• Energy Research

Stem cell vitality is critical for the growth of the developing brain. Growth factors can define the survival of neural stem cells (NSCs) and ethanol can affect growth factor-mediated activities. The present study tested two hypotheses: (a) ethanol causes the apoptotic death of NSCs and (b) this effect is influenced by the ambient growth factor. Monolayer cultures of non-immortalized NS-5 cells were exposed to fibroblast growth factor (FGF) 2 or transforming growth factor (TGF) β1 in the absence or presence of ethanol for 48 h. Ethanol killed NSCs as measured by increases in the numbers of ethidium bromide+ and annexin V+ cells and decreases in the number of calcein AM+ (viable) cells. These toxic effects were promoted by TGFβ1. A quantitative polymerase chain reaction array of apoptosis-related mRNAs revealed an ethanol-induced increase (≥2-fold change; p<0.05) in transcripts involved in Fas ligand (FasL) and tumor necrosis factor (TNF) signaling. These effects, particularly the FasL pathway, were potentiated by TGFβ1. Immunocytochemical analyses of NS-5 cells showed that transcriptional alterations translated into consistent up-regulation of protein expression. Experiments with the neocortical proliferative zones harvested from fetal mice exposed to ethanol showed that ethanol activated similar molecular systems in vivo. Thus, ethanol induces NSC death through two distinct molecular mechanisms, one is initiated by TGFβ1 (FasL) and another (through TNF) which is TGFβ1-independent.

Abstract Background Alcohol use disorders (AUDs) lead to alterations in central nervous system (CNS) architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs) produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. Results Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs) of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP) assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1) was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5) showed a highly significant correlation with AUD-induced decreases in the volume of the left parietal supramarginal gyrus and neuropsychological measures. Conclusions These results demonstrate that alcohol-induced changes in genes related to proliferation, apoptosis, and DNA-repair are observable in the peripheral blood and may serve as a useful biomarker for CNS structural damage and functional performance deficits in human AUD subjects.

J. Neurochem. (2010) 114, 1767–1780.AbstractEthanol inhibits the proliferation of neural precursors by altering mitogenic and anti‐mitogenic growth factor signaling and can affect global methylation activity in the fetus. We tested the hypothesis that epigenetic modification of specific cell cycle genes underlies the ethanol‐induced inhibition of growth factor‐regulated cell cycle progression. Monolayer cultures of neural stem cells (NSCs) were treated with fibroblast growth factor 2 or transforming growth factor (TGF) β1 in the absence or presence of ethanol. Ethanol increased the total length of the cell cycle by elongating the amount of time spent in the gap 1 (G1) and synthesis (S) phases of the cell cycle. Ethanol induced the hypermethylation of multiple cell cycle genes associated with the G1/S and gap 2/mitotic phase (G2/M) checkpoints and increased the expression and activity of DNA methyltransferases. These changes were most pronounced in the presence of TGFβ1. Epigenetic alterations paralleled the down‐regulation of associated transcripts and other checkpoint‐related mRNAs both in vitro (NS‐5 cell culture) and in vivo (fetal mouse cortex). Ethanol‐induced hypermethylation was accompanied by decreases in the proportion of NSCs expressing associated cell cycle proteins. Thus, ethanol disrupts growth factor‐related cell cycle progression by inducing checkpoint restriction at the G1/S transition through a feed‐forward system involving the methylation of G2/M regulators.

Following neurotoxic damage, cells repair their DNA, and survive or undergo apoptosis. This study tests the hypothesis that ethanol induces a DNA damage response (DDR) in neural stem cells (NSCs) that promotes excision repair (ER) and this repair is influenced by the growth factor environment. Non-immortalized NSCs treated with fibroblast growth factor 2 or transforming growth factor (TGF) β1 were exposed to ethanol. Ethanol increased total DNA damage, reactive oxygen species, and oxidized DNA bases. TGFβ1 potentiated these toxic effects. Transcriptional analyses of cultured NSCs revealed ethanol-induced increases in transcripts related to the DDR (e.g., Hus1 and p53), base ER (e.g., Mutyh and Nthl1), and nucleotide ER (e.g., Xpc), particularly in the presence of TGFβ1. Expression and activity of ER proteins were affected by ethanol. Similar changes occurred in proliferating cells of ethanol-treated mouse fetuses. Ethanol-induced DNA repair in NSCs depends on the ambient growth factors. Gene products for DNA repair in stem cells are among the first biomarkers identifying fetal alcohol-induced damage.

There is currently a lack of reliable, minimally invasive biomarkers that could predict the extent of alcoholism-induced CNS damage. Developing such biomarkers may prove useful in reducing the prevalence of alcohol use disorders (AUDs). Extracellular microRNAs (miRNAs) can be informative molecular indicators of changes in neuronal gene expression. In this study, we performed a global analysis of extracellular miRNAs to identify robust biomarkers of early CNS damage in humans diagnosed with DSM-IV AUDs. We recruited a relatively young set of 20 AUD subjects and 10 age-matched controls. They were subjected to comprehensive medical, neuropsychological and neuroimaging tests, followed by comparison of miRNA levels found in peripheral blood serum. Employing a conservative strategy to identify candidate biomarkers, miRNAs were quantified using two independent high-throughput methods: microarray and next-generation RNA-sequencing. This improved our capacity to discover and validate relevant miRNAs.Our results identified several miRNAs with significant and reproducible expression changes in AUD subjects versus controls. Moreover, several significant associations between candidate miRNA biomarkers and various medical, neuropsychological and neuroimaging parameters were identified using Pearson correlation and unbiased hierarchical clustering analyses. Some of the top candidate biomarkers identified, such as mir-92b and mir-96 have established roles in neural development. Cross-species validation of miRNA expression was performed using two different in vivo rat drinking models and two different in vitro mouse neural stem cell exposure models. A systems level analysis revealed a remarkable degree of convergence in the top changes seen in all of these data sets, specifically identifying cell death, cell proliferation and cell cycle processes as most consistently affected. Though not necessarily the same molecules, the affected miRNAs within these pathways clearly influence common genes, such as p53 and TNF, which stand out as potential keystone molecules. Lastly, we also examined the potential tissue origins of these biomarkers by quantifying their levels in 15 different tissue types and show that several are highly-enriched in the brain.Collectively, our results suggest that serum miRNA expression changes can directly relate to alterations in CNS structure and function, and may do so through effects on highly specific cellular pathways.