Stress-related alcohol consumption in heavy drinkers correlates with expression of miR-10a, miR-21, and components of the TAR-RNA-binding protein-associated complex.

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 < 0.05), 79 genes were identified that changed by >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, R(2)  = 0.59, miR-21 R(2)  = 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.

Altered expression of cytokine signaling pathway genes in peripheral blood cells of alcohol dependent subjects: preliminary findings.

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 < 0.05). Two hundred and ninety-one genes differed between AD and MD subjects, 240 differed between AD and HD subjects, but only 6 differed between HD and MD subjects. Pathway analysis using DAVID and GeneGO Metacore(®) software showed that the most affected pathways were those related to T-cell receptor and Janus kinase-Signal transducer and activator of transcription (JAK-Stat) signaling. CONCLUSIONS: These results suggest the transition from heavy alcohol use to dependence is accompanied by changes in the expression of genes involved in regulation of the innate immune response. Such changes may underlie some of the previously described changes in immune function associated with chronic alcohol abuse. Early detection of these changes may allow individuals at high risk for dependence to be identified.

Increased peripheral blood expression of electron transport chain genes in bipolar depression.

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 < 0.05, covaried for age and sex). Of these, 559 genes were up-regulated in BPD subjects and 621 were down-regulated. Surprisingly, there was no difference between medicated (n = 11) and unmedicated (n = 9) subjects with BPD for any of these genes. Pathway analysis using GeneGo MetaCore software showed that the most significantly affected pathway was the mitochondrial electron transport chain (ETC). Of the 85 objects (genes or proteins) in this pathway, 22 were up-regulated and 2 down-regulated in subjects with BPD. qRT-PCR confirmed up-regulation of nuclear encoded ETC genes in complexes I, III, IV, and V and, in addition, demonstrated up-regulation of mitochondrially encoded genes in each of these complexes. CONCLUSION: These results suggest that increased expression of multiple components of the mitochondrial ETC may be a primary deficit in bipolar depression, rather than an effect of medication.

Altered response to antidepressant treatment in FoxG1 heterozygous knockout mice.

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.

Dynamic contribution of nestin-expressing stem cells to adult neurogenesis.

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-CreER(T2)/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-CreER(T2)/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generated in vitro after recombination in 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-CreER(T2)/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-CreER(T2)/R26R-YFP mouse for inducible gene ablation in stem cells and their progeny in vivo in the two major regions of adult neurogenesis.

Rab33A: characterization, expression, and suppression by epigenetic modification.

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.

High-affinity nicotinic acetylcholine receptors are required for antidepressant effects of amitriptyline on behavior and hippocampal cell proliferation.

BACKGROUND: A wide variety of antidepressants act as noncompetitive antagonists of nicotinic acetylcholine receptors (nAChRs), but the relationship between this antagonism and the therapeutic effects of antidepressants is unknown. METHODS: Antidepressant properties of the noncompetitive nAChR antagonist mecamylamine in the forced swim test were tested alone and in combination with the tricyclic antidepressant amitriptyline. Mice lacking high-affinity nAChRs were tested in three behavioral models to determine whether these receptors are required for behavioral effects of amitriptyline in common models of antidepressant action. Finally, the brains of wild-type and knockout animals treated with amitriptyline were examined to determine whether high-affinity nAChRs are required for antidepressant-induced increases in hippocampal cell proliferation. RESULTS: Inhibition of nAChRs by mecamylamine had antidepressant-like effects in the forced swim test and potentiated the antidepressant activity of amitriptyline when the two drugs were used in combination. Mice lacking high-affinity nAChRs showed no behavioral response to amitriptyline. Finally, after chronic treatment with amitriptyline, nAChR knockout mice did not show the increase in hippocampal cell proliferation seen in wild-type mice. CONCLUSIONS: These data support the hypothesis that antagonism of nAChRs is an essential component of the therapeutic action of antidepressants.

Alteration of hippocampal cell proliferation in mice lacking the beta 2 subunit of the neuronal nicotinic acetylcholine receptor.

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.

Nestin promoter/enhancer directs transgene expression to precursors of adult generated periglomerular neurons.

The subventricular zone (SVZ) is a major neurogenic region in the adult brain. Cells from the SVZ give rise to two populations of olfactory bulb interneurons: the granule cells and periglomerular (PG) cells. Currently, little is known about the signaling pathways that direct these newly generated neurons to become either granule or PG neurons. In the present study, we used the nestin promoter and enhancer to direct expression of the tetracycline transactivator (tTA). We generated two independent strains of nestin-tTA transgenic animals and crossed founder mice from both lines to mice containing a tetracycline-regulated transgene (mCREB) whose expression served as a marker for the activity of the nestin-tTA transgene. mCREB expression occurred in a subset of proliferating cells in the SVZ and rostral migratory stream in both lines. Surprisingly, in both lines of nestin-tTA mice transgene expression in the olfactory bulb was limited to PG neurons and was absent from granule cells, suggesting that this nestin promoter construct differentiates between the two interneuronal populations. Transgene expression occurred in several subtypes of PG neurons, including those expressing calretinin, calbindin, GAD67, and tyrosine hydroxylase. These results suggest that a unique subset of SVZ precursor cells gives rise to PG, and not granule cells. The ability to express different transgenes within this subpopulation of neuronal precursors provides a powerful system to define the signals regulating the differentiation and survival of adult-generated neurons in the olfactory bulb.

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription.

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.