Functional Analysis of Pathogenic Human PTEN Variants in Early Neurodevelopment

人类致病性 PTEN 变异在早期神经发育中的功能分析

基本信息

批准号：
10605038
负责人：
Marina Alise Carlson
金额：
$ 4.77万
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：
2022-12-16 至 2025-10-15
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10605038
关键词：
Address Affect Alleles Automobile Driving Behavior Behavior Therapy Behavioral Biological Brain CRISPR/Cas technology Cell Cycle Cell Differentiation process Cell Proliferation Cells Cerebral Ventricles Child Childhood Clinical Computer software Congenital Hydrocephalus Data Disease Drug Targeting Embryo Exhibits Exons FRAP1 gene Fertility Fishes Frameshift Mutation Gene Dosage Genes Genetic Genome Goals Head circumference Heterozygote Human Human Pathology Immunohistochemistry Injections Investigation Larva Libraries Liquid substance Macrocephaly Messenger RNA Metabolic Pathway Methods Modeling Molecular Mutation Neurodevelopmental Disorder Neurons Operative Surgical Procedures Outcome Outcomes Research PTEN gene Pathogenesis Pathogenicity Pathology Pathway interactions Patient-Focused Outcomes Pharmacological Treatment Phenotype Play Pre-Clinical Model Prevalence Process Proliferating Protein Biosynthesis Proto-Oncogene Proteins c-akt Reporting Research Rest Role Screening procedure Sirolimus Startle Reaction Symptoms System Testing United States Variant Western Blotting Zebrafish autism spectrum disorder autistic children behavioral phenotyping brain volume cell growth disorder risk early childhood exome sequencing fetal genetic variant high-throughput drug screening improved in vivo inhibitor insight loss of function mTOR Signaling Pathway member mutant neural neural circuit neurodevelopment neurogenesis new therapeutic target novel novel strategies pharmacologic pleiotropism protein protein interaction risk variant screening targeted treatment teleost

项目摘要

Project Summary/Abstract PTEN is highly associated with autism, macrocephaly, and congenital hydrocephalus, which are increasingly prevalent neurodevelopmental disorders that present in early childhood. Currently, there are no treatments that address the cause of these conditions, despite mounting genetic evidence through large scale whole exome sequencing studies that mutations in specific genes, including PTEN, confer increased risk for these disorders. While these studies produce compelling targets for investigation, the role of PTEN in neurodevelopmental disorders remains poorly understood. We seek to understand how PTEN, through its role as a negative regulator of the greater mTOR pathway, contributes to early neurodevelopment using CRISPR-Cas9 loss-of-function models in zebrafish. We have generated zebrafish mutants with frameshift mutations in exon 5 of both pten alleles present in the teleost duplicated genome: ptena (Δ10) and ptenb (Δ2). We first aim to characterize neurodevelopmental abnormalities in these fish, which preliminary data indicates have significant differences in brain volume, brain activity, brain ventricle size, and startle response. We will further explore the function of PTEN in regulating cell proliferation, differentiation, and establishment of the excitatory and inhibitory circuits in the brain. Secondly, we aim to use this model to provide a new and accessible tool for screening the PTEN variants identified in children with neurodevelopmental disorders, by injecting embryos with human mRNA constructs at the 1-cell stage and evaluating neurodevelopmental changes. Lastly, we will perform a high-throughput drug screen of mTOR pathway inhibitors and additional compounds which oppose or match the behavioral changes observed in our mutant larvae. This will identify new potential pharmacological candidates. Our preliminary data already shows partial phenotype rescue upon treatment with mTORC1-inhibitor sirolimus. In summary, these loss of function lines demonstrate PTEN plays a critical role in early vertebrate neurodevelopment. Furthermore, these models provide a new and accessible tool for screening the PTEN variants identified in neurodevelopmental disorders as well as potential pharmacological candidates.

项目概要/摘要 PTEN 与自闭症、大头畸形和先天性脑积水密切相关，这些疾病越来越多儿童早期普遍存在的神经发育障碍目前尚无治疗方法。尽管通过大规模全外显子组获得了越来越多的遗传证据，但仍需解决这些病症的原因测序研究表明，包括 PTEN 在内的特定基因的突变会增加这些疾病的风险。虽然这些研究产生了令人信服的研究目标，但 PTEN 在神经发育障碍中的作用我们仍然对 PTEN 的负调节作用了解甚少。更大的 mTOR 通路，使用 CRISPR-Cas9 功能丧失模型有助于早期神经发育我们在斑马鱼中产生了两个 pten 等位基因的外显子 5 均具有移码突变的斑马鱼突变体。在硬骨鱼复制基因组中：ptena (Δ10) 和 ptenb (Δ2) 我们首先旨在表征神经发育特征。这些鱼的异常现象，初步数据表明，它们的脑容量、脑我们将进一步探讨PTEN在调节细胞中的功能。其次，我们的目标是大脑中兴奋性和抑制性回路的增殖、分化和建立。使用该模型提供一种新的且易于使用的工具来筛查患有以下疾病的儿童中发现的 PTEN 变异神经发育障碍，通过在 1 细胞阶段向胚胎注射人类 mRNA 构建体最后，我们将对 mTOR 通路进行高通量药物筛选。抑制剂和其他化合物可以对抗或匹配我们突变幼虫中观察到的行为变化。这将确定新的潜在药理学候选者。我们的初步数据已经显示了部分表型。 mTORC1 抑制剂西罗莫司治疗后的挽救总之，这些功能丧失表明。 PTEN 在早期脊椎动物神经发育中发挥着关键作用，此外，这些模型提供了一种新的方法。用于筛选神经发育障碍中发现的 PTEN 变异以及潜在的可用工具药理学候选者。