Disruption of Reelin biosynthesis by de novo missense mutations found in aut

在 aut 中发现从头错义突变破坏 Reelin 生物合成

• 批准号: 8912556
• 负责人: Dawn Brianna Lammert
• 金额: $ 3.35万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2016-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912556
• 关键词: 7q22; Accounting; Adult; Alleles; Anabolism; Area; Arginine; Autistic Disorder; Behavior; Biochemical; Biological; Biological Assay; Birth; Blood; Brain; C-terminal; Candidate Disease Gene; Cell Culture Techniques; Cells; Cellular Stress; Cerebellum; Child; Conserved Sequence; Cytoplasmic Granules; DNA Sequence Alteration; Defect; Detection; Development; Disease; Disease Pathway; Effectiveness; Engineering; Etiology; Event; FDA approved; Future; Genes; Genetic; Genetic study; Glycoproteins; Goals; Health; Health Care Costs; Heat shock proteins; Hela Cells; Impaired cognition; Incidence; Investigation; Lead; Left; Length; Light; Literature; Location; Maintenance; Missense Mutation; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Neurons; Outcome Study; Pathogenesis; Pathology; Patients; Pharmaceutical Preparations; Phosphorylation; Physiologic pulse; Play; Positioning Attribute; Predisposition; Process; Production; Protein Secretion; Proteins; Publishing; Receptor Signaling; Recombinants; Reelin Signaling Pathway; Research; Role; Signal Transduction; Signaling Molecule; Stream; Stress; Structure; Synapses; Syndrome; Techniques; Temperature; Therapeutic; Translating; Work; analog; autism spectrum disorder; beta pleated sheet; biological adaptation to stress; brain research; cell motility; extracellular; falls; granule cell; improved; mouse model; mutant; postnatal; protein expression; receptor; receptor binding; reelin protein; reelin receptor; research study

DESCRIPTION (provided by applicant): Background: The incidence of autism has increased dramatically, and although monogenetic syndromes have been helpful in suggesting pathways of disease pathology, these genes account for only 20% of autism cases. We have identified in the published autism genetics literature a set of mutations in the secreted neurodevelopmental glycoprotein Reelin. Reelin is best known for its role in proper brain lamination, and may function postnatally at the synapse. These mutations occur at arginine residues conserved in a sequence and secondary structure which is repeated throughout the full length Reelin protein. Preliminary studies suggest the disruption of these arginine residues results in a biosynthetic defect, leading to decreased available Reelin and possible build-up of intracellular mutant protein. Objectives: Our research will determine the importance of this conserved sequence and molecular mechanism of Reelin disruption, as well as determine the ability to rescue mutant Reelin levels. Additionally, these investigations will help determine Reelin's role in the etiology of autism. Decreasing cellular stress from aberrant protein and/or increasing Reelin levels to improve signaling may be useful therapeutic strategies. Aim 1: Preliminary studies show mutant Reelin is absent in the media of transfected HeLa cells. Pulse-chase experiments will elucidate whether this is due to a secretion defect, innate protein instability, or degradation. We will then further investigate the intracellular consequences of the aforementioned mutations, whether these behaviors are replicable in granule cell neurons (which secrete endogenous Reelin), and attempt to rescue mutant secretion through cotransfection of heat shock proteins. Finally, as the Orleans mouse is a reeler mutant which is translated but not secreted, we will determine whether Orleans granule cell neuron behavior parallels that of the autistic Reelin mutations. Such a result would suggest the Orleans mouse as a putative autism mouse model. Aim 2: We will establish whether current FDA approved drugs are capable of increasing mutant Reelin secretion. Two mutations fall within the receptor binding location of Reelin, possibly perturbing the receptor-binding. Therefore, although Reelin secretion may be rescued, it is important to establish whether this mutant can stimulate and signal through its receptors. Primary cortical cultures stimulated with recombinant Reelin will be evaluated for Dab1 phosphorylation, the major molecular downstream marker of Reelin signaling.

描述(申请人提供)：背景：自闭症的发病率急剧增加，尽管单基因综合征有助于揭示疾病的病理途径，但这些基因只占自闭症病例的20%。我们已经在发表的自闭症遗传学文献中发现了一组分泌的神经发育糖蛋白Reelin的突变。Reelin最为人所知的是它在正确的大脑分层中的作用，并可能在出生后在突触发挥作用。这些突变发生在精氨酸残基上，这些残基在序列和二级结构中保守，在整个Reelin蛋白全长中重复。初步研究表明，这些精氨酸残基的破坏会导致生物合成缺陷，导致可用Reelin减少，并可能在细胞内积聚突变蛋白。目的：我们的研究将确定这一保守序列的重要性和Reelin干扰的分子机制，并确定拯救突变Reelin水平的能力。此外，这些研究将有助于确定Reelin在病因学中的作用 患有自闭症。减少异常蛋白引起的细胞压力和/或提高Reelin水平以改善信号转导可能是有用的治疗策略。目的1：初步研究表明，HeLa细胞培养上清液中不存在突变的Reelin。脉冲追逐实验将阐明这是由于分泌缺陷、先天蛋白质不稳定还是降解所致。到时候我们会的 进一步研究上述突变的细胞内后果，这些行为是否可以在分泌内源性Reelin的颗粒细胞神经元中复制，并试图通过共转染热休克蛋白来挽救突变的分泌。最后，由于奥尔良小鼠是一种被翻译但不分泌的卷轴突变，我们将确定奥尔良颗粒细胞神经元的行为是否与自闭症Reelin突变的行为相似。这样的结果将表明奥尔良小鼠可能是自闭症小鼠模型。目的2：我们将确定目前FDA批准的药物是否能够增加突变的Reelin的分泌。两个突变落在Reelin的受体结合位置，可能扰乱了受体结合。因此，尽管Reelin的分泌可能会被挽救，但重要的是要确定该突变是否能通过其受体刺激并发出信号。用重组Reelin刺激的原代皮质培养细胞将被评估Dab1的磷酸化，这是Reelin信号的主要分子下游标志。