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）的基因突变导致RETT综合征，RETT综合征，一种严重的后神经系统疾病，以发育消退，自闭症样特征，癫痫发作和运动异常为特征。 MECP2的重复导致MECP2重复综合征，该复制综合征与RETT综合征共享许多功能，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蛋白水平极为敏感，因此双重变化会导致神经精神病，这表明MECP2调节剂的扰动可能通过失调的MECP2引起疾病。但是，关于MECP2调节知之甚少。多腺苷酸化因子的无偏siRNA筛选表明NUDT21在体外调节MECP2替代聚腺苷酸化和蛋白质水平。因此，我假设NUDT21在体内调节MECP2，并且其损失或增益会导致神经精神病，部分是通过改变MECP2水平。为此，我收集了跨NUDT21的神经精神病患者的初步数据，并表明NUDT21-编码的CFIM25和MECP2蛋白水平成反比。此外，当抗NUDT21 siRNA重复患者中CFIM25水平归一化时，MECP2水平恢复，表明单独使用NUDT21调节MECP2水平。 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.为了确认扰动的NUDT21改变了MECP2水平并引起CNV患者的表型，我已经产生了NUDT21敲除小鼠，并获得了带有NUDT21条件等位基因的小鼠系。我将表征它们的行为，细胞和分子表型，包括评估表达和替代多腺苷酸化变化的RNA-Seq。为了确定跨NUDT21的CNV患者的表型由MECP2失调驱动，我将与表达MECP2等位基因的小鼠穿越NUDT21杂合小鼠，该等位基因会导致50％的MECP2降低并显示神经性神经性问题。在双突变体后代中，我可以确定标准化MECP2水平是否会挽救NUDT21杂合小鼠的某些表型，并确定MECP2失调对其表型的贡献。这将证明现在使用MECP2减少药物以治疗NUDT21功能丧失表型的可行性。该提案将检验以下假设：NUDT21功能丧失会导致NUDT21调节体内MECP2的疾病，而MECP2失调驱动NUDT21功能障碍表型的程度。结果可能会为一些神经精神病患者提供诊断和直接治疗策略。