Region & Developmental Stage Specific Deletion of MeCP2 in Mouse Brain

描述(由申请人提供)：这项建议的主要目标是描述MeCP2在特定大脑区域和不同发育时间点在调节雷特综合征(RTT)患者个体观察到的行为表型方面的作用。我们最近开发了一种系统，在该系统中，小鼠大脑中的基因可以通过靶向递送编码Cre重组酶的腺相关病毒(AAV)以区域和时间特异性的方式被敲除。我们假设，MeCP2在特定大脑区域(海马体、杏仁核和运动皮质)的停止表达将导致它们：1)根据目标大脑区域的不同，表现出特定行为的变化；2)提供参与调节RTT各个方面的神经电路的框架。在这些实验中，除了区域特异性，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)提供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脑区MeCP2的缺失是否可以概括与疾病相关的特定症状。出生后4个月(症状出现后的时间点)MeCP2的区域特异性缺失将使我们能够发现症状出现是否存在发育关键期。如果在这种晚期缺失后仍然出现症状，这可能表明MeCP2在任何发育阶段的缺失都会导致功能缺陷，这表明MeCP2除了在神经元发育中发挥作用外，还在急性神经元功能中发挥作用。这一信息很重要，因为它可能提供一个框架，使特定的路径或特定的大脑区域可以通过利用其独特的药理学特征来针对Rett综合征的治疗。雷特综合征(RTT)是一种神经发育障碍，是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影响的人在5-48个月大的时候会经历正常的发育，在这个时候会出现发育问题。大多数RTT缺陷主要表达在中枢神经系统，包括智力低下、孤独症样行为、癫痫发作、睡眠障碍、步态问题和刻板印象的手运动。最近的研究表明，RTT是一种X连锁的显性疾病，在大多数情况下(至少76%)是由甲基CpG结合蛋白(MeCP2)基因突变引起的，这些突变被预测导致MeCP2功能丧失。虽然这些突变已经在大多数RTT病例中被发现，但目前还没有发现MeCP2功能丧失与RTT发病机制之间的直接联系。为了更好地了解MeCP2在调节RTT个体中观察到的行为表型中的作用，我们建议删除大脑特定区域的MeCP2，然后在广泛的行为范式中研究这些动物。我们还将在发育中的小鼠(在行为表型出现之前)和成年小鼠(在行为表型出现之后)中删除MeCP2基因，然后在广泛的行为模型中评估这些动物。这一方法将使我们能够更清楚地解释MeCP2的S在特定脑区和发育时间点调节行为表型的作用，这与在RTT患者中观察到的类似。拟议的研究将增加我们对MeCP2在调节某些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.