Michael C. Saul

A New Mouse Model for Mania Shares Genetic Correlates with Human Bipolar Disorder

Bipolar disorder (BPD) is a debilitating heritable psychiatric disorder. Contemporary rodent models for the manic pole of BPD have primarily utilized either single locus transgenics or treatment with psychostimulants. Our lab recently characterized a mouse strain termed Madison (MSN) that naturally displays a manic phenotype, exhibiting elevated locomotor activity, increased sexual behavior, and higher forced swimming relative to control strains. Lithium chloride and olanzapine treatments attenuate this phenotype. In this study, we replicated our locomotor activity experiment, showing that MSN mice display generationally-stable mania relative to their outbred ancestral strain, hsd:ICR (ICR). We then performed a gene expression microarray experiment to compare hippocampus of MSN and ICR mice. We found dysregulation of multiple transcripts whose human orthologs are associated with BPD and other psychiatric disorders including schizophrenia and ADHD, including: Epor, Smarca4, Cmklr1, Cat, Tac1, Npsr1, Fhit, and P2rx7. RT-qPCR confirmed dysregulation for all of seven transcripts tested. Using a novel genome enrichment algorithm, we found enrichment in genome regions homologous to human loci implicated in BPD in replicated linkage studies including homologs of human cytobands 1p36, 3p14, 3q29, 6p21–22, 12q24, 16q24, and 17q25. Using a functional network analysis, we found dysregulation of a gene system related to chromatin packaging, a result convergent with recent human findings on BPD. Our findings suggest that MSN mice represent a polygenic model for the manic pole of BPD showing much of the genetic systems complexity of the corresponding human disorder. Further, the high degree of convergence between our findings and the human literature on BPD brings up novel questions about evolution by analogy in mammalian genomes.

Genes showing altered expression in the medial preoptic area in the highly social maternal phenotype are related to autism and other disorders with social deficits

BackgroundThe mother-child relationship is the most fundamental social bond in mammals, and previous studies indicate that the medial preoptic area (MPOA) contributes to this increase in sociability. It is possible that the same genes that lead to elevated sociability in one condition (the maternal state) might also be dysregulated in some disorders with social deficits (e.g. autism). In this study, we examined whether there was enrichment (greater than chance overlap) for social deficit disorder related genes in MPOA microarray results between virgin and postpartum female mice. We utilized microarrays to assess large scale gene expression changes in the MPOA of virgin and postpartum mice. The Modular Single Set Enrichment Test (MSET) was used to determine if mental health disorder related genes were enriched in significant microarray results. Additional resources, such as ToppCluster, NIH DAVID, and weighted co-expression network analysis (WGCNA) were used to analyze enrichment for specific gene clusters or indirect relationships between significant genes of interest. Finally, a subset of microarray results was validated using quantitative PCR.ResultsSignificant postpartum MPOA microarray results were enriched for multiple disorders that include social deficits, including autism, bipolar disorder, depression, and schizophrenia. Together, 98 autism-related genes were identified from the significant microarray results. Further, ToppCluser and NIH DAVID identified a large number of postpartum genes related to ion channel activity and CNS development, and also suggested a role for microRNAs in regulating maternal gene expression. WGCNA identified a module of genes associated with the postpartum phenotype, and identified indirect links between transcription factors and other genes of interest.ConclusionThe transition to the maternal state involves great CNS plasticity and increased sociability. We identified multiple novel genes that overlap between the postpartum MPOA (high sociability) and mental health disorders with low sociability. Thus, the activity or interactions of the same genes may be altering social behaviors in different directions in different conditions. Maternity also involves elevated risks for disorders, including depression, psychosis, and BPD, so identification of maternal genes common to these disorders may provide insights into the elevated vulnerability of the maternal brain.

Large Scale Expression Changes of Genes Related to Neuronal Signaling and Developmental Processes Found in Lateral Septum of Postpartum Outbred Mice

Coordinated gene expression changes across the CNS are required to produce the mammalian maternal phenotype. Lateral septum (LS) is a brain region critically involved with aspects of maternal care, and we recently examined gene expression of whole septum (LS and medial septum) in selectively bred maternal mice. Here, we expand on the prior study by 1) conducting microarray analysis solely on LS in virgin and postpartum mice, 2) using outbred mice, and 3) evaluating the role of sensory input on gene expression changes. Large scale changes in genes related to neuronal signaling were identified, including four GABAA receptor subunits. Subunits α4 and δ were downregulated in maternal LS, likely reflecting a reduction in the extrasynaptic, neurosteroid-sensitive α4/δ containing receptor subtype. Conversely, subunits ε and θ were increased in maternal LS. Fifteen K+ channel related genes showed altered expression, as did dopamine receptors Drd1a and Drd2 (both downregulated), hypocretin receptor 1 (Hcrtr1), kappa opioid receptor 1 (Oprk1), and transient receptor potential channel 4 (Trpc4). Expression of a large number of genes linked to developmental processes or cell differentiation were also altered in postpartum LS, including chemokine (C-X-C) motif ligand 12 (Cxcl12), fatty acid binding protein 7 (Fabp7), plasma membrane proteolipid (Pllp), and suppressor of cytokine signaling 2 (Socs2). Additional genes that are linked to anxiety, such as glutathione reductase (Gsr), exhibited altered expression. Pathway analysis also identified changes in genes related to cyclic nucleotide metabolism, chromatin structure, and the Ras gene family. The sensory presence of pups was found to contribute to the altered expression of a subset of genes across all categories. This study suggests that both large changes in neuronal signaling and the possible terminal differentiation of neuronal and/or glial cells play important roles in producing the maternal state.

Development of a versatile enrichment analysis tool reveals associations between the maternal brain and mental health disorders, including autism

BackgroundA recent study of lateral septum (LS) suggested a large number of autism-related genes with altered expression in the postpartum state. However, formally testing the findings for enrichment of autism-associated genes proved to be problematic with existing software. Many gene-disease association databases have been curated which are not currently incorporated in popular, full-featured enrichment tools, and the use of custom gene lists in these programs can be difficult to perform and interpret. As a simple alternative, we have developed the Modular Single-set Enrichment Test (MSET), a minimal tool that enables one to easily evaluate expression data for enrichment of any conceivable gene list of interest.ResultsThe MSET approach was validated by testing several publicly available expression data sets for expected enrichment in areas of autism, attention deficit hyperactivity disorder (ADHD), and arthritis. Using nine independent, unique autism gene lists extracted from association databases and two recent publications, a striking consensus of enrichment was detected within gene expression changes in LS of postpartum mice. A network of 160 autism-related genes was identified, representing developmental processes such as synaptic plasticity, neuronal morphogenesis, and differentiation. Additionally, maternal LS displayed enrichment for genes associated with bipolar disorder, schizophrenia, ADHD, and depression.ConclusionsThe transition to motherhood includes the most fundamental social bonding event in mammals and features naturally occurring changes in sociability. Some individuals with autism, schizophrenia, or other mental health disorders exhibit impaired social traits. Genes involved in these deficits may also contribute to elevated sociability in the maternal brain. To date, this is the first study to show a significant, quantitative link between the maternal brain and mental health disorders using large scale gene expression data. Thus, the postpartum brain may provide a novel and promising platform for understanding the complex genetics of improved sociability that may have direct relevance for multiple psychiatric illnesses. This study also provides an important new tool that fills a critical analysis gap and makes evaluation of enrichment using any database of interest possible with an emphasis on ease of use and methodological transparency.

Gene expression changes in the septum: possible implications for microRNAs in sculpting the maternal brain.

The transition from the non-maternal to the maternal state is characterized by a variety of CNS alterations that support the care of offspring. The septum (including lateral and medial portions) is a brain region previously linked to various emotional and motivational processes, including maternal care. In this study, we used microarrays (PLIER algorithm) to examine gene expression changes in the septum of postpartum mice and employed gene set enrichment analysis (GSEA) to identify possible regulators of altered gene expression. Genes of interest identified as differentially regulated with microarray analysis were validated with quantitative real-time PCR. We found that fatty acid binding protein 7 (Fabp7) and galanin (Gal) were downregulated, whereas insulin-like growth factor binding protein 3 (Igfbp3) was upregulated in postpartum mice compared to virgin females. These genes were previously found to be differentially regulated in other brain regions during lactation. We also identified altered expression of novel genes not previously linked to maternal behavior, but that could play a role in postpartum processes, including glutamate-ammonia ligase (Glul) and somatostatin receptor 1 (Sstr1) (both upregulated in postpartum). Genes implicated in metabolism, cell differentiation, or proliferation also exhibited altered expression. Unexpectedly, enrichment analysis revealed a high number of microRNAs, transcription factors, or conserved binding sites (177 with corrected P-value <0.05) that were significantly linked to maternal upregulated genes, while none were linked to downregulated genes. MicroRNAs have been linked to placenta and mammary gland development, but this is the first indication they may also play a key role in sculpting the maternal brain. Together, this study provides new insights into genes (along with possible mechanisms for their regulation) that are involved in septum-mediated adaptations during the postpartum period.

The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.

Deep evolutionary conservation of autism-related genes

Significance Sociobiological theory proposed that similarities between human and animal societies reflect similar evolutionary origins. We used comparative genomics to test this controversial idea by determining whether superficial behavioral similarities between humans and honey bees reflect shared molecular mechanisms. We found unique and significant enrichment for autism spectrum disorder-related genes in the neurogenomic signatures of a high-level integration center of the insect brain in bees unresponsive to two different salient social stimuli. These results demonstrate deep conservation for genes implicated in autism spectrum disorder in humans and genes associated with social responsiveness in honey bees. Comparative genomics thus provides a means to test theory on the biology of social behavior. E. O. Wilson proposed in Sociobiology that similarities between human and animal societies reflect common mechanistic and evolutionary roots. When introduced in 1975, this controversial hypothesis was beyond science’s ability to test. We used genomic analyses to determine whether superficial behavioral similarities in humans and the highly social honey bee reflect common molecular mechanisms. Here, we report that gene expression signatures for individual bees unresponsive to various salient social stimuli are significantly enriched for autism spectrum disorder-related genes. These signatures occur in the mushroom bodies, a high-level integration center of the insect brain. Furthermore, our finding of enrichment was unique to autism spectrum disorders; brain gene expression signatures from other honey bee behaviors do not show this enrichment, nor do datasets from other human behavioral and health conditions. These results demonstrate deep conservation for genes associated with a human social pathology and individual differences in insect social behavior, thus providing an example of how comparative genomics can be used to test sociobiological theory.

Temporal dynamics of neurogenomic plasticity in response to social interactions in male threespined sticklebacks

Animals exhibit dramatic immediate behavioral plasticity in response to social interactions, and brief social interactions can shape the future social landscape. However, the molecular mechanisms contributing to behavioral plasticity are unclear. Here, we show that the genome dynamically responds to social interactions with multiple waves of transcription associated with distinct molecular functions in the brain of male threespined sticklebacks, a species famous for its behavioral repertoire and evolution. Some biological functions (e.g., hormone activity) peaked soon after a brief territorial challenge and then declined, while others (e.g., immune response) peaked hours afterwards. We identify transcription factors that are predicted to coordinate waves of transcription associated with different components of behavioral plasticity. Next, using H3K27Ac as a marker of chromatin accessibility, we show that a brief territorial intrusion was sufficient to cause rapid and dramatic changes in the epigenome. Finally, we integrate the time course brain gene expression data with a transcriptional regulatory network, and link gene expression to changes in chromatin accessibility. This study reveals rapid and dramatic epigenomic plasticity in response to a brief, highly consequential social interaction.

Behavioral, transcriptomic and epigenetic responses to social challenge in honey bees

Understanding how social experiences are represented in the brain and shape future responses is a major challenge in the study of behavior. We addressed this problem by studying behavioral, transcriptomic and epigenetic responses to intrusion in honey bees. Previous research showed that initial exposure to an intruder provokes an immediate attack; we now show that this also leads to longer‐term changes in behavior in the response to a second intruder, with increases in the probability of responding aggressively and the intensity of aggression lasting 2 and 1 h, respectively. Previous research also documented the whole‐brain transcriptomic response; we now show that in the mushroom bodies (MBs) there are 2 waves of gene expression, the first highlighted by genes related to cytoskeleton remodeling, and the second highlighted by genes related to hormones, stress response and transcription factors (TFs). Overall, 16 of 37 (43%) of the TFs whose cis‐motifs were enriched in the promoters of the differentially expressed genes (DEGs) were also predicted from transcriptional regulatory network analysis to regulate the MB transcriptional response, highlighting the strong role played by a relatively small subset of TFs in the MBs transcriptomic response to social challenge. Whole brain histone profiling showed few changes in chromatin accessibility in response to social challenge; most DEGs were ‘ready’ to be activated. These results show how biological embedding of a social challenge involves temporally dynamic changes in the neurogenomic state of a prominent region of the insect brain that are likely to influence future behavior.

High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice.

Although exercise is critical for health, many lack the motivation to exercise, and it is unclear how motivation might be increased. To uncover the molecular underpinnings of increased motivation for exercise, we analyzed the transcriptome of the striatum in four mouse lines selectively bred for high voluntary wheel running and four non‐selected control lines. The striatum was dissected and RNA was extracted and sequenced from four individuals of each line. We found multiple genes and gene systems with strong relationships to both selection and running history over the previous 6 days. Among these genes were Htr1b, a serotonin receptor subunit and Slc38a2, a marker for both glutamatergic and γ‐aminobutyric acid (GABA)‐ergic signaling. System analysis of the raw results found enrichment of transcriptional regulation and kinase genes. Further, we identified a splice variant affecting the Wnt‐related Golgi signaling gene Tmed5. Using coexpression network analysis, we found a cluster of interrelated coexpression modules with relationships to running behavior. From these modules, we built a network correlated with running that predicts a mechanistic relationship between transcriptional regulation by nucleosome structure and Htr1b expression. The Library of Integrated Network‐Based Cellular Signatures identified the protein kinase C δ inhibitor, rottlerin, the tyrosine kinase inhibitor, Linifanib and the delta‐opioid receptor antagonist 7‐benzylidenenaltrexone as potential compounds for increasing the motivation to run. Taken together, our findings support a neurobiological framework of exercise motivation where chromatin state leads to differences in dopamine signaling through modulation of both the primary neurotransmitters glutamate and GABA, and by neuromodulators such as serotonin.