耳蜗毛细胞PCP4过表达防治噪声性耳蜗突触损伤的机制研究

An effective treatment for synaptopathy in noise-induced hearing loss is not available, and local manipulation of synaptogenesis-related genes is a feasible approach to treating this condition. Results from our previous study showed that overexpression of neurotrophin-3 in hair cells partially promoted the synaptic repair and recovery of auditory function after exposure to noise. However, the protective effects are limited, and other relevant factors need to be explored. Purkinje cell protein 4 (PCP4) regulates synaptic plasticity via Ca2+/calmodulin (CaM) signaling and coordinates with nerve growth factor. The downregulation of PCP4 is associated with a variety of neurodegenerative diseases. Our preliminary experimental results indicate that cochlear PCP4 is highly expressed in the hair cells of neonatal mice, and the expression was significantly decreased after hearing was established. However, the physiological function of cochlear PCP4 is unknown. We hypothesize that PCP4 overexpression in hair cells can protect the cochleae against noise-induced synaptopathy. In this study, the role of the PCP4 gene in synaptic formation and the establishment of hearing was investigated using mouse models of conditional gene knock-out and knock-in in hair cells. Additionally, the protective effects of PCP4 against synaptopathy using PCP4 gene knock-in were evaluated before and after exposure to noise in adult mice, and the underlying mechanism involving Ca2+/CaM signaling was explored. The results from the present study will aid in understanding both the physiological function of PCP4 in cochleae and the protective effects against noise-induced synaptopathy, thus providing new insights into the clinical transformation and treatment of noise-induced hearing loss.

噪声性聋中耳蜗突触损伤尚无有效修复方法，局部调控突触形成相关基因是可行途径。我们前期研究发现毛细胞过表达神经营养因子3能促进噪声暴露后突触修复和听功能部分恢复。但作用尚局限，需探索其他途径。浦肯野细胞细胞蛋白4（PCP4）通过钙离子/钙调蛋白（CaM）信号调控突触可塑性，与神经生长因子有协调作用；其表达下调与多种神经退行性疾病有关。我们预实验提示耳蜗PCP4高表达于新生鼠毛细胞，在听觉建立后表达量显著下降，但其功能未明。我们提出毛细胞PCP4过表达能防治噪声性耳蜗突触损伤的假说。本项研究拟建立PCP4基因毛细胞敲除和敲入小鼠模型，明确其对耳蜗突触形成和听觉建立的作用；利用PCP4在成年小鼠噪声暴露前、后敲入模型，明确其对突触损伤的保护作用，并探索Ca2+/CaM信号在其中的作用。本项目能明确PCP4在耳蜗的生理功能，和对噪声性突触损伤的保护效果，为临床转化防治噪声性聋提供新思路。

耳蜗毛细胞的纤毛及毛细胞与传入神经突触是外周听觉系统易损结构。探索耳蜗毛细胞及其突触的损伤机制是防治各类感音神经性听力损失的基础。位于毛细胞静纤毛根部及表皮板的血影蛋白αII（SPTAN1）表达缺失能造成小鼠早发性进行性听力损伤。我们实验研究发现其表达缺失，导致了听觉建立前的静纤毛形态异常，以及听觉建立后的进行性听力下降。其通过整合素-粘着斑信号通路维持纤毛形态和听觉功能。表达于毛细胞的浦肯野细胞细胞蛋白4（PCP4）能调控突触可塑性，其表达下调与多种神经退行性疾病有关。我们实验提示耳蜗PCP4高表达于新生鼠毛细胞，在听觉建立后表达量显著下降。在首批构建的PCP4条件敲除小鼠中，杂合敲除后代无明显听力下降，而纯合条件敲除后代始终无法育得。目前已重新构建纯合全基因PCP4敲除小鼠，拟明确其对噪声性突触损伤的保护作用。