Search

Background Alterations in stress-related gene expression may play a role in stress-related drinking and the risk of alcohol dependence. Methods Microarrays were used to measure changes in gene expression in peripheral blood in nonsmoking, social drinking subjects exposed to 3 types of personalized imagery: neutral, stressful (but not alcohol related), and alcohol-related cues. Gene expression was measured at baseline, immediately after, and 1 hour after stimulus presentation. Subjects were allowed to drink up to 750 cc of beer in a “taste test” following stimulus presentation in each imagery condition, and the amount of beer consumed was recorded. Gene-expression levels were compared in 2 groups of nonsmoking subjects (n = 11/group): heavy drinkers (HD; defined as regular alcohol use over the past year of at least 8 standard drinks per week for women and at least 15 standard drinks per week for men), and moderate drinkers (MD; defined as up to 7 standard drinks per week for women and 14 standard drinks per week for men). Expression of microRNA-10a (miR-10a) and microRNA-21 (miR-21) was assessed by quantitative real-time polymerase chain reaction. Results After correction for multiple testing (false discovery rate 1.3-fold in the HD group, but not the MD group, following exposure to stress. No changes were observed for any of these genes in either group following exposure to neutral or alcohol-related imagery. Pathway analysis suggested that many of these genes, form part of the transactivation responsive (TAR)-RNA-binding protein (TRBP)-associated complex and are positively regulated by miR-10a and miR-21. Expression of both miR-10a and miR-21 was up-regulated following psychological stress in HD, but not MD subjects; however, the differences between groups were not statistically significant. Expression levels of both microRNAs was correlated (miR-10a, R2 = 0.59, miR-21 R2 = 0.57) with amount drunk in HD, but not MD subjects. Conclusions Expression of miR-10a, miR-21, and several of their target genes is regulated by acute psychological stress and is correlated with stress-induced drinking in a laboratory setting. Alterations in miRNA expression may be one mechanism linking psychological stress with changes in gene expression and increased alcohol intake in binge/HD.

Beech RD, Lowthert L, Leffert JJ, Mason PN, Taylor MM, Umlauf S, Lin A, Lee JY, Maloney K, Muralidharan A, Lorberg B, Zhao H, Newton SS, Mane S, Epperson CN, Sinha R, Blumberg H, Bhagwagar Z. Increased peripheral blood expression of electron transport chain genes in bipolar depression. Bipolar Disord 2010: 12: 813–824. © 2010 The Authors.Journal compilation © 2010 John Wiley & Sons A/S. Objective: To identify specific genetic pathways showing altered expression in peripheral blood of depressed subjects with bipolar disorder (BPD). Methods: Illumina Sentrix BeadChip (Human-6v2) microarrays containing >48,000 transcript probes were used to measure levels of gene expression in peripheral blood from 20 depressed subjects with BPD and in 15 healthy control subjects. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to confirm a subset of these differences. Results: A total of 1,180 genes were differentially expressed between subjects with BPD and healthy controls (fold-change >1.3, false discovery rate-corrected p

Marijuana (both Cannabis indica and Cannabis sativa) was criminalized in the U.S. in 1937, and has been classified as a Schedule I medical plant, meaning it has no currently accepted medical use and a high potential for abuse, since 1970 [1]. Various medical organizations including the AMA, American Academy of Pediatrics and the American Society of Addiction Medicine, have taken positions opposing the use of marijuana for medical purposes. However, the pendulum of public opinion has clearly begun to swing the other direction. At the present time, 23 states and the district of Columbia have approved the use of marijuana to treat various medical conditions, notwithstanding the continuing Federal ban [2]. The conditions for which medical marijuana use has been approved vary from state to state, and in most cases do not meet the standards that would be required for FDA approval for a new medication [3]. In part, this reflects the current paradoxical position of marijuana in the field of medicine where use for specific conditions is being approved on an ad hoc basis by state legislatures, while Federal law prevents the type of large-scale randomized placebo controlled trials that would normally be required for FDA approval.

Evidence from a variety of sources suggests that structural alterations in the brain, including neurogenesis, may play a role in both the pathogenesis of mood disorders and the mechanism of action of antidepressants. Previous studies have implicated both the transforming growth factor-beta (TGF-beta), and the phosphatidyl inositol-3 kinase (PI3K)-Akt pathways in the neurogenesis-promoting and behavioral properties of antidepressants. Forkhead box protein G1 (FoxG1) is a major regulator of both of these pathways, and FoxG1 heterozygous null mice (FoxG1+/-) have previously been reported to have deficits in adult hippocampal neurogenesis and behavioral abnormalities including deficits in contextual fear learning. However the role of FoxG1, if any, in the response to antidepressants has not been previously investigated.To investigate the role of the FoxG1 gene in the behavioral and neurogenic properties of antidepressants, we tested FoxG1+/- mice and littermate controls in two different rodent models of antidepressant action: the tail suspension test and the forced swim test. FoxG1+/- mice showed no response to antidepressants in either of these tests. These results suggest that normal levels of FoxG1 may be required for the behavioral response to antidepressants.

This study was designed to identify genes whose expression in peripheral blood may serve as early markers for treatment response to lithium (Li) in patients with bipolar disorder. Although changes in peripheral blood gene-expression may not relate directly to mood symptoms, differences in treatment response at the biochemical level may underlie some of the heterogeneity in clinical response to Li. Subjects were randomized to treatment with (n=28) or without (n=32) Li. Peripheral blood gene-expression was measured before and 1 month after treatment initiation, and treatment response was assessed after 6 months. In subjects treated with Li, 62 genes were differentially regulated in treatment responders and non-responders. Of these, BCL2L1 showed the greatest difference between Li responders and non-responders. These changes were specific to Li responders (n=9), and were not seen in Li non-responders or patients treated without Li, suggesting that they may have specific roles in treatment response to Li.

Background Preclinical and clinical studies have implicated changes in cytokine and innate immune gene-expression in both the development of and end-organ damage resulting from alcohol dependence. However, these changes have not been systematically assessed on the basis of alcohol consumption in human subjects. Methods Illumina Sentrix Beadchip (Human-6v2) microarrays were used to measure levels of gene-expression in peripheral blood in 3 groups of subjects: those with alcohol dependence (AD, n = 12), heavy drinkers (HD; defined as regular alcohol use over the past year of at least 8 standard drinks/wk for women and at least 15 standard drinks/wk for men, n = 13), and moderate drinkers (MD; defined as up to 7 standard drinks/wk for women and 14 standard drinks/wk for men, n = 17). Results Four hundred and thirty-six genes were differentially expressed among the 3 groups of subjects (false discovery rate corrected p-value

Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreERT2/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreERT2/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generatedin vitroafter recombinationin vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreERT2/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreERT2/R26R-YFP mouse for inducible gene ablation in stem cells and their progenyin vivoin the two major regions of adult neurogenesis.

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal linking proteins. ERM proteins are involved in a wide variety of cellular functions including cell motility, signal transduction, cell-cell interaction and cell-matrix recognition. A recent in situ hybridization study showed that the mRNA encoding ezrin is expressed in neurogenic regions of the mature brain including the subventricular zone (SVZ) and rostral migratory stream (RMS); however, the specific cell types expressing ezrin and their relationship to migrating and proliferating cells in these regions have not been characterized previously. In this study, we used immunocytochemistry to perform double labeling with a variety of cell-type specific markers to characterize the expression of ezrin in the SVZ and RMS of adult mice. Ezrin was expressed at high levels in both the SVZ and RMS where ezrin-immunopositive processes formed a trabecular network surrounding the proliferating and migrating cells. Ezrin-positive cells co-labeled with the glial makers S100beta and GFAP (glial fibrillary acidic protein), but only minimally with the early neuronal markers beta III tubulin and polysialylated form of neural cell adhesion molecule 1 (PSA-NCAM), indicating that ezrin was expressed primarily in the glial tube cells. Ezrin positive cells also expressed beta-catenin, a membrane-complex protein previously implicated in the regulation of stem-cell proliferation and neuronal migration. Glial tube cells act as both precursors of, and a physical channel for, migrating neuroblasts. Bi-directional signals between glial tube cells and migrating neuroblasts have been shown to regulate the rates of both proliferation of the precursor cells and migration of the newly generated neuroblasts. Our finding that ezrin and beta-catenin are both present at the cell membrane of the glial tube cells suggests that these proteins may be involved in those signaling processes.

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.

Adult hippocampal neurogenesis declines with age in parallel with decreased performance on a variety of hippocampal-dependent tasks. We measured the rate of cellular proliferation in the hippocampus of mice lacking the beta 2-subunit of the nicotinic acetylcholine receptor (beta 2-/- mice) at three ages: young adult (3 months old), fully adult (7-10 months old), and aged (22-24 months old). Consistent with previous studies, we observed an age-related decline in hippocampal proliferation in both groups. However, in fully adult beta 2-/- mice a 43% reduction of granule cell proliferation was detected compared to age-matched controls. This was accompanied by a significant decrease in dentate gyrus area/section and the length of the granule cell layer in beta 2-/- mice. These alterations were not the result of a change in plasma corticosterone levels or expression of the neurotrophic factor BDNF in the dentate gyrus, two known regulators of hippocampal cell proliferation. Similarly, there was no increase in gliosis, abnormal myelination, or apoptotic cell death in the beta 2-/- animals, although there was a significant shift in the location of apoptotic cells in the dentate gyrus indicative of a change in neuronal survival. These results suggest that the beta 2-subunit containing nicotinic acetylcholine receptors play an important role in regulating cell proliferation in the hippocampus and that endogenous acetylcholine may act to oppose the negative effects of normal aging and stress on cellular proliferation.

Promoters that govern the transcription of mammalian genes by RNA polymerase II fall broadly into three types: the classical TATA box-dependent promoters, the initiator element (Inr)-dependent promoters, and the G+C-rich promoters, which also have been referred to as CpG islands. Transcription initiation at the TATA box-dependent promoters is dictated by the direct interaction of the TATA box with the TATA-binding protein (TBP) as a first and rate-limiting step (reviewed in reference 8). Likewise, transcription initiation at Inr-dependent promoters occurs when the Inr element interacts with sequence-specific Inr-binding proteins (35, 36). The mechanism by which transcription initiation sites within G+C-rich promoters are recognized by the RNA polymerase II transcription machinery is currently unelucidated. Transcription initiation of a large number of mammalian genes, including most housekeeping and central nervous system genes and many highly regulated genes that control cell growth and differentiation, is under the control of G+C-rich promoters (2, 20, 23, 26, 28, 31). This class of promoter is not found in either the Drosophila melanogaster or yeast genome. Because a common characteristic of this type of promoter is the presence of a noncanonical TATA box and one or more Sp1 binding sites upstream of the major transcription initiation site, several reports have claimed that those sequences are the key functional elements in G+C-rich promoters (6, 18). However, reports that challenged these claims, in some cases even with respect to the same promoter, also have been published (2, 24, 25). The lack of obvious conserved sequence motifs shared among different G+C-rich promoters, with the exception of the Sp1 binding sites and the “noncanonical” TATA boxes, provides further impetus for investigations to elucidate how transcription can initiate nonrandomly at these precise genome locations. The murine Ada gene has an archetypal G+C-rich promoter that contains multiple Sp1 binding sites and the noncanonical TATA-like element TAAAAAA. Neither the Sp1 binding sites nor the TAAAAAA sequence is required for basal or enhancer-activated promoter function (1, 2; C.-Y. Yeung et al., unpublished data). Instead, the minimal self-sufficient promoter activity resides within a 48-bp minimal self-sufficient promoter element (MSPE) that displays an imperfect dyad symmetry (2). Many other G+C-rich promoters contain elements with similar secondary structure-forming potential around their transcription initiation sites (2). We previously demonstrated that there are nuclear protein-binding sites within the Ada gene's MSPE (2). This observation led us to attempt the cloning and characterization of the nuclear protein responsible for directing the assembly of the RNA polymerase II transcription initiation complex at this promoter. A multimerized MSPE probe was used to identify and clone a full-length cDNA that encodes a ubiquitously expressed 66-kDa murine protein. This 66-kDa protein, designated the murine G+C-rich promoter binding protein (mGPBP), can bind specifically to both the murine Ada gene's MSPE and the nonhomologous human Topo IIα gene's G+C-rich promoter, as revealed by competitive gel mobility shift assays. The coding sequence of mGPBP displays domain homologies with those encoding several known transcription factors. Purified recombinant mGPBP generated from Escherichia coli was used to raise polyclonal rabbit antiserum against the protein. In situ immunostaining studies showed that mGPBP is localized in the nucleus. Immunoprecipitation and Western blot analyses revealed that GPBP can form a complex with TBP, transcription fraction IIB (TFIIB), TFIIF, RNA polymerase II, and P300/CAAT binding protein (CBP) both in vitro and in vivo. In cotransfection experiments performed on various mammalian cells, elevated mGPBP expression was found to transactivate a murine Ada gene promoter-controlled reporter gene. Immunoabsorption-induced sequestering of GPBP from HeLa cell nuclear extract resulted in a complete suppression of extract-dependent in vitro transcription directed by the mouse Ada gene's G+C-rich promoter. This suppression was reversed by the replenishment of the nuclear extract with purified recombinant mGPBP. The GPBP requirement for transcription initiated at this G+C-rich promoter is promoter specific. Parallel experiments showed that similar immunoabsorption of GPBP in HeLa nuclear extract had no adverse effect on transcription initiated at the adenovirus major late gene's classical TATA box-dependent promoter and only a partial and reversible suppressive effect on the human Topo IIα gene's G+C-rich promoter, which contains a perfect TATAAA consensus element. These results thus describe the discovery of a novel requisite promoter-specific transactivating transcription factor with demonstrable abilities to both bind a typical G+C-rich promoter and interact with multiple transcription factors that comprise the core mammalian RNA polymerase II transcription initiation complex. Since GPBP is not required for transcription directed by the adenovirus major late gene's classical TATA box-dependent promoter, the results also provide definitive proof that transcription initiated at the murine Ada gene's G+C-rich promoter is mechanistically distinct from that initiated at a classical TATA box-dependent promoter.