Efr3a在小鼠耳蜗神经末梢和螺旋神经元退化变性中的作用及作用机制

Sensorineural hearing loss (SNHL) is one of the most common disease in clinical. Destruction of the sensory hair cells caused by various injurious exposure always leads to pathological changes of the spiral ganglion neurons (SGNs) and their synaptic terminals. The progressive secondary degeneration of SGNs and synaptic terminals that occurs following hair cells loss is morphologically similar to the pathology of the neurites degeneration of neurodegenerative disease. Efr3a may play an important role in the degeneration of neurites of neurodegenerative disorders. Our preliminary studies have shown that the hearing loss of mice with Efr3a knock-out is less following hair cell destruction than that of control mice. Thus, we hypothesized that Efr3a may be related to the degeneration of SGNs and their synaptic terminals. In this study, Efr3a knocking out and over-expression in the inner ear can be achieved by the Cre-LoxP recombination system and local application of lentiviral vector mediated gene transfection, for studying the functions of Efr3a in the kanamycin-induced degeneration of SGNs in mice. Then, the eABR measurement，confocal and electron microscopy and immunocytochemical techniques were used for evaluation of functional and morphological changes of SGNs and their synaptic terminals after kanamycin damage under different expression levels of Efr3a. Further relevant studies were done to investigate the regulating mechanisms and signaling pathways of Efr3a. This project will focus on clarifying the cellular and molecular mechanism involved in degeneration of SGNs and their synaptic terminals following a sensorineural hearing loss, which will provide new insight into the prevention and intervention of spiral ganglion neuron degeneration, the potential future of prevention and treatment of SNH as well as improvement of cochlear implant efficacy.

感音神经性耳聋耳蜗毛细胞破坏后，其耳蜗神经末梢和螺旋神经元常发生退化变性，此种退化变性和神经退行性疾病的神经变性在形态学上相类似。Efr3a 基因在神经退行性疾病的神经末梢变性中起重要作用。前期研究显示，小鼠Efr3a基因敲除后，其毛细胞破坏后的神经功能损伤较轻。我们推测Efr3a在毛细胞破坏后耳蜗神经末梢和螺旋神经元的退化变性中起有重要作用。本研究拟通过移动Cre-LoxP敲除技术和慢病毒载体耳蜗转染过表达技术分别构建小鼠Efr3a基因敲除和过表达动物模型，然后采用卡那霉素破坏动物模型耳蜗毛细胞诱导耳蜗神经末梢和螺旋神经元退化变性，再借助激光共聚焦、电镜、eABR等手段观察Efr3a不同表达丰度时其耳蜗神经末梢和螺旋神经元的形态及功能变化，最后进一步研究其相关作用机制。本项目对阐明感音神经性耳聋时耳蜗神经末梢和螺旋神经元退化变性的发生机制有重要意义，可为感音神经性耳聋的防治提供新的思路。

感音性耳聋中其耳蜗神经末梢和螺旋神经元的退化变性与神经退行性疾病的神经变性在形态学上相类似。研究显示，Efr3a 基因在神经退行性疾病的神经末梢变性中可能起有重要作用。本研究首先通过移动Cre-LoxP敲除技术和转基因技术构建小鼠耳蜗Efr3a基因敲减和过表达动物模型，然后采用耳毒性药物破坏模型动物耳蜗毛细胞以诱导耳蜗神经末梢和螺旋神经元发生退化变性，再借助相关功能及形态技术观察Efr3a不同丰度时小鼠耳蜗神经末梢和螺旋神经元的形态功能变化，最后进一步研究其作用机制。结果：成功构建了Efr3a基因敲除(Efr3a KD)和过表达（Efr3a OE）小鼠；ABR检测显示，Efr3a KD, OE及野生型（WT）成年小鼠听功能无明显差异，Efr3a KD老年小鼠其听功能下降较OE老年小鼠为轻。形态学观察发现，Efr3a KD老年小鼠SGNs存留数明显多于OE老年小鼠。注射卡那霉素和呋塞米后，小鼠耳蜗内外毛细胞及听功能均严重受损,但Efr3a KD小鼠的受损程度较WT小鼠明显为轻。给药破坏毛细胞后5d时，WT小鼠耳蜗螺旋神经节组织中的Efr3a蛋白表达明显增高。耳蜗螺旋神经元计数显示，随着给药后观察时间的延长，其数目逐渐减少。给药30d后，Efr3a KD小鼠耳蜗螺旋神经元的数目明显高于WT小鼠。电镜观察发现，所有小鼠耳蜗毛细胞破坏后其螺旋神经元均出现了退化变性，但Efr3aKD小鼠的变性程度较WT小鼠明显为轻。分子生物学检测结果显示，Efr3a KD小鼠耳蜗神经营养因子及其受体的表达均高于野生型小鼠，p-Akt在Efr3a KD老年小鼠中的表达明显高于老年OE小鼠。结论：Efr3a在小鼠耳蜗螺旋神经元及其神经末梢的退化变性中起有重要作用，它的降低或缺失能够明显减缓螺旋神经元及其神经末梢的退行性变，其机制可能与提高了耳蜗螺旋神经元中神经营养因子及其受体以及p-Akt的表达有关。