Filamin A in TSC

 描述(由申请人提供)：这项提案将检验这样一个假设，即降低结节性硬化症(TSC)中的丝氨酸A(Flna)水平或功能可以防止皮质畸形和相关的癫痫发作活动。TSC是由TSC1或TSC2突变导致mTOR复合体1(MTORC1)过度活动和皮质畸形引起的，与癫痫发作和认知和精神障碍的恶化有关。MTORC1抑制剂雷帕霉素是唯一的治疗选择，不能修复所有的缺陷，并且有轻微到危及生命的并发症，强调需要寻找新的药物靶点。我们最近报道，在Tsc1缺失神经元中，肌动蛋白交联分子Flna的水平增加，这是树突异常的原因。此外，在发育过程中，Flna还调节皮质神经元的迁移。这些发现对于TSC很有吸引力，原因如下：(1)神经元形态异常(包括树突异常)和迁移停滞是TSC相关皮质畸形的特征。(2)在Tsc1缺失神经元以及表达固有活性Rheb(mTORC1规范激活剂)的细胞中，Flna的增加不是mTORC1依赖的，而是ERK1/2依赖的，这为可能的联合治疗打开了一个新的药理学选择；以及(3)我们的初步数据表明，在我们的TSC相关皮质畸形的新模型中，使用shRNA使Flna水平正常化或使用新的Flna调节剂PTI-125可以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，我们将确定在活体内Flna是否控制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时间窗进行旨在预防皮质畸形和减少或消除相关癫痫活动的治疗。我们的新的小鼠局灶性皮质畸形模型与每日惊厥发作的高比率有关。最后，在目标3中，我们将研究Flna水平升高是如何导致畸形发育和迁移停滞的，这可能会发现参与TSC皮质缺陷的新的Flna结合伙伴。大多数实验将使用宫内电穿孔来选择性地操纵第2/3层皮质锥体神经元的发育。这是一项两项绩效指标的拨款。波尔迪实验室将负责目标1和目标2，考尔德伍德实验室将负责目标3。由于目标2和目标3分别需要开发工具(质粒)以及细胞生物学和活体实验，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，而Calderwood博士是一位在FLNA方面拥有丰富专业知识的细胞生物学家。

 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.