Robert D. Beech

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-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 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-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 progeny in vivo in the two major regions of adult neurogenesis.

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.

Increased ratio of anti-apoptotic to pro-apoptotic Bcl2 gene-family members in lithium-responders one month after treatment initiation

BackgroundLithium is considered by many as the gold standard medication in the management of bipolar disorder (BD). However, the clinical response to lithium is heterogeneous, and the molecular basis for this difference in response is unknown. In the present study, we sought to determine how the peripheral blood gene expression profiles of patients with bipolar disorder (BD) changed over time following intitiation of treatment with lithium, and whether differences in those profiles over time were related to the clinical response.MethodsIllumina Sentrix Beadchip (Human-6v2) microarrays containing > 48,000 transcript probes were used to measure levels of expression of gene-expression in peripheral blood from 20 depressed subjects with BD prior to and every two weeks during 8 weeks of open-label treatment with lithium.Changes in gene-expression were compared between treatment responders (defined as a decrease in the Hamilton Depression Rating Scale of 50% or more) and non-responders. Pathway analysis was conducted using GeneGO Metacore software.Results127 genes showed a differential response in responders vs. non-responders. Pathway analysis showed that regulation of apoptosis was the most significantly affected pathway among these genes. Closer examination of the time-course of changes among BCL2 related genes showed that in lithium-responders, one month after starting treatment with lithium, several anti-apoptotic genes including Bcl2 and insulin receptor substrate 2 (IRS2) were up-regulated, while pro-apoptotic genes, including BCL2-antagonist/killer 1 (BAK1) and BCL2-associated agonist of cell death (BAD), were down-regulated. In contrast, in lithium non-responders, BCL2 and IRS2 were down-regulated, while BAK1 and BAD up-regulated at the one-month time-point.ConclusionsThese results suggest that differential changes in the balance of pro- and anti- apoptotic gene-expression following treatment with lithium may explain some of the heterogeneity in clinical response in BD patients.

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. J. Comp. Neurol. 475:128–141, 2004.

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.

Expression of ezrin in glial tubes in the adult subventricular zone and rostral migratory stream.

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.

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.

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‐β), 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.