1574 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1574-1586, 2015 Changes in Dpysl2 expression are associated with prenatally stressed rat offspring and susceptibility to schizophrenia in humans HWAYOUNG LEE1, JAESOON JOO1, SEONG-SU NAH2, JONG WOO KIM3, HYUNG-KI KIM1, JUN-TACK KWON1, HWA-YOUNG LEE4, YOUNG OCK KIM5 and HAK-JAE KIM1,6 1Department of Clinical Pharmacology, 2Division of Rheumatology, Department of Internal Medicine, College of Medicine, Soonchunhyang University, Cheonan; 3Department of Neuropsychiatry, School of Medicine, Kyunghee University, Seoul; 4Department of Psychiatry, College of Medicine, Soonchunhyang University, Cheonan; 5Development of Ginseng and Medical Plants Research Institute, Rural Administration, Eumseong; 6Soonchunhyang Medical Research Institute, College of Medicine, Soonchunhyang University, Cheonan, Republic of Korea Received August 22, 2014; Accepted March 26, 2015 DOI: 10.3892/ijmm.2015.2161 Abstract. Exposure to stress during critical periods of fetal development of schizophrenia. Taken together with previous brain development is an environmental risk factor for the studies investigating the association between the DPYSL2 development of schizophrenia in adult offspring. In the gene and schizophrenia, the present findings may contribute present study, a repeated-variable stress paradigm was applied additional evidence regarding developmental theories of the to pregnant rats during the last week of gestation, which is pathophysiology of schizophrenia. analogous to the second trimester of brain development in humans. Behavioral and proteomic analyses were conducted Introduction in prenatally-stressed (PNS) adult offspring and non-stressed (NS) adult controls. In the behavioral tests, grooming behavior Schizophrenia is a well-known psychiatric disorder with in the social interaction test, line-crossing behavior in the open a developmental etiology that is associated with abnormal field test, and swimming behavior in the forced swimming test mental functions and behaviors, including delusions, hallu- were decreased in the PNS group. Western blot analysis and cinations, disorganized thinking, and negative symptoms. immunohistochemical analysis revealed that the expression Additionally, many patients exhibit concomitant mood symp- of dihydropyrimidinase-like 2 (Dpysl2) or collapsin response toms, such as depression and anxiety, which may contribute mediator protein 2 (Crmp2) was downregulated in the to the 10% lifetime incidence of suicide in schizophrenic prefrontal cortex and hippocampus of rats in the PNS group. patients (1,2). Prenatal stress (PNS) is among the most Subsequently, single-nucleotide polymorphisms (SNPs) of frequently reported environmental risk factors for the develop- the human dihydropyrimidinase-like 2 (DPYSL2) gene were ment of schizophrenia in adults (3-8), and the second trimester analyzed in a population. Two functional SNPs (rs9886448 in of pregnancy in humans seems to be the most vulnerable the promoter region and rs2289593 in the exon region) were period for insults (8). Furthermore, a number of animal studies associated with susceptibility to schizophrenia. The present have shown that maternal exposure to stress during gestation findings demonstrated that the downregulation of genes such elevates glucocorticoids and that this type of stress is associ- as Dpysl2 and Dypsl3 in a rat model of prenatal stress may ated with various biochemical, physiological, and behavioral affect subsequent behavioral changes and that polymorphisms changes in offspring, including reduced birth weight, cardio- of the DPYSL2 gene in humans may be associated with the vascular and neuroendocrinological abnormalities, attentional dysfunction, enhanced anxiety-related behaviors, and cogni- tive deficits (9-18). In studies investigating this connection, pregnant female rats are usually exposed to stressful manipu- lations during the third week of pregnancy, which, in terms of Correspondence to: Professor Hak-Jae Kim, Department of neural development, is equivalent to the second trimester of Clinical Pharmacology, College of Medicine, Soonchunhyang University, 31 Soonchunyang 6-gil, Cheonan 330-721, Republic of human gestation (15-17). Korea Previous findings have demonstrated that PNS or maternal E-mail: [email protected] exposure to glucocorticoids may affect the responsivity of the hypothalamic-pituitary-adrenal (HPA) axis and can induce Key words: dihydropyrimidinase-like 2, social interaction, single cognitive deficits in offspring (10,16,17,19-22). Cognitive nucleotide polymorphism, prenatal stress deficits that result from disrupted hippocampal anatomy and impaired function of the HPA axis are also typically observed in patients with schizophrenia (23,24). In animal studies, PNS LEE et al: Dpysl2 EXPRESSION AND SCHIZOPHRENIA 1575 results in a reduced number of hippocampal synapses as well animal-room husbandry procedures. Following birth, the as fewer neurons in the brain (25). Moreover, the hippocampi dams and their pups were left undisturbed in their cages until of adolescent and adult male rats exposed to PNS exhibit weaning on postnatal day 23. At this time, male and female decreases in dendritic length, spine density, and the number offspring were separated and group-housed in cages with one of neurons relative to non-stressed (NS) controls (26,27). PNS or two same-gender littermates with free access to rat chow also causes variable changes of gene expression in the brains and water. The animals were exposed to normal animal room of rats, including in genes associated with neural development, conditions from that point onwards until experimental use on cell differentiation, and neurotransmitter function (16,28,29). postnatal day 35 (16,17). The frontal poles of PNS rat brains exhibit significant changes in genes associated with postsynaptic density Behavioral measures. Modified behavioral tests, including a complexes and vesicle exocytosis machinery, including the social interaction test, the open field test, and the forced swim N-methyl-D-aspartate (NMDA) receptor 1 and 2A subunits, test, were performed, as previously described (15,30-32). The leucine-rich repeat domain-containing proteins (6), social interaction test was adapted from previous studies brain-enriched guanylate kinase-associated proteins, synap- (15,31,32) and was conducted in a clear Plexiglas chamber tosomal-associated proteins (24), synaphin/complexin, and (77x77x25 cm). The room in which the chamber was located vesicle-associated membrane protein 2 (16). A recent study was darkened during testing, and the chamber was illumi- utilizing microarray-based profiling of the hippocampus nated by a single 25 W red light bulb placed ~100 cm above and frontal cortex investigated changes in gene expression the base of the chamber (subject age, 30 days). The sessions following PNS and identified changes in genes supporting were filmed with a video camera (Samsung, Seoul, Korea) biological processes and/or signal transduction cascades that placed 150 cm above the cage. The investigator remained underlie glutamatergic and γ-aminobutyric acid (GABA)-ergic outside the test room during testing, and the test arena was neurotransmission, mitogen-activated protein kinase (MAPK) cleaned after each test session. Social interaction partners signaling, neurotrophic factor signaling, phosphodiesterase were same-gender siblings who resided in the same cage after (PDE)/cyclic nucleotide signaling, glycogen synthase kinase 3 weaning and were of approximately equal body weight (in (GSK3) signaling, and insulin signaling (28). Furthermore, the few cases in which a same-gender sibling was not avail- Mairesse et al (29) observed alterations in protein expres- able, a playmate from similar conditions was used). Each sion in the hippocampi of PNS rats and confirmed changes session lasted for 20 min and was scored in terms of the total in proteins, such as the LIM and SH3 protein 1 (Lasp1), duration of social play and the number and types of interac- prohibitin, fascin and transferrin. tions. Specifically, a rater blind to the treatment conditions Previous findings such as those have led to the hypothesis scored behaviors as aggressive [fighting (kicking, boxing, and that PNS-induced phenomena alter the expression of schizo- wrestling), aggressive grooming and biting] or non-aggressive phrenia-associated genes via proteomic mechanisms and that (sniffing, following and grooming the partner) based on the these genes ultimately affect susceptibility to schizophrenia in video. Experimental and target rats were not used in this humans. Thus, in the present study, changes in protein expres- paradigm more than once, and the arena was cleaned with sion were examined in the prefrontal cortex and hippocampus 70% ethanol after each trial. of rats exposed to repeated variable PNS. Furthermore, we The open field test was used to assess exploratory activity investigated whether these genes were associated with suscep- and reactivity to a novel environment. On the test day, subjects tibility to schizophrenia in humans using genotyping and were removed from their home cage (subject age, 32 days) functional assays of the polymorphisms. and individually placed in the start box (15x15x20 cm) of the open field arena (77x77x25 cm) for 5 min. The apparatus was Subjects and methods composed of black Polygal, and no background noise was provided. The investigator exited the room, and the behavior Animals and stress paradigm. Previously used, pregnant, of the subject