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 NRF2 gene and the KEAP1 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.

Competing interests: The authors have the following interests: Drs. H. Suganuma and Y. Ushida are employees of Kagome Co, Ltd., which provided SFN-rich supplement. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Funding: This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas of the Ministry of Education, Culture, Sports, Science and Technology, Japan (to K. H., #24116006), and a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the Ministry of Education, Science, Sports and Culture of Japan (to R. H. and K. H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Kagome Co. Ltd. provided support in the form of salaries for authors HS and YS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Considering the potent antioxidant and anti-inflammatory actions of SFN, we hypothesized that SFN might be useful for the prevention or treatment of cognitive impairment in patients with psychiatric diseases. First, we examined whether SFN had prophylactic and therapeutic effects on cognitive deficits in mice after the repeated administration of PCP. Second, we examined whether the dietary intake of 0.1% glucoraphanin (GF: a glucosinolate precursor of SFN) during the juvenile and adolescence could prevent the onset of PCP-induced cognitive deficits at adulthood. Finally, we examined the association between the KEAP1 and NRF2 genes and cognitive function in patients with schizophrenia.

Oxidative stress and inflammation play a key role in the pathophysiology of schizophrenia as well as cognitive impairment in patients with psychiatric diseases such as schizophrenia [ 7 – 15 ]. The potent antioxidant sulforaphane (SFN: 1-isothiocyanato-4-methylsulfinylbutane) is an organosulfur compound derived from a glucosinolate precursor found in cruciferous vegetables, such as broccoli [ 16 – 18 ]. The protection afforded by SFN is thought to be mediated via the activation of the NF-E2-related factor-2 (Nrf2) pathway and the subsequent up-regulation of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the electrophilic-responsive element or the antioxidant-responsive element (ARE)[ 18 – 20 ]. Under normal conditions, Nrf2 is repressed by Keap1 (Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1), which is an adaptor protein for the degradation of Nrf2 [ 21 ]. During oxidative stress, Nrf2 is derepressed and activates the transcription of cytoprotective genes [ 21 ]. Recently, we reported that SFN could prevent behavioral abnormalities and dopaminergic neurotoxicity in mice after the administration of the psychostimulant methamphetamine [ 22 ]. Subsequently, we also reported that SFN could attenuate behavioral abnormalities in mice after the administration of the N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine (PCP)[ 23 ], since a PCP model of schizophrenia has been accepted throughout the world. These findings suggest that SFN could be a potential therapeutic natural compound for neuropsychiatric diseases, including substance abuse and schizophrenia [ 7 , 22 , 23 ].

Cognitive impairment is observed in patients with a number of psychiatric diseases, including schizophrenia, major depressive disorder, bipolar disorder, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive compulsive disorder, attention deficit hyperactivity disorder, and autism spectrum disorder [ 1 ]. Cognitive impairment is also a core feature of schizophrenia, often persisting even when psychotic symptoms have been treated successfully [ 2 , 3 ]. Interestingly, studies on adolescents and young adults at a high risk of developing psychosis have demonstrated cognitive impairment before the onset of psychotic symptoms [ 4 – 6 ]. Since cognitive impairment is a prodromal symptom, early intervention may prevent the onset of psychosis at adulthood [ 7 , 8 ].

Materials and Methods

Animals Male ICR mice (4 or 6 weeks old) weighing 25–30 g were purchased from SLC Japan (Hamamatsu, Shizuoka, Japan). The mice were housed in clear polycarbonate cages (22.5×33.8×14.0 cm) in groups of 5 or 6 individuals under a controlled 12-h light/12-h dark cycle (lights on from 7:00 AM to 7:00 PM), with the room temperature kept at 23°C ± 1°C and the humidity at 55% ± 5% to acclimatize the mice before the behavioral experiments. The mice were given free access to water and food pellets (CE-2; CLEA Japan, Inc., Tokyo, Japan). The experimental procedure was approved by the Chiba University Institutional Animal Care and Use Committee (Permission number: 26–24).

Prophylactic effect of SFN on PCP-induced cognitive deficits The treatment protocol for repeated PCP administration to induce cognitive deficits in mice has been previously reported [24–26]. Forty-six mice (6 weeks old) were divided into the following four groups: a vehicle (10 mL/kg/day, i.p., water in 10% corn oil) + saline (10 mL/kg/day, s.c.) group; a SFN (30 mg/kg/day, i.p.; LKT Laboratories, Inc., St. Paul, MN) + saline (10 mL/kg/day, s.c.) group; a vehicle (10 mL/kg/day, i.p.) + PCP (10 mg/kg/day as a hydrochloride salt, s.c.; synthesized by K. Hashimoto) group; and a SFN (30 mg/kg/day, i.p.) + PCP (10 mg/kg/day, s.c.) group. The interval between the first injection and the second injection was 30 min. In this study, we used a 30 mg/kg dose of SFN in the mice, as this was the most effective dose in previously reported experiments evaluating PCP-induced hyperlocomotion and PPI deficits [23]. Treatment was performed for 10 days (once daily on days 1–5 and 8–12). The NORT was performed on days 15 and 16.

Therapeutic effect of SFN on PCP-induced cognitive deficits Thirty-eight mice (6 weeks old) were divided into the following four groups: a saline + vehicle group; a saline + SFN group; a PCP + vehicle group; and a PCP + SFN group. Saline (10 mL/kg/day, s.c.) or PCP (10 mg/kg/day, s.c.) was administered on days 1–5 and days 8–12. Subsequently, SFN (30 mg/kg/day, i.p.) or the vehicle (10 mL/kg/day, i.p., water in 10% corn oil) was administered once daily on days 15–28. The NORT was performed on days 29 and 30.

Prophylactic effect of the dietary intake of 0.1% glucoraphanin (GF) during the juvenile and adolescence on PCP-induced cognitive deficits at adulthood Food pellets (CE-2; Japan CLEA, Ltd., Tokyo, Japan) containing 0.1% glucoraphanin (GF) were prepared as follows. Broccoli sprout extract powder containing SFN precursor GF was industrially produced by KAGOME CO., LTD. In brief, broccoli sprout was grown from specially selected seeds (Brassica Protection Products LLC., Baltimore, MD) for 1 day after the germination. The 1 day broccoli sprout was plunged into boiling water and maintained at 95°C for 30 minutes, and the sprout residues was removed by filtration. The boiling water extract was mixed with a waxy corn starch dextrin and then spray dried to yield the broccoli sprout extract powder containing 135 mg (approx. 0.31 mmol) of GF per gram. For preparing the animal diet containing 0.1% GF (approx. 2.3 mmol GF per 1 kg-diet), the extract powder was mixed with a basal diet CE-2 (CLEA Japan Inc., Tokyo, Japan), and then pelletized at a processing facility (Oriental Yeast Co., ltd., Tokyo, Japan). The GF content in the diet was determined by high performance liquid chromatography as previously described [27, 28]. Forty-three mice (4 weeks old) were divided into a normal food pellet group and a 0.1% GF-containing pellet group. The mice were given free access to water and both food pellets specifically designed for mice for 4-weeks (days 1–28). Subsequently, the mice were divided into the four groups: (1) a normal food + vehicle (10 mL/kg/day, s.c.) group; (2) a normal food + PCP (10 mg/kg/day, s.c.) group; (3) a 0.1% GF-containing food + vehicle (10 mL/kg/day, s.c.) group; and (4) a 0.1% GF-containing food + PCP (10 mg/kg/day, s.c.) group. Saline (10 mL/kg/day, s.c.) or PCP (10 mg/kg/day, s.c.) was administered on days 29–33 and days 36–40, as reported previously (24). In addition, normal food (CE-2) was given to the four groups on days 29–44. The NORT was performed on days 43 and 44.

Novel object recognition test (NORT) The NORT was performed as previously reported [24–26]. The apparatus for this task consisted of a black open-field box (50.8 × 50.8 × 25.4 cm). Before the test, mice were habituated to the box for 3 days. During the training session, two objects (various objects were used that differed with respect to shape and color, but that were similar in size) were placed in the box at a 35.5 cm distance from each other, and in a symmetrical fashion, and each animal was allowed to explore the interior of the box for 10 min (5 min × 2). The Animals were considered to be investigating the object when the head of the animal was either facing the object and was located within an inch of the object, or if any part of the body, except for the tail, was touching the object. After the training, the mice ware immediately returned to their home cages, and the box and objects ware cleaned with 75% ethanol to avoid any possible pheromonal cues. The retention test session was carried out one day after the respective training sessions. During each retention test session, each mouse was placed back into the same box it had previously encountered, but in which one of the two objects used during training session had been replaced by a novel object. The mice ware then allowed to freely explore the interior for 5 min, and the time spent exploring each object was again recorded. Throughout the experiments, the objects were used in a counter-balanced manner in terms of their physical complexity. In order to measure memory performance, a preference index was used, i.e., the ratio of the amount of time the mouse spent exploring any one of the two objects (training session) or the novel object (retention session) to total time spent exploring both objects.

Golgi staining Golgi staining was performed using the FD Rapid GolgiStain Kit (FD Neuro Technologies, Inc., Columbia, MD, USA), following the manufacturer's instructions, as previously reported [29, 30]. Mice were deeply anesthetized with sodium pentobarbital, and brains were removed from the skull and rinsed in double distilled water. Brains were immersed in the impregnation solution, made by mixing equal volumes of Solution A and B, overnight and then stored in fresh solution, for 2 weeks in the dark. Brains were transferred into Solution C overnight and then stored in fresh solution at 4°C for 1 week, in the dark. Coronal brain sections (100 μm thickness) were cut on a cryostat (3050S, Leica Microsystems AG, Wetzlar, Germany), with the chamber temperature set at -20°C. Each section was mounted in Solution C, on saline-coated microscope slides. After absorption of excess solution, sections were dried naturally, at room temperature. Dried sections were processed following the manufacturer's instructions. Briefly, images of dendrites within medial prefrontal cortex (mPFC), hippocampal CA1, CA3, and dentate gyrus (DG), nucleus accumbens (NAc)-core, NAc-shell, striatum and ventral tegmental area (VTA) were captured using a 100× objective with a Keyence BZ-9000 GenerationⅡmicroscope (Osaka, Japan). Spine density in these regions was counted as previously reported [29–31]. For spine density measurements, all clearly evaluable areas containing 50–100 μm of secondary dendrites from each imaged neuron were used. To determine relative spine density, spines on multiple dendritic branches from a single neuron were counted to obtain an average spine number per 10 μm. For spine number measurements, only spines that emerged perpendicular to the dendritic shaft were counted. Two to Three neurons per section, three sections per animal and seven to eight animals were analyzed. The average value for each region, in each individual was obtained. These individual averages were then combined to yield a grand average for each region.

Immunohistochemistry for 8-oxo-dG Immunohistochemistry of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) was performed by the previous reports [32, 33] with a slight modification. Mice ware anesthetized with sodium pentobarbital (50 mg/kg) and perfused transcardially with 10 mL of isotonic saline, followed by 40 mL of ice-cold, 4% paraformaldehyde in 0.1%M phosphate buffer (pH 7.4). Brains ware removed from the skulls and postfixed overnight at 4°C in the same fixative. For the immunohistochemical analysis, 50 μm-thick serial, coronal sections of brain tissue were cut in ice-cold, 0.01M phosphate buffered saline (pH 7.5) using a vibrating blade microtome (VT1000s, Leica Microsystems AG, Wetzlar, Germany). The VectorⓇ Mouse on Mouse (MOM) Immunodetection Kit (Catalog No. PK-2200, Vector Laboratories, Inc., Burlingame, CA) was used. Free-floating sections were treated with 0.3% H 2 O 2 in 50 mM Tris-HCL saline (TBS) for 30 min and rinsed two times in TBS and were blocked in TBS containing 0.2% Triton X-100 (TBST), 0.1% bovine serum albumin (BSA), and the MOM Ig blocking reagent for 1 h at room temperature. The sections ware quickly washed two times in TBS. The samples were then incubated in MOM diluent (add 600 μL of Protein Concentrate stock solution to 7.5 mL of TBS) for 5 min at room temperature. The samples were then incubated with mouse anti-8-oxo-dG antibody (1:250; Catalog # 4354-MC-050, TREVIGEN, Gaithersburg, USA) in the MOM Diluent for 30 min. The sections were washed quickly two times in TBS and then processed using the avidin-biotin-peroxidase method. The sections were incubated in biotinylated anti-mouse IgG reagent in MOM diluent. The sections were washed two times in TBS and incubated with avidin-biotin-peroxidase complex in TBS (add 2 drops of Reagent A to 2.5 mL of TBS, mix, and add 2 drops of Reagent B, mix) for 10 min. The sections were rinsed two times. Sections ware incubated for 3 min in a solution of 0.25 mg/mL diaminobenzidine (DAB) containing 0.01% H 2 O 2 . Then, sections were mounted on gelatinized slides, dehydrated, cleared, and coverslipped under PermountⓇ (Fisher Scientific, Fair Lawn, NJ, USA). The sections were imaged, and the staining intensity of 8-oxo-dG immunoreactivity in the middle prefrontal cortex (mPFC), hippocampus (CA1, CA3, DG) was analyzed using a light micro-scope equipped with a CCD camera (Olymups IX70) and the SCION IMAGE software package. Images of sections within the mPFC and hippocampal CA1, CA3, and DG were captured using a 100× objective with a Keyence BZ-9000 GenerationⅡmicroscope (Osaka, Japan).

Immunohistochemistry for parvalbumin (PV) Mice ware anesthetized with sodium pentobarbital (50 mg/kg) and perfused transcardially with 10 mL of isotonic saline, followed by 40 mL of ice-cold, 4% paraformaldehyde in 0.1%M phosphate buffer (pH 7.4). Brains ware removed from the skulls and postfixed overnight at 4°C in the same fixative. For the immunohistochemical analysis, 50 μm-thick serial, coronal sections of brain tissue were cut in ice-cold, 0.001M phosphate buffered saline (pH 7.5) using a vibrating blade microtome (VT1000s, Leica Microsystems AG, Wetzlar, Germany). Free-floating sections were treated with 0.3% H 2 O 2 in 50 mM Tris-HCL saline (TBS) for 30min and ware blocked in TBS containing 0.2% Triton X-100 (TBST) and 1.5% normal serum for 1 h at room temperature. The samples ware then incubated for 24 h at 4°C with rabbit polyclonal anti-parvalbumin (PV) antibody (1:2,500, Swant, Bellinzona, Switzerland). The sections were washed three times in TBS and then processed using the avidin-biotin-peroxidase method (Vectastain Elite ABC, Vector Laboratories, Inc., Burlingame, CA, USA). Sections ware incubated for 3 min in a solution of 0.25 mg/mL DAB containing 0.01% H 2 O 2 . Then, sections were mounted on gelatinized slides, dehydrated, cleared, and cover slipped under PermountⓇ(Fisher Scientific, Fair Lawn, NJ, USA). The sections were imaged, and the staining intensity of PV immunoreactivity in the mPFC, hippocampus (CA1, CA3, DG) was analyzed using a light micro-scope equipped with a CCD camera (Olymups IX70) and the SCION IMAGE software package. Images of sections within mPFC and hippocampal CA1, CA3, DG regions were captured using a 100× objective with a Keyence BZ-9000 GenerationⅡmicroscope (Osaka, Japan).

Gene analysis of the NRF2 and KEAP1 gene variants in humans All subjects were biologically unrelated within the second-degree of relationship and of Japanese descent. Subjects were excluded if they had neurological or medical conditions that could potentially affect the central nervous system, as previously described [34, 35]. Cases were recruited from the Osaka University Hospital. Each patient with schizophrenia had been diagnosed by at least two trained psychiatrists according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) based on the Structured Clinical Interview for DSM-IV (SCID). Controls were recruited through local advertisements at Osaka University. Psychiatrically, medically and neurologically healthy controls were evaluated using the non-patient version of the SCID to exclude individuals who had current or past contact with psychiatric services or received psychiatric medication. The mean age and gender ratio did not differ significantly between cases and controls (P > 0.10), while the years of education and estimated premorbid IQ were significantly lower in the patients with schizophrenia than the controls (P < 0.001) (Table 1). Written informed consent was obtained from all subjects after the procedures had been fully explained. This study was conducted in accordance with the World Medical Association’s Declaration of Helsinki and approved by the Research Ethical Committee of Osaka University (Permission number: 379). PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Demographic information for patients with schizophrenia and healthy controls. https://doi.org/10.1371/journal.pone.0127244.t001 Venous blood was collected from the subjects, and genomic DNA was extracted from whole blood according to standard procedures. We selected three single nucleotide polymorphisms (SNPs); rs10930781 from NRF2 gene and rs1048290 and rs11545829 from KEAP1 gene. It has been reported that rs6721961 located in the promoter region of the NRF2 gene affects the transcriptional activity of NRF2 and the minor allele of rs6721961 SNP diminishes promoter activity of the gene [36]. As the rs6721961 did not exist in our genotyped Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA)[37], we selected proxy SNP rs10930781 of the rs6721961 in Japanese population (JPT) (r2 = 0.86). The rs10930781 was located in the intron region of the NRF2 gene. The genotyping data was extracted from the array. Next, we selected two possible functional synonymous polymorphisms from exons in the KEAP1 gene because no functional SNP affecting the KEAP1 function has been reported. The selected rs1048290 and rs11545829 were Leu471Leu and Tyr537Tyr, respectively. As these SNPs of the KEAP1 gene did not exist in the Affymetrix array or there was no proxy SNP around these SNPs in the array, these SNPs were genotyped using the TaqMan 5’-exonuclease allelic discrimination assay (Assay ID: rs1048290; C_9323035_1_, rs11545829; C_34043047_10, Applied Biosystems, Foster City, California, USA) as previously described [38,39]. Detailed information on the PCR conditions is available upon request. No deviation from the Hardy-Weinberg equilibrium (HWE) was detected in the examined SNPs in the patients or controls (P>0.05). According to these genotyping data, we divided subjects into two sets of the four groups that minimized minor allele carriers due to small sample size in some cells: i) major allele homozygotes (NRF2 CC-KEAP1 rs1048290 GG), NRF2 major allele homozygotes plus KEAP1 rs1048290 minor allele carriers (NRF2 CC-KEAP1 rs1048290 CG/CC), NRF2 minor allele carriers plus KEAP1 rs1048290 major allele homozygotes (NRF2 CT/TT-KEAP1 rs1048290 GG), and minor allele carriers (NRF2 CT/TT-KEAP1 rs1048290 CG/CC), ii) major allele homozygotes (NRF2 CC-KEAP1 rs11545829 CC), NRF2 major allele homozygotes plus KEAP1 rs11545829 minor allele carriers (NRF2 CC-KEAP1 rs11545829 CT/TT), NRF2 minor allele carriers plus KEAP1 rs11545829 major allele homozygotes (NRF2 CT/TT-KEAP1 rs11545829 CC), and minor allele carriers (NRF2 CT/TT-KEAP1 rs11545829 CT/TT). To assess intellectual functions remarkably impaired in patients with schizophrenia [40], we used the full-scale IQ and the four subscales; Verbal Comprehension, Perceptual Organization, Working Memory and Processing Speed, of the Japanese version of the Wechsler Adult Intelligence Scale (WAIS)-third edition [41]. The subjects were assessed by trained clinical psychologists to obtain the scores on the WAIS.