Molecular analysis of glutamatergic neurons derived from iPSCs containing PPM1D truncating mutations found in Jansen de Vries Syndrome

Jansen de Vries 综合征中发现的含有 PPM1D 截短突变的 iPSC 衍生的谷氨酸能神经元的分子分析

• 批准号: 10573782
• 负责人: HERBERT M LACHMAN
• 金额: $ 21万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10573782
• 关键词: ATAC-seq; Achievement; Acute; Address; Alleles; Appearance; Architecture; Attention; Autoimmune; Behavior; Biological; Biological Models; Blood; Brain; C-terminal; CRISPR/Cas technology; Ca(2+)-Calmodulin Dependent Protein Kinase; Catalytic Domain; Cell Line; Cell physiology; Cells; Cerebrum; Child; Childhood; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; DNA Repair Pathway; Development; Diameter; Disease; Doctor of Medicine; Drug Targeting; Eating; Engineering; Exons; Family; First Pregnancy Trimester; Frameshift Mutation; Genes; Genetic; Glutamates; Impairment; Individual; Induced pluripotent stem cell derived neurons; Inflammatory; Intellectual functioning disability; Knock-out; Knowledge; Leukocytes; Literature; Magnesium; Malignant - descriptor; Malignant Neoplasms; Mediating; Microscopic; Modernization; Molecular; Molecular Analysis; Mutate; Mutation; Neurites; Neurons; Nonsense Mutation; Obsessive-Compulsive Disorder; Organoids; Outcome; Pain Threshold; Pathological anxiety; Patients; Pharmaceutical Preparations; Phenotype; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Principal Investigator; Protein Serine/Threonine Phosphatase; Protein Truncation; Protein phosphatase; Proteins; Proteomics; Quality of life; RNA; Reporting; Research; Research Personnel; Resistance; Resources; SARS-CoV-2 B.1.617.2; Stem Cell Development; Symptoms; Syndrome; TP53 gene; Techniques; Testing; Therapeutic; Therapeutic Effect; Therapeutic Intervention; Transcript; Tumor Suppressor Genes; Variant; anticancer research; autism spectrum disorder; brain cell; cancer subtypes; cell type; delta protein; differential expression; experimental study; feeding; fetal; gain of function; gastrointestinal; improved; individuals with autism spectrum disorder; induced pluripotent stem cell; induced pluripotent stem cell technology; inhibitor; interest; loss of function; loss of function mutation; member; millimeter; molecular marker; molecular phenotype; multiple omics; mutant; neuropsychiatry; novel; novel therapeutics; null mutation; overexpression; pharmacologic; phenotypic biomarker; phosphoproteomics; programs; public health relevance; small molecule; stem cell model; targeted treatment; three dimensional structure; tool; transcriptome sequencing; transcriptomics

Program Director/Principal Investigator (Lachman, Herbert M) Abstract Mutations in PPM1D (Protein Phosphatase Magnesium-Dependent 1 Delta), which codes for a member of the protein serine-threonine phosphatase family, have recently been found in individuals with Jansen-De Vries Syndrome (JdVS), a condition characterized by intellectual disability (ID), severe anxiety, oppositional behavior, attention problems, high pain threshold, restricted eating, and gastrointestinal problems. So far, several dozen children with JdVS have been reported in the literature. Remarkably, each has a de novo germline nonsense or frameshift mutation in exon 5 or 6 that predicts the formation of a protein truncated at the C-terminal end. The phosphatase domain is spared. A key question is whether the truncating mutations result in gain- or loss- of PPM1D function in neurons and other brain cells. PPM1D is also a tumor suppressor gene that is over-expressed in a variety of cancer subtypes, contributing to malignant transformation through its action as a negative regulator of the p53-mediated DNA repair pathway. In cancers, somatic, truncating mutations in exons 5 and 6 like those found in JdVS, but somatic in origin, result in gain-of-function (GOF) effects because PPM1D degradation is reduced. Yet, loss-of-function (LOF) variants in the catalytic domain have also been found in some cancers, suggesting that the effects of PPM1D on cellular function could be context-dependent. Whether the germline truncating mutations in children with JdVS cause GOF or LOF is a critical question to address because PPM1D inhibitors being developed by cancer researchers could potentially have therapeutic value in JdVS if the former is correct. On the other hand, if they cause LOF, drugs targeting over-phosphorylated downstream targets caused by reduced PPM1D phosphatase activity, would be a more feasible approach. To address these fundamental questions, we have established an induced pluripotent stem cell (iPSC) model for JdVS using patient-specific and CRISPR-engineered lines, each containing exon 5 or exon 6 truncating mutations. Preliminary RNA-seq and proteomics experiments on glutamatergic neurons derived from iPSCs identified a few functional signatures, including deficits in neurite outgrowth, altered chromatin architecture, and calmodulin kinase 2 (CAMK2) phosphorylation that suggest a GOF effect. However, molecular studies need to be repeated and expanded in additional lines to firmly establish these as legitimate phenotypes, and to identify novel makers that can be used to both test the GOF hypothesis and identify downstream targets for therapeutic intervention. This will be accomplished by a multi- OMICs approach on glutamatergic neurons that includes proteomics, phosphoproteomics, RNA-seq, and ATAC-seq. Considering the uniqueness of the PPM1D mutations in the development of JdVS, we also hypothesize that truncated PPM1D proteins have powerful effects on cellular function that could make them resistant to typical PPM1D inhibitors. To test these hypotheses, we will knockout either the mutant allele or wild type PPM1D allele by introducing a null mutation using CRISPR-Cas9 editing, after which, rescue of dysregulated cellular and molecular phenotypes will be assessed. The ability of small molecule PPM1D inhibitors to rescue these phenotypes will also be evaluated. The experiments will show whether JdVS associated PPM1D variants have a GOF effect on glutamatergic neurons, and whether generalized reduction in PPM1D phosphatase activity or targeted inhibition of the truncated protein will be suitable targets for therapeutic intervention.

计划总监/首席研究员（Lachman，Herbert M） 抽象的 PPM1D的突变（蛋白质磷酸酶镁依赖性1 Delta），该突变代码为成员 蛋白质丝氨酸 - 苏氨酸磷酸酶家族最近在Jansen-de Vries的个体中发现 综合征（JDVS），这种疾病以智力残疾（ID），严重的焦虑，对立为特征 行为，注意力问题，高疼痛阈值，饮食有限和胃肠道问题。迄今为止， 文献中已经报道了几十个患有JDV的儿童。值得注意的是，每个人都有从头开始 外显子5或6中的生殖线胡说八道或移码突变，该突变预测形成在该蛋白 C末端。磷酸酶结构域幸免。一个关键问题是截断突变是否结果 在神经元和其他脑细胞中PPM1D功能的增益或损失中。 PPM1D也是肿瘤抑制基因 这在多种癌症亚型中都过表达，导致恶性转化 作为p53介导的DNA修复途径的负调节剂的作用。在癌症中，躯体，截断 外显子5和6中的突变就像在JDV中发现的突变，但起源于躯体，导致功能获得（GOF） 效果是因为PPM1D降解降低。然而，催化域中的功能丧失（LOF）变体 在某些癌症中也发现了，这表明PPM1D对细胞功能的影响可能是 与上下文有关。 JDVS儿童的种系截断突变是否导致GOF或LOF是一个 要解决的关键问题，因为癌症研究人员开发的PPM1D抑制剂可以 如果前者是正确的，则可能具有JDV的治疗价值。另一方面，如果它们引起LOF，毒品 靶向由降低的PPM1D磷酸酶活性引起的过度磷酸化的下游靶标将是 一种更可行的方法。为了解决这些基本问题，我们已经建立了一个感应的 使用患者特异性和CRISPR工程线的JDV的多能干细胞（IPSC）模型，每条 包含外显子5或外显子6截断突变。初步的RNA-seq和蛋白质组学实验 源自IPSC的谷氨酸能神经元确定了一些功能特征，包括神经突的缺陷 出生，改变染色质结构和钙调蛋白激酶2（CAMK2）磷酸化，这表明 GOF效应。但是，分子研究需要重复并在其他线上扩展，以牢固 将它们确立为合法的表型，并确定可用于测试GOF的新型制造商 假设并确定治疗干预的下游靶标。这将通过多个 谷氨酸能神经元的OMICS方法，包括蛋白质组学，磷酸蛋白质组学，RNA-SEQ和 atac-seq。考虑到JDV的开发中PPM1D突变的独特性，我们也 假设截短的PPM1D蛋白对细胞功能具有强大的影响，这可能使它们成为 对典型的PPM1D抑制剂的抗性。为了检验这些假设，我们将淘汰突变等位基因或野生 类型PPM1D等位基因通过使用CRISPR-CAS9编辑引入无效突变，此后， 将评估细胞和分子表型失调。小分子PPM1D的能力 还将评估营救这些表型的抑制剂。实验将显示JDV是否 相关的PPM1D变体对谷氨酸能神经元具有GOF作用，以及是否概括 在PPM1D磷酸酶活性或靶向抑制截短蛋白的活性中，将是适合的目标 治疗干预。