DLK介导自噬障碍促使毛细胞衰老在感音型老年性耳聋听力损失加速进展中的作用及机制

The phenomenon of accelerated process of sensorineural age-related hearing loss has been discovered, while the underlying mechanism is still unknown. In our previous studies, it was found that in C57BL/6J mice, which showed a typical characteristic of early presbycusis. The neurodegenerative related molecule DLK, as well as its downstream proteins JNK and VPS34 were significantly increased in the cochlear hair cells of C57BL/6J mice. When DLK was overexpressed in normal hair cell, the cellular senescence could be accelerated and autophagic dysfunction was observed. These results suggested that DLK might play an irreplaceable role during the aging process of cochlear hair cells in sensorineural age-related hearing loss. As a result, we speculate that DLK is involved in autophagic dysfunction during the aging of cochlear hair cells and it is through the JNK/VPS34 signaling pathway accelerating the progression of sensorineural hearing loss. This project is firstly aimed to observe the dynamic changes of DLK, JNK and VPS34, and autophagic level in hair cells in C57BL6/J mice. Then, the results of manipulating DLK and autophagy on the aging hair cells will be explored. In addition, we will generate model mice with DLK conditional knockout and overexpression in hair cells and focus on the morphological changes of hair cells and auditory function of these model mice during their aging process. Finally, whether DLK-mediated autophagic dysfunction is through JNK/VPS34 pathway that leads to aging of hair cells will be revealed. We firmly believe that this project can provide theoretical basis and intervention target for the prevention and treatment of sensorineural age-related hearing loss.

感音型老年性耳聋听力下降过程中存在加速进展现象，其发生机制不明。我们前期研究发现，早老性耳聋小鼠C57BL/6J听力损失加速进展时，神经退变相关分子DLK、其下游JNK、以及VPS34在耳蜗毛细胞中显著升高；将正常毛细胞过表达DLK时，可加速细胞衰老，并存在自噬障碍现象。提示DLK在老年性耳聋耳蜗毛细胞衰老中起有重要作用。我们推测，DLK通过JNK/VPS34信号诱导自噬障碍介入毛细胞衰老从而促使听力损失的加速进展。本项目拟首先观察C57BL/6J小鼠耳聋加速进展前后其耳蜗毛细胞中DLK、JNK、VPS34、自噬的动态变化，然后探究操控DLK及自噬对毛细胞衰老的影响，再构建毛细胞DLK条件性敲除和过表达小鼠模型，探索模型小鼠增龄过程中毛细胞形态和听功能的变化，最后再深入研究DLK是否通过JNK/VPS34介导自噬障碍导致毛细胞衰老，为老年性耳聋听力损失加速进展的防治提供理论依据及干预靶点。

耳蜗毛细胞衰老、死亡是人类老年性耳聋的主要病理表现之一。在临床观察中发现，老年性耳聋患者的听力损失大致可以分为三个时期：缓慢发展期、加速进展期及听力损失稳定期。我们对不同月龄C57BL/6J小鼠及CBA小鼠进行听力检测时发现，两种小鼠亦存在着人类老年性耳聋进程的特点。通过对小鼠的老年性耳聋不同发展阶段进行基因测序，我们锁定了变化差异最大的基因DLK（MAP3K12）。DLK是经典衰老信号通路MAPK的上游信号，也是神经元衰老和应激反应的重要感知因子，在一些神经退行性疾病的研究中已初步显示其出可能具有重要的干预价值。本项目拟探索操控小鼠耳蜗DLK/JNK通路能否延缓或减轻其老年性耳聋的快速进展及其机制。我们选用1月龄、6月龄、9月龄、12月龄的C57BL/6J小鼠作为实验对象，通过听性脑干反应（ABR）、耳蜗基底膜铺片观察在小鼠衰老过程中听力的下降与耳蜗毛细胞丢失情况，通过免疫组化观察DLK及其下游JNK通路在小鼠耳蜗衰老过程中的变化。用D-半乳糖诱导HEI-OC1毛细胞系构建衰老模型，并用慢病毒载体构建DLK过表达的毛细胞模型，采用western blot、免疫荧光、小分子抑制剂等手段观察操控DLK/JNK通路后耳蜗毛细胞的自噬水平变化及其衰老状态，以及操控其自噬水平后毛细胞的衰老状态变化。最后采用DLK抑制剂GNE3511对6月龄小鼠连续给药3月，观察是否能减轻小鼠衰老过程中其耳蜗毛细胞的自噬失调、毛细胞的丢失和听力阈值的升高。结果：在小鼠老年性耳聋的发生发展过程中，耳蜗组织伴随有DLK及下游JNK通路的显著上调。在毛细胞衰老模型中，其DLK/JNK通路及自噬有显著激活；抑制DLK/JNK通路后，其自噬水平降低。上调正常毛细胞的DLK，其自噬及衰老水平显著上升；抑制其自噬则可以减轻DLK上调导致的毛细胞衰老。将小鼠在体给与DLK抑制剂后，则可显著延缓小鼠耳蜗的衰老进展及其听力的快速损失进程。结论：在C57小鼠老年性耳聋的发生发展过程中，DLK是启动耳蜗毛细胞衰老的一个关键分子，DLK显著上调引起的自噬水平失调能导致毛细胞受损和衰老，使用DLK抑制剂可改善毛细胞的自噬失调，延缓衰老导致的小鼠听力快速损失。

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