The role of MafB in auditory synapse development

MafB 在听觉突触发育中的作用

• 批准号: 8598807
• 负责人: Wei-Ming Yu
• 金额: $ 6.1万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598807
• 关键词: Adult; Affect; American; Auditory; Auditory Brainstem Responses; Auditory Threshold; Behavioral; Biological Preservation; Brain; Brain Stem; CCL21 gene; Cell physiology; Cells; Cochlea; Cochlear Implants; Cochlear nucleus; Data; Defect; Development; Ear; Electrophysiology (science); Exhibits; Future; Ganglia; Gene Expression; Genes; Genetic Programming; Genetic Recombination; Goals; Hair Cells; Hearing; Human; Lead; Link; LoxP-flanked allele; Measures; Mediating; Microglia; Mitotic; Molecular; Morphology; Mouse Strains; Mus; Mutant Strains Mice; Nature; Nervous system structure; Neurons; Newborn Infant; Patients; Pattern; Phenotype; Physiological; Play; Population; Process; Property; Proteins; Relative (related person); Role; Sensory; Severities; Signal Transduction; Susceptibility Gene; Synapses; Synaptic Transmission; System; Testing; Time; Vestibular ganglion; Work; base; chemokine; deafness; density; design; effective therapy; electrical property; hair cell regeneration; hearing impairment; improved; mutant; mutant mouse model; neural circuit; neuron development; neuronal survival; novel; overexpression; postnatal; postsynaptic; public health relevance; relating to nervous system; repaired; research study; response; ribbon synapse; sound; spiral ganglion; synaptic function; synaptogenesis; transcription factor; transcriptome sequencing; transmission process

DESCRIPTION (provided by applicant): Hearing impairment is the most common sensory defect in humans. To faithfully transmit information from hair cells to the brain, developing spira ganglion neurons (SGNs) form different types of specialized synapses. Understanding how these auditory synapses develop may lead to the development of new therapies and broaden opportunities for patient treatment. We have identified the transcription factor MafB as a potential master regulator of auditory synapse development based on its expression pattern, functions in other developing systems, and ability to control the expression of known synaptic molecules. To investigate the function of MafB in auditory synapse development, we generated a floxed allele of MafB and specifically disrupted MafB protein in SGNs. MafB conditional knock-outs (MafBCKO) are viable and exhibit no obvious behavioral abnormalities. Analysis of auditory function in MafBCKO mice by auditory brainstem responses shows that the mutants can still detect sound, consistent with preserved hair cell function. In contrast, the neural response was significantly decreased and delayed relative to controls, suggesting that MafB is required for proper SGN activity. To further dissect a role for MafB, we created a strain of mice (MafBOE) that overexpress MafB upon Cre-mediated recombination. Immunostaining for the ribbon synapse marker RIBEYE/CtBP2 revealed that the number of synaptic ribbons is reduced in MafBCKO and conversely, is increased in MafBOE, suggesting that MafB is required for normal formation of synapses between hair cells and SGNs in the cochlea. These effects may depend in part on the neuronal chemokine CCL21, which activates microglia in other regions of the nervous system. In MafBCKO SGNs, CCL21 expression is dramatically reduced. We will use MafB and CCL21 mutant mouse models to further define the cellular and molecular functions of MafB during auditory synapse development. To achieve these goals, we propose the following experiments. First, we will perform detailed histological and physiological analyses of the MafBCKO and MafBOE mice to evaluate the morphologies of ribbon synapses and the function of SGNs. Second, we will investigate whether MafB works through CCL21 to control microglia and promote synapse maturation. Third, we will use RNA-seq to conduct differential expression analysis and identify MafB downstream genes. Identification of downstream targets will help us understand how MafB coordinates the networks of genes that underlie auditory neuron maturation and synaptogenesis. Together, these studies will provide the opportunity to elucidate the molecular mechanisms of auditory synaptogenesis and may identify novel deafness susceptibility genes.

描述（由申请人提供）：听力障碍是人类最常见的感觉缺陷。为了将信息从毛细胞忠实地传递到大脑，发育中的螺旋神经节神经元（SGN）形成不同类型的专门突触。了解这些听觉突触如何发育可能会导致新疗法的开发并拓宽患者治疗的机会。基于转录因子 MafB 的表达模式、在其他发育系统中的功能以及控制已知突触分子表达的能力，我们已将转录因子 MafB 确定为听觉突触发育的潜在主调节因子。为了研究 MafB 在听觉突触发育中的功能，我们生成了 MafB 的 floxed 等位基因，并特异性破坏了 SGN 中的 MafB 蛋白。 MafB 条件敲除 (MafBCKO) 是可行的，并且没有表现出明显的行为异常。通过听觉脑干反应对 MafBCKO 小鼠的听觉功能进行分析表明，突变体仍然可以检测到声音，这与保留的毛细胞功能一致。相反，相对于对照组，神经反应显着降低和延迟，这表明 MafB 是适当的 SGN 活性所必需的。为了进一步剖析 MafB 的作用，我们创建了一种在 Cre 介导的重组后过度表达 MafB 的小鼠品系 (MafBOE)。带状突触标记 RIBEYE/CtBP2 的免疫染色显示，MafBCKO 中突触带的数量减少，相反，MafBOE 中突触带的数量增加，这表明 MafB 是耳蜗毛细胞和 SGN 之间突触正常形成所必需的。这些效应可能部分取决于神经元趋化因子 CCL21，它激活神经系统其他区域的小胶质细胞。在 MafBCKO SGN 中，CCL21 表达显着降低。我们将使用 MafB 和 CCL21 突变小鼠模型来进一步定义 MafB 在听觉突触发育过程中的细胞和分子功能。为了实现这些目标，我们提出以下实验。首先，我们将对 MafBCKO 和 MafBOE 小鼠进行详细的组织学和生理分析，以评估带状突触的形态和 SGN 的功能。其次，我们将研究 MafB 是否通过 CCL21 来控制小胶质细胞并促进突触成熟。第三，我们将使用RNA-seq进行差异表达分析并鉴定MafB下游基因。下游靶标的识别将帮助我们了解 MafB 如何协调听觉神经元成熟和突触发生的基因网络。总之，这些研究将为阐明听觉突触发生的分子机制提供机会，并可能鉴定新的耳聋易感基因。