Targeted rescue of synaptic plasticity improves cognitive decline in sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a frequent complication of severe systemic infection resulting in delirium, premature death, and long-term cognitive impairment. We closely mimicked SAE in a murine peritoneal contamination and infection (PCI) model. We found long-lasting synaptic pathology in the hippocampus including defective long-term synaptic plasticity, reduction of mature neuronal dendritic spines, and severely affected excitatory neurotransmission. Genes related to synaptic signaling, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and members of the transcription-regulatory EGR gene family, were downregulated. At the protein level, ARC expression and mitogen-activated protein kinase signaling in the brain were affected. For targeted rescue we used adeno-associated virus-mediated overexpression of ARC in the hippocampus in vivo. This recovered defective synaptic plasticity and improved memory dysfunction. Using the enriched environment paradigm as a non-invasive rescue intervention, we found improvement of defective long-term potentiation, memory, and anxiety. The beneficial effects of an enriched environment were accompanied by an increase in brain-derived neurotrophic factor (BDNF) and ARC expression in the hippocampus, suggesting that activation of the BDNF-TrkB pathway leads to restoration of the PCI-induced reduction of ARC. Collectively, our findings identify synaptic pathomechanisms underlying SAE and provide a conceptual approach to target SAE-induced synaptic dysfunction with potential therapeutic applications to patients with SAE.

Behavioural and functional evidence revealing the role of RBFOX1 variation in multiple psychiatric disorders and traits.

Common variation in the gene encoding the neuron-specific RNA splicing factor RNA Binding Fox-1 Homolog 1 (RBFOX1) has been identified as a risk factor for several psychiatric conditions, and rare genetic variants have been found causal for autism spectrum disorder (ASD). Here, we explored the genetic landscape of RBFOX1 more deeply, integrating evidence from existing and new human studies as well as studies in Rbfox1 knockout mice. Mining existing data from large-scale studies of human common genetic variants, we confirmed gene-based and genome-wide association of RBFOX1 with risk tolerance, major depressive disorder and schizophrenia. Data on six mental disorders revealed copy number losses and gains to be more frequent in ASD cases than in controls. Consistently, RBFOX1 expression appeared decreased in post-mortem frontal and temporal cortices of individuals with ASD and prefrontal cortex of individuals with schizophrenia. Brain-functional MRI studies demonstrated that carriers of a common RBFOX1 variant, rs6500744, displayed increased neural reactivity to emotional stimuli, reduced prefrontal processing during cognitive control, and enhanced fear expression after fear conditioning, going along with increased avoidance behaviour. Investigating Rbfox1 neuron-specific knockout mice allowed us to further specify the role of this gene in behaviour. The model was characterised by pronounced hyperactivity, stereotyped behaviour, impairments in fear acquisition and extinction, reduced social interest, and lack of aggression; it provides excellent construct and face validity as an animal model of ASD. In conclusion, convergent translational evidence shows that common variants in RBFOX1 are associated with a broad spectrum of psychiatric traits and disorders, while rare genetic variation seems to expose to early-onset neurodevelopmental psychiatric disorders with and without developmental delay like ASD, in particular. Studying the pleiotropic nature of RBFOX1 can profoundly enhance our understanding of mental disorder vulnerability.

ADHD-associated PARK2 copy number variants: A pilot study on gene expression and effects of supplementary deprivation in patient-derived cell lines.

Recent studies show an association of Parkin RBR E3 ubiquitin protein ligase (PARK2) copy number variations (CNVs) with attention deficit hyperactivity disorder (ADHD). The aim of our pilot study to investigate gene expression associated with PARK2 CNVs in human-derived cellular models. We investigated gene expression in fibroblasts, hiPSC and dopaminergic neurons (DNs) of ADHD PARK2 deletion and duplication carriers by qRT PCR compared with healthy and ADHD cell lines without PARK2 CNVs. The selected 10 genes of interest were associated with oxidative stress response (TP53, NQO1, and NFE2L2), ubiquitin pathway (UBE3A, UBB, UBC, and ATXN3) and with a function in mitochondrial quality control (PINK1, MFN2, and ATG5). Additionally, an exploratory RNA bulk sequencing analysis in DNs was conducted. Nutrient deprivation as a supplementary deprivation stress paradigm was used to enhance potential genotype effects. At baseline, in fibroblasts, hiPSC, and DNs, there was no significant difference in gene expression after correction for multiple testing. After nutrient deprivation in fibroblasts NAD(P)H-quinone-dehydrogenase 1 (NQO1) expression was significantly increased in PARK2 CNV carriers. In a multivariate analysis, ubiquitin C (UBC) was significantly upregulated in fibroblasts of PARK2 CNV carriers. RNA sequencing analysis of DNs showed the strongest significant differential regulation in Neurontin (NNAT) at baseline and after nutrient deprivation. Our preliminary results suggest differential gene expression in pathways associated with oxidative stress, ubiquitine-proteasome, immunity, inflammation, cell growth, and differentiation, excitation/inhibition modulation, and energy metabolism in PARK2 CNV carriers compared to wildtype healthy controls and ADHD patients.

Hippocampal overexpression of NOS1AP promotes endophenotypes related to mental disorders.

BACKGROUND: Nitric oxide synthase 1 adaptor protein (NOS1AP; previously named CAPON) is linked to the glutamatergic postsynaptic density through interaction with neuronal nitric oxide synthase (nNOS). NOS1AP and its interaction with nNOS have been associated with several mental disorders. Despite the high levels of NOS1AP expression in the hippocampus and the relevance of this brain region in glutamatergic signalling as well as mental disorders, a potential role of hippocampal NOS1AP in the pathophysiology of these disorders has not been investigated yet. METHODS: To uncover the function of NOS1AP in hippocampus, we made use of recombinant adeno-associated viruses to overexpress murine full-length NOS1AP or the NOS1AP carboxyterminus in the hippocampus of mice. We investigated these mice for changes in gene expression, neuronal morphology, and relevant behavioural phenotypes. FINDINGS: We found that hippocampal overexpression of NOS1AP markedly increased the interaction of nNOS with PSD-95, reduced dendritic spine density, and changed dendritic spine morphology at CA1 synapses. At the behavioural level, we observed an impairment in social memory and decreased spatial working memory capacity. INTERPRETATION: Our data provide a mechanistic explanation for a highly selective and specific contribution of hippocampal NOS1AP and its interaction with the glutamatergic postsynaptic density to cross-disorder pathophysiology. Our findings allude to therapeutic relevance due to the druggability of this molecule. FUNDING: This study was funded in part by the DFG, the BMBF, the Academy of Finland, the NIH, the Japanese Society of Clinical Neuropsychopharmacology, the Ministry of Education of the Russian Federation, and the European Community.

Knockdown of the ADHD Candidate Gene Diras2 in Murine Hippocampal Primary Cells.

Objective: The DIRAS2 gene is associated with ADHD, but its function is largely unknown. Thus, we aimed to explore the genes and molecular pathways affected by DIRAS2. Method: Using short hairpin RNAs, we downregulated Diras2 in murine hippocampal primary cells. Gene expression was analyzed by microarray and affected pathways were identified. We used quantitative real-time polymerase chain reaction (qPCR) to confirm expression changes and analyzed enrichment of differentially expressed genes in an ADHD GWAS (genome-wide association studies) sample. Results:Diras2 knockdown altered expression of 1,612 genes, which were enriched for biological processes involved in neurodevelopment. Expression changes were confirmed for 33 out of 88 selected genes. These 33 genes showed significant enrichment in ADHD patients in a gene-set-based analysis. Conclusion: Our findings show that Diras2 affects numerous genes and thus molecular pathways that are relevant for neurodevelopmental processes. These findings may further support the hypothesis that DIRAS2 is linked to etiological processes underlying ADHD.

Lithium-induced gene expression alterations in two peripheral cell models of bipolar disorder.

Objectives: The aim of our study was to investigate molecular mechanisms of lithium action by studying the gene expression profile of peripheral cell models generated from bipolar patients (BD) and healthy controls (HC). Methods: EBV-immortalised lymphoblastoid cells (LCLs) and fibroblast cells from BD and HC were incubated with either lithium chloride or plain medium for 3 weeks. We first conducted a microarray gene expression study. The most promising differentially regulated genes in terms of lithium-associated or disorder-associated pathways were then replicated by quantitative real-time PCR (qRT-PCR). Results: The pooled microarray analysis showed 459 genes to be differentially regulated in BD compared to HC and 58 due to lithium treatment in LCLs, and 295 genes to be differentially regulated in BD compared to HC and five due to lithium treatment in fibroblasts. After correction for multiple comparison, EPHB1 disorder × treatment interactions remained significant in LCLs validated by qRT-PCR. In the control group, lithium influenced the expression of ANP32E, PLEKHA2, KCNK1, PRKCH, ST3GAL6 and AIF1. In bipolar and control fibroblast cells lithium treatment decreased FGF9 expression. Conclusions: The differentially regulated genes in our study add evidence for the relevance of inflammation, neuronal/glial development, phosphatidylinositol second-messenger pathway and ion channels in the mode of action of lithium.

Expression of the ADHD candidate gene Diras2 in the brain.

The distinct subgroup of the Ras family member 2 (DIRAS2) gene has been found to be associated with attention-deficit/hyperactivity disorder (ADHD) in one of our previous studies. This gene is coding for a small Ras GTPase with unknown function. DIRAS2 is highly expressed in the brain. However, the exact neural expression pattern of this gene was unknown so far. Therefore, we investigated the expressional profile of DIRAS2 in the human and murine brain. In the present study, qPCR analyses in the human and in the developing mouse brain, immunocytological double staining on murine hippocampal primary cells and RNA in situ hybridization (ISH) on brain sections of C57BL/6J wild-type mice, have been used to reveal the expression pattern of DIRAS2 in the brain. We could show that DIRAS2 expression in the human brain is the highest in the hippocampus and the cerebral cortex, which is in line with the ISH results in the mouse brain. During mouse brain development, Diras2 levels strongly increase from prenatal to late postnatal stages. Co-expression studies indicate Diras2 expression in glutamatergic and catecholaminergic neurons. Our findings support the idea of DIRAS2 as a candidate gene for ADHD as the timeline of its expression as well as the brain regions and cell types that show Diras2 expression correspond to those assumed to underlie the pathomechanisms of the disease.

The regulation of tetraspanin 8 gene expression-A potential new mechanism in the pathogenesis of bipolar disorder.

In a previous study, we identified the single nucleotide polymorphism (SNP) rs4500567, located in the upstream region of tetraspanin 8 (TSPAN8), to be associated with bipolar disorder (BD). Due to its proximal position, the SNP might have an impact on promoter activity, thus on TSPAN8 gene expression. We investigated the impact of rs4500567 on TSPAN8 expression in vitro with luciferase-based promoter assays in human embryonic kidney (HEK293) and neuroblastoma cells (SH-SY5Y), and its effect on expression of downstream associated genes by microarray-based transcriptome analyses. Immunohistochemical localization studies on murine brain slices served to identify possible target regions of altered TSPAN8 expression in the brain. Promoter assays revealed decreased TSPAN8 expression in presence of the minor allele. Transcriptome analyses of TSPAN8-knockdown cells, mirroring the effects of putatively reduced TSPAN8 expression in minor allele carriers, resulted in 231 differentially expressed genes with enrichments of relevant signaling pathways for psychiatric disorders and neuronal development. Finally, we demonstrate Tspan8 abundance in mouse cerebellum and hippocampus. These findings point to a role of TSPAN8 in neuronal function or development. Considering a rather protective effect of the minor allele of rs4500567, our findings reveal a possible novel mechanism that contributes to the development of BD.

Functional Impact of An ADHD-Associated DIRAS2 Promoter Polymorphism.

The DIRAS2 gene is coding for a small Ras GTPase with so far unknown function. In a previous study, we described the association of DIRAS2 rs1412005, as well as a haplotype containing this polymorphism and located in the promoter region of this gene with attention-deficit/hyperactivity disorder (ADHD). In the present study, we searched for rare variants within or near the DIRAS2 gene that might be associated with ADHD using next-generation sequencing. As we were not able to detect any rare variants associated with the disease, we sought to establish a functional role of DIRAS2 rs1412005 on the molecular or systems level. First, we investigated whether it has an influence on gene expression by means of a luciferase-based promoter assay. We could demonstrate that the minor risk allele goes along with the increased expression of the reporter gene. Next, we aimed to identify differences in response inhibition between risk-allele and non-risk allele carriers in children suffering from ADHD and healthy control individuals by analyzing event-related potentials in the electroencephalogram during a Go/NoGo task. Risk-allele carriers showed a changed NoGo anteriorization. Therefore, our results suggest an impact of the investigated polymorphism on the prefrontal response control in children with ADHD. These results imply that the promoter polymorphism is indeed the associated as well as in itself a causal variant. Further research is thus warranted to clarify the mechanisms linking DIRAS2 to ADHD.