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.

The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus.

Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.

On the surface of most animal and other eukaryotic cells are small rod-like protrusions known as primary cilia. Each cilium is encased by a specialized membrane which is enriched in protein complexes that help the cell sense its local environment. Some of these complexes help transport ions in out of the cell, while others act as receptors that receive chemical signals called ligands. A unique ion channel known as the polycystin complex is able to perform both of these roles as it contains a receptor called PC-1 in addition to an ion channel called PC-2. Various mutations in the genes that code for PC-1 and PC-2 can result in autosomal dominant polycystic kidney disease (ADPKD), which is the most common monogenetic disease in humans. However, due to the small size of primary cilia ­ which are less than a thousandth of a millimeter thick ­ little is known about how polycystin complexes are regulated and how mutations lead to ADPKD. To overcome this barrier, Ha et al. modified kidney cells grown in the lab so that PC-1 and PC-2 form a working channel in the plasma membrane which surrounds the entire cell. As the body of a cell is around 10,000 times bigger than the cilium, this allowed the movement of ions across the polycystin complex to be studied using conventional techniques. Experiments using this newly developed assay revealed that a region at one of the ends of the PC-1 protein, named the C-type lectin domain, is essential for stimulating polycystin complexes. Ha et al. found that this domain of PC-1 is able to cut itself from the protein complex. Further experiments showed that when fragments of PC-1, which contain the C-type lectin domain, are no longer bound to the membrane, they can activate the polycystin channels in cilia as well as the plasma membrane. This suggests that this region of PC-1 may also act as a secreted ligand that can activate other polycystin channels. Some of the genetic mutations that cause ADPKD likely disrupt the activity of the polycystin complex and reduce its ability to transport ions across the cilia membrane. Therefore, the cell assay created in this study could be used to screen for small molecules that can restore the activity of these ion channels in patients with ADPKD.

Extending the vulnerability-stress model of mental disorders: three-dimensional NPSR1 × environment × coping interaction study in anxiety.

BACKGROUND: The general understanding of the 'vulnerability-stress model' of mental disorders neglects the modifying impact of resilience-increasing factors such as coping ability. AIMS: Probing a conceptual framework integrating both adverse events and coping factors in an extended 'vulnerability-stress-coping model' of mental disorders, the effects of functional neuropeptide S receptor gene (NPSR1) variation (G), early adversity (E) and coping factors (C) on anxiety were addressed in a three-dimensional G × E × C model. METHOD: In two independent samples of healthy probands (discovery: n = 1403; replication: n = 630), the interaction of NPSR1 rs324981, childhood trauma (Childhood Trauma Questionnaire, CTQ) and general self-efficacy as a measure of coping ability (General Self-Efficacy Scale, GSE) on trait anxiety (State-Trait Anxiety Inventory) was investigated via hierarchical multiple regression analyses. RESULTS: In both samples, trait anxiety differed as a function of NPSR1 genotype, CTQ and GSE score (discovery: ß = 0.129, P = 3.938 × 10-8; replication: ß = 0.102, P = 0.020). In A allele carriers, the relationship between childhood trauma and anxiety was moderated by general self-efficacy: higher self-efficacy and childhood trauma resulted in low anxiety scores, and lower self-efficacy and childhood trauma in higher anxiety levels. In turn, TT homozygotes displayed increased anxiety as a function of childhood adversity unaffected by general self-efficacy. CONCLUSIONS: Functional NPSR1 variation and childhood trauma are suggested as prime moderators in the vulnerability-stress model of anxiety, further modified by the protective effect of self-efficacy. This G × E × C approach - introducing coping as an additional dimension further shaping a G × E risk constellation, thus suggesting a three-dimensional 'vulnerability-stress-coping model' of mental disorders - might inform targeted preventive or therapeutic interventions strengthening coping ability to promote resilient functioning.

Orexin in the anxiety spectrum: association of a HCRTR1 polymorphism with panic disorder/agoraphobia, CBT treatment response and fear-related intermediate phenotypes.

Preclinical studies point to a pivotal role of the orexin 1 (OX1) receptor in arousal and fear learning and therefore suggest the HCRTR1 gene as a prime candidate in panic disorder (PD) with/without agoraphobia (AG), PD/AG treatment response, and PD/AG-related intermediate phenotypes. Here, a multilevel approach was applied to test the non-synonymous HCRTR1 C/T Ile408Val gene variant (rs2271933) for association with PD/AG in two independent case-control samples (total n = 613 cases, 1839 healthy subjects), as an outcome predictor of a six-weeks exposure-based cognitive behavioral therapy (CBT) in PD/AG patients (n = 189), as well as with respect to agoraphobic cognitions (ACQ) (n = 483 patients, n = 2382 healthy subjects), fMRI alerting network activation in healthy subjects (n = 94), and a behavioral avoidance task in PD/AG pre- and post-CBT (n = 271). The HCRTR1 rs2271933 T allele was associated with PD/AG in both samples independently, and in their meta-analysis (p = 4.2 × 10-7), particularly in the female subsample (p = 9.8 × 10-9). T allele carriers displayed a significantly poorer CBT outcome (e.g., Hamilton anxiety rating scale: p = 7.5 × 10-4). The T allele count was linked to higher ACQ sores in PD/AG and healthy subjects, decreased inferior frontal gyrus and increased locus coeruleus activation in the alerting network. Finally, the T allele count was associated with increased pre-CBT exposure avoidance and autonomic arousal as well as decreased post-CBT improvement. In sum, the present results provide converging evidence for an involvement of HCRTR1 gene variation in the etiology of PD/AG and PD/AG-related traits as well as treatment response to CBT, supporting future therapeutic approaches targeting the orexin-related arousal system.

Human BDNF rs6265 polymorphism as a mediator for the generalization of contextual anxiety.

The Met allele of the human brain-derived neurotrophic factor (BDNF) gene might be a risk factor for anxiety disorders and is associated with reduced hippocampal volume. Notably, hippocampus plays a crucial role in contextual learning and generalization. The role of the BDNF gene variation in human context-conditioning and generalization is still unknown. We investigated 33 carriers of the Met allele (18 females) and 32 homozygous carriers of the Val allele (15 females) with a virtual-reality context-conditioning paradigm. Electric stimulations (unconditioned stimulus, US) were unpredictably delivered in one virtual office (CTX+), but never in another virtual office (CTX-). During generalization, participants revisited CTX+ and CTX- and a generalization office (G-CTX), which was a mix of the other two. Rating data indicated successful conditioning (more negative valence, higher arousal, anxiety and contingency ratings for CTX+ than CTX-), and generalization of conditioned anxiety by comparable ratings for G-CTX and CTX+. The startle data indicated discriminative learning for Met allele carriers, but not for Val homozygotes. Moreover, a trend effect suggests that startle responses of only the Met carriers were slightly potentiated in G-CTX versus CTX-. In sum, the BDNF polymorphism did not affect contextual learning and its generalization on a verbal level. However, the physiological data suggest that Met carriers are characterized by fast discriminative contextual learning and a tendency to generalize anxiety responses to ambiguous contexts. We propose that such learning may be related to reduced hippocampal functionality and the basis for the risk of Met carriers to develop anxiety disorders.

Plasticity of Functional MAOA Gene Methylation in Acrophobia.

Epigenetic mechanisms have been proposed to mediate fear extinction in animal models. Here, MAOA methylation was analyzed via direct sequencing of sodium bisulfite-treated DNA extracted from blood cells before and after a 2-week exposure therapy in a sample of n = 28 female patients with acrophobia as well as in n = 28 matched healthy female controls. Clinical response was measured using the Acrophobia Questionnaire and the Attitude Towards Heights Questionnaire. The functional relevance of altered MAOA methylation was investigated by luciferase-based reporter gene assays. MAOA methylation was found to be significantly decreased in patients with acrophobia compared with healthy controls. Furthermore, MAOA methylation levels were shown to significantly increase after treatment and correlate with treatment response as reflected by decreasing Acrophobia Questionnaire/Attitude Towards Heights Questionnaire scores. Functional analyses revealed decreased reporter gene activity in presence of methylated compared with unmethylated pCpGfree_MAOA reporter gene vector constructs. The present proof-of-concept psychotherapy-epigenetic study for the first time suggests functional MAOA methylation changes as a potential epigenetic correlate of treatment response in acrophobia and fosters further investigation into the notion of epigenetic mechanisms underlying fear extinction.

Cellular aspect ratio and cell division mechanics underlie the patterning of cell progeny in diverse mammalian epithelia.

Cell division is essential to expand, shape, and replenish epithelia. In the adult small intestine, cells from a common progenitor intermix with other lineages, whereas cell progeny in many other epithelia form contiguous patches. The mechanisms that generate these distinct patterns of progeny are poorly understood. Using light sheet and confocal imaging of intestinal organoids, we show that lineages intersperse during cytokinesis, when elongated interphase cells insert between apically displaced daughters. Reducing the cellular aspect ratio to minimize the height difference between interphase and mitotic cells disrupts interspersion, producing contiguous patches. Cellular aspect ratio is similarly a key parameter for division-coupled interspersion in the early mouse embryo, suggesting that this physical mechanism for patterning progeny may pertain to many mammalian epithelia. Our results reveal that the process of cytokinesis in elongated mammalian epithelia allows lineages to intermix and that cellular aspect ratio is a critical modulator of the progeny pattern.

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.

CRHR1 promoter hypomethylation: An epigenetic readout of panic disorder?

The corticotropin releasing hormone receptor 1 (CRHR1) is crucially involved in the hypothalamic-pituitary-adrenal axis and thus a major regulator of the stress response. CRHR1 gene variation is associated with several mental disorders including anxiety disorders. Studies in rodents have demonstrated epigenetic regulation of CRHR1 gene expression to moderate response to stressful environment. In the present study, we investigated CRHR1 promoter methylation for the first time regarding its role in panic disorder applying a case-control approach (N=131 patients, N=131 controls). In an independent sample of healthy volunteers (N=255), CRHR1 methylation was additionally analyzed for association with the Beck Anxiety Inventory (BAI) score as a dimensional panic-related intermediate phenotype. The functional relevance of altered CRHR1 promoter methylation was investigated by means of luciferase-based reporter gene assays. In panic disorder patients, a significantly decreased CRHR1 methylation was discerned (p<0.001). Accordingly, healthy controls with high BAI scores showed significantly decreased CRHR1 methylation. Functional analyses revealed an increased gene expression in presence of unmethylated as compared to methylated pCpGl_CRHR1 reporter gene vectors. The present study identified a potential role of CRHR1 hypomethylation - conferring increased CRHR1 expression - in panic disorder and a related dimensional intermediate phenotype. This up-regulation of CRHR1 gene expression driven by de-methylation might constitute a link between the stress response and panic disorder risk.

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.

Influence of 5-HTT variation, childhood trauma and self-efficacy on anxiety traits: a gene-environment-coping interaction study.

Environmental vulnerability factors such as adverse childhood experiences in interaction with genetic risk variants, e.g., the serotonin transporter gene linked polymorphic region (5-HTTLPR), are assumed to play a role in the development of anxiety and affective disorders. However, positive influences such as general self-efficacy (GSE) may exert a compensatory effect on genetic disposition, environmental adversity, and anxiety traits. We, thus, assessed childhood trauma (Childhood Trauma Questionnaire, CTQ) and GSE in 678 adults genotyped for 5-HTTLPR/rs25531 and their interaction on agoraphobic cognitions (Agoraphobic Cognitions Questionnaire, ACQ), social anxiety (Liebowitz Social Anxiety Scale, LSAS), and trait anxiety (State-Trait Anxiety Inventory, STAI-T). The relationship between anxiety traits and childhood trauma was moderated by self-efficacy in 5-HTTLPR/rs25531 LALA genotype carriers: LALA probands maltreated as children showed high anxiety scores when self-efficacy was low, but low anxiety scores in the presence of high self-efficacy despite childhood maltreatment. Our results extend previous findings regarding anxiety-related traits showing an interactive relationship between 5-HTT genotype and adverse childhood experiences by suggesting coping-related measures to function as an additional dimension buffering the effects of a gene-environment risk constellation. Given that anxiety disorders manifest already early in childhood, this insight could contribute to the improvement of psychotherapeutic interventions by including measures strengthening self-efficacy and inform early targeted preventive interventions in at-risk populations, particularly within the crucial time window of childhood and adolescence.