Mathias Rhein

High-affinity neurotrophin receptors and ligands promote leukemogenesis.

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR), we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target.

Insertional Mutagenesis by Replication‐Deficient Retroviral Vectors Encoding the Large T Oncogene

Abstract:  Insertion sites of replication‐deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor‐suppressor proteins p53 and Rb by virtue of a chaperone‐like activity. Primary hematopoietic stem/progenitor cells transduced with retroviral vectors encoding TAg‐induced histiocytic sarcoma (HS) or myeloid leukemia (ML) in transplanted mice (average survival of 21 weeks). Retrovirally introducing TAg into pretransformed 32D cells generated a monocytic leukemia, with faster kinetics (∼8 weeks). Leukemic clones showed retroviral insertions in genes contributing to all known TAg cooperation pathways, acting mitogenic and/or modulating apoptosis (such as BclX, Crk, Pim2, Csfr1/Pdgfrb, Osm/Lif, Axl, Fli, Sema4b, Sox4). 32D‐derived monocytic leukemias showed hits in Pim2 and Max proto‐oncogenes, or the chaperone Hspa4, plus additional signaling genes. Vector‐mediated insertional mutagenesis thus revealed a broad spectrum of potential TAg complementation genes. These findings have important implications for the use of retroviral transgenesis in cancer research, and the expression of signaling genes in somatic gene therapy.

Alcohol-Induced Changes in Methylation Status of Individual CpG Sites, and Serum Levels of Vasopressin and Atrial Natriuretic Peptide in Alcohol-Dependent Patients during Detoxification Treatment

Disturbances of volume-regulating peptides like vasopressin (AVP) and atrial natriuretic peptide (ANP) have been described in early abstinent alcohol-dependent patients. In a longitudinal approach, we investigated whether changes in AVP and ANP serum levels correlated to cytosine-phosphatidyl-guanine (CpG) methylation of the respective gene promoters on days 1, 7 and 14 of alcohol withdrawal. We analyzed the blood samples of 99 patients suffering from alcohol dependence alongside age- and BMI-matched controls. Concerning AVP promoter methylation, we observed an interaction between time of measurement and CpG loci with CpG 2 showing a significant increase in methylation from day 1 to 14. Serum levels of AVP were significantly decreased in the patient group. Compared to healthy controls, promoter-related DNA methylation of the ANP promoter was significantly reduced on days 7 and 14. Moreover, we detected a significant interaction between CpG position and group. In both cases the difference was mainly observed at CpG 1. The present study shows significant changes in the methylation status of individual CpG sites of AVP and ANP. Observing respective alterations of AVP serum protein levels in alcohol-dependent patients during detoxification treatment, we consider methylation as a possible mode of regulation for these proteins during alcohol detoxification.

Do changes in the BDNF promoter methylation indicate the risk of alcohol relapse

The neurotrophic growth factor brain derived neurotrophic factor (BDNF) was linked to the risk of alcohol relapse in clinical studies. In this study we investigated alterations in the methylation of the BDNF gene during alcohol withdrawal (day 1, 7 and 14) in 99 male alcohol-dependent patients compared to age matched healthy males (n=33). In particular, we aimed to investigate a possible association between the BDNF promoter methylation and the self-reported duration of alcohol abstinence before relapse. Mean methylation of the BDNF promoter was significantly increased in alcohol-dependent patients compared to the healthy controls (F=10.014, p<0.001) and decreased significantly during alcohol-withdrawal (F=10.014, p<0.001). Moreover, mean methylation was associated with depressive (F=2.014, p<0.001) and anxious symptoms in the alcohol-dependent patients (F=2.228, p<0.001). On day 14 of alcohol-withdrawal we found significantly higher methylation rates in those patients who abstained longer before relapse compared to those patients who abstained shorter (F=9.938, p<0.001). Our results suggest an association between BDNF expression and the symptomatology of alcohol withdrawal and imply that changes in the methylation of the BDNF IV gene may contribute to alcohol consumption.

Hypomethylation of neuregulin in rats selectively bred for reduced sensorimotor gating

Low prepulse inhibition (PPI) of startle is associated with reduced sensorimotor gating found in schizophrenia. In rats with breeding-induced low PPI neuregulin (NRG1) methylation was significantly decreased in brain regions associated with this phenotype and with schizophrenia, i.e., the medial prefrontal cortex, the nucleus accumbens, and the ventral hippocampus, while methylation in the amygdala and dorsal hippocampus was less affected. The dopamine D2 receptor (DRD2) promoter region showed negligible changes between groups. Rats with low PPI may be used to understand the reduced epigenetic regulation found in schizophrenia, and eventually lead to the development of novel therapies.

Behavioral differences of male Wistar rats from different vendors in vulnerability and resilience to chronic mild stress are reflected in epigenetic regulation and expression of p11

Outbred rat lines such as Wistar rats are commonly used for models of depressive disorders. Such rats arise from random mating schedules. Hence, genetic drift occurs in outbred populations which could lead to genotypic and phenotypic heterogeneity between rats from different vendors. Additionally, vendor specific rearing conditions could contribute to intrastrain variability. In the present study differences in behavioral responses to the chronic mild stress (CMS) model of depression within Wistar rat strains from different vendors are described. DNA methylation studies and mRNA expression analysis of p11 revealed that the behavioral differences between the substrains are reflected at the epigenetic and genetic level. The results suggest that there are breeder-dependent differences in vulnerability to stress in the CMS model of depression, which might bear on the validity of the model and contribute to contradictory findings and difficulties of replication between laboratories. P11 mRNA expression seems to be differently regulated depending on the quality of the stress response evoked by CMS exposure.

DNA methylation results depend on DNA integrity – role of post mortem interval

Major questions of neurological and psychiatric mechanisms involve the brain functions on a molecular level and cannot be easily addressed due to limitations in access to tissue samples. Post mortem studies are able to partly bridge the gap between brain tissue research retrieved from animal trials and the information derived from peripheral analysis (e.g., measurements in blood cells) in patients. Here, we wanted to know how fast DNA degradation is progressing under controlled conditions in order to define thresholds for tissue quality to be used in respective trials. Our focus was on the applicability of partly degraded samples for bisulfite sequencing and the determination of simple means to define cut-off values. After opening the brain cavity, we kept two consecutive pig skulls at ambient temperature (19–21°C) and removed cortex tissue up to a post mortem interval (PMI) of 120 h. We calculated the percentage of degradation on DNA gel electrophoresis of brain DNA to estimate quality and relate this estimation spectrum to the quality of human post mortem control samples. Functional DNA quality was investigated by bisulfite sequencing of two functionally relevant genes for either the serotonin receptor 5 (SLC6A4) or aldehyde dehydrogenase 2 (ALDH2). Testing our approach in a heterogeneous collective of human blood and brain samples, we demonstrate integrity of measurement quality below the threshold of 72 h PMI. While sequencing technically worked for all timepoints irrespective of conceivable DNA degradation, there is a good correlation between variance of methylation to degradation levels documented in the gel (R2 = 0.4311, p = 0.0392) for advancing post mortem intervals (PMI). This otherwise elusive phenomenon is an important prerequisite for the interpretation and evaluation of samples prior to in-depth processing via an affordable and easy assay to estimate identical sample quality and thereby comparable methylation measurements.

Promoter Polymorphism rs886205 Genotype Interacts With DNA Methylation of the ALDH2 Regulatory Region in Alcohol Dependence

Aims Aldehyde dehydrogenase 2 (ALDH2) protects cells from ethanol toxicity by metabolizing acetaldehyde. We studied the single nucleotide polymorphism (SNP) rs886205s located between a negative and a positive regulating promoter element in the ALDH2 gene. The negative regulatory region was already associated with differential DNA methylation in the two allele variations of rs886205 SNP. Another CpG island, in the positive regulatory region of the ALDH2 promoter, extends through the SNP rs886205 and a nuclear receptor response element. Methods We assessed rs886305 genotype and DNA methylation using bisulfite sequencing in a cohort of 83 male alcohol-dependent patients undergoing detoxification treatment (Days 1, 7 and 14) and in 33 male age-matched controls. Luciferase reporter assays were performed to address the functional significance of genotype and methylation. Results We observed a higher methylation in alcohol-dependent patients compared to controls. Patients with AA (n = 52) or GG/GA (n = 31) genotype differed significantly in baseline methylation levels as well as in methylation kinetics during withdrawal. AA carriers display an increase in methylation from low baseline levels while GG/GA showed the inverse pattern. The reporter gene assays corroborate these data by showing a significant effect of genotype on ALDH2 expression as well as an interaction between genotype and methylation. Conclusion Our results describe a new regulatory role of rs886205 in the methylation of ALDH2 promoter region and provide additional insight into the complex regulation of ALDH2 under the condition of alcohol dependence. Short summary Genetic variations have been described to influence DNA promoter methylation of various genes. We investigated the association between the polymorphism rs886205, located on ALDH2 promoter and methylation kinetics of the neighboring CpG island in alcohol-dependent patients. Luciferase reporter assays showed functional significance of genotype, methylation and a genotype-epigenotype interaction in vitro.

Impaired Regulation of ALDH2 Protein Expression Revealing a Yet Unknown Epigenetic Impact of rs886205 on Specific Methylation of a Negative Regulatory Promoter Region in Alcohol-Dependent Patients.

Acetaldehyde, the carcinogenic metabolite of ethanol known to provoke aversive symptoms of alcohol consumption, is predominantly eliminated by aldehyde dehydrogenase 2 (ALDH2). Reduced ALDH2 activity correlates with low alcohol tolerance and low risk for alcohol dependence. The ALDH2 promoter polymorphism rs886205 (A>G) is associated with decreased promoter activity, but a molecular mechanism and allele-dependent ALDH2 protein expression has not been described yet. On the basis of allele-dependent epigenetic effects, we analyzed the rs886205 genotype, methylation rates of cytosine-phosphatidyl-guanine (CpG)-sites within a regulatory promoter region and ALDH2 protein levels in 82 alcohol-dependent patients during a 2-week withdrawal and compared them to 34 matched controls. Patients without the G-allele of rs886205 showed higher methylation of the promoter region than controls and readily adapted epigenetically as well as on protein level during withdrawal, while patients with the G-allele displayed retarded methylation readjustment and no change in ALDH2 protein levels. Our data provide novel insights into an unknown genetic-epigenetic interaction, revealing impaired ALDH2 protein expression in patients with the G-allele of rs886205. Additionally, we checked for an association between rs886205 and protection against alcohol dependence and found a trend association between the G-allele and protection against alcohol dependence that needs replication in a larger Caucasian cohort.

Stable-isotope dilution GC-MS method for ethanol in vapour ethanol and microdialysis systems based on carbonate-catalyzed extractive pentafluorobenzoylation

Common ethanol detection methods are not applicable to cell culture media and microdialysates due to interference with medium constituents including amino acids and pH indicators. We present a novel GC-MS method for the accurate and precise analysis of ethanol in cell cultures and microdialysates. The method is based on the carbonate-catalyzed extractive pentafluorobenzoylation of ethanol and deuterium-labelled ethanol serving as the internal standard and on their GC-MS analysis in the electron-capture negative-ion chemical ionization mode. The method was used to optimize experimental conditions in a custom-made ethanol vapour system utilized for studies examining ethanol influences on neuronal cell lines and in microdialysis.