Andrea Rotter

Orexin expression and promoter-methylation in peripheral blood of patients suffering from major depressive disorder

Orexins are endogenous neuropeptides synthesized in hypothalamic neurones; they play a major role in the regulation of feeding, drinking, endocrine function and sleep/wakefulness that is often disturbed in major depression. The aim of this study was to explore Orexin A expression and promotermethylation in peripheral blood cells of 29 patients (14 male and 15 female patients at three different time points during antidepressive treatment) suffering from major depressive disorder (MDD) by quantitative RT-PCR and bisulfite sequencing. There was a measurable difference between Orexin A expression on admission in comparison to the Orexin mRNA expression in the healthy control group (T=1.53; df=39; p=0.135) that failed to reach statistical significance. An inverse correlation between Orexin A mRNA expression on admission and the HAMD scores at all measurement dates each week over 6 weeks could be detected. A cluster of methylated CPG sites within the promoter region of the Orexin A gene could be identified that was positively correlated with Delta CT values of the mRNA expression 14 days after hospital admission (r=0.625; p=0.072) and 4 weeks afterwards (r=0.582; p=0.1). Considering only the methylation of the 7 CPGs within the CPG island in the promoter 4 weeks after treatment onset a statistically significant relation between the cluster of CPG sites within the island and body weight (r=0.55; p=0.034) as well as BMI (r=0.474; p=0.074) could be detected. Furthermore, this methylation pattern 4 weeks after treatment onset was positively associated with mRNA expression on admission, 2 and 4 weeks afterwards. In sum, these results are an indicator of a link between social stresses, disruptions in energy homeostasis and changes in body weight in relation to depressive disorders that are possibly linked to Orexin dysregulation.

Activity of secretory sphingomyelinase is increased in plasma of alcohol-dependent patients.

BACKGROUND Acid sphingomyelinase (ASM, EC 3.1.4.12) hydrolyzes sphingomyelin to ceramide and represents a major regulator of sphingolipid metabolism. Increased activity of ASM has been observed in a variety of human diseases, and a critical contribution of ASM to medical conditions was demonstrated in several mouse models. In agreement with increased ASM activity in cell lines treated with ethanol, we have recently found higher levels of ASM activity in peripheral blood cells of active drinkers. However, the influence of ethanol on secretory ASM (S-ASM) has not been investigated so far. METHODS ASM activity and routine blood parameters were determined in plasma samples of 27 patients with alcohol dependence during physical withdrawal and compared to a group of 36 healthy volunteers. RESULTS Compared to the control group, patients with alcohol dependence had S-ASM activity increased by about 3-fold (141 ± 69 vs. 428 ± 220 pmol/ml/h; p < 0.001) at the beginning of physical withdrawal. During withdrawal, S-ASM activity decreased by about 50% (p < 0.001; day 0 vs. day 7 to 10) and finally approximated nearly normal values. On the day of admission, S-ASM activity correlated positively with levels of carbohydrate-deficient transferrin (r = 0.410, p = 0.034) and high-density lipoprotein cholesterol (r = 0.440, p = 0.022) and inversely with body mass index (r = -0.509; p = 0.007), glucose (r = -0.480; p = 0.011), triglycerides (r = -0.592; p = 0.001), and large unstained cells (r = -0.526; p = 0.017). CONCLUSIONS Activity of S-ASM is increased in alcohol-dependent patients and correlates with established biomarkers of excessive drinking. The increased S-ASM activity is implicated in alcohol-induced lipid alterations and might be relevant for the occurrence of alcohol-related disorders.

Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system

Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pKa value was associated with a more rapid uptake. The drug‐induced displacement of LysoTracker was neither caused by elevation of intra‐lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra‐lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi‐drug resistance by altering subcellular drug distribution. J. Cell. Physiol. 224:152–164, 2010

Orexin A expression and promoter methylation in patients with alcohol dependence comparing acute and protracted withdrawal

The orexins (hypocretins) are neuropeptides deriving from the lateral hypothalamus and may be of importance within the context of drug craving, withdrawal, and relapse. Therefore, the orexin A expression and promoter methylation in peripheral blood cells of 68 patients (41 male and 27 female patients at three different time points during withdrawal and 27 patients during stationary dehabituation therapy) suffering from alcohol dependence were assessed by quantitative reverse transcription-polymerase chain reaction and bisulfite sequencing. There was a statistically significant difference of orexin A expression between the three time points of withdrawal and long-term (LT) abstinence (F=4.16, P=.011). This difference was most prominent in comparison with LT abstinence (t=-3.08, P=.0032). Expression was significantly associated with the severity of withdrawal symptoms measured with the Withdrawal Syndrome Scale for Alcohol and Related Psychoactive Drugs (WSA) (t=2.17, P=.0356). The stronger the withdrawal symptoms, the lower the orexin A expression (F=4.69, P=.036). Body mass index (t=2.15, P=.041), the severity of withdrawal measured with the WSA (t=2.595, P=.0133), craving measured either by the Obsessive Compulsive Drinking Scale (t=2.77, P=.0085) or the Lübecker Craving Questionnaire (t=-2.23, P=.0314) had a significant influence on orexin A expression taking into account mean methylation of the CpG island of the orexin A promoter during withdrawal. Orexin A may be a possible candidate to further elucidate mechanisms of alcohol withdrawal taking into account energy homoeostasis in the circuit of reward and motivation.

Neurokinin3 receptor as a target to predict and improve learning and memory in the aged organism

Significance Cognitive decline during aging impairs life quality and may lead to dementia. It is associated with a dysfunction of the brain acetylcholinergic system. Here we demonstrate that pharmacological stimulation of neurokinin3 receptors improves learning and memory in aged rats by enhancing acetylcholinergic function in the brain. In a human association study we show that a single-nucleotide polymorphism in the neurokinin3-receptor–coding gene TACR3 can predict learning and memory in elderly patients with cognitive impairments and their hippocampus volume. These findings suggest the neurokinin3 receptor as a potential biomarker and treatment target for cognitive enhancement in the elderly. Impaired learning and memory performance is often found in aging as an early sign of dementia. It is associated with neuronal loss and reduced functioning of cholinergic networks. Here we present evidence that the neurokinin3 receptors (NK3-R) and their influence on acetylcholine (ACh) release may represent a crucial mechanism that underlies age-related deficits in learning and memory. Repeated pharmacological stimulation of NK3-R in aged rats was found to improve learning in the water maze and in object-place recognition. This treatment also enhanced in vivo acetylcholinergic activity in the frontal cortex, hippocampus, and amygdala but reduced NK3-R mRNA expression in the hippocampus. Furthermore, NK3-R agonism incurred a significantly higher increase in ACh levels in aged animals that showed superior learning than in those that were most deficient in learning. Our findings suggest that the induced activation of ACh, rather than basal ACh activity, is associated with superior learning in the aged. To test whether natural variation in NK3-R function also determines learning and memory performance in aged humans, we investigated 209 elderly patients with cognitive impairments. We found that of the 15 analyzed single single-nucleotide ploymorphism (SNPs) of the NK3-R–coding gene, TACR3, the rs2765 SNP predicted the degree of impairment of learning and memory in these patients. This relationship could be partially explained by a reduced right hippocampus volume in a subsample of 111 tested dementia patients. These data indicate the NK3-R as an important target to predict and improve learning and memory performance in the aged organism.

Abuse potential and adverse cognitive effects of mitragynine (kratom)

Mitragynine is the major psychoactive alkaloid of the plant kratom/ketum. Kratom is widely used in Southeast Asia as a recreational drug, and increasingly appears as a pure compound or a component of ‘herbal high’ preparations in the Western world. While mitragynine/kratom may have analgesic, muscle relaxant and anti‐inflammatory effects, its addictive properties and effects on cognitive performance are unknown. We isolated mitragynine from the plant and performed a thorough investigation of its behavioural effects in rats and mice. Here we describe an addictive profile and cognitive impairments of acute and chronic mitragynine administration, which closely resembles that of morphine. Acute mitragynine has complex effects on locomotor activity. Repeated administration induces locomotor sensitization, anxiolysis and conditioned place preference, enhances expression of dopamine transporter‐ and dopamine receptor‐regulating factor mRNA in the mesencephalon. While there was no increase in spontaneous locomotor activity during withdrawal, animals showed hypersensitivity towards small challenging doses for up to 14 days. Severe somatic withdrawal signs developed after 12 hours, and increased level of anxiety became evident after 24 hours of withdrawal. Acute mitragynine independently impaired passive avoidance learning, memory consolidation and retrieval, possibly mediated by a disruption of cortical oscillatory activity, including the suppression of low‐frequency rhythms (delta and theta) in the electrocorticogram. Chronic mitragynine administration led to impaired passive avoidance and object recognition learning. Altogether, these findings provide evidence for an addiction potential with cognitive impairments for mitragynine, which suggest its classification as a harmful drug.

Changes of cytokine profiles during electroconvulsive therapy in patients with major depression.

Objectives Electroconvulsive therapy (ECT) is an effective treatment of depression, but its mechanism of action still remains unknown. Some studies emphasize that epileptic seizures result in cerebral production of cytokines, including the cytokine network in association with the pathophysiology of depression. We hypothesized that depressed patients would show a dysregulated profile of peripheral cytokines before and after ECT treatment. Methods Fifteen hospitalized subjects with major depressive disorder were recruited. Human cytokine array IV was used to determine the profile of cytokines in the serum during the course of ECT. Positive results of the cytokine assay were verified by reverse transcriptase-polymerase chain reaction. Depressive symptoms were evaluated before and after ECT series. Results The signal intensity of eotaxin-3 and interleukin (IL)-5 changed statistically significantly between the first ECT and 24 hours after the last ECT. Furthermore, there were significant correlations between the signal intensities of eotaxin-3, bone morphogenetic protein 6, IL-5, and transforming growth factor-&bgr; and the severity of depression. The results of Cytoray assays were confirmed partly by reverse transcriptase-polymerase chain reaction. The changes of tumor necrosis factor &bgr; in pre-post comparison of ECT and the correlation of the Montgomery-Asberg Depression Scale score with tumor necrosis factor &bgr;, IL-5, and bone morphogenetic protein 6 expression could be verified. Only the relative signal intensity of IL-16 correlated significantly with the clinically as well as electroencephalographically measurable seizure duration. Conclusion Electroconvulsive therapy treatment seems to change the expression of various cytokines in relation to changes of affective states such as mood. Therefore, cytokines might play a specific role within the treatment and pathogenesis of affective disorders.

Antidepressant drugs modulate growth factors in cultured cells

BackgroundDifferent classes of antidepressant drugs are used as a treatment for depression by activating the catecholinergic system. In addition, depression has been associated with decrease of growth factors, which causes insufficient axonal sprouting and reduced neuronal damage repair. In this study, antidepressant treatments are analyzed in a cell culture system, to study the modulation of growth factors.ResultsWe quantified the transcription of several growth factors in three cell lines after application of antidepressant drugs by real time polymerase chain reaction. Antidepressant drugs counteracted against phorbolester-induced deregulation of growth factors in PMA-differentiated neuronal SY5Y cells. We also found indications in a pilot experiment that magnetic stimulation could possibly modify BDNF in the cell culture system.ConclusionThe antidepressant effects antidepressant drugs might be explained by selective modulation of growth factors, which subsequently affects neuronal plasticity.

CB1 and CB2 Receptor Expression and Promoter Methylation in Patients with Cannabis Dependence

CB1 and CB2 receptors are influenced via exogenous and endogenous cannabinoids. To date, little is known regarding changes in receptor expression and methylation in THC (tetrahydrocannabinol) dependence. Therefore, the CB1 and CB2 receptor mRNA expression levels and promoter methylation status in the peripheral blood cells of 77 subjects (36 with THC dependence, 21 cigarette smokers and 20 nonsmokers) were assessed by quantitative real-time PCR and methylation-specific PCR. There was a significant difference in CB1 receptor expression levels between the three groups (ANOVA, p < 0.001, d.f. = 2, F = 71.3). The mean promoter methylation (%) was significantly negatively correlated with CB1 receptor mRNA expression levels (Spearman’s rho: r = –0.37; p = 0.002). Using a mixed general linear model, it was demonstrated that the CB1 mRNA expression (as the dependent variable) was associated with the satisfaction with life scale (SWLS) (r = 0.101; T = 2.8; p = 0.007), craving (as measured with the VAS; r = –0.023; T = –2.3; p = 0.023) and the WHO-Assist Subscale for Cannabis consumption (r = –0.068; T = –2.4; p = 0.02). CB1 receptor expression levels and methylation status appear to be altered in subjects with THC dependence.

Orexin A expression and promoter methylation in patients with cannabis dependence in comparison to nicotine-dependent cigarette smokers and nonsmokers.

Background: The orexins (hypocretins) are neuropeptides with an origin in the lateral hypothalamus. They have been found to be crucial within the context of drug craving, withdrawal und relapse. Methods: Therefore, orexin A gene expression and promoter methylation in peripheral blood cells of 77 subjects [36 with tetrahydrocannabinol (THC) dependence, 20 nicotine-dependent cigarette smokers and 21 nonsmokers] were assessed by quantitative real-time PCR and methylation-specific digestion PCR. Results: There was a statistically significant difference in orexin A expression between the three groups [p = 0.000, F = 131.4, d.f. = 2, analysis of variance (ANOVA)]. Orexin A gene expression was statistically significantly correlated with the Satisfaction with Life Scale (r = –0.28, p = 0.018), a visual analogue scale of craving (r = 0.734, p = 0.000) and three subscales of the World Health Organization Alcohol, Smoking and Substance Involvement Screening Test, i.e. nicotine consumption (r = 0.388, p = 0.001), alcohol consumption (r = 0.354, p = 0.002) and cannabis consumption (r = 0.783, p = 0.000). The mean promoter methylation (as a percentage) was not statistically related to orexin gene expression. However, there was a statistically significant difference in promoter methylation with regard to body mass index in general (F = 2.37, d.f. = 54, p = 0.016, ANOVA). Conclusions: Orexin might be a possible target in THC as well as nicotine dependence, taking into account the effect of THC on energy homeostasis in the circuit of reward and motivation and its impact on appetite and body weight.