Exploration of Connexin26 Genotypes, Phenotypes, and Gene Replacement

摘要： 常染色体隐性遗传性耳聋最常见的形式是由于连接蛋白26（Cx 26）的丢失。 蛋白质（由人类GJB 2基因编码）。在小鼠中，Cx 26（Gjb 2）的缺失导致葡萄糖转运受损 在胎盘和早期胚胎死亡，而条件损失限于内耳的结果， 听力损伤和耳蜗上皮细胞和神经元细胞死亡。Cx 26减少的小鼠模型 表现出许多在Gjb 2突变人类中发现的特征，但模型显示出更严重的 比人类的表型。具体而言，小鼠的病理学发作较早，听力损失是进行性的， 更严重的是，发生螺旋神经节神经元（SGN）变性。这些潜在的机制 表型差异尚不清楚，但可能与发育或细胞类型特异性表达有关 和/或Cx 26的功能。为一些小鼠模型选择的全耳囊缺失也可以是 导致了极其严重的小鼠表型在这里，我们建议使用我们拥有的小鼠模型， 最近产生的（Sox 10 Cre-Gjb 2），其中Cre由支持细胞基因Sox 10的启动子驱动， 用于删除Gjb 2。Sox 10 Cre-Gjb 2小鼠表现出听力损失和耳蜗上皮细胞变性， 和SGN，其看起来不像其他现有模型那么严重。使用我们的老鼠，我们将描述关键的 Cx 26在听力开始前耳蜗发育过程中的作用，与其在功能中的作用相比， 以及毛细胞、支持细胞和神经元在成熟内耳中的存活。我们的调查团队 一个强大的历史，富有成效的合作，将测试全球假设，Cx 26表现出关键的 促进感觉上皮和神经元功能和完整性的要求是暂时的（即， 胚胎vs.出生后早期vs.成人）和细胞类型特异性，Cx 26缺失的功能影响可能是 通过基因替换来纠正。我们有三个具体的目标：（1）表征内耳结构/功能， Cx 26在支持细胞中缺失的小鼠，（2）测试Cx 26是否表现出时间和细胞类型特异性 促进耳蜗上皮细胞和螺旋神经节细胞功能和完整性的要求，和（3） 确定Ad.CX26-GFP是否足以（a）恢复听觉上皮中的功能性间隙连接， 通过光漂白后荧光恢复（FRAP）和免疫细胞化学测定，（B）改善 ABR阈值和（c）拯救Gjb 2缺陷小鼠的毛细胞、支持细胞和神经元。结果 这些研究有望提高对耳部Gjb 2突变病理生理学的了解，并且 加速开发针对人类Cx 26相关耳聋的特异性和有效的基因治疗。

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.