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

• 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
• 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

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.

项目总结/摘要 PTEN与自闭症、大头畸形和先天性脑积水高度相关，这些疾病越来越多地被发现。 儿童早期普遍存在的神经发育障碍。目前，没有治疗方法， 解决这些条件的原因，尽管越来越多的遗传证据，通过大规模的整个外显子组 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 仍然知之甚少。我们试图了解PTEN是如何通过其作为一个负调节因子的作用， 更大的mTOR通路，有助于使用CRISPR-Cas9功能丧失模型的早期神经发育 在斑马鱼中。我们已经产生了斑马鱼突变体的移码突变外显子5的两个pten等位基因存在 在硬骨鱼重复基因组中：ptena（Δ10）和ptenb（Δ2）。我们的目标首先是描述神经发育 这些鱼的异常，初步数据表明，在脑容量，脑 活动、脑室大小和惊吓反应。我们将进一步探讨PTEN在调节细胞凋亡中的作用， 增殖、分化以及大脑中兴奋性和抑制性回路的建立。第二，我们的目标 使用该模型提供一种新的和可访问的工具，用于筛选在患有癌症的儿童中鉴定的PTEN变体。 神经发育障碍，通过在1-细胞阶段注射人mRNA构建体的胚胎， 评估神经发育变化。最后，我们将进行mTOR通路的高通量药物筛选， 抑制剂和其他化合物，其对抗或匹配在我们的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物。我们的初步数据已经显示了部分表型 在用mTORC 1抑制剂西罗莫司治疗后挽救。总之，这些功能丧失线表明 PTEN在早期脊椎动物神经发育中起关键作用。此外，这些模型提供了一个新的和 用于筛选在神经发育障碍中鉴定的PTEN变体以及潜在的 药理学候选人