Microarray Analysis of Gene Expression Changes in Neuroplastin 65-Knockout Mice: Implications for Abnormal Cognition and Emotional Disorders

Neurosci. Bull. https://doi.org/10.1007/s12264-018-0251-5 www.neurosci.cn www.springer.com/12264 ORIGINAL ARTICLE Microarray Analysis of Gene Expression Changes in Neuroplastin 65-Knockout Mice: Implications for Abnormal Cognition and Emotional Disorders Huanhuan Li1 • Jiujiang Zeng1 • Liang Huang1 • Dandan Wu1 • Lifen Liu1 • Yutong Liu2 • Qionglan Yuan1 Received: 26 December 2017 / Accepted: 26 April 2018 Ó The Author(s) 2018 Abstract Neuroplastin 65 (Np65) is an immunoglobulin superfamily cell adhesion molecule involved in synaptic formation and plasticity. Our recent study showed that Np65-knockout (KO) mice exhibit abnormal cognition and emotional disorders. However, the underlying mechanisms remain unclear. In this study, we found 588 differentiallyexpressed genes in Np65-KO mice by microarray analysis. RT-PCR analysis also revealed the altered expression of genes associated with development and synaptic structure, such as Cdh1, Htr3a, and Kcnj9. In addition, the expression of Wnt-3, a Wnt protein involved in development, was decreased in Np65-KO mice as evidenced by western blotting. Surprisingly, MRI and DAPI staining showed a significant reduction in the lateral ventricular volume of Np65-KO mice. Together, these findings suggest that ablation of Np65 influences gene expression, which may contribute to abnormal brain development. These results provide clues to the mechanisms underlying the altered brain functions of Np65-deficient mice. Keywords Neuroplastin 65  Microarray analysis  Gene expression profile  Htr3a  Wnt Huanhuan Li, Jiujiang Zeng and Liang Huang have contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12264-018-0251-5) contains supplementary material, which is available to authorized users. & Qionglan Yuan 1 Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China 2 Department of Radiology, University of Nebraska Medical Center, Omaha, NE 68198, USA Introduction Neuroplastin (Np) is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules and exists in two isoforms, Np65 and Np55 [1]. Np65 contains extracellular Ig1-2-3 modules, while Np55 only contains extracellular Ig2-3. Thus, Np65 can be differentiated from Np55 by its extracellular Ig1. Np55 is expressed in various organs and cell types, whereas the expression of Np65 is brain-specific and restricted to neurons. Np65 undergoes trans- and cis-homophilic bindings as well as several heterophilic bindings with fibroblast growth factor receptors, the a1 or a2 subunit of GABAA receptors, and the basigin-monocarboxylate transporter [2–4]. Np65 has been implicated in the regulation of synaptic plasticity and the maintenance of excitatory/inhibitory balance. Antibodies specific for Np65 or recombinant Np65 block long-term potentiation (LTP) in the hippocampal CA1. The induction of LTP also increases the expression of Np65 in postsynaptic densities [5]. In addition, Nptn-deficient neurons exhibit impaired inhibitory transmission [6]. Previous studies have suggested that Np65 is associated with cognition and emotional states. Polymorphisms in the human NPTN gene have been shown to correlate with cortical thickness and intellectual abilities in adolescents as well as in patients with schizophrenia [7, 8]. Nptn-deficient mice exhibit retrograde amnesia, depressive-like behaviors, and decreased social interactions [9]. In addition, mutation of the Nptn gene results in deafness in mice, suggesting that NPTN is a novel deafness gene [10, 11]. We have previously demonstrated that Np65 knock-out (KO) mice exhibit enhanced hippocampal-dependent spatial memory in the Morris water maze and step-through passive avoidance tests [12], but the underlying mechanisms were unclear. In this study, we used custom-designed microarray 123 Neurosci. Bull. analysis to profile differentially-expressed genes in Np65KO mice, in order to explain the altered brain functions in Np65-deficient mice. before all replicates were combined. Genes with a foldchange of [ 2.0 and a P value \ 0.05 were selected for Gene Ontology (GO) and pathway analysis. Gene Ontology and Pathway Analysis Materials and Methods Animals The homozygous Np65-KO mice were obtained from engineered mouse models; this caused Np65-Ig1 deficiency in single chromosome as previously described [12]. Wildtype (WT) littermates served as controls. Animals were housed in a temperature-controlled environment under a 12 h light/dark cycle (08:00–20:00) with food and water ad libitum. All protocols complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Ethics Committee of Tongji University School of Medicine, and conformed to Directive 2010/63/EU and NIH guidelines. Microarray Experiments Microarray analysis was performed as previously described [13]. Briefly, animals were sacrificed after deep anesthesia with intraperitoneal (i.p.) injection of 1% pentobarbital sodium (30 mg/kg). Hippocampi from adult Np65-KO mice (3 months old) and age-matched WT mice (n = 3/genotype) were dissected and immediately frozen in liquid nitrogen. The samples were stored at -80°C until use. Total RNA was extracted from the hippocampal tissue using TRIzol (15596026, Thermo Fisher Scientific, Waltham, MA) and further purified with an RNeasy Mini Kit (74104, Qiagen, Hilden, Germany). RNA concentration and quality were evaluated by spectrophotometry (NanoDrop ND-1000, Thermo Fisher Scientific, Waltham, MA). One microgram of total RNA was amplified and labeled with a One-Color Quick Amp Labeling Kit (5190-0442, Agilent Technologies, Santa Clara, CA). The fluorescencelabeled cRNA was hybridized onto the Whole Mouse Genome Oligo Microarray (4 9 44K, Agilent Technologies, Santa Clara, CA) using the Agilent Gene Expression Hybridization Kit (5188-5242, Agilent Technologies, Santa Clara, CA). Chips were washed and scanned by a microarray scanner (G2565BA, Agilent Technologies, Santa Clara, CA). Raw data were then normalized and analyzed using the GeneSpring GX Software Package (v11.5, Agilent Technologies). The microarray experiment was performed with 3 biological and experimental repeats. Normalized values were used to screen for differentiallyexpressed genes from biological and experimental repeats 123 The fold-changes of differential expression were determined by the abundance ratio of Np65-KO and WT mice. Hierarchical clustering was used to analyze the differentially-expressed genes. GO analysis was applied to analyze the cellular components, biological functions, and biological processes of the differentially-expressed genes (www. geneontology.org). Pathway analysis was used to reveal significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the differentially-expressed genes. Quantitative Real-Time Reverse-Transcription PCR Quantitative (...truncated)