胆红素通过谷氨酸兴奋毒性作用诱发听神经病谱系障碍

Auditory neuropathy spectrum disorder (ANSD) is known as special performance of the hearing disorder and close connection with neonatal hyperbilirubinemia, while the underlying mechanisms and characteristics of damages in the auditory system remain unclear. Cochlear hair cell-specific synaptic structure (Ribbon bodies) is full of glutamate synaptic vesicles. When the envelope protein Otoferlin is activated by calcium, the process of releasing glutamate exocytosis start, which in turn regulate type I afferent synaptic endings and auditory nerve excitability strength. Excessive glutamate release will lead to excitotoxic damage to the target cells. Our early experiments confirmed that bilirubin induces ANSD in neonatal guinea pigs via myelin sheath lesions of auditory nerve fiber and decreasing of type 1 afferent endings beneath inner hair cells which fitting glutamate induced excitotoxicity damages. We speculate that bilirubin could activate excessive release of glutamate vesicles, there by induce excitotoxicity and ANSD by upregulating the calcium concentration and activating protein Otoferlin in hair cells. The purpose of this proposal is to explore the important role of glutamate excitotoxicity pathway in bilirubin-induced ANSD by using auditory evoked potential testing, ultrastructural morphology and quantitative observations, functional protein expression observations, calcium concentration by immunofluorescence test and patch clamp recording techniques. Our studies may shed light on the potential pathophysiological mechanisms and provide an efficient management of ANSD in clinical.

胆红素是导致听神经病谱系障碍(ANSD)的常见因素之一，发病机制有待明确。耳蜗毛细胞Ribbon突触富含大量谷氨酸囊泡，当囊膜蛋白Otoferlin被钙离子激活时，启动胞吐过程释放谷氨酸，继而调控I型传入突触末梢、听神经的兴奋强度。过量谷氨酸释放将导致靶细胞的兴奋毒性损害。我们新近发现，高胆红素血症豚鼠出现ANSD的典型听功能损害；I型传入突触末梢空泡化及听神经纤维数量减少系其病理基础，且损害特征拟合兴奋毒性表现。据此我们推测，胆红素通过上调毛细胞钙离子浓度激活Otoferlin蛋白，导致谷氨酸囊泡过度释放，诱发谷氨酸兴奋毒性和ANSD。本课题将联合听觉诱发电位测试、超微结构形态与定量观察、功能蛋白表达、免疫荧光毛细胞内钙离子浓度测试、膜片钳记录囊泡释放等技术，揭示谷氨酸兴奋毒性在胆红素所致ANSD中的重要作用。课题的完成将有助于揭示胆红素耳毒性机制，并为深化ANSD研究提供模型平台。

1）高胆红素血症是导致听神经病谱系障碍(ANSD)的常见因素之一，且感音神经性聋是高胆红素血症（黄疸）的常见后遗症，但损伤机制不明，使得临床缺乏有效监控及防治策略。我们运用多种研究方法，探索了胆红素所致ANSD 中的耳毒性机制。.2）主要研究内容包括：1.观察胆红素对离体培养的新生大鼠耳蜗和前庭器官的损伤效应，探索其损伤机制；2.谷氨酸兴奋毒性在胆红素耳毒性中意义的研究；3.胆红素对听觉中枢核团损害的形态学研究； 4. 胆红素对嗅觉中枢神经元兴奋性的影响及机制。.3）主要研究结果包括：1.胆红素在内耳前庭器官培养系统表现出对耳蜗和前庭神经元及其纤维和末梢损伤的作用机制（已刊发论文 Neurotoxicology，2018）；2.验证了谷氨酸兴奋毒性参与了胆红素耳毒性；3.通过胆红素对于脑干听觉核团神经元凋亡的研究结果，论证了胆红素对听觉中枢核团的神经毒性机制；4.胆红素对嗅觉中枢神经元兴奋性的影响及机制（已刊发论文，Scientific Reports， 2016）.4）科学意义：上述研究探索了胆红素所致听觉损害和前庭损害的潜在机制，为防止胆红素所致听力及前庭损害提供了新思路。同时也揭示了听神经病和前庭神经病的潜在发病机制。