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 α1 or α2 subunit of GABA
A 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 analysis to profile differentially-expressed genes in
Np65-KO mice, in order to explain the altered brain functions in
Np65-deficient mice.
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]. Wild-type (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 fluorescence-labeled cRNA was hybridized onto the Whole Mouse Genome Oligo Microarray (4 × 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 differentially-expressed genes from biological and experimental repeats before all replicates were combined. Genes with a fold-change 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™ 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 μL 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. β-actin was used as the reference gene. The 2
− ΔΔCt method was used to determine the relative expression levels of genes.
Table 1
Primers used in RT-PCR.
Cdh1
| NM_009864 | CAGCCGGTCTTTGAGGGATT | TGACGATGGTGTAGGCGATG |
Cdh4
| NM_009867 | ACAACCGTCCCGAGTTCATC | TCATCTGCATCGTTGGCTGT |
Htr3a
| NM_013561 | CAGACCACCTCCTGGCTAAC | GATGCTGTCTGTGGGGATGG |
Htr4
| NM_008313 | ACGTCCTCATGCCCATTTCC | ACCACTGCAAGGAACGTGAG |
Kcnj9
| NM_008429 | TCTTCTTCGTGCTCGCCTAC | CGAAGCCGTTGAGGTTGTTG |
Pla2g4e
| NM_177845 | CTCCAACTGCCTACACCCAG | CCTCTGGGTTGAGTGGGAAC |
Xaf1
| NM_001037713 | AGAGCCCATCCCAGAGTCAA | CAGATTGCTAAGCTGCACGG |
Lactb
| NM_030717 | GGCTATGCAGACGTGGAGAA | CAGTTTAGCCAGAGCCACCA |
Actb
| NM_007393 | GCTGTATTCCCCTCCATCGTG | AGTCCTTCTGACCCATTCCCA |
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.
Magnetic Resonance Imaging (MRI)
A separate cohort of mice (4 months old, n = 5/genotype) was used in MRI analysis. MRI data were acquired with a 7.0 T animal MRI scanner (PharmaScan, Bruker Biospin GmbH, Germany) with 4-channel phased array coil. T2-weighed (T2-wt) MRI was performed using a rapid acquisition with relaxation enhancement (RARE) sequence with TR/TE = 4200/36 ms, RARE factor = 8, and averaging number = 3. The geometric parameters for the scan were: slice number = 18, slice thickness = 0.5 mm, matrix = 256 × 256, and FOV = 21 × 21 mm2.
4′,6-Diamidino-2-Phenylindole (DAPI) Staining
Adult Np65-KO and WT mice (n = 4, 2 months old) were anesthetized with 1% pentobarbital sodium intraperitoneally and perfused with 4% paraformaldehyde. The brain was removed, postfixed for 10 h–16 h, and cryoprotected in 20% sucrose. Coronal sections (10 μm, at the level of the lateral ventricle, 0 mm–2 mm from bregma) were prepared for DAPI staining. In brief, the sections were blocked in 5% BSA (B2064-100G, Sigma-Aldrich, St. Louis, MO) with 0.3% Triton X-100 (ST795, Beyotime), then incubated with DAPI (1:300, Beyotime) diluted in 1% BSA with 0.3% Triton X-100 for 10 min at room temperature. The sections were then rinsed with PBS and covered with Permount for fluorescent microscopy (Eclipse 80i, Nikon Corp., Tokyo, Japan).
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.
Discussion
Np65 is specifically expressed in the brain and has been reported to mediate several cellular processes including cell-cell 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 Aβ (1–42) [
23]. In addition, central 5-HT activity is closely associated with anxiety [
24‐
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.