Early Behavioral Abnormalities and Perinatal Alterations of PTEN/AKT Pathway in Valproic Acid Autism Model Mice

RESEARCH ARTICLE Early Behavioral Abnormalities and Perinatal Alterations of PTEN/AKT Pathway in Valproic Acid Autism Model Mice Eun-Jeong Yang1, Sangzin Ahn1,2, Kihwan Lee1, Usman Mahmood3*, Hye-Sun Kim1,3,4* 1 Department of Pharmacology and Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea, 2 Department of Pharmacology, Inje Univeirsity College of Medicine, Busan, Republic of Korea, 3 Interdisciplinary Program in Brain Sciences, Seoul National University College of Natural Sciences, Seoul, Republic of Korea, 4 Seoul National University Bundang Hospital, Seoul National University College of Medicine, Sungnam, Republic of Korea a11111 * (UM); (HSK) Abstract OPEN ACCESS Citation: Yang E-J, Ahn S, Lee K, Mahmood U, Kim H-S (2016) Early Behavioral Abnormalities and Perinatal Alterations of PTEN/AKT Pathway in Valproic Acid Autism Model Mice. PLoS ONE 11(4): e0153298. doi:10.1371/journal.pone.0153298 Editor: Valerie W Hu, The George Washington University, UNITED STATES Received: June 27, 2015 Accepted: March 28, 2016 Published: April 12, 2016 Copyright: © 2016 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was financially supported by grants from the Korea Healthcare Technology R&D Project (HI3C1451) of Ministry for Health, Welfare and Family Affairs of the Republic of Korea, by the National Research Foundation of Korea (NRF) through the Ministry of Education, Science and Technology (NRF-2011-0021866). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Exposure to valproic acid (VPA) during pregnancy has been linked with increased incidence of autism, and has repeatedly been demonstrated as a useful autism mouse model. We examined the early behavioral and anatomical changes as well as molecular changes in mice prenatally exposed to VPA (VPA mice). In this study, we first showed that VPA mice showed developmental delays as assessed with self-righting, eye opening tests and impaired social recognition. In addition, we provide the first evidence that primary cultured neurons from VPAtreated embryos present an increase in dendritic spines, compared with those from control mice. Mutations in phosphatase and tensin homolog (PTEN) gene are also known to be associated with autism, and mice with PTEN knockout show autistic characteristics. Protein expression of PTEN was decreased and the ratio of p-AKT/AKT was increased in the cerebral cortex and the hippocampus, and a distinctive anatomical change in the CA1 region of the hippocampus was observed. Taken together, our study suggests that prenatal exposure to VPA induces developmental delays and neuroanatomical changes via the reduction of PTEN level and these changes were detectable in the early days of life. Introduction Autism spectrum disorder (ASD) is a group of developmental disabilities characterized by social interaction, verbal and nonverbal communication, and stereotyped behaviors and interests [1]. Its prevalence is as high as 0.7–1.1% in the general population and is four times more common in males than females [2–4]. Abnormal development is often observed in autistic patients in the early stages of life, including weight fluctuation [5, 6], abnormal brain development [7–9], disruption in synaptic connection and hyperactive neuronal connections resulting in behavioral complexities [10–12]. While up to 25% of ASD cases are identified to carry inheritable single genes or rare gene mutations [13–16], population studies suggest that environmental factors during the prenatal period also contribute to an increased incidence of autism [4, 17, 18]. PLOS ONE | DOI:10.1371/journal.pone.0153298 April 12, 2016 1 / 17 Early Behavior and PTEN/AKT Alterations in VPA Mice Competing Interests: The authors have declared that no competing interests exist. Valproic acid (VPA), an antiepileptic agent used to treat epilepsy and bipolar disorder, is also associated with an increased risk for congenital malformations and delayed cognitive development in offsprings [19–21]. Prospective and retrospective studies have demonstrated that the exposure to VPA during pregnancy is associated with a three-fold rate of major anomalies and dysmorphic features as well as decreased intrauterine growth [22]. Epidemiological data has been successfully implanted into research as animal studies using male VPA-exposed mice have shown repeatedly core behavioral signs of autism as well as molecular changes linked to the disorder [23–26]. The underlying molecular mechanisms of VPA-treated mice have been explored to imply autism-related genes including brain-derived neurotrophic factor [26], neuroligin 1 [27], neuroligin 3 [28, 29], and monoamine synaptic transmission [30, 31]. Phosphatase and tensin homolog (PTEN), a gene located on chromosome 10q23, is involved in a wide variety of cellular processes relevant to brain growth and circuit function [32, 33]. PTEN, previously recognized as a tumor suppressor gene mutated in many human cancers [34], has recently gained traction in its association with ASD [32, 35–39]. PTEN mutation was recently documented as a causative factor and its conditional knockout studies are validating the link between autism and PTEN [32, 37, 38]. Pten gene is considered as susceptible for autism as Fragile X protein (FXS) and Tuberous sclerosis protein complex 1 and 2 (TSC1/2 complex), and PTEN mutations may account as much as 5% of autism associated with macrocephaly and 1% of autism [40]. Perturbation in downstream pathway of PTEN, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway, results in behavioral abnormalities and is expected to play a significant role in ASD [35, 37, 38, 41]. In order to gain insight in an environmental inducer of autism, we explored the possibility of VPA’s in utero exposure in relations to PTEN expression. Although ASD is generally considered to be a developmental disorder, behavioral alteration in the early postnatal phase have yet to be extensively studied in the VPA-induced autism model. In this study, we focused on the early behavioral, anatomical, and molecular changes similar to those found in previously reported PTEN conditional knockout mice [37, 38]. In addition, we analyzed the changes in dendritic spine density by employing primary neuronal cultures from VPA-exposed mice. Materials and Methods Experimental animals Fourteen pregnant BALB/c (Central Lab Animal Inc., Korea) pregnant mice were randomly assigned to VPA-injected (VPA group, n = 9) or saline-injected (SAL group, n = 5) groups. The VPA (...truncated)