The role of MafB in auditory synapse development

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）形成了不同类型的特殊突触。了解这些听觉突触如何发育可能会导致新疗法的发展，并拓宽患者治疗的机会。基于其表达模式、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，我们已经确定转录因子MafB是听觉突触发育的潜在主调节器。为了研究mab在听觉突触发育中的功能，我们在sgn中产生了mab的一个floxed等位基因，并特异性地破坏了mab蛋白。MafB条件敲除（MafBCKO）是可行的，没有表现出明显的行为异常。通过听觉脑干反应分析MafBCKO小鼠的听觉功能表明，突变体仍然可以检测声音，与保留的毛细胞功能一致。相比之下，与对照组相比，神经反应明显减少和延迟，这表明mab是SGN正常活动所必需的。为了进一步剖析MafB的作用，我们创建了一株在cre介导的重组中过表达MafB的小鼠（MafBOE）。带状突触标记物RIBEYE/CtBP2的免疫染色显示，在MafBCKO中突触带的数量减少，相反，在MafBOE中突触带的数量增加，这表明耳蜗毛细胞和sgn之间突触的正常形成需要MafB。这些作用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的表达显著降低。我们将使用MafB和CCL21突变小鼠模型进一步确定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学分析，以评估带状突触的形态和sgn的功能。其次，我们将研究mab是否通过CCL21控制小胶质细胞并促进突触成熟。第三，我们将利用RNA-seq进行差异表达分析，鉴定MafB下游基因。鉴定下游目标将有助于我们了解mab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，并可能确定新的耳聋易感基因。

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.