Howell, Brian; Lammert, Dawn (2017)
      Autism spectrum disorder (ASD) affects approximately 1 in 45 people, and is characterized by deficits in social communication and repetitive behaviors. Sequencing advancements have enabled the identification of numerous candidate genes, but precisely how these genes contribute to ASD remains largely unknown. RELNis consistently implicated as a candidate gene for autism. The encoded secreted glycoprotein, Reelin is important for proper brain developmental and postnatal synapse function. Here we examine the molecular and cellular consequences of the de novo RELNmutation R2290C. This mutation falls in a conserved arginine-amino acid-arginine (RXR) motif that is found within the Reelin subrepeat structure. Several other ASD patient mutations fall with in this consensus and all examined reduce Reelin secretion. Based on this we tested two hypothesis: (1) that the mutations reduce Reelin signaling and (2) that they have a gain-of-function consequence, such as ER stress. Using an engineered cell line with a heterozygous RELNR2290C mutation and the RELN Orleans (Orl) mouse line that produces nearly full length Reelin that is defective for secretion, we found evidence for both increased Dab1 and increased PDIA1 expression. Since, like most genes implicated in ASD RELNlikely acts in a multifactorial manner, we investigated whether second site mutations might contribute to ASD-related behaviors. Towards this end we crossed the heterozygous Orl and Shank3b mice to model two hits that are present in at least one ASD proband. We found that the resulting double heterozygousmice had impaired socialization and altered ultrasonic vocalizations. Furthermore, forebrain and cerebellar lysates showed increased PSD-95, identifying a potentially common mechanism and therapeutic target for ASD. These studies are the first to investigate the biological relevance of RELNcoding mutations in ASD.

      Knox, Barry; ZHUO, XINMING (2013)
      Rod photoreceptors are a group of specialized retinal neurons that convert light into a neuronal signal in low light condition. The phototransduction function of rodsrequires expression of a group of rod genes. The homeostasis of these genes is primarily regulated by a photoreceptor regulatory network, which contains two major retina-specific transcription factors, neural retina leucine zipper (Nrl) and cone-rod homeobox(Crx). Nrl and Crx synergistically activate the expression of several phototransduction genes, most notably rhodopsin. Base on the studies done in cell culture, the synergy is theconsequence of interaction of Nrl and Crx. However, the interaction of Nrl and Crx has not been studied in live rods. The goal of thesis is to develop methods that can be used for studying Nrl and Crx interaction in live rods.In order to study Nrl and Crx in live rods without altering cell fate and inducing cell degeneration, I developed a novel inducible system, G3U, which can regulate gene expression in live rods. In this chapter, we investigated the characters of G3U by using rhodopsin-mCherry as a reporter and monitoring the induction response in transgenic Xenopus. The results of live rod imaging suggest that the inducible system has negligible background expression before induction and significant fold increase of expression after induction. Moreover, the induction response of G3U is reproducible in rods. These findings suggest that G3U system is a good candidate for expressing Nrl and Crx temporally in rods. In the second part of my thesis, I developed a flow cytometry based FRET method, FC-FRET, to study Nrl and Crx interaction in live cells. This method allows non-invasively analysis of protein-protein interaction in large population of live cells in a very short time. Furthermore, researchers will be able to analyze the concentration effect of FRET conveniently. In this study, I investigated the orientation of Nrl-Nrl, Crx-Crx and Nrl-Crx interactions. Our studies revealed that the Nrl-Nrl homodimer has a head-to-head and tail-to-tai l conformation. Both the Crx-Crx and the Nrl-Crx dimers have a head-to-head conformation in interacting complex. I also performed structure-function studies on both Nrl and Crx and classified the role of their different domains in the interactions. In addition to the known Nrl basic leucine zipper domain (b-ZIP) and Crx homeobox domain (HD), we found that the Nrl extended homology domain (EHD) plays an important role in the Nrl-Crx interaction. Two methods presented in this thesis are my major achievement during my graduate studies. The G3U inducible can be used to generate transgenic animals carrying inducible Nrl and Crx in rods. These transgenic animals allow researchers to study the interaction of Nrl and Crx in live rods by FC-FRET assay.