激活Akt信号诱导的代谢重编程促进Müller细胞逆分化

Structural and functional recovery after retinal ganglion cells (RGC) injury is the ultimate goal of regenerative research in glaucoma. It has been reported that Müller cells can be stimulated dedifferentiation and participate in endogenous regeneration, although the capacity of proliferation is limited. During nuclear reprogramming study, we demonstrated that activation of PI3K/Akt signaling upregulated the expression of several key enzymes related to glycolysis, and facilitated the transition of metabolism from somatic to stem cells (named metabolic reprogramming). It is necessary to study whether manipulation of metabolic reprogramming will improve Müller cells dedifferentiation since the process of dedifferentiation is similar to nuclear reprogramming. .In this proposal, using luciferase reporter system, we attempt to screen activator/inhibitor from Akt pathway which can activate metabolic reprogramming. In vitro, employing primary cultured Müller cells, we will explore the transition of metabolism and supermicro structure, cell proliferation, dedifferentration and the ability of differentiation into RGC after activation of Akt pathway. In vivo, we will also take advantage of lineage tracing techniques mediated by Cre-loxP recombination system to study the dedifferentration and endogenous regeneration of Müller cells. Our study will provide a better understanding of the endogenous regeneration for glaucoma.

视网膜神经节细胞(RGC)的结构与功能修复是青光眼神经再生的最终目标。研究发现，Müller细胞可被激活实现逆分化，参与内源性再生，但其增殖能力有限。我们在核重编程研究中发现，激活PI3K/Akt信号，能促进核重编程过程中糖酵解相关酶类表达，有助于体细胞实现代谢方式向干细胞转换（即代谢重编程）。Müller细胞的逆分化与核重编程重返多潜能干细胞状态相似，因此在Müller细胞中，调控代谢重编程是否有助于逆分化值得深入探讨。本项目采用荧光素酶报告系统，从Akt信号通路筛选具有活化代谢重编程的激活/抑制剂。在培养的Müller细胞中，通过激活Akt信号诱导代谢重编程，研究其对Müller细胞代谢、线粒体超微结构转变、细胞增殖、逆分化及其体外向RGC分化能力。在体内，通过Cre酶介导的谱系示踪，探讨Müller细胞的在体逆分化与内源性再生，为青光眼患者神经节细胞内源性再生提供理论和实验基础。

在本项目中，我们分析了视网膜祖细胞成熟过程中的代谢转变，初步探讨了视网膜祖细胞内源性再生的可能性。在对神经祖细胞的研究中，我们报道了Wnt信号通路对于调节神经干细胞内源性再生的关键作用，持续的Wnt信号激活将导致神经祖细胞延迟分化成熟。通过体内分子遗传手段，我们观察到Lgr5+细胞能产生新的视网膜神经元和Müller细胞, 提示Wnt通路对于视网膜神经干细胞的发育分化以及Amacrine 细胞的命运决定具有重要意义。总之，我们对神经祖细胞增殖分化、能量代谢，信号通路调控以及成体细胞重编程的研究，加深了对神经祖细胞生物特性的认识。本研究的相关报道为进一步探索视网膜神经内源性再生提供良好基础。