In vitro study methodologies to investigate genetic aspects and effects of drugs used in attention-deficit hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common psychiatric disorders in children and adolescents, with up to 5% affected worldwide. Twin and family studies on ADHD show its high familiality with heritability estimated around 70%, but, to date, no specific polymorphism or gene was found to be specifically affected. Psychostimulants (amphetamine, methylphenidate) and non-psychostimulants (atomoxetine) are used successfully in ADHD therapy, but many of their mechanisms of action and their adverse effects are not yet fully understood. Therefore, both genetic findings and therapeutic interventions should be further investigated. One easy platform for such studies is in vitro analyses, which encompass neuronal cell culture studies, transfections of genetic constructs, binding and electrophysiology analyses. In this review, different methods will be referred in particular to ADHD findings, and new techniques will be mentioned for future studies of drug or genetic effects in vitr

In vitro study methodologies to investigate genetic aspects and effects of drugs used in attention-deficit hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common psychiatric disorders in children and adolescents, with up to 5 % affected worldwide. Twin and family studies on ADHD show its high familiality with heritability estimated around 70 %, but, to date, no specific polymorphism or gene was found to be specifically affected. Psychostimulants (amphetamine, methylphenidate) and non-psychostimulants (atomoxetine) are used successfully in ADHD therapy, but many of their mechanisms of action and their adverse effects are not yet fully understood. Therefore, both genetic findings and therapeutic interventions should be further investigated. One easy platform for such studies is in vitro analyses, which encompass neuronal cell culture studies, transfections of genetic constructs, binding and electrophysiology analyses. In this review, different methods will be referred in particular to ADHD findings, and new techniques will be mentioned for future studies of drug or genetic effects in vitro

Opioid and non-opioid activities of the dynorphins

Endogenous opioid peptides a -neoendorphin, dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B, collectively known as dynorphins are derived from the precursor protein prodynorphin (PDYN). Dynorphins regulate pain processing and memory acquisition and modulate reward induced by intake of addictive substances. Such actions are generally mediated through the k -opioid receptors. However, excitotoxic effects of Dyn A and Big Dyn relevant for neuropathic pain are non-opioid; the mechanisms of these effects are unknown but glutamate receptors are apparently involved. PDYN mRNA and dynorphin peptides have been extensively characterized in the rat and human brain, whereas little is known about PDYN, its biochemical properties, and localization and processing in the brain. The general aim of the present study was (1) to characterize mechanisms of non-opioid interactions of dynorphins with cells, (2) to identify non-opioid biochemical targets for these peptides, (3) to characterize biochemical properties, intracellular localization and distribution in the brain of the precursor protein PDYN, and (4) to assess role of dynorphins in human neurodegenerative disorders including Alzheimer disease. Our analysis revealed similarity of dynorphins with cell-penetrating peptides (CPPs) capable of translocating into cells. The ability of dynorphins to translocate across the plasma membrane was tested using immunofluorescence, confocal fluorescence microscopy, and fluorescence correlation spectroscopy on fixed and live cells. Big Dyn and Dyn A but not Dyn B were found to be capable to penetrate into cells. Big Dyn showed higher translocation potential compared with that of Dyn A, while Dyn A and transportan-10, a prototypical CPP translocated into cells with similar efficacy. The translocated dynorphins were predominantly located in the cytoplasm where they were associated with the endoplasmic reticulum. Fluorescence and circular dichroism spectroscopy imply that two structural features, the ability to form a -helix and high positive charge, may in conjunction determine the membrane translocation potential of Big Dyn and Dyn A. Translocation of dynorphins into cells followed by interactions with intracellular targets might represent an evolutionary ancient mechanism of intercellular communications and signal transduction (Paper I). For identification of intracellular dynorphin targets - non-opioid binding sites in neuronal cells and brain, radioligand-binding assay was used. A novel soluble factor that binds Dyn A and Big Dyn with high specificity and affinity (IC 50 5-10 nM) was found. Binding of Dyn A to the factor was virtually irreversible and resulted in conversion of Dyn A into Leu-enkephalin, suggesting that the Dyn A-binding factor (DABF) functions as an oligopeptidase. This enzyme may potentially degrade other neuropeptides. Dynorphins may regulate this degradation acting as competitors when they bind to the enzyme (Paper II). Dynorphins generation may be regulated at the levels of gene transcription, translation, protein trafficking and processing. As the first step in the analysis of these processes, the biochemical properties of PDYN, its intracellular localization and distribution in the rat and human brain were characterized. The focus was on structures where PDYN is synthesized and where mature dynorphins are located. PDYN distribution pattern in rat brain determined by immunohistochemistry and western blot was similar to that of dynorphin peptides with highest levels in the amygdala, hippocampus and striatum and lower levels in the cerebral cortex. PDYN in unprocessed form was also present in the ventral tegmental area (VTA) and the hippocampal CA3 region that do not have cell bodies of neurons producing PDYN but contain axons and axon terminals originating from PDYN-ergic neurons. Thus PDYN is transported to and stored in axon terminals. PDYN in axon terminals may be processed to mature peptides prior to their release from cells. This notion is supported by the observation that K +-evoked depolarization of PDYN producing cells increases the total amounts of dynorphins in cells and medium demonstrating that PDYN processing is activated. Stimulation of PDYN processing in axon terminals and dendrites by neuronal activity and extracellular signals may represent a mechanism for the local regulation of synaptic transmission (Paper III). Dyn A through non-opioid glutamate receptor-mediated mechanism may induce excitotoxicity resulting in neuronal death (related paper 1). Neuronal death in Alzheimer disease (AD) and other neurodegenerative diseases may be associated with upregulation of Dyn A, which may potentially contribute to neurodegeneration. To assess the relationship between neurodegeneration and dynorphins, the status of the dynorphin system was evaluated in AD. The levels of Dyn A and Dyn B and the related neuropeptide nociceptin in the inferior parietal lobule (Brodmann area 7) of control, AD, Parkinson disease (PD) and cerebro-vascular disease (CVD) groups were determined. AD subjects displayed robustly elevated levels of Dyn A, whereas the levels of Dyn B and nociceptin were unaltered. Subjects with PD and CVD showed no changes in all three peptides. The levels of PDYN and the activity of DABF did not change in AD, whereas the levels of the processing enzyme pro-hormone convertase 2 (PC2), which processes PDYN, were elevated. The levels of Dyn A correlated with the density of neuritic plaques. These results suggest a non-opioid role of Dyn A in the pathogenesis of AD (Paper IV)

Editorial of the Special Issue “Neurobiological Mechanisms Implicated in Stress-Related Psychiatric Disorders”

Mental disorders may seriously impair the quality of life of affected individuals and cause a significant public health burden [...

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

The hallucinogen 2,5-dimethoxy-4-iodoamphetamine hydrochloride activates neurotrophin receptors in a neuronal cell line and promotes neurites extension

Decreased neurotrophic factors expression and neurotrophin receptors signalling have repeatedly been reported in association with stress, depression, and neurodegenerative disorders. We have previously identified the hallucinogen 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) as protective against trophic deprivation-induced cytotoxicity in human neuroblastoma SK-N-SH cells and established the dependence of this effect on the 5-HT2A receptor, tyrosine kinases activity, and the extracellular signal-regulated kinase pathway. In the current study, we investigated the effect of DOI on tropomyosin-related kinase receptor A (TrkA) phosphorylation. Treatment with DOI increased TrkA tyrosine phosphorylation in SK-N-SH cells, determined by immunoprecipitation with TrkA antibody and immunoblotting with anti-phosphotyrosine- and TrkA-antibodies. Analysis of DOI's effect on individual TrkA residues in SK-N-SH cells showed that it increases TrkA Tyr490 phosphorylation (177 ± 23% after 5 μM DOI for 30 min compared to vehicle). Furthermore, DOI treatment increased the percentage of SK-N-SH cells extending neurites in a TrkA-dependent manner (17.2 ± 2.2% after 5 μM DOI treatment for 6 days compared to 5.6 ± 1.7% after vehicle). In a different cell model-lymphoblastoid cell lines-DOI treatment increased tropomyosin-related kinase receptor B (TrkB) phosphorylation, determined by immunoprecipitation with TrkB antibody and immunoblotting with anti-phosphotyrosine antibody and total Trk antibody. Our results identify the Trk receptors as a downstream target of the hallucinogen DOI. In light of the known involvement of Trk receptors in mental diseases, their participation in DOI-mediated effects warrants further investigation

Effects of oxytocin and arginine vasopressin on the proliferation and differentiation of a serotonergic cell line

The neuropeptides oxytocin and arginine vasopressin (AVP) are involved in the regulation of social behavior and cognition. The current study analyzed the effect of oxytocin and AVP on proliferation and differentiation of serotonergic neurons (RN46A cells). Oxytocin did not affect, while 5-10 μM AVP decreased RN46A proliferation. Oxytocin did not significantly alter, while 10 μM AVP decreased the number of cells extending neurites. We found divergent effects of oxytocin and AVP in serotonergic neurons, underscoring their functional differences

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

ERAWATCH Country Reports 2009: Bulgaria. Analysis of policy mixes to foster R&D investment and to contribute to the ERA

The main objective of the ERAWATCH Policy Mix Country reports 2009 is to characterise and assess in a structured manner the evolution of the national policy mixes in the perspective of the Lisbon goals, with a particular focus on the national R&D investments targets and on the realisation and better governance of the European Research Area. The reports were produced for all EU Member State and five Associated States to support the mutual learning process and the monitoring of Member and Associated States' efforts by DG-RTD in the context of the Lisbon Strategy and the European Research Area. The country reports 2009 build and extend on the analysis provided by analytical country reports 2008 and on a synthesis of information from the ERAWATCH Research Inventory and other important available information sources.JRC.J.3-Knowledge for Growt

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) (25nM to 5μM) 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 DOI's protective effect. An antibody array suggested that 1μM 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