Exploration of Connexin26 Genotypes, Phenotypes, and Gene Replacement

摘要： 最常见的常染色体隐性遗传性耳聋是由于连接蛋白26(Cx26)的缺失 蛋白质(由人类GJB2基因编码)。在小鼠中，Cx26(Gjb2)的缺失导致葡萄糖转运受损 在胎盘和早期胚胎死亡中，而条件性损失仅限于内耳导致 耳蜗内上皮细胞和神经细胞的听力损伤和死亡。减少Cx26的小鼠模型 显示出许多在携带Gjb2突变的人类中发现的特征，但模型显示出更严重的 比人类的表型要好。具体地说，小鼠的病理发病较早，听力损失是进行性的， 病情越重，螺旋神经节神经元(SGN)就会发生变性。这些问题的根本机制是 表型的差异尚不清楚，但可能与发育或细胞类型特异性表达有关 和/或Cx26的功能。为某些小鼠模型选择的泛耳囊缺失也可能是 对极其严重的小鼠表型负责。在这里，我们建议使用我们已有的小鼠模型 最近产生的(Sox10Cre-Gjb2)，其中Cre由支持细胞基因Sox10的启动子驱动， 用于删除Gjb2。Sox10Cre-Gjb2小鼠表现出听力损失和耳蜗上皮变性， 以及看起来没有其他现有模型那么严重的SGN。使用我们的老鼠，我们将描述关键的 Cx26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 成熟内耳的毛细胞、支持细胞和神经元存活。我们的调查团队拥有 卓有成效的协作历史将检验Cx26显示出关键的全球假说 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，且Cx26丢失的功能影响可能是 通过基因替换来纠正的。我们有三个具体的目标：(1)表征内耳的结构/功能 支持细胞中Cx26缺失的小鼠，(2)检测Cx26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)恢复听觉上皮细胞的功能性缝隙连接 通过光漂白后荧光恢复(FRAP)和免疫细胞化学确定，(B)改善 ABR阈值和(C)挽救Gjb2缺陷小鼠的毛细胞、支持细胞和神经元。结果来自 这些研究有望提高对耳朵中Gjb2突变的病理生理学的了解，以及 加快开发针对人类Cx26相关性耳聋的特异和有效的基因治疗方法。

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.