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: 8744641
• 负责人: Dawn Brianna Lammert
• 金额: $ 3.31万
• 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/8744641
• 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; Defect; Detection; Development; Disease; Disease Pathway; Effectiveness; Engineering; Etiology; Event; FDA approved; Future; Gene Mutation; Genes; Genetic; Glycoproteins; Goals; 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; public health relevance; 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（由申请人提供）： 背景资料：自闭症的发病率急剧增加，虽然单基因综合征有助于提示疾病病理学途径，但这些基因仅占自闭症病例的20%。我们已经确定在已发表的自闭症遗传学文献中的一组突变分泌的神经发育糖蛋白Reelin。Reelin最为人所知的是其在适当的大脑分层中的作用，并且可能在出生后在突触中起作用。这些突变发生在序列和二级结构中保守的精氨酸残基处，其在整个全长Reelin蛋白中重复。初步研究表明，这些精氨酸残基的破坏导致生物合成缺陷，导致可用的Reelin减少和细胞内突变蛋白的可能积累。目的：我们的研究将确定这种保守序列的重要性和Reelin破坏的分子机制，以及确定拯救突变体Reelin水平的能力。此外，这些调查将有助于确定Reelin在病因学中的作用 自闭症的症状减少来自异常蛋白质的细胞应激和/或增加Reelin水平以改善信号传导可能是有用的治疗策略。目的1：初步研究表明，在转染HeLa细胞的培养基中不存在突变体Reelin。脉冲追踪实验将阐明这是否是由于分泌缺陷，先天蛋白质不稳定性或降解。然后我们将 进一步研究上述突变的细胞内结果，这些行为是否可在颗粒细胞神经元（其分泌内源性Reelin）中复制，并试图通过热休克蛋白的共转染来拯救突变体分泌。最后，由于Orleans小鼠是一种翻译但不分泌的reeler突变体，我们将确定Orleans颗粒细胞神经元行为是否与自闭症Reelin突变相似。这样的结果表明奥尔良小鼠是一种假定的自闭症小鼠模型。目的2：我们将确定目前FDA批准的药物是否能够增加突变体Reelin分泌。两个突变落在Reelin的受体结合位置内，可能干扰受体结合。因此，虽然Reelin分泌可能被拯救，但重要的是要确定这种突变体是否可以通过其受体刺激和信号传导。将评价用重组Reelin刺激的原代皮质培养物的Dab 1磷酸化，Dab 1磷酸化是Reelin信号传导的主要分子下游标志物。