Genetic variation and early adverse environmental events work together to increase risk for schizophrenia. γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in adult mammalian brain, plays a major role in normal brain development, and has been strongly implicated in the pathobiology of schizophrenia. GABA synthesis is controlled by two glutamic acid decarboxylase (GAD) genes, GAD1 and GAD2, both of which produce a number of alternative transcripts. Genetic variants in the GAD1 gene are associated with increased risk for schizophrenia, and reduced expression of its major transcript in the human dorsolateral prefrontal cortex (DLPFC). No consistent changes in GAD2 expression have been found in brains from patients with schizophrenia. In this work, with the use of RNA sequencing and PCR technologies, we confirmed and tracked the expression of an alternative truncated transcript of GAD2 (ENST00000428517) in human control DLPFC homogenates across lifespan besides the well-known full length transcript of GAD2. In addition, using quantitative RT-PCR, expression of GAD2 full length and truncated transcripts were measured in the DLPFC of patients with schizophrenia, bipolar disorder and major depression. The expression of GAD2 full length transcript is decreased in the DLPFC of schizophrenia and bipolar disorder patients, while GAD2 truncated transcript is increased in bipolar disorder patients but decreased in schizophrenia patients. Moreover, the patients with schizophrenia with completed suicide or positive nicotine exposure showed significantly higher expression of GAD2 full length transcript. Alternative transcripts of GAD2 may be important in the growth and development of GABA-synthesizing neurons as well as abnormal GABA signaling in the DLPFC of patients with schizophrenia and affective disorders.

Brain oscillations play a critical role in information processing and may, therefore, be essential to uncovering the mechanisms of cognitive impairment in neurological disease. In Dravet syndrome (DS), a mutation in SCN1A, coding for the voltage-gated sodium channel Nav1.1, is associated with severe cognitive impairment and seizures. While seizure frequency and severity do not correlate with the extent of impairment, the slowing of brain rhythms may be involved. Here we investigate the role of Nav1.1 on brain rhythms and cognition using RNA interference. We demonstrate that knockdown of Nav1.1 impairs fast- and burst-firing properties of neurons in the medial septum in vivo. The proportion of neurons that fired phase-locked to hippocampal theta oscillations was reduced, and medial septal regulation of theta rhythm was disrupted. During a working memory task, this deficit was characterized by a decrease in theta frequency and was negatively correlated with performance. These findings suggest a fundamental role for Nav1.1 in facilitating fast-firing properties in neurons, highlight the importance of precise temporal control of theta frequency for working memory, and imply that Nav1.1 deficits may disrupt information processing in DS via a dysregulation of brain rhythms.

A subset of human neocortical neurons harbors complex karyotypes wherein megabase-scale copy-number variants (CNVs) alter allelic diversity. Divergent levels of neurons with complex karyotypes (CNV neurons) are reported in different individuals, yet genome-wide and familial studies implicitly assume a single brain genome when assessing the genetic risk architecture of neurological disease. We assembled a brain CNV atlas using a robust computational approach applied to a new dataset (>800 neurons from 5 neurotypical individuals) and to published data from 10 additional neurotypical individuals. The atlas reveals that the frequency of neocortical neurons with complex karyotypes varies widely among individuals, but this variability is not readily accounted for by tissue quality or CNV detection approach. Rather, the age of the individual is anti-correlated with CNV neuron frequency. Fewer CNV neurons are observed in aged individuals than in young individuals.

Antipsychotics are the most widely used medications for the treatment of schizophrenia spectrum disorders. While such drugs generally ameliorate positive symptoms, clinical responses are highly variable in terms of negative symptoms and cognitive impairments. However, predictors of individual responses have been elusive. Here, we report a pharmacogenetic interaction related to a core cognitive dysfunction in patients with schizophrenia. We show that genetic variations reducing dysbindin-1 expression can identify individuals whose executive functions respond better to antipsychotic drugs, both in humans and in mice. Multilevel ex vivo and in vivo analyses in postmortem human brains and genetically modified mice demonstrate that such interaction between antipsychotics and dysbindin-1 is mediated by an imbalance between the short and long isoforms of dopamine D2 receptors, leading to enhanced presynaptic D2 function within the prefrontal cortex. These findings reveal one of the pharmacodynamic mechanisms underlying individual cognitive response to treatment in patients with schizophrenia, suggesting a potential approach for improving the use of antipsychotic drugs.

Brain-derived neurotrophic factor (Bdnf) transcription is controlled by several promoters, which drive expression of multiple transcripts encoding an identical protein. We previously reported that BDNF derived from promoters I and II is highly expressed in hypothalamus and is critical for regulating aggression in male mice. Here we report that BDNF loss from these promoters causes reduced sexual receptivity and impaired maternal care in female mice, which is concomitant with decreased oxytocin (Oxt) expression during development. We identify a novel link between BDNF signaling, oxytocin, and maternal behavior by demonstrating that ablation of TrkB selectively in OXT neurons partially recapitulates maternal care impairments observed in BDNF-deficient females. Using translating ribosome affinity purification and RNA-sequencing we define a molecular profile for OXT neurons and delineate how BDNF signaling impacts gene pathways critical for structural and functional plasticity. Our findings highlight BDNF as a modulator of sexually-dimorphic hypothalamic circuits that govern female-typical behaviors.

Although transposable element (TE) derived DNA accounts for more than half of mammalian genomes and initiates a significant proportion of RNA transcripts, high throughput methods are rarely leveraged specifically to detect expression from interspersed repeats.

Phosphatase and tensin homolog (Pten) catalyzes the reverse reaction of PI3K by dephosphorylating PIP3 to PIP2. This negatively regulates downstream Akt/mTOR/S6 signaling resulting in decreased cellular growth and proliferation. Co-injection of a lentivirus knocking Pten down with a control lentivirus allows us to compare the effects of Pten knockdown between individual neurons within the same animal. We find that knockdown of Pten results in neuronal hypertrophy by 21 days post-injection. This neuronal hypertrophy is correlated with increased p-S6 and p-mTOR in individual neurons. We used this system to test whether an environmental factor that has been implicated in cellular hypertrophy could influence the severity of the Pten knockdown-induced hypertrophy. Implantation of mini-osmotic pumps delivering fatty acids results in increased neuronal hypertrophy and p-S6/p-mTOR staining. These hypertrophic effects were reversed in response to rapamycin treatment. However, we did not observe a similar increase in hypertrophy in response to dietary manipulations of fatty acids. Thus, we conclude that by driving growth signaling with fatty acids and knocking down a critical regulator of growth, Pten, we are able to observe an additive morphological phenotype of increased soma size mediated by the mTOR pathway.

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.

Brain development and function depend on the precise regulation of gene expression. However, our understanding of the complexity and dynamics of the transcriptome of the human brain is incomplete. Here we report the generation and analysis of exon-level transcriptome and associated genotyping data, representing males and females of different ethnicities, from multiple brain regions and neocortical areas of developing and adult post-mortem human brains. We found that 86 per cent of the genes analysed were expressed, and that 90 per cent of these were differentially regulated at the whole-transcript or exon level across brain regions and/or time. The majority of these spatio-temporal differences were detected before birth, with subsequent increases in the similarity among regional transcriptomes. The transcriptome is organized into distinct co-expression networks, and shows sex-biased gene expression and exon usage. We also profiled trajectories of genes associated with neurobiological categories and diseases, and identified associations between single nucleotide polymorphisms and gene expression. This study provides a comprehensive data set on the human brain transcriptome and insights into the transcriptional foundations of human neurodevelopment.

Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599)--both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val--had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3' SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.

Differential expression analysis of RNA sequencing (RNA-seq) data typically relies on reconstructing transcripts or counting reads that overlap known gene structures. We previously introduced an intermediate statistical approach called differentially expressed region (DER) finder that seeks to identify contiguous regions of the genome showing differential expression signal at single base resolution without relying on existing annotation or potentially inaccurate transcript assembly.We present the derfinder software that improves our annotation-agnostic approach to RNA-seq analysis by: (i) implementing a computationally efficient bump-hunting approach to identify DERs that permits genome-scale analyses in a large number of samples, (ii) introducing a flexible statistical modeling framework, including multi-group and time-course analyses and (iii) introducing a new set of data visualizations for expressed region analysis. We apply this approach to public RNA-seq data from the Genotype-Tissue Expression (GTEx) project and BrainSpan project to show that derfinder permits the analysis of hundreds of samples at base resolution in R, identifies expression outside of known gene boundaries and can be used to visualize expressed regions at base-resolution. In simulations, our base resolution approaches enable discovery in the presence of incomplete annotation and is nearly as powerful as feature-level methods when the annotation is complete.derfinder analysis using expressed region-level and single base-level approaches provides a compromise between full transcript reconstruction and feature-level analysis. The package is available from Bioconductor at www.bioconductor.org/packages/derfinder.

Pten, a gene associated with autism spectrum disorder, is an upstream regulator of receptor tyrosine kinase intracellular signaling pathways that mediate extracellular cues to inform cellular development and activity-dependent plasticity. We therefore hypothesized that Pten loss would interfere with activity dependent dendritic growth. We investigated the effects of this interaction on the maturation of retrovirally labeled postnatally generated wild-type and Pten knockout granule neurons in male and female mouse dentate gyrus while using chemogenetics to manipulate the activity of the perforant path afferents. We find that enhancing network activity accelerates the dendritic outgrowth of wild-type, but not Pten knockout, neurons. This was specific to immature neurons during an early developmental window. We also examined synaptic connectivity and physiological measures of neuron maturation. The input resistance, membrane capacitance, dendritic spine morphology, and frequency of spontaneous synaptic events were not differentially altered by activity in wild-type versus Pten knockout neurons. Therefore, Pten and its downstream signaling pathways regulate the activity-dependent sculpting of the dendritic arbor during neuronal maturation.

Brain-derived neurotrophic factor (BDNF) signals through its cognate receptor tropomyosin receptor kinase B (TrkB) to promote the function of several classes of inhibitory interneurons. We previously reported that loss of BDNF-TrkB signaling in cortistatin (Cort)-expressing interneurons leads to behavioral hyperactivity and spontaneous seizures in mice. We performed bulk RNA sequencing (RNA-seq) from the cortex of mice with disruption of BDNF-TrkB signaling in cortistatin interneurons, and identified differential expression of genes important for excitatory neuron function. Using translating ribosome affinity purification and RNA-seq, we define a molecular profile for Cort-expressing inhibitory neurons and subsequently compare the translatome of normal and TrkB-depleted Cort neurons, revealing alterations in calcium signaling and axon development. Several of the genes enriched in Cort neurons and differentially expressed in TrkB-depleted neurons are also implicated in autism and epilepsy. Our findings highlight TrkB-dependent molecular pathways as critical for the maturation of inhibitory interneurons and support the hypothesis that loss of BDNF signaling in Cort interneurons leads to altered excitatory/inhibitory balance.

In this study, we established induced pluripotent stem (iPS) cell lines from postmortem dura-derived fibroblasts of four control individuals with low polygenic risk score for psychiatric disorders including schizophrenia and bipolar disorder. The fibroblasts were reprogrammed into iPS cells using episomal vectors carrying OCT3/4, SOX2, KLF4, L-Myc, LIN28 and shRNA-p53. All iPS cell lines showed the same genotype with parental postmortem brain tissues, expressed pluripotency markers, and exhibited the differentiation potency into three embryonic germ layers.

Genetic studies of hippocampal granule neuron development have been used to elucidate cellular functions of Pten and Fmr1. While mutations in each gene cause neurodevelopmental disorders such as autism and fragile X syndrome, how Pten and Fmr1 function alone or together during normal development is not known. Moreover, Pten mRNA is bound by the fragile X mental retardation protein (FMRP) RNA binding protein, but how this physical interaction impinges on phosphatase and tensin homolog protein (PTEN) expression is not known. To understand the interaction of PTEN and FMRP, we investigated the dentate gyrus granule neuron development in Pten and Fmr1 knockout (KO) mice. Interestingly, heterozygosity of Pten restored Fmr1 KO cellular phenotypes, including dendritic arborization, and spine density, while PTEN protein expression was significantly increased in Fmr1 KO animals. However, complete deletion of both Pten and Fmr1 resulted in a dramatic increase in dendritic length, spine density, and spine length. In addition, overexpression of PTEN in Fmr1 KO Pten heterozygous background reduced dendritic length, arborization, spine density, and spine length including pS6 levels. Our findings suggest that PTEN levels are negatively regulated by FMRP, and some Fmr1 KO phenotypes are caused by dysregulation of PTEN protein. These observations provide evidence for the genetic interaction of PTEN and FMRP and a possible mechanistic basis for the pathogenesis of Fmr1-related fragile X neurodevelopmental disorders.

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are due to sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

Hyperactivation of PI3K/PTEN-mTOR signaling during neural development is associated with focal cortical dysplasia (FCD), autism, and epilepsy. mTOR can signal through two major hubs, mTORC1 and mTORC2, both of which are hyperactive following PTEN loss of function (LOF). Here, we tested the hypothesis that genetic inactivation of the mTORC2 complex via deletion of Rictor is sufficient to rescue morphologic and electrophysiological abnormalities in the dentate gyrus caused by PTEN loss, as well as generalized seizures. An established, early postnatal mouse model of PTEN loss in male and female mice showed spontaneous seizures that were not prevented by mTORC2 inactivation. This lack of rescue occurred despite the normalization or amelioration of many morphologic and electrophysiological phenotypes. However, increased excitatory connectivity proximal to dentate gyrus granule neuron somas was not normalized by mTORC2 inactivation. Further studies demonstrated that, although mTORC2 inactivation largely rescued the dendritic arbor overgrowth caused by PTEN LOF, it increased synaptic strength and caused additional impairments of presynaptic function. These results suggest that a constrained increase in excitatory connectivity and co-occurring synaptic dysfunction is sufficient to generate seizures downstream of PTEN LOF, even in the absence of characteristic changes in morphologic properties.SIGNIFICANCE STATEMENT Homozygous deletion of the Pten gene in neuronal subpopulations in the mouse serves as a valuable model of epilepsy caused by mTOR hyperactivation. To better understand the physiological mechanisms downstream of Pten loss that cause epilepsy, as well as the therapeutic potential of targeted gene therapies, we tested whether genetic inactivation of the mTORC2 complex could improve the cellular, synaptic, and in vivo effects of Pten loss in the dentate gyrus. We found that mTORC2 inhibition improved or rescued all morphologic effects of Pten loss in the dentate gyrus, but synaptic changes and seizures persisted. These data suggest that synaptic dysfunction can drive epilepsy caused by hyperactivation of PI3K/PTEN-mTOR, and that future therapies should focus on this mechanistic link.

Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

Gene co-expression networks capture biological relationships between genes and are important tools in predicting gene function and understanding disease mechanisms. We show that technical and biological artifacts in gene expression data confound commonly used network reconstruction algorithms. We demonstrate theoretically, in simulation, and empirically, that principal component correction of gene expression measurements prior to network inference can reduce false discoveries. Using data from the GTEx project in multiple tissues, we show that this approach reduces false discoveries beyond correcting only for known confounders.

Increased African-American research participation is critical to the applicability and generalizability of biomedical research, as population diversity continues to increase both domestically and abroad. Yet numerous studies document historical origins of mistrust, as well as other barriers that may contribute to resistance in the African-American community towards participation in biomedical research. However, a growing body of more recent scientific evidence suggests that African-Americans value research and are willing to participate when asked. In the present study, we set out to determine factors associated with research participation of African-American families in postmortem human brain tissue donation for neuropsychiatric disorders as compared with Caucasian families, from same-day medical examiner autopsy referrals. We retrospectively reviewed brain donation rates, as well as demographic and clinical factors associated with donation in 1,421 consecutive referrals to three medical examiner's offices from 2010-2015. Overall, 69.7% of all next-of-kin contacted agreed to brain donation. While Caucasian families consented to donate brain tissue at a significantly higher rate (74.1%) than African-American families (57.0%) (p<0.001), African-American brain donation rates were as high as 60.5% in referrals from Maryland. Neither African-American nor Caucasian donors differed significantly from non-donors on any demographic or clinical factors ascertained, including age, sex, diagnosis of the donor, or in the relationship of the next-of-kin being contacted (p>0.05). However, Caucasian donors were significantly older, had more years of education, were more likely to be referred for study due to a psychiatric diagnosis, more likely to have comorbid substance abuse, and more likely to have died via suicide, as compared with African-American donors (p<0.05). When African-American participants are identified and approached, African-American families as well as Caucasian families are indeed willing to donate brain tissue on the spot for neuropsychiatric research, which supports the belief that African-American attitudes towards biomedical research may be more favorable than previously thought.