调控TFEB质核穿梭阻止螺旋神经元及耳蜗神经末梢退化变性的研究

The retrogression of spiral ganglion neurons (SGNs) and synaptic terminals following hair cell loss is one of the important changes in the morphology of the sensorineural hearing loss (SNHL). Unfortunately, the mechanism about the SGN retrogression is still unknown. In our preliminary studies, we found that some interesting changes such as lipofuscin accumulation, an autophagy degradation substrate P62 increase, lysosomal enzymes decline, and TFEB aggregation within the cytoplasm of the SGNs could be observed in the process of SGN and synaptic terminal degeneration. Based on our results, we hypothesized that autophagy dysfunction may be involved in the process of the SGN and synaptic terminal degeneration, and probably resulted from the disrupted TFEB nuclear translocation. Therefore, we consider that the improvement of the TFEB nuclear translocation may block or alleviate the SGN and synaptic terminal degeneration. In this study, the regulation of the TFEB nuclear translocation within the SGNs can be achieved by the round window application of the ERK2 siRNA or recombinant adeno-associated virus vector in the mice with SGN degeneration established with kanamycin. Then, following the regulation of TFEB nuclear translocation, the changes of autophagy-lysosome pathway were tested with the molecular biology and morphological technology, and the functional and morphological changes of SGNs and synaptic terminals were evaluated by the EABR measurement, confocal and electron microscopy. Further relevant studies were done to investigate the mechanisms and signaling pathways of the improvement of TFEB nuclear translocation blocking the SGN degeneration. This study would provide a new vision of the prevention and intervention of SGN degeneration and the potential future of prevention and treatment of SNHL.

耳蜗毛细胞破坏后螺旋神经元及耳蜗神经末梢退化变性是感音神经性耳聋的一个重要病理改变，其发生机制不明。前期研究发现：耳蜗毛细胞破坏后螺旋神经元内脂褐素积聚增多，自噬降解底物P62升高，溶酶体关键消化酶表达下降，TFEB集聚于胞质中不能入核。提示螺旋神经元及耳蜗神经末梢在其退化变性过程中有自噬失调现象存在，它可能为TFEB的质核穿梭障碍所导致，改善TFEB质核穿梭有望能够阻止或减轻螺旋神经元及耳蜗神经末梢的退化变性。本项目拟采用ERK2基因内耳转染过表达和RNA干扰技术调控耳蜗螺旋神经元退行性变模型小鼠螺旋神经元的TFEB质核穿梭，然后借用分子生物学和相关形态学方法研究TFEB质核穿梭调控后自噬-溶酶体途径相关因子的变化，再借助EABR、激光共聚焦、电镜等手段观察调控TFEB质核穿梭后螺旋神经元及耳蜗神经末梢形态与功能的改变，最后进一步研究其分子机制。本项目可为感音神经性耳聋的防治提供新思路。

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，此种退化变性在病理形态学上与许多神经退行性变疾病相类似。TFEB 为调控溶酶体功能及自噬形成等自噬-溶酶体降解通路的关键因子，调控TFEB 可改变神经退行性变疾病的进程。本研究在观察耳蜗毛细胞丧失后螺旋神经元退化变性过程中自噬失调、自噬-溶酶体信号轴、TFEB 动态变化的基础上，研究促进TFEB 入核穿梭改善SGNs 退化变性过程中的自噬失调及氧化应激，减轻耳蜗SGNs 的退化变性。采用耳毒性药物破坏小鼠耳蜗毛细胞诱导螺旋神经元退化变性，然后借助透射电镜、冰冻切片自发荧光观察等手段观察耳蜗SGNs 退化变性过程中其胞体内脂褐素的动态变化，Western blot、冰冻切片染色激光共聚焦观察检测耳蜗SGNs 退化变性过程中自噬水平蛋白LC3、Beclin-1、自噬底物蛋白p62、泛素化蛋白表达、溶酶体功能调控转录因子TFEB 质核分布的动态变化，Real-time PCR 检测溶酶体膜蛋白基因Lamp-1、溶酶体组织消化酶CSTB、CSTD mRNA 表达水平的动态变化，以了解耳蜗螺旋神经元退化变性过程中其自噬溶酶体信号通路和TFEB 穿梭入核的受损程度。然后利用mTOR抑制剂CCI-779 促进耳蜗SGNs 内TFEB 的入核穿梭，观察TFEB穿梭入核改善后能否减轻退化变性SGNs 的自噬障碍，降低其氧化应激水平，阻止或减轻耳蜗毛细胞破坏后SGNs 的退化变性。结果：小鼠耳蜗毛细胞破坏后SGNs 退化变性过程中其螺旋神经元胞体内有许多脂褐素堆积及自噬泡形成，表明有自噬参与其病理过程。随着耳蜗SGNs 退化变性的加重，其自噬水平升高，但自噬底物p62、及泛素化蛋白增多，TFEB 的细胞核质比不断下降，溶酶体生成及其主要消化酶的表达下降明显，显示自噬流受到了抑制。采用CCI-779 促进退化变性SGNs 胞体内的TFEB 穿梭入核后，其自噬受损现象明显改善，氧化应激水平降低，存留的SGNs数目显著增多，ABR 阈值降低。结论：小鼠耳蜗毛细胞丧失后SGNs 退化变性过程中存在TFEB 入核障碍和自噬受损。利用CCI-779 促进TFEB 穿梭入核后，能明显改善其自噬障碍，降低氧化应激水平，减轻SGNs 的退化变性。

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