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Role of mTORC1 dependent translation in neurological deficits of TSC

mTORC1 依赖性翻译在 TSC 神经功能缺陷中的作用

基本信息

批准号：
10689021
负责人：
Matthew Binder
金额：
$ 1.55万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10689021
关键词：
Acceleration Address Anatomy Attenuated Axon Behavior Behavioral Binding Proteins Bioinformatics Brain CRISPR/Cas technology Caregivers Clinical Complex Corpus striatum structure Development Disease Electrophysiology (science)Electroporation Epilepsy Etiology Eukaryotic Initiation Factors FRAP1 gene Genes High Prevalence Hyperactivity Hypertrophy Incidence Individual Knowledge Laboratories Laboratory Finding Lead Light Medial Microscopy Modeling Molecular Mus Mutation Neurologic Neurologic Deficit Neurons Pathologic Patients Phenotype Population Prefrontal Cortex Property Ras homolog enriched in brain Research Role SDZ RAD Seizures Signal Pathway Signal Transduction Social Behavior Social Interaction Synapses TSC1 gene TSC2 gene Testing Therapeutic Transgenic Model Translations Tuberous Sclerosis Ultrasonics Western Blotting autism spectrum disorder autistic axon growth behavioral phenotyping comorbidity design developmental disease effective therapy hippocampal pyramidal neuron in utero inhibitor insight mouse model neural novel prevent small hairpin RNA social synaptic function trait transcriptome sequencing translatome vocalization

项目摘要

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.

摘要多发性硬化症（TSC）是由TSC 1或TSC 2突变引起的发育障碍。基因TSC基因突变导致雷帕霉素复合物1的机制靶点超活化（mTORC 1），一种对发育至关重要的信号通路。TSC患者的高发病率神经系统疾病，其中最普遍的是癫痫和自闭症谱系障碍（ASD）。虽然mTORC 1抑制剂可以有效地减弱癫痫发作，但它们不能改善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。我们实验室已经开发了一种TSC模型，其中组成型活性Rheb在发育中的皮质中表达内侧前额叶皮层（mPFC）的神经元，这是一个在TSC中失调并与ASD有关的区域。该模型允许比转基因模型更精确地激活mTORC 1，因此理想地适合于解析复杂的病因使用这个模型，我们的实验室发现了局部mPFC连接的改变， TSC自闭症共病的病理变化。此外，当异常的帽依赖性翻译被激活时，通过激活翻译阻遏物使其正常化，可以防止观察到的改变。而在过度活跃的mTORC 1状态下，在mPFC内检查了连接，伴随的行为表型尚未评估，也没有mTORC 1对特定的皮质下投射的影响，前额叶皮层。为此，纹状体是一个引人注目的目标，因为它与mPFC密切相关，社会沟通行为的组成部分（ASD的核心缺陷）。然而，TSC中的轴突连接是研究不足，尚未评估mPFC-纹状体投射。此外，在规范翻译的同时，可以减轻过度活跃mTORC 1条件下的细胞改变，特异性下游分子解释这些变化的改变是未知的，它们减弱行为缺陷的功效也是未知的。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mTORC 1 会导致社交障碍2）的情况。确定早期增加mTORC 1活性 mPFC的发展导致皮质-纹状体投射的改变，从而导致兴奋-抑制纹状体的不平衡3）。确定是否改变了帽依赖性翻译和下游分子有助于轴突连接和社会交流缺陷的改变。为了实现这些目标，电穿孔（IUE）将用于增加mPFC的锥体神经元中的mTORC 1。发声和社会互动测试将评估表型，而光片显微镜，轴突追踪，电生理学将评估mPFC-纹状体连接。潜在的候选分子下游将通过Trap鉴定控制轴突生长的翻译，并设计shRNA以评估其作用正常化识别的基因对社会沟通的缺陷和mPFC-纹状体连接的改变。