Role of mTORC1 dependent translation in neurological deficits of TSC

摘要 结节性硬化症(TSC)是一种由TSC1或TSC2突变引起的发育障碍 吉恩。TSC基因突变导致雷帕霉素复合体1的机制靶点过度激活 (MTORC1)，一条对发育至关重要的信号通路。患有TSC的个体具有较高的发病率 神经系统疾病，其中最常见的是癫痫和自闭症谱系障碍(ASD)。 虽然mTORC1抑制剂可以有效地减轻癫痫发作，但它们并不能改善TSC的自闭症共病。 因此，迫切需要更好地了解自闭症特征的病因，以确定新的治疗方法。我们的 实验室已经建立了TSC的模型，其中成分活性的Rheb在发育中的皮质中表达 内侧前额叶皮质(MPFC)的神经元，该区域在TSC中调节失调，并与ASD有关。 这种模型允许比转基因模型更精确地激活mTORC1，因此非常适合于 解析出复杂的病因。使用这个模型，我们的实验室发现了本地mPFC连接的变化，类似于 自闭症共病的病理改变。此外，当异常的大小写依赖的翻译是 通过激活翻译抑制物而正常化，防止了观察到的改变。而当 已在超活动mTORC1状态下检查了mPFC内的连接性，随之而来的行为 尚未评估表型，也未评估mTORC 1‘S对特定皮质下投射的影响 MPFC。为此，纹状体是一个引人注目的靶子，因为它与mPFC密切相关，并且 社会交往行为(自闭症的核心缺陷)的组成部分。然而，TSC内的轴突连接是 未充分研究和mPFC-纹状体投射尚未得到评估。此外，在规范化翻译的同时 可以减轻细胞在高度活跃的mTORC1条件下的变化，特定的下游分子 解释这些变化的变化是未知的，它们减轻行为缺陷的有效性也是未知的。 因此，具体目标是：1)。确定mPFC-纹状体投射中是否存在过度活跃的mTORC1 造成社会沟通障碍。2)。确定是否在早期增加mTORC1活动 MPFC的发育导致皮质-纹状体投射的改变，导致兴奋抑制 纹状体不平衡。3)。确定改变的帽子依赖的翻译和下游分子 导致轴突连通性改变和社会沟通障碍。为了达到这些目标，在子宫内 电穿孔(IUE)将被用来增加mPFC锥体神经元的mTORC1。发声和发声 社会互动测试将评估表型，而光片显微镜、轴突追踪和 电生理学将评估mPFC-纹状体的连接性。潜在的候选分子下游 将通过Trap确定控制轴突生长的翻译，并将设计shRNA来评估其影响 关于社会沟通缺陷和mPFC-纹状体连接性改变的已确认基因的正常化。

ABSTRACT Tuberous sclerosis complex (TSC) is a developmental disorder caused by a mutation in the TSC1 or TSC2 gene. Mutations in TSC genes result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1), a signaling pathway vital for development. Individuals with TSC present with a high incidence of neurological conditions, among the most prevalent of which are epilepsy and autism spectrum disorder (ASD). While mTORC1 inhibitors can effectively attenuate seizures, they do not improve TSC’s autistic comorbidity. Thus, there is a critical need to better understand the etiology of autism traits to identify novel treatments. Our laboratoryhasdevelopedamousemodelofTSCin which constitutively active Rheb is expressed in developing cortical neurons of the medial prefrontal cortex (mPFC), a region that is dysregulated in TSC and implicated in ASD. This model allows for a more precise activation of mTORC1 than transgenic models and is thus ideally suited to parse out complex etiology. Using this model, our lab found alterations in local mPFC connectivity that resemble the pathological changes in TSC’s autistic comorbidity. Moreover, when aberrant cap dependent translation was normalized via the activation of a translational repressor, the observed alterations were prevented. While connectivity has been examined within the mPFC in a hyperactive mTORC1 state, the accompanying behavioral phenotype has not been assessed nor have mTORC1’s effects on specific subcortical projections from the mPFC. To this end, the striatum is a compelling target, as it is heavily interconnected with the mPFC and is integral to socio-communicative behaviors (core deficits in ASD). However, axonal connectivity in TSC is understudied and mPFC-striatal projections have not been assessed. Furthermore, while normalizing translation can mitigate cellular alterations in hyperactive mTORC1 conditions, the specific downstream molecular alterations that account for these changes are unknown, as are their efficacy to attenuate behavioral deficits. Therefore, the specific aims are to: 1). Determine whether hyperactive mTORC1 in mPFC-striatal projections contributes to socio-communicative deficits. 2). Determine whether increasing mTORC1 activity during early mPFC development leads to alterations in cortico-striatal projections resulting in an excitation-inhibition imbalance in the striatum. 3). Determine whether altered cap-dependent translation and downstream molecules contribute to alterations in axonal connectivity and socio-communicative deficits. To address these aims, in utero electroporation (IUE) will be used to increase mTORC1 in pyramidal neurons of the mPFC. Vocalization and social interaction tests will assess the phenotype, whereas lightsheet microscopy, axonal tracing, and electrophysiology will assess mPFC-striatal connectivity. Potential candidate molecules downstream of translation that control axonal growth will be identified via Trap, and shRNA will be designed to assess the effects of normalizing the identified genes on socio-communicative deficits and alterations in mPFC-striatal connectivity.