Exploration of Connexin26 Genotypes, Phenotypes, and Gene Replacement

Connexin26 基因型、表型和基因替换的探索

• 批准号: 9185684
• 负责人: Donna M. Martin
• 金额: $ 48.82万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185684
• 关键词: Accounting; Address; Adult; Aging; Alleles; Auditory; Cell Death; Cell physiology; Cells; Cessation of life; Cochlea; Cochlear duct; Collaborations; Development; Ear; Embryo; Environmental Exposure; Epithelial; Epithelial Cells; Epithelium; Exhibits; Fluorescence Recovery After Photobleaching; Functional disorder; GJB2 gene; Gap Junctions; Genes; Genetic; Genotype; Hair Cells; Hearing; Hereditary Disease; Histology; Human; Immunohistochemistry; Inherited; Injection of therapeutic agent; Laboratories; Labyrinth; Maintenance; Measures; Mesenchymal; Modeling; Mus; Mutation; Myelin; Nerve Degeneration; Neuroglia; Neurons; Nomenclature; Pathology; Pattern; Phenotype; Placenta; Predisposition; Proteins; Recording of previous events; Research; Research Personnel; Role; Schwann Cells; Sensory; Severities; Stria Vascularis; Structure; Supporting Cell; Tamoxifen; Testing; Therapeutic; abstracting; cell type; deafness; ganglion cell; gene replacement; gene replacement therapy; gene therapy; glucose transport; hearing impairment; immunocytochemistry; improved; mouse model; neuron development; novel; otoconia; postnatal; promoter; research study; spiral ganglion; stem cell therapy; transdifferentiation

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.

抽象的： 常染色体隐性遗传性耳聋的最常见形式是由于连接蛋白26的丧失（CX26） 蛋白质（由人GJB2基因编码）。在小鼠中，CX26（GJB2）的损失导致葡萄糖转运受损 在胎盘和早期胚胎死亡中，而有条件的损失仅限于内耳导致 耳蜗上上皮和神经元细胞的听力障碍和死亡。减少CX26的鼠标模型 展示了具有GJB2突变的人类中发现的许多功能，但模型显示出更严重的 表型比人类。具体而言，小鼠病理的发作较早，听力损失是进步的，并且 发生更严重，螺旋神经神经元（SGN）变性。这些的基本机制 表型的差异尚不清楚，但可能与发育类型或细胞类型的表达有关 和/或CX26的功能。为某些鼠标模型选择的PAN-粒细胞缺失也可能是 负责极度严重的小鼠表型。在这里，我们建议使用鼠标模型 最近生成的（SOX10CRE-GJB2），其中由支持细胞基因SOX10的启动子驱动的CRE为 用于删除GJB2。 SOX10CRE-GJB2小鼠表现出听力损失和人工耳蜗上皮的变性， 和SGN看起来不如其他现有模型严重。使用我们的老鼠，我们将表征关键 在听力发作之前，CX26的角色与其在功能中的作用相比 毛细胞的存活，支持成熟的内耳中的细胞和神经元。我们的调查人员团队与 有效的合作历史将测试CX26表现出关键的全球假设 促进感觉上皮和神经元功能和完整性的要求（即 胚胎与早期产后与成人的早期）和细胞类型特异性，以及CX26丢失的功能效应可以是 通过基因置换纠正。我们有三个具体的目的：（1）表征内耳的结构/功能 支持细胞中CX26损失的小鼠（2）测试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，（3） 确定AD.CX26-GFP是否足以（a）（a）恢复听觉上皮中的功能间隙连接 通过光漂白（FRAP）和免疫细胞化学后的荧光恢复确定，（b）改善 ABR阈值和（C）在GJB2缺乏小鼠中拯救毛细胞，支撑细胞和神经元。结果 这些研究有望提高对耳朵中GJB2突变的病理生理学的理解，以及 加快针对人CX26相关耳聋的特定和有效基因疗法的发展。