TFEB/TFE3介导的溶酶体功能代偿对脊髓损伤的促修复作用及调控机制研究

Spinal cord injury is a common traumatic disease of the central nervous system. However, an effective target to repair spinal cord injury hasn't been found in the world. It was reported that the stimulation of autophagy can promote spinal cord injury recovery. It was also found that lysosomal dysfunction can inhibit autophagy activity directly. But there are few studies on lysosome in spinal cord injury. Our preliminary data showed that the activities of lysosome and autophagy enhanced in the moderate injury of spinal cord compression, but depressed in the severe one. To further understand the mechanism of this phenomenon, we will verify whether reactive oxygen species (ROS) can result in the compensation/ decompensation of lysosome activity induced by the aggravation of spinal cord injury. On the other hand, it was found that transcriptional factors TFEB and TFE3 regulation can reduce the level of ROS and promote the compensation of lysosome activity. Therefore, we will regulate the activities of TFEB and TFE3 to enhance the compensation of lysosome activity and further promote the repair of spinal cord injury. Furthermore, we will explore whether the activities of TFEB and TFE3 can be affected by the regulation of AMPK-SPK2-CARM1 signaling pathway and AMPK-mTOR signaling pathway in the nucleus and outside the nucleus, respectively. This study is to elucidate a pathological mechanism of lysosome functional compensation/decompensation in spinal cord injury, and to provide a new strategy of taking the regulation of TFEB and TFE3 activities to enhance the compensation of lysosome activity as a target to promote spinal cord injury recovery.

脊髓损伤是常见的中枢神经创伤性疾病，尚缺乏有效治疗靶点。自噬激活能促进脊髓损伤的修复。溶酶体功能障碍则直接抑制自噬，但脊髓损伤中关于溶酶体的研究尚少。前期发现，脊髓压迫伤后溶酶体及自噬的活性，在中度损伤中增强，在重度损伤中抑制。为明确该现象的病理机制，本研究将在脊髓损伤中，验证活性氧簇（ROS）介导损伤加剧所致的溶酶体功能代偿/失代偿。转录因子TFEB/TFE3调控能降低ROS水平，并促进溶酶体活性代偿。且前期发现TFEB/TFE3活性在脊髓损伤后均显著增强。本研究将探究通过分别调控TFEB/TEF3增进溶酶体功能代偿，以促进脊髓损伤的修复。并验证脊髓损伤后TFEB/TFE3活性受细胞核内外AMPK-SPK2-CARM1及AMPK-mTOR通路调控。本项目旨在阐明脊髓损伤中溶酶体功能代偿/失代偿的病理机制，并提出基于TFEB/TFE3活性调控增强溶酶体功能代偿，以促进脊髓损伤修复的新策略。

脊髓损伤（spinal cord injury, SCI）是中枢神经系统的一种灾难性疾病，可导致受累部位出现不可逆性的感觉和运动功能丧失。脊髓损伤包括原发性和继发性损伤两大病理阶段。原发性损失在瞬间发生，对神经组织的破坏严重且不可逆转；继发性损伤是原发损伤后的一系列病理性事件，如缺血、炎症反应及钙离子超载等，最终导致神经细胞的凋亡及坏死。该过程可逆且可被调控的，其转归对损伤修复有重要意义。近年来，自噬在脊髓损伤中扮演的角色受到越来越多的关注。自噬调控能通过改善炎症环境及抑制神经元死亡，促进脊髓损伤结构和功能的综合修复。然而，脊髓损伤后可引发自噬性细胞死亡，此时上调自噬水平反而加重脊髓损伤的神经破坏程度。在细胞病理状态下，溶酶体的功能障碍往往造成自噬流的阻滞。目前，在脊髓损伤中，对神经细胞溶酶体及自噬活性进行综合评估的研究尚少。本课题组前期研究脊髓损伤严重程度是神经细胞溶酶体功能及自噬流代偿与否的关键因素，大鼠在重度脊髓压迫伤后，应用TFEB及TFE3 活性激动剂海藻糖，能激活溶酶体相关膜蛋白、蛋白组织酶、质子泵V型H+-ATP 酶，以促进溶酶体功能代偿，进而促进自噬流增强及脊髓损伤的修复，同时在大鼠脊髓损伤后早期，病灶脊髓中磷酸化的 AMPK水平呈现显著的上升。本项目旨在阐明脊髓损伤中溶酶体活性代偿/失代偿的病理机制，并提出基于TFEB/TFE3 的活性调控，以增进溶酶体功能代偿，从而促进脊髓损伤修复的新策略，为将来的临床应用提供理论依据。本项目发现在压迫性脊髓损伤中，神经细胞内溶酶体活性及自噬流强度在中度损伤后增强，在重度损伤后抑制。脊髓损伤中 ROS 可能介导损伤加剧所致的溶酶体功能代偿/失代偿。同时通过促进溶酶体功能代偿，改善自噬溶酶体途径，对脊髓损伤具有神经修复作用。此外，本研究通过构建TFEB KI 小鼠及 TFE3 KI 小鼠模型分别调控转录因子 TFEB 及TFE3 增强溶酶体功能代偿，以促进脊髓损伤后的神经修复。同时激活核内外AMPK-SPK2- CARM1及 AMPK-mTOR 信号通路，协同促进 TFEB 及 TFE3 活性的增强。为今后脊髓损伤治疗的临床转化应用提供理论依据。