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

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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.