts as well as that of m.7501A > G in tRNASer(UCN) were predicted. CONCLUSIONS: The m.904C > T variant was found to be a new candidate mutation associated with hearing loss. The m.1105T > C variant is unlikely to be pathogenic. The pathogenicity of the homoplasmic m.7501T > A variant awaits further study.

Mesenchymal stem cell transplantation accelerates hearing recovery through the repair of injured cochlear fibrocytes.

Cochlear fibrocytes play important roles in normal hearing as well as in several types of sensorineural hearing loss attributable to inner ear homeostasis disorders. Recently, we developed a novel rat model of acute sensorineural hearing loss attributable to fibrocyte dysfunction induced by a mitochondrial toxin. In this model, we demonstrate active regeneration of the cochlear fibrocytes after severe focal apoptosis without any changes in the organ of Corti. To rescue the residual hearing loss, we transplanted mesenchymal stem cells into the lateral semicircular canal; a number of these stem cells were then detected in the injured area in the lateral wall. Rats with transplanted mesenchymal stem cells in the lateral wall demonstrated a significantly higher hearing recovery ratio than controls. The mesenchymal stem cells in the lateral wall also showed connexin 26 and connexin 30 immunostaining reminiscent of gap junctions between neighboring cells. These results indicate that reorganization of the cochlear fibrocytes leads to hearing recovery after acute sensorineural hearing loss in this model and suggest that mesenchymal stem cell transplantation into the inner ear may be a promising therapy for patients with sensorineural hearing loss attributable to degeneration of cochlear fibrocytes.

Wnt signaling and a Hox protein cooperatively regulate psa-3/Meis to determine daughter cell fate after asymmetric cell division in C. elegans.

Asymmetric cell division is a mechanism for achieving cellular diversity. In C. elegans, many asymmetric cell divisions are controlled by the Wnt-MAPK pathway through POP-1/TCF. It is poorly understood, however, how POP-1 determines the specific fates of daughter cells. We found that nob-1/Hox, ceh-20/Pbx, and a Meis-related gene, psa-3, are required for asymmetric division of the T hypodermal cell. psa-3 expression was asymmetric between the T cell daughters, and it was regulated by POP-1 through a POP-1 binding site in the psa-3 gene. psa-3 expression was also regulated by NOB-1 and CEH-20 through a NOB-1 binding sequence in a psa-3 intron. PSA-3 can bind CEH-20 and function after the T cell division to promote the proper fate of the daughter cell. These results indicate that cooperation between Wnt signaling and a Hox protein functions to determine the specific fate of a daughter cell.

Components of the transcriptional Mediator complex are required for asymmetric cell division in C. elegans.

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, the Wnt signaling pathway regulates the asymmetric divisions of a number of cells including the T blast cell. We found that the let-19 and dpy-22 mutants have defects in their T-cell lineage, and lineage analyses showed that the defects were caused by disruption in the asymmetry of the T-cell division. We found that let-19 and dpy-22 encode homologs of the human proteins MED13/TRAP240 and MED12/TRAP230, respectively, which are components of the Mediator complex. Mediator is a multi-component complex that can regulate transcription by transducing the signals between activators and RNA polymerase in vitro. We also showed that LET-19 and DPY-22 form a complex in vivo with other components of Mediator, SUR-2/MED23 and LET-425/MED6. In the let-19 and dpy-22 mutants, tlp-1, which is normally expressed asymmetrically between the T-cell daughters through the function of the Wnt pathway, was expressed symmetrically in both daughter cells. Furthermore, we found that the let-19 and dpy-22 mutants were defective in the fusion of the Pn.p cell, a process that is regulated by bar-1/beta-catenin. Ectopic cell fusion in bar-1 mutants was suppressed by the let-19 or dpy-22 mutations, while defective cell fusion in let-19 mutants was suppressed by lin-39/Hox mutations, suggesting that let-19 and dpy-22 repress the transcription of lin-39. These results suggest that LET-19 and DPY-22 in the Mediator complex repress the transcription of Wnt target genes.

Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells.

Embryonic stem (ES) cells differentiate into dopamine (DA)-producing neurons when co-cultured with PA6 stromal cells, but the resulting cultures contain a variety of unidentified cells. In order to label live DA neurons in mixed populations, we introduced a GFP reporter under the control of the tyrosine hydroxylase (TH) gene promoter into ES cells. GFP expression was observed in TH-immunoreactive cells that differentiated from the ES cells that carried the TH-GFP reporter gene. DA neurons expressing GFP were sorted from the mixed cell population by fluorescence-activated cell sorting of cells exhibiting GFP fluorescence, and the sorted GFP(+) cells obtained were transplanted into a rat model of Parkinson's disease. Some of these cells survived and innervated the host striatum, resulting in a partial recovery from parkinsonian behavioral defects. This strategy of isolation and transplantation of ES-cell-derived DA neurons should be useful for cellular and molecular studies of DA neurons and for clinical application in the treatment of Parkinson's disease.