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

基本信息

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
项目类别：
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

项目摘要

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：自闭症的发病率显着增加，尽管单基因综合症有助于暗示疾病病理的途径，但这些基因仅占自闭症病例的20％。我们已经在已发表的自闭症遗传学文献中确定了一组分泌神经发育糖蛋白reelin中的突变。 Reelin以其在适当的脑层压中的作用而闻名，并且可能在突触中产生产后发挥作用。这些突变发生在精氨酸残基中以序列和二级结构保守的，该残基在整个全长reelin蛋白中都重复。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，从而导致可用的reelin降低，并可能积累细胞内突变蛋白。目的：我们的研究将确定这种保守序列和reelin破坏的分子机制的重要性，并确定拯救突变体reelin水平的能力。此外，这些调查将有助于确定reelin在病因中的作用自闭症。从异常蛋白质和/或增加reelin水平以改善信号传导的细胞应激可能是有用的治疗策略。 AIM 1：初步研究表明，转染的HeLa细胞介质中不存在突变relin。脉搏追踪实验将阐明这是由于分泌缺陷，先天蛋白质的不稳定性或降解所致。然后我们会进一步研究上述突变的细胞内后果，这些行为是否在颗粒细胞神经元中可复制（分泌内源性relin），并试图通过热激蛋白的共转染来挽救突变体分泌。最后，由于奥尔良小鼠是一种翻译但不分泌的卷轴突变体，因此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突变的相似。这样的结果将表明奥尔良小鼠是推定的自闭症小鼠模型。 AIM 2：我们将确定当前的FDA批准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，可能会扰动受体结合。因此，尽管可以挽救reelin的分泌，但重要的是要确定该突变体是否可以通过其受体刺激和发信号。将通过重组reelin刺激的原代皮质培养物评估DAB1磷酸化，这是reelin信号的主要分子下游标记。