Epigenetic regulation of neuronal morphogenesis in development and regeneration

描述（申请人提供）：发育过程中的神经元形态发生，包括神经元迁移、极化、轴突生长/引导和树突发育，是形成功能性神经回路的关键步骤。这些过程的中断是许多神经发育障碍的基础。成人神经系统损伤后需要类似的神经元形态发生步骤来再生和修复受损的神经回路。不幸的是，哺乳动物中枢神经系统（CNS）的神经元失去了支持神经再生的内在能力。基因表达的协调调控不仅控制着发育过程中神经元的形态发生，而且是成熟中枢神经系统神经元内在再生能力丧失的基础。表观遗传调控，包括DNA甲基化和组蛋白修饰，正成为调控基因表达的主要细胞机制。然而，迄今为止，很少有研究调查了神经发育和再生过程中神经元形态发生是如何在表观遗传水平上调控的。最近的两项研究发现，组蛋白甲基转移酶EZH2是导致韦弗综合征的突变基因，韦弗综合征是一种发育障碍，表现为智力残疾和大脑体积增大（大头畸形）。EZH2使组蛋白赖氨酸27 （H3K27）甲基化。我们的初步研究表明，EZH2在有丝分裂后神经元的发育过程中高度表达。在功能上，EZH2是发育中的皮质神经元轴突生长所必需的，并调节几种神经元形态发生相关基因，如Slit2和Pten。此外，分化神经元中EZH2的条件缺失似乎会导致更大的大脑。在成人神经系统中，EZH2在中枢神经系统中的水平很低。然而，在周围神经损伤后，它在成人感觉神经元中急剧上调，同时H3K27去甲基化酶JMJD3/UTX下调。在功能上，敲低EZH2可能通过上调Pten和KLF4这两种众所周知的神经再生抑制因子，从而损害体外和体内的感觉轴突再生。相反，去甲基化酶JMJD3的异位下调足以促进体内轴突再生。基于这些结果，我们推测在神经发育过程中，EZH2和H3K27甲基化是神经元形态发生的关键表观遗传调控因子，在成人神经系统中，它们通过抑制神经再生抑制因子来支持神经再生的内在能力。在本研究中，我们将通过有丝分裂后神经元中EZH2的有条件敲除或过表达来研究EZH2在发育过程中对神经元形态发生的调控作用；2)研究EZH2和JMJD3/UTX对H3K27甲基化调控在哺乳动物轴突再生中是否必要和充分；3)阐明EZH2和H3K27甲基化调控发育和再生过程中基因表达的分子机制。

DESCRIPTION (provided by applicant): Neuronal morphogenesis during development, including neuronal migration, polarization, axon growth/guidance, and dendrite development, are crucial steps for forming the functional neural circuits. Disruption in these processes underlies many neurodevelopmental disorders. Similar neuronal morphogenesis steps are required in adult nervous system after injuries to regenerate and restore damaged neural circuits. Unfortunately, neurons in the mammalian central nervous system (CNS) lose their intrinsic ability to support neural regeneration. Coordinated regulation of gene expression not only controls neuronal morphogenesis during development, but also underlies the loss of intrinsic regeneration ability of mature CNS neurons. Epigenetic regulation, including DNA methylation and histone modification, is becoming a major cellular mechanism for regulation of gene expression. To date, however, very few studies have investigated how neuronal morphogenesis during neural development and regeneration is regulated at the epigenetic level. Two recent studies have identified the histone methyltransferase EZH2, which methylates histone 3 at lysine 27 (H3K27), as the mutant gene that causes the Weaver Syndrome, a developmental disorder showing intellectual disability and enlarged brain size (macrocephaly). Our preliminary studies have revealed that EZH2 is highly expressed in post-mitotic neurons during development. Functionally, EZH2 is necessary for axon growth of developing cortical neurons, and regulates several neuronal morphogenesis-associated genes, such as Slit2 and Pten. Moreover, conditional deletion of EZH2 in differentiated neurons seems to result in bigger brain. In the adult nervous system, the level of EZH2 is very low in the CNS. However, it is drastically up regulated in adult sensory neurons upon peripheral nerve injury, together with down regulation of H3K27 demethylases JMJD3/UTX. Functionally, knocking down EZH2 impairs sensory axon regeneration both in vitro and in vivo, probably through up regulation of Pten and KLF4, two well-known repressors of neural regeneration. Conversely, ectopically down regulation of the demethylase JMJD3 is sufficient to promote axon regeneration in vivo. Based on these results, we hypothesize that during neural development EZH2 and H3K27 methylation are key epigenetic regulators of neuronal morphogenesis, and in the adult nervous system they function to support the intrinsic neural regeneration ability through repressing neural regeneration repressors. In this proposed study we will 1) investigate the roles of EZH2 in regulation of neuronal morphogenesis during development by conditionally knock out or overexpress EZH2 in post- mitotic neurons, 2) investigate if regulation of H3K27 methylation by EZH2 and JMJD3/UTX is necessary and sufficient for mammalian axon regeneration, and 3) elucidate the molecular mechanisms by which EZH2 and H3K27 methylation regulate gene expression during development and regeneration.