The role of MafB in auditory synapse development

描述(申请人提供)：听力障碍是人类最常见的感官缺陷。为了将信息从毛细胞忠实地传递到大脑，发育中的脊神经节神经元(SGN)形成不同类型的专门突触。了解这些听觉突触是如何发展的，可能会导致新疗法的开发，并扩大患者治疗的机会。根据转录因子MafB的表达模式、在其他发育系统中的功能以及对已知突触分子表达的调控能力，我们已确定MafB转录因子是听觉突触发育的潜在主要调节因子。为了研究MafB在听觉突触发育中的作用，我们产生了MafB的等位基因，并特异性地破坏了SGN中的MafB蛋白。MafB条件性基因敲除(MafBCKO)是可行的，没有明显的行为异常。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，与对照组相比，神经反应显著降低和延迟，这表明MafB是正常SGN活动所必需的。为了进一步剖析MafB的作用，我们创造了一种小鼠(MafBOE)，它在Cre介导的重组中过表达MafB。突触标记Ribeye/CtBP2的免疫组织化学染色显示，MafBCKO的突触带数量减少，而MafBOE的突触带数量增加，这表明MafB是正常形成耳蜗毛细胞和SGN之间突触所必需的。这些效应可能在一定程度上依赖于神经趋化因子CCL21，它可以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突变小鼠模型来进一步确定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，我们提出了以下实验。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MafB是否通过CCL21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。下游靶点的识别将帮助我们理解MafB如何协调构成听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，并可能识别新的耳聋易感基因。

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.