Region & Developmental Stage Specific Deletion of MeCP2 in Mouse Brain

描述（由申请人提供）：本提案的主要目的是描述MeCP 2在特定脑区域和不同发育时间点介导Rett综合征（RTT）个体患者中观察到的行为表型的作用。我们最近开发了一种系统，其中小鼠脑中的基因可以通过以区域和时间特异性方式靶向递送编码Cre重组酶的腺相关病毒（AAV）来敲除。我们假设MeCP 2在特定脑区（海马、杏仁核和运动皮层）表达的停止将导致它们：1）根据靶向脑区表现出特定行为的改变; 2）提供参与RTT介导方面的神经回路框架。在这些实验中，除了区域特异性之外，我们将能够在两个不同的发育阶段（出生后1个月或出生后4个月）递送AAV-CRE。出生后一个月对应于小鼠出现症状之前的时间。通过这种方式，我们将能够看到出生后特定大脑区域中MeCP 2的缺失是否可以重现与疾病相关的特定症状。出生后四个月（症状出现后的时间点）MeCP 2的区域特异性缺失将使我们能够发现是否存在症状出现的发育关键期。如果在这种晚期缺失后仍然出现症状，这将表明MeCP 2在任何发育阶段的缺失都会导致功能缺陷，这表明MeCP 2除了在神经元发育中的作用之外，还在急性神经元功能中发挥作用。这一信息很重要，因为它可以提供一个框架，通过利用其独特的药理学特征，可以靶向特定的通路或特定的大脑区域来治疗Rett综合征。雷特综合征（RTT）是一种神经发育障碍，是导致女性智力低下和自闭症行为的主要原因之一。一般来说，受RTT影响的个体在5-48个月的年龄之前经历正常发育，此时发育问题发生。大多数RTT缺陷主要在CNS中表达，包括精神发育迟滞、自闭症样行为、癫痫发作、睡眠障碍、步态问题和刻板的手部运动。最近的研究表明，RTT是一种X连锁显性遗传疾病，在大多数情况下（至少76%）是由甲基CpG结合蛋白（MeCP 2）基因突变引起的，这些突变预计会导致MeCP 2功能丧失。虽然在大多数RTT病例中已经鉴定出这些突变，但目前MeCP 2功能丧失与RTT发病机制之间没有直接联系。为了更好地理解MeCP 2在介导RTT个体中观察到的行为表型中的作用，我们建议在大脑的特定区域中删除MeCP 2，然后在广泛的行为范式中检查这些动物。我们还将删除发育中小鼠（行为表型出现之前）和成年小鼠（行为表型出现之后）的MeCP 2基因，然后在广泛的行为模型中评估这些动物。这种方法将允许更清楚地解释MeCP 2在特定大脑区域以及发育时间点中介导与RTT患者中观察到的行为表型相似的行为表型中的作用。拟议的研究应该增加我们对MeCP 2在介导某些RTT相关行为中的作用的理解，并确定介导这些异常的神经回路。

DESCRIPTION (provided by applicant): The major objective of this proposal is to delineate the role of MeCP2 in specific brain regions and at distinct developmental time points in mediating behavioral phenotypes observed in individuals patients afflicted with Rett Syndrome (RTT). We have recently developed a system in which genes in the mouse brain can be knocked out by targeted delivery of an adeno-associated virus (AAV) encoding Cre recombinase in a regional and temporal specific manner. We hypothesize that cessation of MeCP2 expression in specific brain regions (hippocampus, amygdala and motor cortex) will cause them to: 1) exhibit alterations in specific behaviors depending on the brain region targeted; and 2) provide a framework of the neural circuitry that is involved in mediating aspects of RTT. In these experiments, in addition to regional specificity, we will be able to deliver AAV- CRE at two distinct stages of development (1 month after birth or 4 months after birth.). One month after birth corresponds to a time before appearance of symptoms in mice. In this way, we will be able to see whether deletion of MeCP2 in a specific brain region after birth can recapitulate specific symptoms associated with the disease. Region specific deletion of MeCP2 four months after birth (a time point after emergence of symptoms) will enable us to find out if there is a developmental critical period for appearance of the symptoms. If symptoms still emerge after this late deletion, this would suggest that loss of MeCP2 at any developmental stage causes functional deficiencies indicating a role for MeCP2 in acute neuronal function in addition to a role in neuronal development. This information is important because it may provide a framework whereby particular pathways or specific brain regions can be targeted for the treatment of Rett Syndrome by taking advantage of their distinctive pharmacological profiles. Rett's syndrome (RTT) is a neurodevelopmental disorder that accounts for one of the leading causes of mental retardation and autistic behavior in females. In general, individuals affected with RTT experience normal development up to the age of 5-48 months at which time developmental problems occur. Most RTT defects are predominantly expressed in the CNS, including mental retardation, autism-like behavior, seizures, disturbances of sleep, problems with gait, and stereotypical hand movements. Recent work has demonstrated that RTT is an X-linked dominant disorder that in most instances (at least 76%) results from mutations in the Methyl-CpG-binding protein (MeCP2) gene that are predicted to result in loss of function of MeCP2. While these mutations have been identified in the majority of RTT cases, there is currently no direct link between loss of function of MeCP2 and the pathogenesis of RTT. To better understand the role of MeCP2 in mediating the behavioral phenotypes observed in RTT individuals, we propose to delete MeCP2 in specific regions of the brain and then examine these animals in a broad array of behavioral paradigms. We will also delete the MeCP2 gene in developing mice (before the appearance of a behavioral phenotype) and in adult mice (after the appearance of a behavioral phenotype) and then assess these animals in a broad array of behavioral models. This approach will allow a clearer interpretation of MeCP2's role in specific brain regions as well as in developmental time points in mediating behavioral phenotypes similar to those observed in RTT patients. The proposed studies should increase our understanding of the role of MeCP2 in mediating certain RTT associated behaviors as well as identify neural circuits that mediate these abnormalities.