<i>In silico</i> analysis of the SNP rs13438494 located in intron 24 of <i>PCLO.</i>

<p>The allele of rs13438494 is indicated in bold.</p><p>These algorithms are included in the analysis of Human Splice Finder (v.2.4).</p

Physical map of PCLO gene locus and SNP rs13438494 location in <i>PCLO.</i>

<p><i> PCLO</i> is located on chromosome 7 and transcribed in opposite direction. This gene spans 409 kb and comprises 25 exons. The position of rs13438494 in intron 24 of <i>PCLO</i> is indicated.</p

Primers used for cloning of the <i>PCLO</i> minigene and site-directed mutagenesis.

<p>Restriction site targets introduced to allow sequential cloning of the PCR-amplified fragments are underlined. The nucleotide replaced by site-directed mutagenesis is indicated in bold.</p

A Naturally Occurring Null Variant of the NMDA Type Glutamate Receptor NR3B Subunit Is a Risk Factor of Schizophrenia

<div><p>Hypofunction of the <i>N</i>-methyl-D-aspartate type glutamate receptor (NMDAR) has been implicated in the pathogenesis of schizophrenia. Here, we investigated the significance of a common human genetic variation of the NMDAR NR3B subunit that inserts 4 bases within the coding region (insCGTT) in the pathogenesis of schizophrenia. The cDNA carrying this polymorphism generates a truncated protein, which is electrophysiologically non-functional in heterologous expression systems. Among 586 schizophrenia patients and 754 healthy controls, insCGTT was significantly overrepresented in patients compared to controls (odds ratio = 1.37, <i>p</i> = 0.035). Among 121 schizophrenia patients and 372 healthy controls, genetic analyses of normal individuals revealed that those carrying insCGTT have a predisposition to schizotypal personality traits (<i>F_1,356</i> = 4.69, <i>p</i> = 0.031). Furthermore, pre-pulse inhibition, a neurobiological trait disturbed in patients with schizophrenia, was significantly impaired in patients carrying insCGTT compared with those with the major allele (<i>F_1,116</i> = 5.72, <i>p</i> = 0.018, <i>F_1,238</i> = 4.46, <i>p</i> = 0.036, respectively). These results indicate that a naturally occurring null variant in NR3B could be a risk factor of schizophrenia.</p></div

Effect of insCGTT type on distribution of NR3B in HEK293T cells.

<p><b>A</b>, HEK293T cells transfected with GluR1 tagged with GFP at the extracellular N-terminus or intracellular C-terminus were stained with a GFP antibody under non-permeabilized conditions on ice, as verification of the specificity of cell surface receptor detection using immunostaining. <b>B</b>, HEK293T cells transfected with GFP-tagged NR3B major type or insCGTT type (green) were stained with GFP antibody under non-permeabilized condition to detect the cell surface population (surface GFP, red). <b>C</b>, Quantification of the surface levels of NR3B major type and insCGTT type with and without NR1. Because expression level of major type and insCGTT were different, the level was normalized by total GFP fluorescence in each cell. Data are expressed as a normalized value to that of major type alone. n = 55, 52, 59, and 52 for GFP-NR3B major type only, GFP-NR3B major type with NR1, GFP-NR3B insCGTT type only, and GFP-NR3B insCGTT type with NR1, respectively. *: p < 0.05; **: p < 0.01; ns: not significant. <b>D</b>, Surface biotinylation of NR3B major type and insCGTT type. GFP-tagged NR3B major type or insCGTT type was coexpressed with NR1 in HEK293T cells and the surface population was labeled by biotin. NR3B types in both total (T) and biotinylated surface population (S) were detected by anti-GFP antibody. Total fractions represent 1/8 of the starting material applied in surface fractions. α-Tubulin (Tub), an intracellular protein, was not biotinylated, confirming specificity of surface biotinylation.</p

Glutamate-induced whole-cell current recorded from HEK293T cells expressing major type or insCGTT type NR3B.

<p><b>A</b>, Sample traces of glutamate-induced whole-cell current recorded in HEK293T cells coexpressing NR1 and NR2A with or without NR3B major type or insCGTT type. The recordings were performed at -60 mV in the presence of 10 μM glycine and in the absence of Mg²⁺. <b>B</b>, The normalized current amplitude from cells without NR3B, with NR3B major type or insCGTT type. The major type NR3B suppressed the current to a statistically significant level, whereas insCGTT type did not show such suppression. The time course of current decay reflects the clearance of puffed glutamate from extracellular fluid by perfusion rather than the kinetics of channel opening and closing. n = 14, each. **: p<0.01 from control, *: p<0.05 from major type, ns: not significant.</p

Dietary Intake of Sulforaphane-Rich Broccoli Sprout Extracts during Juvenile and Adolescence Can Prevent Phencyclidine-Induced Cognitive Deficits at Adulthood

<div><p>Oxidative stress and inflammation play a role in cognitive impairment, which is a core symptom of schizophrenia. Furthermore, a hallmark of the pathophysiology of this disease is the dysfunction of cortical inhibitory γ-aminobutyric acid (GABA) neurons expressing parvalbumin (PV), which is also involved in cognitive impairment. Sulforaphane (SFN), an isothiocyanate derived from broccoli, is a potent activator of the transcription factor Nrf2, which plays a central role in the inducible expressions of many cytoprotective genes in response to oxidative stress. Keap1 is a cytoplasmic protein that is essential for the regulation of Nrf2 activity. Here, we found that pretreatment with SFN attenuated cognitive deficits, the increase in 8-oxo-dG-positive cells, and the decrease in PV-positive cells in the medial prefrontal cortex and hippocampus after repeated administration of phencyclidine (PCP). Furthermore, PCP-induced cognitive deficits were improved by the subsequent subchronic administration of SFN. Interestingly, the dietary intake of glucoraphanin (a glucosinolate precursor of SFN) during the juvenile and adolescence prevented the onset of PCP-induced cognitive deficits as well as the increase in 8-oxo-dG-positive cells and the decrease in PV-positive cells in the brain at adulthood. Moreover, the <i>NRF2</i> gene and the <i>KEAP1</i> gene had an epistatic effect on cognitive impairment (e.g., working memory and processing speed) in patients with schizophrenia. These findings suggest that SFN may have prophylactic and therapeutic effects on cognitive impairment in schizophrenia. Therefore, the dietary intake of SFN-rich broccoli sprouts during the juvenile and adolescence may prevent the onset of psychosis at adulthood.</p></div

Genetic analyses of insCGTT type in patients with schizophrenia and controls.

<p><b>A</b>, The association between the NR3B insCGTT type and SPQ total score and the three factors in control individuals. -/-, n = 317; insCGTT carrier, n = 44. <b>B</b>, The association between NR3B insCGTT type and PPI in schizophrenia patients. -/-, n = 107; insCGTT carrier, n = 14 in patients with schizophrenia. <b>C</b>, The association between NR3B insCGTT type and WCST score in control individuals. -/-, n = 243; insCGTT carrier, n = 31. *: p<0.05.</p

NR3B with insCGTT type generates a truncated protein that accumulates intracellularly.

<p><b>A</b>, Schematic drawing of NR3B illustrating the position of insCGTT type. SP: signal peptide, AT-D, amino-terminal domain; S1 and S2, S1 and S2 lobes of ligand binding domain; M1-4, transmembrane and membrane associated regions; CT-D, carboxyl-terminal domain. <b>B</b>, Western blot of total lysate of HEK293T cells expressing either NR3B major type (NR3B) or insCGTT type. <b>C</b>, Western blot of lysate of HEK293T cells expressing major type NR3B, GFP-tagged major type NR3B, insCGTT type, and GFP-tagged insCGTT type, blotted with anti-NR3B N-terminus antibody. Both GFP tagged major type and insCGTT type constructs generated bands that were detected at ∼27 kD more than the respective untagged proteins. <b>D</b>, Double immunostaining of HEK293T cells expressing GFP-NR3B with GFP antibody (green) and an antibody against ER marker calreticulin (CRT, red). NT: no transfected cells. <b>E</b>, Co-immunoprecipitation of NR3B and NR1 from HEK293T cells. GFP-tagged NR3B major type or insCGTT was coexpressed with NR1 and immunoprecipitated by anti-GFP antibody. The immunoprecipitate were blotted with anti-NR1 (middle), and anti-GFP (bottom) antibodies. Immunoblot of NR1 in total cell lysate is also shown (top). The results demonstrate that both NR3B major type and insCGTT types interact with NR1 to a similar degree. <b>F</b>, Extraction of ER and Golgi luminal proteins using sodium carbonate buffer. Crude membrane fractions were treated with sodium carbonate buffer and centrifuged. Western blots were performed using the pelleted membrane fraction (P) and supernatant (S) with indicated antibodies. The detection of CTR, an intraluminal protein, but not calnexin (CNX), a representative integral luminal membrane protein, in the supernatant, indicates the successful extraction of luminal proteins. Under these conditions, both major type and insCGTT type remained in the membrane fraction and were not detect in the supernatant.</p

Demographic information for patients with schizophrenia and healthy controls.

<p>Data are the mean ± SD. Significant <i>P</i> values are shown in boldface.</p><p>^a χ² test. Complete demographic information was not obtained for all subjects (estimated premorbid IQ in patients, n = 179; PANSS, n = 182). PANSS, Positive and Negative Syndrome Scale; CPZ-eq., chlorpromazine equivalent of total antipsychotics.</p><p>Demographic information for patients with schizophrenia and healthy controls.</p