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

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 differentially-expressed 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. Electronic supplementary material The online version of this article (10.1007/s12264-018-0251-5) contains supplementary material, which is available to authorized users.


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 GABA A receptors, and the basigin-monocarboxylate transporter [2][3][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 analysis to profile differentially-expressed genes in Np65-KO mice, in order to explain the altered brain functions in Np65-deficient mice.

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 (Nano-Drop 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 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
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 real-time reverse-transcription PCR (RT-PCR) was performed as previously described [14]. Adult Np65-KO and WT mice (n = 4/genotype) were sacrificed after anesthesia with 1% pentobarbital sodium (30 mg/kg, i.p.) and the forebrain was harvested to extract total RNA using TRIzol (15596026, Thermo Fisher Scientific, Waltham, MA). RNA concentration and quality were determined by NanoDrop (ND-1000, Thermo Fisher Scientific, Waltham, MA). cDNA was generated using reverse transcriptase (PrimeScript TM RT reagent Kit, RR0747Q, Takara Bio, Tokyo, Japan). The first-strand cDNA was used as a template for RT-PCR analysis. The primers for RT-PCR analysis (Table 1) were designed by the NCBI primer designing tool [15] and synthesized by Sangon (Shanghai, China). Each RT-PCR reaction was carried out in a 20 lL volume using SYBR Green Master Mix (RR820Q, Takara Bio, Tokyo, Japan), started at 30 s at 95°C for initial denaturation, followed by 40 cycles of 5 s at 95°C and 34 s at 60°C in the ABI 7500 Real-Time PCR System. A total of 3 independent samples per subject were run in duplicate for RT-PCR. b-actin was used as the reference gene. The 2 -DDCt method was used to determine the relative expression levels of genes.

Western Blotting
Adult Np65-KO and WT mice (4 months old, n = 3/genotype) were used for western blotting. Briefly, animals were decapitated after deep anesthesia with 1% pentobarbital sodium (30 mg/kg i.p.). Forebrains were collected and frozen in nitrogen and then stored at -80°C until use. The total proteins were extracted using RIPA lysis buffer (P0013B, Beyotime) with 1 mmol/L PMSF (ST506, Beyotime). Protein concentrations were measured using the BCA Protein Assay Kit (P0010, Beyotime, Jiangsu, China), then 10 ng of total protein was separated by SDS-PAGE and transferred to the PVDF membrane. After blocking with 5% bovine serum albumin (BSA), the membranes were incubated overnight at 4°C with primary antibody against Wnt-3 (1:1,000, Santa Cruz Biotechnology, Dallas, TX) and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000, Santa Cruz Biotechnology, Dallas, TX). Subsequently, the membranes were incubated with HRP-conjugated goat anti-mouse secondary antibody (1:1,000, Beyotime) for 2 h at room temperature. The labeled proteins were detected by using the ImageQuant LAS 4000mini system (GE Healthcare Life Sciences, Chicago, IL). The protein levels were normalized to that of GAPDH from three independent experiments.

Statistics
Statistics were calculated using SPSS Statistics software (v22.0, IBM). All data are presented as mean ± SEM. Independent samples were tested by the unpaired Student's t-test (two-tailed). The Mann-Whitney U test was used to determine the significance of data with an abnormal distribution or unequal variance. Statistical significance was set at P \ 0.05.

Results
Microarray Analysis of Differentially-Expressed Genes in the Hippocampus of Np65-KO Mice All genes are shown in a scatter plot with normalized intensity in Fig. 1A (details in Table S1). Of the 34397 targeted genes by the Mouse 4 9 44K Gene Chip, 481 (1.4%) were up-regulated and 418 (1.2%) were downregulated by 2-fold in the Np65-KO mice (Fig. 1B). Using P \ 0.05 as the criterion, 367 genes were significantly higher and 221 genes were significantly lower in Np65-KO mice as compared to age-matched WT mice ( Fig. 1C and D, Table S2). These differentially-expressed genes were primarily located on chromosomes 7, 9 and 11. Notably, the NPTN gene resides on chromosome 9 (Fig. 1E).

Gene Ontology and Pathway Analysis of Differentially-Expressed Genes
Using the criterion of P \ 0.001, GO analysis showed that the upregulated genes were involved in several cellular components, including extracellular region, plasma membrane, and desmosome ( Fig. 2A). The molecular functions of the upregulated genes were mainly associated with co-receptor activity. The down-regulated genes were involved in various binding actions, such as ankyrin binding, lipoprotein binding, and Wnt-protein binding (Fig. 2B). The main biological processes were the cellular response to interferon-beta, embryonic hindlimb morphogenesis, negative regulation of neuron differentiation, cellular process, positive regulation of epidermis development, and negative regulation of transmembrane receptor protein serine/threonine kinase signaling pathway. The downregulated genes were associated with several biological processes, including post-transcriptional regulation of gene expression, developmental growth involved in morphogenesis, regulation of translation, cell adhesion, developmental growth, and response to stimuli (Fig. 2C).
According to the pathway analysis, 8 pathways were significantly up-regulated in Np65-KO mice, including glycerophospholipid metabolism, pancreatic secretion, and regulation of actin cytoskeleton. Among the 9 downregulated pathways, the most prominent was cytokinecytokine receptor interaction. The other downregulated pathways were involved in bladder cancer, chemical carcinogenesis, and drug metabolism (Fig. 2D).
The expressions of genes related to the structure and function of synapses were also altered in Np65-KO mice. Notably, expression of the serotonin receptor 4 (Htr4) gene was significantly increased, whereas the expression of serotonin receptor 3A (Htr3a) was significantly decreased in Np65-KO mice. Moreover, two ion channel-related genes displayed significant downregulations in Np65-KO mice: Clchannel Ca 2? activated 5 (Clca5) and K ? inwardlyrectifying channel subfamily J member 9 (Kcnj9) ( Table 2).
MAPK signaling is essential for various physiological and pathological processes, such as neural plasticity and memory. We found that the expression of mitogenactivated protein kinase kinase 7 (Map2k7) was significantly decreased, while phospholipase A2, group IVE (Pla2g4e), serine/threonine kinase 38 like (Stk38l), and  protein phosphatase 6, regulatory subunit 1 (Ppp6r1) were significantly increased in Np65-KO mice. In addition, the expression level of X-linked inhibitor of apoptosis protein associated factor 1 (Xaf1), an apoptosis-promoting factor, was decreased. Most notably, b-lactamase (Lactb), which is involved in mitochondrial metabolism, was also significantly down-regulated in Np65-KO mice (24.30-fold, P \ 0.001) ( Table 2).

Decreased Expression of Wnt-3 in Np65-KO Mice
Microarray and RT-PCR analysis showed that the expression levels of several genes associated with development were altered in Np65-KO mice. Some differentiallyexpressed genes, such as Wif1 and Cdh1, are involved in Wnt signaling. Wnt signaling is a crucial regulator of many developmental processes, such as cell proliferation, maintenance of stem cells, and cell fate determination [16]. Therefore, we examined the protein level of Wnt-3 in Np65-KO mice by western blotting. The results showed that the protein level of Wnt-3 was significantly lower in the forebrain of Np65-KO mice than in WT mice (Fig. 4).

Reduced Lateral Ventricles in Np65-KO Mice
Given that the dysfunction of Wnt signaling may influence brain development, we then assessed whether ablation of Np65 affects the brain morphology of mice. T2-wt images were used to assess region-specific volume changes. The gross brain architecture was not affected in Np65-KO mice.  MRI morphometry also showed normal anatomy of the cerebral cortex, hippocampus, thalamus, hypothalamus, basal ganglia, and caudatoputamen of Np65-KO mice. However, the lateral ventricular volume was significantly reduced compared to WT mice (Fig. 5A), and this was further confirmed by DAPI staining (Fig. 5B). Thus, these results suggested that the absence of Np65 leads to altered architecture of the mouse brain.

Discussion
Np65 is specifically expressed in the brain and has been reported to mediate several cellular processes including cellcell adhesion, neurite outgrowth, and synaptic plasticity [2,5,17,18]. Our previous studies have shown that Np65-KO mice exhibit abnormal cognitive and emotional behaviors [12]. To investigate the underlying mechanisms, we further analyzed the gene expression profiles in Np65-KO mice in this study. Our microarray analysis demonstrated a large number of differentially-expressed genes in Np65-KO mice; these genes are crucially involved in development, ion channels, neurotransmission, and signal transduction.
Our study identified many differentially-expressed genes involved in neuronal development, such as the decreased expressions of Cdh1, Ccr5, Foxo3, Mbp, Wif1, and Mef2c, as well as upregulation of Ntf3, Gcm1, and Trp73, implying that Np65 deletion affects the configuration of the brain. Coincidently, T2-wt MRI morphometry and brain slices stained with DAPI showed a significant reduction in lateral ventricular volume in Np65-KO mice compared to WT mice. The expression of Wnt-3 was significantly decreased in Np65-KO mice. Wnt signaling is a crucial regulator of developmental processes like cell proliferation and cell fate determination [16]. Dysregulation of Wnt signaling may contribute to neuropsychiatric disorders, such as depression and schizophrenia [19]. In this study, our findings suggested that Np65 deletion affects the Wnt signaling pathway by decreasing Wnt expression. Together, these differentially-expressed genes associated with development may contribute, at least in part, to changes in the ventricles and abnormal behaviors in Np65-KO mice.
Recent studies have reported that mutation of the NPTN gene results in deafness in mice [10]. It has been reported that Np65 may regulate the properties of synapses connecting the inner hair cells with spiral ganglion neurons [10]. Intriguingly, Zeng et al. reported that Np55 is expressed in stereocilia of outer but not inner hair cells and affects interactions of stereocilia with the tectorial membrane and cochlear amplification in mice with NPTN mutation [11]. Together, these recent findings clearly confirm NPTN as a novel deafness gene. Consistent with their reports, our microarray analysis showed that the genes associated with inner ear receptor cell development, myosin VIIA (Myo7a) and collagen triple helix repeat containing 1 (Cthrc1) were significantly decreased in Np65-KO mice, supporting the hypothesis that Np65 is involved in hearing.
It has been shown that Np65 is linked with ribbon synapse formation in the plexiform layers of the rat retina [20]. Retinal function, as assessed using the electroretinogram, is unaffected by the absence of NPTN [10]. Surprisingly, the involvement of Np65 in vision was demonstrated using the pupillary light reflex and flash visual evoked potentials (our unpublished data). In agreement with our finding, our microarray analysis showed that the expression of eye development-related genes, including Aldh1a3, Rpgrip1, Crb1, Vsx1, Krt12, and Sfrp5, was significantly downregulated in Np65-KO mice. Although these alterations in eye-development genes need to be confirmed, the reduced amplitude of the pupil in the pupillary light reflex and reduced first negative and positive amplitude of flash visual evoked potentials (our unpublished data) suggest that Np65 plays roles in vision.
Our previous studies have shown that Np65-KO mice appear to show enhanced memory in the Morris water maze and increased anxiety [12]. Central 5-hydroxytryptamine (5-HT) activity is involved in emotional and cognitive activities [21,22]. Generally, stimulation of central 5-HT activity impairs cognition, while its inhibition enhances cognition in rodent models. Tropisetron, a selective 5-HT 3 receptor antagonist, has been confirmed to reverse the cognitive deficit in rats injected with Ab (1-42) [23]. In addition, central 5-HT activity is closely associated with anxiety [24][25][26][27]. Among the 5-HT receptors, 5-HT 3 is the only ligand-gated ion channel that increases intracellular cations such as Ca 2? , Na ? , and K ? . Stimulation of 5-HT 3 receptors induces the rapid and transient depolarization of neurons. 5-HT receptor 3A-null mice exhibit anxiolytic behaviors, indicating that this receptor influences anxiety-like behavior [28]. More surprisingly, microarray and RT-PCR analysis demonstrated that Htr3a mRNA was significantly reduced in Np65-KO mice. How deletion of Np65 affects the expression of Htr3a remains to be determined. To date, the decreased expression of Htr3a may explain, at least in part, the changed cognitive and anxiety behaviors in Np65-KO mice.
In conclusion, the present study demonstrates that a large number of genes are differentially expressed in Np65-KO mice. Notably, microarray analysis in Np65-KO mice revealed altered expression of Htr3a and genes associated with development, hearing, and vision, which may provide important insights for understanding the role of Np65 in brain development as well as brain functions like cognition and emotion.