Exploration of Connexin26 Genotypes, Phenotypes, and Gene Replacement

文摘:

Abstract: The most common form of autosomal recessive hereditary deafness is due to loss of the Connexin 26 (Cx26) protein (encoded by the human GJB2 gene). In mice, loss of Cx26 (Gjb2) leads to impaired glucose transport in the placenta and early embryonic death, whereas conditional loss restricted to the inner ear results in hearing impairment and death of epithelial and neuronal cells in the cochlea. Mouse models for reduced Cx26 exhibit many of the features found in humans with Gjb2 mutations, but the models display a more severe phenotype than humans. Specifically, onset of pathology in mice is earlier, hearing loss is progressive and more severe, and spiral ganglion neuron (SGN) degeneration occurs. The underlying mechanisms for these differences in phenotypes are unclear, but may be related to developmental or cell type-specific expression and/or functions of Cx26. The pan-otocyst deletions selected for some mouse models may also be responsible for the extremely severe mouse phenotypes. Here, we propose to use a mouse model we have recently generated (Sox10Cre-Gjb2) in which Cre, driven by the promoter of the supporting cell gene Sox10, is used for deleting Gjb2. Sox10Cre-Gjb2 mice exhibit hearing loss and degeneration of the cochlear epithelium, and SGNs which appear less severe than other existing models. Using our mice, we will characterize critical roles for Cx26 during the development of the cochlea prior to the onset of hearing, versus its roles in function and survival of hair cells, supporting cells and neurons in the mature inner ear. Our team of investigators with a strong history of productive collaboration, will test the global hypothesis that Cx26 exhibits critical requirements for promotion of sensory epithelial and neuronal function and integrity that are temporal (i.e. embryonic vs. early postnatal vs adult) and cell type-specific, and that functional effects of loss of Cx26 can be corrected by gene replacement. We have three Specific Aims: (1) Characterize inner ear structure/function in mice with loss of Cx26 in supporting cells, (2) Test whether Cx26 exhibits temporal and cell type-specific requirements for promotion of cochlear epithelial cell and spiral ganglion cell function and integrity, and (3) Determine if Ad.CX26-GFP is sufficient to (a) restore functional gap junctions in the auditory epithelium as determined by fluorescence recovery after photobleaching (FRAP) and immunocytochemistry, (b) improve ABR thresholds and (c) rescue hair cells, supporting cells, and neurons in Gjb2 deficient mice. Results from these studies are poised to improve understanding of the pathophysiology of Gjb2 mutations in the ear, and accelerate development of specific and effective gene-based therapies for human Cx26 related deafness.