The role of Nudt21 in MeCP2 regulation and neuropsychiatric disease

Nudt21 在 MeCP2 调节和神经精神疾病中的作用

• 批准号: 9189900
• 负责人: Callison Edward Alcott
• 金额: $ 4.38万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189900
• 关键词: 3' Untranslated Regions; Affect; Alleles; Animals; Autistic Disorder; Behavioral; Binding; Binding Sites; Biological Assay; Birth; Brain; Cessation of life; Copy Number Polymorphism; Data; Development; Diagnosis; Disease; Dose; Embryo; Epilepsy; Female; Future; Genes; Hela Cells; Human; In Vitro; Individual; Knockout Mice; Knowledge; Lead; Length; Life; Mediating; Messenger RNA; Methyl-CpG-Binding Protein 2; MicroRNAs; Modeling; Molecular; Motor Seizures; Mus; Mutation; Neurons; Pathogenicity; Patients; Pharmaceutical Preparations; Phenotype; Polyadenylation; Pre-mRNA Polyadenylation Factor; Protein Isoforms; Proteins; Regulation; Research; Rett Syndrome; Role; Schizophrenia; Severities; Site; Small Interfering RNA; Syndrome; Testing; Translating; Work; X-linked intellectual disability; early onset; gain of function; in vivo; knock-down; loss of function; loss of function mutation; malformation; molecular phenotype; mouse model; mutant; nervous system disorder; neurobehavioral; neuropsychiatric disorder; neuropsychiatry; novel; offspring; postnatal; premature; targeted treatment; therapy development; transcriptome sequencing; treatment strategy

Mutations in the gene encoding methyl CpG-binding protein 2 (MECP2) cause Rett syndrome, a severe post- natal neurological disease, marked by developmental regression, autism-like features, seizures, and motor abnormalities. Duplications of MECP2 cause MECP2 duplication syndrome, which shares a number of features with Rett syndrome, including autism and epilepsy, and is a leading cause of X-linked intellectual disability. Animal studies show that the brain is extremely sensitive to MeCP2 protein levels such that a twofold change up or down causes neuropsychiatric disease, suggesting that perturbations of MeCP2 modulators could cause disease by dysregulating MeCP2. However, little is known about MeCP2 regulation. An unbiased siRNA screen of polyadenylation factors showed that NUDT21 regulates MECP2 alternative polyadenylation and protein levels in vitro. Thus, I hypothesize that NUDT21 regulates MeCP2 in vivo, and that its loss or gain causes neuropsychiatric disease, partly by altering MeCP2 levels. To this end I gathered preliminary data from neuropsychiatric patients with copy-number variations (CNVs) spanning NUDT21, and showed that NUDT21- encoded CFIm25 and MeCP2 protein levels inversely correlate. Moreover, when CFIm25 levels are normalized in duplication patients by anti-NUDT21 siRNA, MeCP2 levels are restored, showing that NUDT21 alone regulates MeCP2 levels. I also found MECP2 mRNA increases in both the NUDT21 deletion and duplications patients, but that the increase in the deletion patient comes from an increase in short MECP2 isoforms, whereas the elevated mRNA from the duplication patients comes largely from long MECP2 isoforms, which harbor known regulatory miRNA binding sites, and are inefficiently translated. To confirm that perturbed NUDT21 alters MeCP2 levels and causes the phenotypes in patients with the CNVs, I have generated Nudt21 knockout mice, and have acquired a mouse line with a Nudt21 conditional allele. I will characterize their behavioral, cellular, and molecular phenotypes, including RNA-seq to assess expression and alternative poly- adenylation changes. To determine the degree that the phenotypes of patients with CNVs spanning NUDT21 are driven by MeCP2 dysregulation, I will cross Nudt21 heterozygous mice with mice expressing a Mecp2 allele that causes 50% reduction of MeCP2 and display neurobehavioral problems. In the double mutant offspring, I can determine if normalizing MeCP2 levels will rescue some phenotypes of the Nudt21 heterozygous mice and ascertain the contribution of MeCP2 dysregulation to their phenotypes. This will demonstrate the feasibility of using MeCP2 reducing drugs now in development for the treatment of NUDT21 loss-of-function phenotypes. This proposal will test the hypothesis that NUDT21 loss of function causes disease that NUDT21 regulates MeCP2 in vivo, and the degree to which MeCP2 dysregulation drives the Nudt21 loss-of-function phenotypes. The results will potentially provide some neuropsychiatric patients with a diagnosis, and direct future treatment strategies.

编码甲基 CpG 结合蛋白 2 (MECP2) 的基因突变会导致 Rett 综合征，这是一种严重的产后神经系统疾病，其特点是发育退化、自闭症样特征、癫痫发作和运动异常。 MECP2 重复导致 MECP2 重复综合征，该综合征与 Rett 综合征有许多共同特征，包括自闭症和癫痫，并且是 X 连锁智力障碍的主要原因。动物研究表明，大脑对 MeCP2 蛋白水平极其敏感，因此双重变化会导致神经精神疾病，这表明 MeCP2 调节剂的扰动可能会通过 MeCP2 失调而导致疾病。然而，人们对 MeCP2 的调控知之甚少。多腺苷酸化因子的无偏 siRNA 筛选表明，NUDT21 在体外调节 MECP2 替代多腺苷酸化和蛋白质水平。因此，我假设 NUDT21 在体内调节 MeCP2，并且其丢失或增加会导致神经精神疾病，部分原因是通过改变 MeCP2 水平。为此，我收集了跨 NUDT21 拷贝数变异 (CNV) 的神经精神病患者的初步数据，并表明 NUDT21 编码的 CFIm25 和 MeCP2 蛋白水平呈负相关。此外，当重复患者中的CFIm25水平通过抗NUDT21 siRNA恢复正常时，MeCP2水平恢复，表明NUDT21单独调节MeCP2水平。我还发现 NUDT21 缺失和重复患者中 MECP2 mRNA 均增加，但缺失患者的增加来自短 MECP2 亚型的增加，而重复患者的 mRNA 升高主要来自长 MECP2 亚型，这些亚型含有已知的长 MECP2 亚型。调控 miRNA 结合位点，且翻译效率低下。为了确认受干扰的 NUDT21 会改变 MeCP2 水平并导致 CNV 患者出现表型，我培育了 Nudt21 敲除小鼠，并获得了具有 Nudt21 条件等位基因的小鼠品系。我将描述它们的行为、细胞和分子表型，包括 RNA-seq 来评估表达和替代性多腺苷酸化变化。为了确定跨 NUDT21 的 CNV 患者的表型由 MeCP2 失调驱动的程度，我将 Nudt21 杂合小鼠与表达 Mecp2 等位基因的小鼠杂交，该等位基因导致 MeCP2 减少 50% 并表现出神经行为问题。在双突变后代中，我可以确定 MeCP2 水平正常化是否会挽救 Nudt21 杂合小鼠的一些表型，并确定 MeCP2 失调对其表型的影响。这将证明使用目前正在开发的 MeCP2 还原药物治疗 NUDT21 功能丧失表型的可行性。该提案将检验NUDT21功能丧失导致NUDT21体内调节MeCP2疾病的假设，以及MeCP2失调驱动Nudt21功能丧失表型的程度。研究结果可能会为一些神经精神病患者提供诊断，并指导未来的治疗策略。