TRPML1招募动力蛋白介导溶酶体与自噬体融合促进BDNF信号核转录在AD轴突营养不良的作用和机制研究

Axonal dystrophy is closely related to AD cognitive impairment, but its mechanism remains unclear. The applicant first reported that TRPML1 improves cognitive impairment of AD by regulating autophagy. Recent studies reported that TRPML1 is involved in the centripetal movement of lysosomes, and their fusion with autophagosomes. Preliminary experiment found that bafilomycin A1 inhibits lysosomes fusion with autophagosome, which blocks BDNF signal nuclear transcription, and induces axonal dystrophy. Over-expression of TRPML1 can improve axonal dystrophy. The CoIP experiment found that TRPML1 forms a protein complex with p62 and dynein. Hence, it is hypothesized that recruitment of dynein by combining TRPML1 with p62 can mediate centripetal movement of lysosomes as well as their fusion with autophagosomes, to promote BDNF signal nuclear transcription, repair axonal lesion and inhibit the progression of AD. To prove this hypothesis, we used the AD cell model and the transgenic mouse model to identify the effect of TRPML1 on the centripetal movement of lysosomes, their fusion with autophagosomes, BDNF signal nuclear transcription, axon and cellular function, using gene expression interference, electron and confocal microscopy as well as molecular and cell biology techniques , in order to elucidate its molecular mechanism in the regulation of AD axonal dystrophy through p62 and dynein. and thus provide new targets and theoretical and experimental basis for the treatment of AD.

轴突营养不良与AD认知障碍密切相关，其机制尚不明确。申请人首次报道TRPML1通过调节自噬改善AD认知障碍。新近文献显示TRPML1参与溶酶体向心运动及溶酶体与自噬体融合。预实验发现巴弗洛霉素A1抑制溶酶体融合自噬体后，BDNF信号核转录受抑制，轴突发生营养不良；过表达TRPML1能改善轴突营养不良；CoIP证实TRPML1与p62和动力蛋白形成蛋白复合体。故提出假说：TRPML1结合p62招募动力蛋白介导溶酶体向心运动及融合自噬体，促进BDNF信号核转录，修复轴突病变，阻止AD进展。为证明该假说，我们利用AD细胞模型和转基因小鼠模型，采用基因表达干预，电镜、激光共聚焦、分子细胞生物学技术明确TRPML1对溶酶体向心运动、溶酶体与自噬体融合、BDNF信号核转录、轴突和细胞功能的作用，阐明其通过p62和动力蛋白调控AD轴突营养不良分子机制。为AD治疗提供新的作用靶点及理论和实验依据。

轴突营养不良及线粒体自噬障碍是AD早期病理特征，其导致囊泡运输障碍、神经元死亡，是构成AD认知障碍的关键事件。我们体外研究 TRPML1 介导溶酶体与自噬体融合调控 BDNF 信号核转录对 AD 轴突营养不良的功能作用；TRPML1结合p62 招募动力蛋白调控溶酶体与自噬体融合对轴突营养不良的分子作用机制；在体验证 TRPML1 对 AD 轴突营养不良的功能和作用机制。体外研究BRUCE和STX17相互作用调控BDNF信号核转录对AD轴突营养不良的功能作用；miR-204靶向调节BRUCE和STX17之间相互作用对 AD 轴突营养不良的分子作用机制；在体验证上调BRUCE介导BDNF/TrkB信号通路在AD轴突营养不良的功能和作用机制。体外研究 miR-204靶向结合并调控TRPML1表达对线粒体损伤、ROS产生和线粒体自噬的功能作用；STATS介导miR-204/TRPLM1活性调节线粒体损伤、ROS产生和线粒体自噬的分子作用机制；在体验证下调miR-204抑制线粒体自噬和ROS产生在AD中的作用机制。基于上述研究结果表明，TRPML1与p62相互作用募集动力蛋白，促进自噬体-溶酶体融合，介导BDNF核转位，阻止AD小鼠轴突营养不良。TRPML1激活介导TRPML1/p62/dynein网络调节是防止神经轴突营养不良所必需的。抑制miR-204靶向上调BRUCE与STX17的相互作用，促进自噬体-溶酶体融合，介导BDNF核转位，预防轴突营养不良，通过miR-204/BRUCE/ STX17/BDNF轴介导自噬体-溶酶体融合影响AD轴突营养不良。激活STAT3介导下调miR-204靶向上调TRPML1表达，抑制AD中ROS的产生和线粒体自噬。通过激活STAT3/miR-204/TRPML1轴减少线粒体自噬和ROS产生抑制AD进展。为AD的治疗提供新的作用靶点及理论和实验依据。

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