The role of MafB in auditory synapse development

MafB 在听觉突触发育中的作用

• 批准号: 8787938
• 负责人: Wei-Ming Yu
• 金额: $ 6.42万
• 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/8787938
• 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; Health; 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; differential expression; effective therapy; electrical property; hair cell regeneration; hearing impairment; improved; mutant; mutant mouse model; neural circuit; neuron development; neuronal survival; novel; overexpression; postnatal; postsynaptic; 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。为了忠实地将信息从毛细胞传递到大脑，发育中的螺旋神经节神经元（SGN）形成不同类型的特化突触。了解这些听觉突触是如何发展的，可能会导致新疗法的发展，并扩大患者治疗的机会。我们已经确定了转录因子MafB作为一个潜在的主调节听觉突触发育的基础上，其表达模式，在其他发展系统中的功能，并能够控制已知的突触分子的表达。为了研究MafB在听觉突触发育中的功能，我们产生了MafB的floxed等位基因，并特异性地破坏SGN中的MafB蛋白。MafB条件性敲除（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的分析表明，突变体仍然可以检测到声音，这与保留的毛细胞功能一致。相比之下，相对于对照，神经反应显著降低和延迟，表明MafB是适当SGN活性所必需的。为了进一步剖析MafB的作用，我们创建了一种小鼠品系（MafBOE），其在Cre介导的重组后过表达MafB。带状突触标记物RIBEYE/CtBP 2的免疫染色显示，MafBCKO中突触带状物的数量减少，相反，MafBOE中突触带状物的数量增加，这表明MafB是耳蜗中毛细胞和SGN之间突触正常形成所需的。这些作用可能部分取决于神经元趋化因子CCL 21，它激活神经系统其他区域的小胶质细胞。在MafBCKO SGN中，CCL 21表达显著降低。我们将使用MafB和CCL 21突变小鼠模型，以进一步确定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CCL 21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。下游靶点的鉴定将帮助我们了解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，并可能发现新的耳聋易感基因。