DNA Hypomethylation and Cortical Neuronal Degeneration

描述（由申请人提供）：在哺乳动物中，DNA胞嘧啶甲基化是调节许多细胞事件（包括发育基因表达和基因组印记）的主要表观遗传因素之一。DNA甲基化机制的改变与几种精神发育迟滞疾病有关，包括Rett、ICF、脆性X和ATRX综合征，表明甲基化对神经元发育和功能很重要。为了研究DNA甲基化在中枢神经系统（CNS）中的作用，我们最近应用cre/loxP系统删除了CNS中唯一的维持DNA甲基转移酶Dnmtl基因。通过将Emx 1-cre转基因与Dnmtl条件等位基因（Dnmt 2lox）杂交，我们获得了Dnmtl缺陷仅限于皮质和海马的条件敲除小鼠。Emx 1-cre介导的Dnmtl基因缺失起始于胚胎（E）9-10天的皮层前体细胞，导致胚胎和出生后皮层投射神经元的DNA低甲基化。突变小鼠在成年后仍能存活，但表现出明显的行为缺陷，如活动过度和尾巴悬挂时后肢紧握。形态学研究表明，Emx 1-cre; Dnmtl突变小鼠表现出大量的皮质体积损失，从而成为研究DNA低甲基化对皮质变性影响的有价值的动物模型。在这个提议中，我们计划首先检查皮层神经元细胞死亡的时间过程，并确定突变小鼠的总体组织学和行为缺陷。设计进一步的实验以确定DNA低甲基化对前体细胞的增殖和分化以及Dnmtl-/-神经元的树突状分支的影响。最后，我们计划确定DNA低甲基化诱导突变皮层神经细胞死亡的机制。众所周知，DNA甲基化水平随着年龄的增长和与年龄相关的神经退行性疾病如阿尔茨海默病而下降。了解低甲基化皮层神经元的细胞死亡机制可能有助于我们开发治疗策略，以防止低甲基化诱导的神经元变性。

DESCRIPTION (provided by applicant): In mammals DNA cytosine methylation is one of the major epigenetic factors that regulate many cellular events including developmental gene expression and genomic imprinting. Alternations in DNA methylation machinery have been linked to several mental retardation disorders, including Rett, ICF, Fragile-X, and ATRX syndromes, suggesting that methylation is important for neuronal development and function. To investigate the function of DNA methylation in the central nervous system (CNS), we have recently applied the cre/loxP system to delete the maintenance DNA methyltransferase Dnmtl gene exclusively in the CNS. By crossing the Emx1-cre transgene with the Dnmtl conditional allele (Dnmt2lox), we have obtained conditional knockout mice with Dnmtl deficiency restricted to the cortex and hippocampus. Emx1-cre mediated Dnmtl gene deletion is initiated in pallial cortical precursor cells at embryonic day (E) 9-10, resulting in DNA hypomethylation in embryonic and postnatal cortical projection neurons. Mutant mice are viable in adulthood but exhibit obvious behavioral defects such as hyperactivity and hind limb clasping upon tail suspension. Morphological studies indicate that Emx1-cre; Dnmtl mutant mice exhibit massive loss of cortical volume, thus become a valuable animal model for studying the effect of DNA hypomethylation on cortical degeneration. In this proposal, we plan to first examine the time course of cortical neuronal cell death and determine the gross histological and behavioral defects in the mutant mice. Further experiments are designed to determine the effect of DNA hypomethylation on the proliferation and differentiation of precursor cells, as well as dendritic arborization of Dnmtl-/- neurons. Finally, we plan to determine the mechanism by which DNA hypomethylation induces neuronal cell death in the mutant cortex. It is known that levels of DNA methylation decrease with aging and in age-related neurodegenerative disorders such as Alzheimer's disease. Understanding the mechanism of cell death in hypomethylated cortical neurons may help us develop therapeutic strategies to prevent hypomethylation-induced neuronal degeneration.