A re-review of the association between the NOTCH4 locus and schizophrenia.

NOTCH4 has long been identified as a candidate susceptibility gene for schizophrenia, but the collective body of genetic association studies of this gene has been less than conclusive. Recently a variant in NOTCH4 was implicated as one of the most reliably associated polymorphisms observed in a genome-wide association scan of the disorder, and the collective evidence for this polymorphism now surpasses criteria for genome-wide significance. To place these developments in context, we now summarize the initial work identifying NOTCH4 as a candidate gene for schizophrenia. The results of the genome-wide association studies that have confirmed this as a risk gene, and novel bioinformatics analyses that reveal potential functional profiles of the most likely risk-conferring polymorphisms. These analyses suggest that the NOTCH4 polymorphisms most strongly associated with schizophrenia exert their effects on susceptibility by altering the efficiency and/or alternative splicing of Notch4 transcripts. Further experimental evidence should be pursued to clarify the NOTCH4-regulated molecular and cellular phenotypes of relevance to the disorder, and the functional consequences of the implicated polymorphisms in the gene.

Dysfunctional gene splicing as a potential contributor to neuropsychiatric disorders.

Alternative pre-mRNA splicing is a major mechanism by which the proteomic diversity of eukaryotic genomes is amplified. Much akin to neuropsychiatric disorders themselves, alternative splicing events can be influenced by genetic, developmental, and environmental factors. Here, we review the evidence that abnormalities of splicing may contribute to the liability toward these disorders. First, we introduce the phenomenon of alternative splicing and describe the processes involved in its regulation. We then review the evidence for specific splicing abnormalities in a wide range of neuropsychiatric disorders, including psychotic disorders (schizophrenia), affective disorders (bipolar disorder and major depressive disorder), suicide, substance abuse disorders (cocaine abuse and alcoholism), and neurodevelopmental disorders (autism). Next, we provide a theoretical reworking of the concept of "gene-focused" epidemiologic and neurobiologic investigations. Lastly, we suggest potentially fruitful lines for future research that should illuminate the nature, extent, causes, and consequences of alternative splicing abnormalities in neuropsychiatric disorders.

Environmental Enrichment Rescues Social Behavioral Deficits and Synaptic Abnormalities in Pten Haploinsufficient Mice.

Pten germline haploinsufficient (Pten+/-) mice, which model macrocephaly/autism syndrome, show social and repetitive behavior deficits, early brain overgrowth, and cortical-subcortical hyperconnectivity. Previous work indicated that altered neuronal connectivity may be a substrate for behavioral deficits. We hypothesized that exposing Pten+/- mice to environmental enrichment after brain overgrowth has occurred may facilitate adaptation to abnormal "hard-wired" connectivity through enhancing synaptic plasticity. Thus, we reared Pten+/- mice and their wild-type littermates from weaning under either standard (4-5 mice per standard-sized cage, containing only bedding and nestlet) or enriched (9-10 mice per large-sized cage, containing objects for exploration and a running wheel, plus bedding and nestlet) conditions. Adult mice were tested on social and non-social assays in which Pten+/- mice display deficits. Environmental enrichment rescued sex-specific deficits in social behavior in Pten+/- mice and partially rescued increased repetitive behavior in Pten+/- males. We found that Pten+/- mice show increased excitatory and decreased inhibitory pre-synaptic proteins; this phenotype was also rescued by environmental enrichment. Together, our results indicate that environmental enrichment can rescue social behavioral deficits in Pten+/- mice, possibly through normalizing the excitatory synaptic protein abundance.

Pten haploinsufficiency disrupts scaling across brain areas during development in mice.

Haploinsufficiency for PTEN is a cause of autism spectrum disorder and brain overgrowth; however, it is not known if PTEN mutations disrupt scaling across brain areas during development. To address this question, we used magnetic resonance imaging to analyze brains of male Pten haploinsufficient (Pten+/-) mice and wild-type littermates during early postnatal development and adulthood. Adult Pten+/- mice display a consistent pattern of abnormal scaling across brain areas, with white matter (WM) areas being particularly affected. This regional and WM enlargement recapitulates structural abnormalities found in individuals with PTEN haploinsufficiency and autism. Early postnatal Pten+/- mice do not display the same pattern, instead exhibiting greater variability across mice and brain regions than controls. This suggests that Pten haploinsufficiency may desynchronize growth across brain regions during early development before stabilizing by maturity. Pten+/- cortical cultures display increased proliferation of glial cell populations, indicating a potential substrate of WM enlargement, and provide a platform for testing candidate therapeutics. Pten haploinsufficiency dysregulates coordinated growth across brain regions during development. This results in abnormally scaled brain areas and associated behavioral deficits, potentially explaining the relationship between PTEN mutations and neurodevelopmental disorders.

RNA sequencing of transformed lymphoblastoid cells from siblings discordant for autism spectrum disorders reveals transcriptomic and functional alterations: Evidence for sex-specific effects.

Genome-wide expression studies of samples derived from individuals with autism spectrum disorder (ASD) and their unaffected siblings have been widely used to shed light on transcriptomic differences associated with this condition. Females have historically been under-represented in ASD genomic studies. Emerging evidence from studies of structural genetic variants and peripheral biomarkers suggest that sex-differences may exist in the biological correlates of ASD. Relatively few studies have explicitly examined whether sex-differences exist in the transcriptomic signature of ASD. The present study quantified genome-wide expression values by performing RNA sequencing on transformed lymphoblastoid cell lines and identified transcripts differentially expressed between same-sex, proximal-aged sibling pairs. We found that performing separate analyses for each sex improved our ability to detect ASD-related transcriptomic differences; we observed a larger number of dysregulated genes within our smaller set of female samples (n = 12 sibling pairs), as compared with the set of male samples (n = 24 sibling pairs), with small, but statistically significant overlap between the sexes. Permutation-based gene-set analyses and weighted gene co-expression network analyses also supported the idea that the transcriptomic signature of ASD may differ between males and females. We discuss our findings in the context of the relevant literature, underscoring the need for future ASD studies to explicitly account for differences between the sexes. Autism Res 2017, 10: 439-455. © 2016 International Society for Autism Research, Wiley Periodicals, Inc.

Characterization of a Novel Mutation in SLC1A1 Associated with Schizophrenia.

We have recently described a hemi-deletion on chromosome 9p24.2 at the SLC1A1 gene locus and its co-segregation with schizophrenia in an extended Palauan pedigree. This finding represents a point of convergence for several pathophysiological models of schizophrenia. The present report sought to characterize the biological consequences of this hemi-deletion. Dual luciferase assays demonstrated that the partially deleted allele (lacking exon 1 and the native promoter) can drive expression of a 5'-truncated SLC1A1 using sequence upstream of exon 2 as a surrogate promoter. However, confocal microscopy and electrophysiological recordings demonstrate that the 5'-truncated SLC1A1 lacks normal membrane localization and glutamate transport ability. To identify downstream consequences of the hemi-deletion, we first used a themed qRT-PCR array to compare expression of 84 GABA and glutamate genes in RNA from peripheral blood leukocytes in deletion carriers (n = 11) versus noncarriers (n = 8) as well as deletion carriers with psychosis (n = 5) versus those without (n = 3). Then, targeted RNA-Seq (TREx) was used to quantify expression of 375 genes associated with neuropsychiatric disorders in HEK293 cells subjected to either knockdown of SLC1A1 or overexpression of full-length or 5'-truncated SLC1A1. Expression changes of several genes strongly implicated in schizophrenia pathophysiology were detected (e.g. SLC1A2, SLC1A3, SLC1A6, SLC7A11, GRIN2A, GRIA1 and DLX1).

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats.

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT3 agonist-induced inhibition of electrically evoked [3H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT2, 5-HT3 and D2 receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT3 agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 microM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 microM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 microM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT2 receptor antagonist. ICS-205,930, a 5-HT3 receptor antagonist or sulpiride, a D2 receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 microM) and was completely reversed by sulpiride (1 microM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT2, 5-HT3 and D2 receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D2 receptors and high concentrations of the 5-HT3 receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.

Acute prenatal exposure to a moderate dose of valproic acid increases social behavior and alters gene expression in rats.

Prenatal exposure to moderate doses of valproic acid (VPA) produces brainstem abnormalities, while higher doses of this teratogen elicit social deficits in the rat. In this pilot study, we examined effects of prenatal exposure to a moderate dose of VPA on behavior and on transcriptomic expression in three brain regions that mediate social behavior. Pregnant Long Evans rats were injected with 350 mg/kg VPA or saline on gestational day 13. A modified social interaction test was used to assess social behavior and social preference/avoidance during early and late adolescence and in adulthood. VPA-exposed animals demonstrated more social investigation and play fighting than control animals. Social investigation, play fighting, and contact behavior also differed as a function of age; the frequency of these behaviors increased in late adolescence. Social preference and locomotor activity under social circumstances were unaffected by treatment or age. Thus, a moderate prenatal dose of VPA produces behavioral alterations that are substantially different from the outcomes that occur following exposure to a higher dose. At adulthood, VPA-exposed subjects exhibited transcriptomic abnormalities in three brain regions: anterior amygdala, cerebellar vermis, and orbitofrontal cortex. A common feature among the proteins encoded by the dysregulated genes was their ability to be modulated by acetylation. Analysis of the expression of individual exons also revealed that genes involved in post-translational modification and epigenetic regulation had particular isoforms that were ubiquitously dysregulated across brain regions. The vulnerability of these genes to the epigenetic effects of VPA may highlight potential mechanisms by which prenatal VPA exposure alters the development of social behavior.

Transcriptomic analysis of postmortem brain identifies dysregulated splicing events in novel candidate genes for schizophrenia.

The diverse spatial and temporal expression of alternatively spliced transcript isoforms shapes neurodevelopment and plays a major role in neuronal adaptability. Although alternative splicing is extremely common in the brain, its role in mental illnesses such as schizophrenia has received little attention. To examine this relationship, postmortem brain tissue was obtained from 20 individuals with schizophrenia (SZ) and 20 neuropsychiatrically normal comparison subjects. Gray matter samples were extracted from two brain regions implicated in the disorder: Brodmann Area 10 and caudate. Affymetrix Human Gene 1.0 ST arrays were used on four subjects per group to attain an initial profile of differential expression of transcribed elements within and across brain regions in SZ. Numerous genes of interest with altered mRNA transcripts were identified by microarray through the differential expression of particular exons and 3' untranslated regions (UTRs) between diagnostic groups. Select microarray results--including dysregulation of ENAH exon 11a and CPNE3 3'UTR--were verified by qRTPCR and replicated in the remaining independent sample of 16 SZ patients and 16 normal comparison subjects. These results, if further replicated, clearly illustrate the importance of Identifying transcriptomic variants in expression studies, and implicate novel candidate genes in the disorder.

Prenatal cocaine exposure decreases nigrostriatal dopamine release in vitro: effects of age and sex.

The present study examined the effects of prenatal cocaine (PCOC) exposure, age, sex, and estrous phase on the functional development of nigrostriatal dopamine (DA) neurons. Striatal tissue was obtained from prepubescent and adult rats of both sexes after bidaily exposure to saline (1 ml/kg) or cocaine (20 mg/kg/ml saline) from embryonic days 15-21. Tissue levels, basal release, and electrically evoked (1 or 8 Hz) overflow of endogenous DA and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as their efflux in response to superfusion with the DA transport blocker, nomifensine (10 microM), were measured from superfused striatal slices. Generally, these measures were highest in tissue from males and adults. Tissue DA and DOPAC levels and the rate of DA turnover were unaffected by PCOC exposure. Slices from PCOC-exposed juvenile and adult male rats exhibited significantly reduced basal and electrically evoked DA release at both stimulation intensities, in conjunction with higher levels of presynaptic DA reuptake. Female rats were largely spared from the effects of PCOC exposure, and measures did not vary with estrous phase. These findings demonstrate that the effects of PCOC exposure on various parameters of nigrostriatal DA neuronal function are not uniform across age, sex, or phases of the estrous cycle. These novel alterations in nigrostriatal DA transmission are in need of independent replication, but they may have profound implications for behavioral activities regulated by these neurons and, thus, may provide a basis for sex-selective effects of PCOC in exposed humans. Possible mechanisms of deleterious effects of PCOC exposure in select groups are discussed.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation.

Smad4, the common Smad, is central for transforming growth factor (TGF)-beta superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-beta and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-beta-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.