mTOR Hyperactivity Disrupts the Molecular Framework of Inhibitory Synapses

项目摘要 脑硬化综合征（TSC）是由TSC 1或TSC 2基因的功能缺失突变引起的， 蛋白质产物结合并抑制雷帕霉素复合物1（mTORC 1）的哺乳动物靶标。反过来，mTOR 结果是过度活跃，这被认为是TSC的驱动病理特征。TSC患者遭受 从癫痫和自闭症谱系障碍（ASD），和TSC研究人员假设，癫痫发作的早期， 童年会导致ASD。目前的治疗方法旨在降低癫痫发作的可能性，但这些 药物有严重的副作用。因此，确定 分子机制，有助于癫痫发作，并提供新的药物治疗。是 表明兴奋和抑制之间的不平衡是癫痫发作和ASD的基础。具体地说， 抑制性突触蛋白表达在这些疾病中被破坏，并且可能是功能障碍的共同中心 自闭症和癫痫本研究旨在探讨抑制性突触在TSC中的失调。我们 假设TSC中mTOR过度活性通过以下途径抑制抑制性突触的形成 mTORC 1依赖性mRNA抑制。利用无偏见的生物信息学方法，我们确定了一个 推定的mTOR敏感抑制性突触蛋白，囊泡GABA转运蛋白（vGAT）-突触前蛋白 它将GABA和甘氨酸（抑制性神经递质）包装到突触囊泡中。我们将确定是否 RNA结合蛋白DJ-1的水平升高，在体内结合并抑制vGAT mRNA的翻译， 与TSC 1杂合子小鼠相比，TSC 1野生型小鼠的皮质。此外，通过从头蛋白质合成 通过检测，我们将检验mTORC 1抑制vGAT mRNA翻译的假设;我们将确定是否 雷帕霉素处理（mTORC 1抑制）和DJ-1敲低增加了Aim 1中vGAT的蛋白质合成。 最后，在目标2中，我们将利用两种互补的方法：体外电生理学和一种新的体外电生理学。 突触测量测定以确定在TSC中vGAT-桥蛋白突触的数目是否减少1 基因剂量依赖性损失，以及mTORC 1抑制是否挽救了这种缺陷。总的来说，这两个相关的 独立的目标将有助于阐明我们对蛋白质合成缺陷如何损害 抑制性突触的形成。

Project Summary Tuberous sclerosis complex (TSC) develops from loss of function mutations in the TSC1 or TSC2 gene, whose protein products associate and inhibit mammalian target of rapamycin complex 1 (mTORC1). In turn, mTOR hyperactivity results, which is considered to be the driving pathological feature of TSC. TSC patients suffer from epilepsy and autism spectrum disorders (ASDs), and TSC researchers hypothesize that seizures in early childhood can cause ASD. Current treatments aim to decrease the likelihood of seizure generation, but these medications have serious side effects. Therefore, it has become increasingly important to identify the molecular mechanism that contributes to seizures and to provide new pharmacological treatments. It is suggested that the imbalance between excitation and inhibition underlies seizures and ASD. Specifically, inhibitory synaptic protein expression is disrupted in these disorders and may be a common hub of dysfunction in ASDs and epilepsy. This study investigates how the inhibitory synapse is dysregulated in TSC. We hypothesize that mTOR hyperactivity in TSC represses the formation of inhibitory synapses through mTORC1-dependent mRNA repression. Utilizing an unbiased bioinformatics approach, we identified a putative mTOR-sensitive inhibitory synaptic protein, vesicular GABA transporter (vGAT)- a presynaptic protein which packages GABA and glycine (inhibitory neurotransmitters) into synaptic vesicles. We will determine if elevated levels of an RNA-binding protein, DJ-1, binds and represses vGAT mRNA translation in vivo in the cortex of TSC1 wildtype compared to TSC1 heterozygous mice. Additionally, with a de novo protein synthesis assay, we will test the hypothesis that mTORC1 represses mRNA translation of vGAT; we will determine if rapamycin treatment (mTORC1 inhibition) and DJ-1 knockdown increase protein synthesis of vGAT in Aim 1. Finally, in Aim 2, we will utilize two complimentary approaches: in vitro electrophysiology and a novel in vitro synapse measuring assay to determine if the number of vGAT-gephyrin synapses are decreased in a TSC1 gene dosage dependent loss, and if mTORC1 inhibition rescues this deficit. Collectively, these two related yet independent aims will help elucidate our mechanistic understanding of how protein synthesis deficits impair the formation of inhibitory synapses.