Functional Analysis of Pathogenic Human PTEN Variants in Early Neurodevelopment

项目摘要/摘要 PTEN与自闭症、巨头症和先天性脑积水高度相关，这些疾病的发病率越来越高。 普遍存在于儿童早期的神经发育障碍。目前，还没有任何治疗方法可以 解决这些疾病的原因，尽管通过大规模的整个外显子组获得了越来越多的遗传证据 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 人们对此仍然知之甚少。我们试图理解PTEN是如何通过其作为负向调节因子的作用 使用CRISPR-Cas9功能丧失模型，更大的mTOR通路有助于早期神经发育 在斑马鱼身上。我们已经产生了斑马鱼突变体，两个PTEN等位基因的外显子5都存在移码突变 在硬骨复制基因组中：Ptena(Δ10)和Ptenb(Δ2)。我们首先的目标是描述神经发育的特征 这些鱼的异常，初步数据表明，它们在脑体积、脑 活动性、脑室大小和惊吓反应。我们将进一步探讨PTEN在细胞调节中的作用 脑部的增殖、分化以及兴奋和抑制回路的建立。第二，我们的目标是 为了使用这个模型提供一个新的和可访问的工具来筛查在儿童中发现的PTEN变异体 神经发育障碍，通过在1-细胞期向胚胎注射人类mRNA构建物和 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，与我们在突变幼虫中观察到的行为变化相反或匹配。 这将确定新的潜在药理候选药物。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抢救。总而言之，这些功能行损失说明 PTEN在脊椎动物早期神经发育中起关键作用。此外，这些模型提供了一种新的和 可用于筛选神经发育障碍中已识别的PTEN变异体的工具以及潜在的 药理学候选人。

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.