Filamin A in TSC

 描述（由申请人提供）：本提案将检验结节性硬化症（TSC）中细丝蛋白A（FLNA）水平或功能降低可预防皮质畸形和相关癫痫发作活动的假设。TSC是由TSC 1或TSC 2突变引起的，导致mTOR复合物1（mTORC 1）活动过度和与癫痫发作以及认知和精神缺陷恶化相关的皮质畸形。mTORC 1抑制剂雷帕霉素是唯一的治疗选择，不能挽救所有的缺陷，并具有轻度至危及生命的并发症，强调需要找到新的药物靶点。我们最近报道，肌动蛋白交联分子FLNA的水平增加Tsc 1 null神经元，这种增加是负责树突异常。此外，FLNA在发育过程中调节皮质神经元的迁移。这些发现对于TSC是有吸引力的，原因如下：（1）神经元畸形（包括树突异常）和停滞的迁移是TSC相关的皮质畸形的标志。(2)FLNA在Tsc 1缺失神经元以及表达组成性活性Rheb的细胞中增加（mTORC 1典型激活剂）不是mTORC 1依赖性的，而是ERK 1/2依赖性的，为可能的联合治疗开辟了一种新的药理学选择，和（3）我们的初步数据显示，使用shRNA或施用新的FLNA调节剂PTI-125，在我们的新的TSC相关的皮质畸形模型中，在发育过程中，防止神经元错位和畸形发生。为了解决我们的假设，我们有以下三个目标。在目标1中，我们将确定FLNA是否控制体内皮质锥体神经元的发育，以及在发育过程中降低FLNA水平是否可以防止皮质畸形。在目标2中，我们将检查新生儿是否有一个时间窗，用于预防皮质畸形和减少或消除相关癫痫发作活动的治疗。我们的新的局灶性皮质畸形小鼠模型与每日惊厥发作的高发生率相关。最后，在目标3中，我们将研究FLNA水平的增加如何导致畸形和停滞的迁移，这可能会发现新的FLNA结合伴侣参与TSC的皮质缺陷。大多数实验将使用子宫内电穿孔来选择性地操纵2/3层皮质锥体神经元的发育。这是一个2 PI的补助金。Bordey实验室将负责目标1和目标2，Calderwood实验室将负责目标3。由于需要工具（质粒）开发以及需要分别在目标2和3中进行细胞生物学和体内实验，这两个实验室将每周进行大量互动。Bordey博士是神经元发育和TSC方面的专家，Calderwood博士是一位细胞生物学家，在FLNA方面具有广泛的专业知识。

 DESCRIPTION (provided by applicant): This proposal will test the hypothesis that decreasing Filamin A (FLNA) levels or function in tuberous sclerosis complex (TSC) prevents cortical malformations and associated seizure activity. TSC is caused by mutations in TSC1 or TSC2 leading to mTOR complex 1 (mTORC1) hyperactivity and cortical malformations associated with seizures and worsening of cognitive and psychiatric deficits. The mTORC1 inhibitor rapamycin is the only therapeutic option, does not rescue all defects, and has mild to life-threatening complications emphasizing the need to find novel drug targets. We recently reported that the level of the actin cross-linking molecule FLNA is increased in Tsc1null neurons and that this increase is responsible for dendrite abnormalities. In addition, FLNA regulates the migration of cortical neurons during development. These findings are attractive with respect to TSC for the following reasons: (1) Neuronal dysmorphogenesis (including dendritic abnormality) and stalled migration are hallmarks of TSC-related cortical malformations. (2) FLNA increase in Tsc1null neurons as well as in cells expressing a constitutively active Rheb (the mTORC1 canonical activator) was not mTORC1-dependent but rather ERK1/2-dependent, opening a novel pharmacological option for a possible combination therapy, and (3) Our preliminary data show that normalizing FLNA levels using shRNA or administrating the new FLNA modulator, PTI-125, during development prevents neuronal misplacement and dysmorphogenesis in our new model of TSC-related cortical malformations. To address our hypothesis we have the following three aims. In Aim 1, we will determine whether FLNA controls development of cortical pyramidal neurons in vivo and whether decreasing FLNA levels during development prevents cortical malformations. In Aim 2, we will examine whether there is a time-window in neonates for treatments aimed at preventing cortical malformations and reducing or eliminating associated seizure activity. Our new murine model of focal cortical malformations is associated with a high rate of daily convulsive seizures. Finally in Aim 3, we will investigate how increased FLNA levels leads to dysmorphogenesis and stalled migration, which may identify novel FLNA binding partners involved in cortical defects in TSC. Most experiments will use in utero electroporation to selectively manipulate the development of layer 2/3 cortical pyramidal neurons. This is a 2 PIs grant. The Bordey lab will be in charge of Aim 1 and 2, and the Calderwood lab will be in charge of Aim 3. Both labs will heavily interact on a weekly basis due to the need for tool (plasmid) development and the need for cell biology and in vivo experiments in Aim 2 and 3, respectively. Dr. Bordey is an expert on neuronal development and TSC, and Dr. Calderwood is a cell biologist with extensive expertise on FLNA.