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.

项目总监/主要研究者（Lachman，赫伯特M） 摘要 PPM 1D（蛋白磷酸酶镁依赖性1 δ）中的突变，其编码 蛋白丝氨酸-苏氨酸磷酸酶家族，最近在Jansen-De弗里斯病个体中发现 综合征（JdVS），一种以智力残疾（ID）、严重焦虑、对立 行为、注意力问题、高痛阈、限制进食和胃肠道问题。到目前为止， 文献中报道了几十名患有JdVS的儿童。值得注意的是，每一个都有一个新的 外显子5或6中的种系无义突变或移码突变，其预测蛋白质在 C-末端。磷酸酶结构域被保留。一个关键的问题是， 在神经元和其他脑细胞中获得或丧失PPM 1D功能。PPM 1D也是一种肿瘤抑制基因 在多种癌症亚型中过度表达，通过其 作为p53介导的DNA修复途径的负调节剂。在癌症中，体细胞，截短 外显子5和6中的突变与JdVS中发现的突变相似，但起源于体细胞，导致功能获得性（GOF） 效果，因为PPM 1D降解减少。然而，催化结构域中的功能丧失（LOF）变体 在一些癌症中也发现了这种现象，这表明PPM 1D对细胞功能的影响可能是 上下文相关。JdVS患儿的生殖系截短突变是否会导致GOF或LOF是一个重要因素。 一个关键的问题需要解决，因为癌症研究人员正在开发的PPM 1D抑制剂可以 如果前者是正确的，则可能在JdVS中具有治疗价值。另一方面，如果他们导致LOF，药物 针对PPM 1D磷酸酶活性降低引起的过度磷酸化下游靶点，将是 更可行的方法。为了解决这些基本问题，我们建立了一个诱导 使用患者特异性和CRISPR工程化细胞系的JdVS多能干细胞（iPSC）模型， 含有外显子5或外显子6截短突变。初步RNA-seq和蛋白质组学实验 来自iPSC的神经元识别出一些功能特征，包括神经突缺陷， 生长，改变染色质结构，钙调蛋白激酶2（CAMK 2）磷酸化，表明 GOF效应。然而，分子研究需要重复和扩大在额外的线，以坚定 建立这些作为合法的表型，并确定新的标记，可用于测试GOF 假设并确定治疗干预下游靶点。这将通过一个多- OMICs方法对多巴胺能神经元，包括蛋白质组学，磷酸化蛋白质组学，RNA-seq， ATAC-seq.考虑到JdVS发展中PPM 1D突变的独特性，我们还 假设截短的PPM 1D蛋白对细胞功能有强大的影响， 对典型的PPM 1D抑制剂有抗性。为了验证这些假设，我们将敲除突变等位基因或野生等位基因。 通过使用CRISPR-Cas9编辑引入无效突变来分型PPM 1D等位基因，之后， 将评估失调的细胞和分子表型。小分子PPM 1D的能力 还将评价挽救这些表型的抑制剂。实验将显示JdVS是否 相关的PPM 1D变体对多巴胺能神经元有GOF效应， 在PPM 1D磷酸酶活性或截短蛋白的靶向抑制中， 治疗干预