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（OLs）是唯一的髓鞘产生细胞。髓鞘提供绝缘 和营养支持的RGC轴突，并允许正常的视力。OL死亡和视神经脱髓鞘 神经脱髓鞘疾病的标志，往往损害视力，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 但将这些营养线索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，我通过将转录因子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的OL谱系细胞中高度表达 神经。我产生了一种新的TFEB条件小鼠系，并表明在小鼠大脑中，TFEB强烈地 通过特异性地促进髓鞘形成前OL细胞凋亡来拮抗髓鞘形成，同时 抑制OL成熟。我开发了一种基于活体成像的测定方法来模拟体内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， 这两个关键通路以前与脱髓鞘疾病有关。基于这些发现，我 我建议测试TFEB作为髓鞘形成前OL的关键传感器的假设， 细胞凋亡的轴突接触或营养支持的情况下，和细胞外信号调节 TFEB表达/活性通过GPCR-PKA轴控制OL成熟、髓鞘形成和髓鞘再生。 我将利用啮齿动物的视神经作为模型系统来测试这些假设。在我的K99阶段，我将 TFEB在视神经髓鞘形成中作用及其在视神经髓鞘再生中的作用 神经炎动物模型，利用我已经产生的细胞类型特异性TFEB条件突变体。我会 进行全基因组RNA测序实验，以确定TFEB调节的候选途径， 并将进一步验证这些途径与优化的体外培养系统和体内病毒 操纵工具包。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。最后作为 作为一名独立的研究者，我将采用无偏见的基因组和生物化学方法来确定直接的 TFEB的基因靶点和相互作用蛋白。我将利用K99阶段的动物手术技术培训 和病毒的方法来调节TFEB在促进髓鞘修复中的功能，以及在小鼠中的训练。 视觉行为，以评估视神经髓鞘再生中的视觉功能恢复。拟议的研究将 描述了一种调节视神经髓鞘形成的新途径，并将确定潜在的机制。 通过开发靶向TFEB通路的新药，我的目标是减轻视神经损伤， 视神经炎和多发性硬化患者的髓鞘再生和视力恢复。 !

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. !