Zoya Marinova

Trio study and meta-analysis support the association of genetic variation at the serotonin transporter with early-onset obsessive–compulsive disorder

Despite compelling evidence for major genetic contributions to the etiology of obsessive-compulsive disorder (OCD), few genetic variants have been consistently associated with this debilitating illness. Molecular genetic studies in children and adolescents with OCD are of particular interest, since early onset of the disease has been observed to be associated with increased familiality. We replicate here for the first time in early-onset OCD patients, a previously reported association of OCD with the common gain-of-function LA allele at the serotonin transporter linked polymorphic region known as 5-HTTLPR in a collection of parent-offspring trios. The present meta-analysis of this recently refined serotonin transporter gene variant revealed further support for the LA allele conferring increased genetic susceptibility to OCD. We conclude that the 5-HTTLPR is currently the single best supported risk variant for OCD, in regards of early-onset OCD, albeit of modest effect size and the possibility that the conferred risk might not be specific to OCD.

Biological correlates of complex posttraumatic stress disorder*state of research and future directions

Complex posttraumatic stress disorder (PTSD) presents with clinical features of full or partial PTSD (re-experiencing a traumatic event, avoiding reminders of the event, and a state of hyperarousal) together with symptoms from three additional clusters (problems in emotional regulation, negative self-concept, and problems in interpersonal relations). Complex PTSD is proposed as a new diagnostic entity in ICD-11 and typically occurs after prolonged and complex trauma. Here we shortly review current knowledge regarding the biological correlates of complex PTSD and compare it to the relevant findings in PTSD. Recent studies provide support to the validity of complex PTSD as a separate diagnostic entity; however, data regarding the biological basis of the disorder are still very limited at this time. Further studies focused on complex PTSD biological correlates and replication of the initial findings are needed, including neuroimaging, neurobiochemical, genetic, and epigenetic investigations. Identification of altered biological pathways in complex PTSD may be critical to further understand the pathophysiology and optimize treatment strategies.

Expression of D-Amino Acid Oxidase (DAO/DAAO) and D-Amino Acid Oxidase Activator (DAOA/G72) during Development and Aging in the Human Post-mortem Brain

In the brain, D-amino acid oxidase (DAO/DAAO) mainly oxidizes D-serine, a co-agonist of the N-methyl-D-aspartate (NMDA) receptors. Thus, DAO can regulate the function of NMDA receptors via D-serine breakdown. Furthermore, DAO activator (DAOA)/G72 has been reported as both DAOA and repressor. The co-expression of DAO and DAOA genes and proteins in the human brain is not yet elucidated. The aim of this study was to understand the regional and age span distribution of DAO and DAOA (mRNA and protein) in a concomitant manner. We determined DAO and DAOA mRNA and protein expression across six brain regions in normal human post-mortem brain samples (16 weeks of gestation to 91 years) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found higher expression of DAO mRNA in the cerebellum, whereas lower expression of DAO protein in the cerebellum compared to the other brain regions studied, which suggests post-transcriptional regulation. We detected DAOA protein but not DAOA mRNA in all brain regions studied, suggesting a tightly regulated expression. To understand this regulation at the transcriptional level, we analyzed DNA methylation levels at DAO and DAOA CpG sites in the cerebellum and frontal cortex of control human post-mortem brain obtained from Gene Expression Omnibus datasets. Indeed, DAO and DAOA CpG sites in the cerebellum were significantly more methylated than those in the frontal cortex. While investigating lifespan effects, we found that DAO mRNA levels were positively correlated with age <2 years in the cerebellum and amygdala. We also detected a significant positive correlation (controlled for age) between DAO and DAOA protein in all of the brain regions studied except for the frontal cortex. In summary, DAO and DAOA expression in the human brain are both age and brain region dependent.

5-HT2A serotonin receptor agonist DOI alleviates cytotoxicity in neuroblastoma cells: Role of the ERK pathway

Disturbances of serotonergic signaling, including the serotonin 2A (5-HT2A) receptor, have been implicated in neuropsychiatric and neurodegenerative disorders. The aim of the present study was to characterize the effect of a 5-HT2A receptor agonist on cytotoxicity in a neuronal cell line and address the involved mechanism. HTR2A mRNA and protein expression in human neuroblastoma SK-N-SH cells was confirmed. Cells were subjected to serum deprivation and cell viability was monitored continuously with xCELLigence. In a dose-response study the 5-HT2A agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (25 nM to 5 μM) protected against serum deprivation cytotoxicity. The selective 5-HT2A receptor antagonist MDL 11,939, the general protein tyrosine kinase inhibitor genistein, and the extracellular signal-regulated kinase (ERK) pathway MEK inhibitor U0126, all attenuated DOIs protective effect. An antibody array suggested that 1 μM DOI affected phosphorylation of several tyrosine kinases. Western blot further confirmed that DOI transiently increased ERK phosphorylation, indicating its activation. Finally, protective concentrations of DOI increased cellular mitochondrial mass, an effect prevented by pretreatment with U0126. In conclusion, our results suggest that DOI protects SK-N-SH cells against serum deprivation through ERK pathway activation. They imply 5-HT2A receptor modulation as a potential target for neuroprotection.

Region-specific regulation of the serotonin 2A receptor expression in development and ageing in post mortem human brain.

The serotonin 2A receptor (HTR2A) is widely expressed in the brain and involved in the modulation of fear, mood, anxiety and other symptoms. HTR2A and HTR2A gene variations are implicated in depression, schizophrenia, anxiety and obsessive‐compulsive disorder. To understand HTR2A signalling changes in psychiatric or neurodegenerative disorders, its normal pattern of brain expression and region specificity during development and ageing needs to be clarified. The aim of the present study was to assess HTR2A expression through developmental and ageing stages in six brain regions in post mortem human brain samples from individuals with no clinical or neuropathological evidence of neuropsychiatric disorders and to investigate the interaction with the rs6311 HTR2A promoter polymorphism.

Are Epigenetic Factors Implicated in Chronic Widespread Pain

Background Chronic widespread musculoskeletal pain (CWP) is the cardinal symptom of fibromyalgia and affects about 12% of the general population. Familial aggregation of CWP has been repeatedly demonstrated with estimated heritabilities of around 50%, indicating a genetic susceptibility. The objective of the study was to explore genome-wide disease-differentially methylated positions (DMPs) for chronic widespread pain (CWP) in a sample of unrelated individuals and a subsample of discordant monozygotic (MZ) twins. Methodology/Principle Findings A total of N = 281 twin individuals from the TwinsUK registry, including N = 33 MZ twins discordant for self-reported CWP, were part of the discovery sample. The replication sample included 729 men and 756 women from a subsample of the KORA S4 survey–an independent population-based cohort from Southern Germany. Epigenome-wide analysis of DNA methylation was conducted using the Illumina Infinium HumanMethylation 450 DNA BeadChip in both the discovery and replication sample. Of our 40 main loci that were carried forward for replication, three CPGs reached significant p-values in the replication sample, including malate dehydrogenase 2 (MDH2; p-value 0.017), tetranectin (CLEC3B; p-value 0.039), and heat shock protein beta-6 (HSPB6; p-value 0.016). The associations between the collagen type I, alpha 2 chain (COL1A2) and monoamine oxidase B (MAOB) observed in the discovery sample–both of which have been previously reported to be biological candidates for pain–could not be replicated. Conclusion/Significance Our results may serve as a starting point to encourage further investigation in large and independent population-based cohorts of DNA methylation and other epigenetic changes as possible disease mechanisms in CWP. Ultimately, understanding the key mechanisms underlying CWP may lead to new treatments and inform clinical practice.

A pilot investigation on DNA methylation modifications associated with complex posttraumatic symptoms in elderly traumatized in childhood

ObjectiveComplex posttraumatic stress disorder (CPTSD) is a newly proposed diagnosis in the International Classification of Diseases-version 11, which is currently intensively investigated. Childhood trauma is regarded as main source of CPTSD symptoms, even in later life. Induction of DNA methylation changes by childhood trauma may contribute to its long-lasting adverse health consequences. The current study analyzed the correlation of genome-wide DNA methylation profiles with complex posttraumatic sequelae in buccal epithelial cells from 31 elderly former indentured child laborers (Verdingkinder) using the Infinium Illumina 450k Human DNA methylation chip.ResultsDNA methylation modifications indicated experiment-wide significant associations with the following complex posttraumatic symptom domains: dissociation, tension reduction behavior and dysfunctional sexual behavior. Differentially methylated CpG sites were mapped to the genes huntington associated protein 1 (HAP1), RAN binding protein 2 (RANBP2) and proteasome subunit alpha 4 (PSMA4), respectively. In addition, the methylation of cg07225277 located in carnosine synthase 1 (CARNS1) correlated with trauma symptom complexity. Our pilot data suggest correlation of DNA methylation modifications with complex posttraumatic symptoms in elderly individuals subjected to prolonged and complex childhood trauma. More comprehensive and elaborated studies should be carried out to analyze epigenetic modifications associated with CPTSD.

Real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations - most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.