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.

抽象的结节性硬化症复合物（TSC）是由TSC1或TSC2突变引起的发育障碍基因。 TSC基因的突变导致雷帕霉素复合物的机械靶标的过度激活1 （MTORC1），一种对开发至关重要的信号通路。具有TSC的人出现了高事件神经疾病，其中最普遍的是癫痫和自闭症谱系障碍（ASD）。尽管MTORC1抑制剂可以有效地减弱癫痫发作，但它们并不能改善TSC令人印象深刻的合并症。这是迫切需要更好地理解自闭症特征的病因以识别新型治疗方法。我们的实验室Hasdevelvedmousemodeloftscin，在开发皮质中表达了组成型活性的Rheb 培养基前额叶皮层（MPFC）的神经元，该区域在TSC中失调并与ASD有关。该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复杂的病因。使用此模型，我们的实验室发现了类似于本地MPFC连接的变化 TSC自闭症合并症的病理变化。而且，当异常帽依赖性翻译是通过激活翻译表示，预防了观察到的改变。尽管已在MPFC中检查了连通性，以多动MTORC1状态（参与行为）尚未评估表型，也没有对MTORC1对特定皮质下项目的影响 MPFC。为此，纹状体是一个引人注目的目标，因为它与MPFC紧密相互联系，并且社会通信行为不可或缺（核心在ASD中定义）。但是，TSC中的轴突连接性为尚未评估正在研究和MPFC-纹状体项目。此外，同时将翻译标准化可以减轻过度活跃MTORC1条件下的细胞改变，特异性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效率也是如此。因此，具体目的是：1）。确定MPFC-纹状体项目中的多动MTORC1是否有助于社会通信缺陷。 2）。确定早期MTORC1活性是否增加 MPFC的开发导致皮质 - 纹状体项目的改变，导致令人兴奋的抑制作用纹状体不平衡。 3）。确定帽依赖性翻译和下游分子是否改变有助于改变轴突连通性和社会通信缺陷。为了解决这些目标，在子宫里电穿孔（IUE）将用于增加MPFC锥体神经元中的MTORC1。发声和社交互动测试将评估表型，而灯表显微镜，轴突跟踪和电生理学将评估MPFC-纹状体连通性。潜在的候选分子下游翻译控制轴突生长将通过陷阱确定，SHRNA将被设计为评估效果将确定的基因在社会通信防御和MPFC-纹状体连通性的变化方面进行标准化。