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

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提示疾病病理途径，但这些基因仅占自闭症病例的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神经发育糖蛋白Reelin的突变。Reelin以其在大脑层压中的作用而闻名，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上，在整个长度的Reelin蛋白中重复。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减少和细胞内突变蛋白的可能积聚。目的：我们的研究将确定这个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

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.