Identification of a novel pathway that regulates optic nerve myelination and remyelination

项目总结 在视神经中，少突胶质细胞是唯一的髓鞘生成细胞。髓磷脂提供隔热 以及对RGC轴突的营养支持，并允许正常视力。白斑狼疮的死亡和视神经脱髓鞘 神经是通常损害视力的脱髓鞘疾病的特征，包括视神经炎和多发性 硬化症。控制OL存活和脱髓鞘的信号机制仍然知之甚少。 以前的研究已经确定了无数的细胞外信号和OL细胞表面受体介导OL 生存和分化，但将这些营养信号与下游事件联系起来的内在途径仍然存在 难以捉摸。在我的初步研究中，我有了一个惊人的发现，将转录因子EB(TFEB)鉴定为 缺失的一环。我发现TFEB在包括视神经在内的中枢神经系统中的OL系细胞中高度表达 很有胆量。我产生了一种新的TFEB条件性小鼠株系，并在小鼠的大脑中显示出TFEB强大的 通过特异性促进髓鞘形成前OL细胞的凋亡和同时 抑制OL成熟。我开发了一种基于活体成像的分析方法来模拟活体OL的发育， 证明TFEB直接调节整合应激反应和GPCR-PKA的基因表达 途径，两个关键途径，以前涉及脱髓鞘疾病。基于这些发现，我 建议检验TFEB在髓鞘前白斑中作为关键感受器的假设，以促进 在缺乏轴突接触或营养支持的情况下细胞凋亡，以及细胞外信号调节 TFEB通过GPCR-PKA轴的表达/活性控制OL的成熟、髓鞘形成和再髓鞘形成。 我将利用啮齿类动物的视神经作为模型系统来检验这些假说。在我的K99阶段，我会 探讨TFEB在发育过程中视神经髓鞘形成中的作用及其在视神经再髓鞘形成中的作用 神经炎动物模型，利用我已经产生的细胞类型特异性TFEB条件突变。这就做 进行全基因组RNA测序实验以确定TFEB调控的候选途径， 并将利用优化的体外培养系统和体内病毒进一步验证这些途径 操作工具包。为了评估视神经髓鞘再生和修复的功能效果，我将获得 来自指导实验室的动物视觉行为和立体定位注射技术方面的专业知识。最后，由于 作为一名独立的研究人员，我将使用公正的基因组和生化方法来鉴定直接 TFEB的基因靶点和相互作用蛋白。我将利用K99阶段的动物外科技术培训 以及病毒途径调节TFEB功能促进髓鞘修复，以及在小鼠身上的训练 评估视神经髓鞘重建术中视功能恢复的视觉行为。拟议的研究将 描述一种新的调节视神经髓鞘形成的途径，并将确定潜在的机制。 通过开发针对TFEB途径的新药，我的目标是减轻视神经损伤并促进 视神经炎和多发性硬化症患者的重新髓鞘形成和视力恢复。 好了！

Project Summery In the optic nerve, oligodendrocytes (OLs) are sole myelin-producing cells. Myelin provides insulation and trophic support for RGC axons and allows for normal vision. Death of OLs and demyelination in the optic nerve are the hallmarks of demyelinating diseases that often impair vision, including optic neuritis and multiple sclerosis. The signaling mechanisms that controls OL survival and demyelination are still poorly understood. Previous studies have identified a myriad of extracellular cues and OL cell surface receptors that mediate OL survival and differentiation but the intrinsic pathways that link these trophic cues to downstream events remain elusive. In my preliminary studies, I made a striking discovery by identifying Transcription Factor EB (TFEB) as the missing link. I discovered that TFEB is highly expressed by OL lineage cells in the CNS including the optic nerve. I generated a novel TFEB conditional mouse line and showed that in the mouse brain TFEB powerfully antagonizes myelination by specifically promoting premyelinating OL cell apoptosis and simultaneously inhibiting OL maturation. I developed a live-imaging based assay to model in vivo OL development, and demonstrated that TFEB directly regulates gene expression of the integrated stress response and GPCR-PKA pathways, two critical pathways previously implicated in demyelinating diseases. Based on these findings, I propose to test the hypotheses that TFEB serves as the critical sensor in premyelinating OLs that facilitates cell apoptosis in the absence of axonal contact or trophic support, and that extracellular signals modulate TFEB expression/activity through the GPCR-PKA axis to control OL maturation, myelination, and remyelination. I will utilize the rodent optic nerve as a model system to test these hypotheses. In my K99 phase I will investigate TFEB function in optic nerve myelination during development and its roles in remyelination in optic neuritis animal models, utilizing cell-type specific TFEB conditional mutants that I have already generated. I will perform whole-genome RNA sequencing experiments to identify the candidate pathways that TFEB regulates, and will further validate these pathways with the optimized in vitro culture system and the in vivo viral manipulation toolkits. To assess the functional efficacy of optic nerve remyelination and repair, I will acquire expertise in animal visual behaviors and stereotaxic injection techniques from the mentoring labs. Finally, as an independent investigator, I will employ unbiased genomic and biochemical approaches to identify the direct gene targets and interacting proteins of TFEB. I will leverage K99 phase training in animal surgery techniques and viral approaches to modulate TFEB function in promoting myelin repair, as well as the training in mouse visual behaviors to assess visual function recovery in optic nerve remyelination. The proposed research will characterize a novel pathway regulating optic nerve myelination and will determine the underlying mechanisms. By developing new drugs that target the TFEB pathway, my goal is to lessen optic nerve damage and promote remyelination and visual recovery in patients with optic neuritis and multiple sclerosis. !