Gene Expression In Neuronal Cells Research Articles

Exposure of Zebrafish Embryos to Urea Affects NOS1 Gene Expression in Neuronal Cells

Nitrogen-based fertilizers represent the most common fertilization tools, particularly used in crop food agriculture, despite the low cost-efficiency and the high negative environmental impact. At present, there is still inadequate information available about the effects of urea on human health; nevertheless, previous studies in animals observed that high urea concentration exposure can damage different tissues, including the brain. In several vertebrates, a crucial factor involved in neuronal cell formation is represented by the gas molecule, nitric oxide (NO), derived from the conversion of arginine to citrulline through the enzymatic activity of nitric oxide synthases (NOS). In zebrafish, three different isoforms of the NOS gene are known: nos1, nos2a, and nos2b. In the present study we show that nos1 represents the unique isoform with a stable high expression in the brain and spinal cord during all the embryonic stages of zebrafish development. Then, by using a specific transgenic zebrafish line, Tg(HuC:GFP), to mark neuronal cells, we observed nos1 to be specifically expressed in neurons. Interestingly, we observed that urea exposure at sub-lethal doses affected cell proliferation and the number of nos1-expressing cells, inducing apoptosis. Consistently, brain NO levels were observed to be reduced in urea-treated animals compared to untreated ones. This finding represents the first evidence that urea exposure affects the expression of a key gene involved in neuronal cell formation during embryonic development.

Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.

The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.

Propionic acid induces mitochondrial dysfunction and affects gene expression for mitochondria biogenesis and neuronal differentiation in SH-SY5Y cell line

Studies in animal models have shown that the short-chain fatty acid, propionic acid (PPA), interferes with mitochondrial metabolism leading to mitochondrial dysfunction and behavioral abnormalities. The aim of this study was to investigate the effects of PPA on mitochondrial function and gene expression in neuronal cells. SH-SY5Y cells and normal human neural progenitor (NHNP) cells were exposed to 1, 5 mM PPA for 4 or 24 h and we found that the mitochondrial potential measured in SH-SY5Y cells decreased in a dose-dependent manner after PPA treatment. Electron microscopy analysis revealed that the size of the mitochondria was significantly reduced following PPA treatment. A dose-dependent increase in the mitochondrial DNA copy number was observed in the PPA-treated cells. The expression of the mitochondrial biogenesis-related proteins PGC-1α, TFAM, SIRT3, and COX4 was significantly increased after PPA treatment. Transcriptome analysis revealed that mRNA expression in the notch signaling-related genes ASCL1 and LFNG changed after PPA treatment and the positive correlated protein expression changes were also observed. These results revealed that PPA treatment may affect neurodevelopment by altering mitochondrial function and notch signaling-related gene expression.

SUN-014 Identification of a New SUMOylation Process Regulating Nuclear Receptor Nor-1

Nuclear receptors are transcriptional factors controlling critical gene expression networks essential to the developmental and homeostatic regulation of several physiological processes. Transcriptional control of nuclear receptors is achieved by interaction with specific ligands and in response to cellular signalling pathways, which often results in post-translational modifications of receptors. Among such modifications, SUMOylation has emerged as a key process regulating nuclear receptor function. Our recent work has revealed an atypical SUMOylation process that requires phosphorylation, thereby defining a novel SUMO conjugating motif termed pSuM. Functional pSuM has been described for estrogen nuclear receptor ERβ and bile acid receptor FXR, and sequence analysis has indicated a conserved pSuM in the orphan nuclear receptor Nor-1. This study aims to characterize the function of Nor-1 pSuM-mediated SUMOylation. Nor-1 has various functions in the nervous system, including a role in neuronal survival and hippocampal development. In line with an implication of the SUMOylation process in neurodegenerative diseases, we speculate that pSuM SUMOylation might regulate Nor-1 activity and participate in the maintenance of neuronal integrity. The SUMOylation potential of Nor-1 and the mechanism regulating this process were investigated using immunoprecipitation and Western blot analysis. Our results indicate that Nor-1 was SUMOylated at the pSuM motif as well as at another consensus SUMOylation motif, thereby conjugating SUMO-1 and SUMO-2. We also found that the phosphorylation of the pSuM motif was essential for pSuM activation and SUMO conjugation. Furthermore, phosphorylation of adjacent residues to the pSuM appeared to potentiate the SUMOylation of Nor-1, suggesting a role for pSuM extension. This demonstrates the implication of specific kinase pathways required to promote pSuM activation. Using luciferase reporter assays and qPCR analysis, we also showed that SUMOylation regulates Nor-1 transcriptional activity and responsive gene expression in neuronal cells. Our findings provide new insights on the mechanistic regulation of Nor-1 function by phosphorylation and SUMOylation. We also identify a new atypical and highly regulated SUMOylation process as an additional targeting possibility for SUMO conjugation to coordinate transcriptional competence. This study contributes to expand the intricate dynamics of the SUMOylation process used by incoming signals to govern gene networks by nuclear receptors and possibly other transcription factors. Sources of Research Support: This work is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institute of Health Research (CIHR) award, and Fonds de la recherche en santé du Québec (FRQS) award.

Carbamoylated erythropoietin induces a neurotrophic gene profile in neuronal cells

Erythropoietin (EPO), a cytokine molecule, is best-known for its role in erythropoiesis. Preclinical studies have demonstrated that EPO has robust neuroprotective effects that appear to be independent of erythropoiesis. It is also being clinically tested for the treatment of neuropsychiatric illnesses due to its behavioral actions. A major limitation of EPO is that long-term administration results in excessive red blood cell production and increased blood viscosity. A chemical modification of EPO, carbamoylated erythropoietin (CEPO), reproduces the behavioral response of EPO in animal models but does not stimulate erythropoiesis. The molecular mechanisms involved in the behavioral effects of CEPO are not known. To obtain molecular insight we examined CEPO induced gene expression in neuronal cells. PC-12 cells were treated with CEPO followed by genome-wide microarray analysis. We investigated the functional significance of the gene profile by unbiased bioinformatics analysis. The Ingenuity pathway analysis (IPA) software was employed. The results revealed activation of functions such as neuronal number and long-term potentiation. Regulated signaling cascades included categories such as neurotrophin, CREB, NGF and synaptic long-term potentiation signaling. Some of the regulated genes from these pathways are CAMKII, EGR1, FOS, GRIN1, KIF1B, NOTCH1. We also comparatively examined EPO and CEPO-induced gene expression for a subset of genes in the rat dentate gyrus. The CEPO gene profile shows the induction of genes and signaling cascades that have roles in neurogenesis and memory formation, mechanisms that can produce antidepressant and cognitive function enhancing activity.

Hyperforin and Miquelianin from St. Johnʼs Wort Attenuate Gene Expression in Neuronal Cells After Dexamethasone-Induced Stress

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis plays an important part in the development of depressive symptoms. In this study, the effects of a commercial St.John's wort extract (STW3-VI), hyperforin, miquelianin, and the selective serotonin reuptake inhibitor citalopram on the expression of genes relevant to HPA axis function were investigated in human neuronal cells. SH-SY5Y cells were treated with STW3-VI (20 µg/mL), hyperforin (1 µM), miquelianin (10 µM), or citalopram (10 µM) in the presence of the glucocorticoid receptor agonist dexamethasone (DEX,10 µM) for 6 h and 48 h, respectively. Quantitative real-time polymerase chain reaction was used to determine the expression of FKBP5 (FK506 binding protein 51), CREB (cAMP responsive element binding protein), GRIK4 (glutamate ionotropic receptor kainate type subunit 4), VEGF (vascular endothelial growth factor), NET (norepinephrine transporter), and ARRB (β-arrestins), promising biomarkers of antidepressant therapy. Using DEX to mimic stress conditions, it was shown that the gene expression pattern of FKBP5, CREB, GRIK4, VEGF, NET, and ARRB2 in SH-SY5Y cells is time- and treatment-dependent. Most pronounced effects were observed for FKBP5: after 6 h of co-incubation, only STW3-VI could reverse the DEX-induced increase in FKBP5 expression, and after 48 h, citalopram, miquelianin, and hyperforin also reversed the glucocorticoid-induced increase in FKBP5 mRNA expression. The effects observed on FKBP5, CREB, GRIK4, VEGF, NET, and ARRB2 are in good correlation with published data, suggesting that this in vitro model could be used to screen the responsiveness of antidepressants under stress conditions.

Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage

BackgroundThe LH-induced folliculo-luteal transformation is connected with alterations of the gene expression profile in cells of the granulosa layer. It has been described that hypoxic conditions occur during luteinization, thus favoring the formation of L-lactate within the follicle. Despite being a product of anaerobic respiration, L-lactate has been shown to act as a signaling molecule affecting gene expression in neuronal cells. During the present study, we tested the hypothesis that L-lactate may influence differentiation of follicular granulosa cells (GC).MethodsIn a bovine granulosa cell culture model effects of L- and D-lactate, of increased glucose concentrations and of the lactate transport inhibitor UK5099 were analyzed. Steroid hormone production was analyzed by RIA and the abundance of key transcripts was determined by quantitative real-time RT-PCR.ResultsL-lactate decreased the production of estradiol and significantly affected selected genes of the folliculo-luteal transition as well as genes of the lactate metabolism. CYP19A1, FSHR, LHCGR were down-regulated, whereas RGS2, VNN2, PTX3, LDHA and lactate transporters were up-regulated. These effects could be partly or completely reversed by pre-treatment of the cells with UK5099. The non-metabolized enantiomer D-lactate had even more pronounced effects on gene expression, whereas increased glucose concentrations did not affect transcript abundance.ConclusionsIn summary, our data suggest that L-lactate specifically alters physiological and molecular characteristics of GC. These effects critically depend on L-lactate uptake, but are not triggered by increased energy supply. Further, we could show that L-lactate has a positive feedback on the lactate metabolism. Therefore, we hypothesize that L-lactate acts as a signaling molecule in bovine and possibly other monovular species supporting differentiation during the folliculo-luteal transformation.

Protective Mechanisms of Flavonoids in Parkinson's Disease.

Parkinson's disease is a chronic, debilitating neurodegenerative movement disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta region in human midbrain. To date, oxidative stress is the well accepted concept in the etiology and progression of Parkinson's disease. Hence, the therapeutic agent is targeted against suppressing and alleviating the oxidative stress-induced cellular damage. Within the past decades, an explosion of research discoveries has reported on the protective mechanisms of flavonoids, which are plant-based polyphenols, in the treatment of neurodegenerative disease using both in vitro and in vivo models. In this paper, we have reviewed the literature on the neuroprotective mechanisms of flavonoids in protecting the dopaminergic neurons hence reducing the symptoms of this movement disorder. The mechanism reviewed includes effect of flavonoids in activation of endogenous antioxidant enzymes, suppressing the lipid peroxidation, inhibition of inflammatory mediators, flavonoids as a mitochondrial target therapy, and modulation of gene expression in neuronal cells.

Amyloid-clearing proteins and their epigenetic regulation as a therapeutic target in Alzheimer’s disease

Abnormal elevation of amyloid β-peptide (Aβ) levels in the brain is the primary trigger for neuronal cell death specific to Alzheimer’s disease (AD). It is now evident that Aβ levels in the brain are manipulable due to a dynamic equilibrium between its production from the amyloid precursor protein (APP) and removal by amyloid clearance proteins. Clearance can be either enzymic or non-enzymic (binding/transport proteins). Intriguingly several of the main amyloid-degrading enzymes (ADEs) are members of the M13 peptidase family (neprilysin (NEP), NEP2 and the endothelin converting enzymes (ECE-1 and -2)). A distinct metallopeptidase, insulin-degrading enzyme (IDE), also contributes to Aβ degradation in the brain. The ADE family currently embraces more than 20 members, both membrane-bound and soluble, and of differing cellular locations. NEP plays an important role in brain function terminating neuropeptide signals. Its decrease in specific brain areas with age or after hypoxia, ischaemia or stroke contribute significantly to the development of AD pathology. The recently discovered mechanism of epigenetic regulation of NEP (and other genes) by the APP intracellular domain (AICD) and its dependence on the cell type and APP isoform expression suggest possibilities for selective manipulation of NEP gene expression in neuronal cells. We have also observed that another amyloid-clearing protein, namely transthyretin (TTR), is also regulated in the neuronal cell by a mechanism similar to NEP. Dependence of amyloid clearance proteins on histone deacetylases and the ability of HDAC inhibitors to up-regulate their expression in the brain opens new avenues for developing preventive strategies in AD.

Epigenetic gene expression dynamics induced by singing in a critical period of vocal learning

Event Abstract Back to Event Epigenetic gene expression dynamics induced by singing in a critical period of vocal learning Kazuhiro Wada1*, Masahiko Kobayashi1 and Wan-Chun Liu2 1 Hokkaido Universituy, Department of Biological Sciences, Japan 2 The Rockefeller University, United States A complex animal behavior is not only genetically developed but also is affected with influence of environmental factors. Regulation of gene expression in the brain is controlled by species-specific genome information modified under epigenetic influences. Epigenetic molecular mechanisms, such as DNA methylation and histone modification, involve in regulation of gene expression in neuronal cells of the brain. Songbirds learn species-specific song pattern from a tutor during the critical period of vocal learning. For vocal learning and production, songbirds possess specific neural pathways called song system including vocal motor pathway (VMP) and anterior forebrain pathway (AFP). The VMP is critical for vocal production and the AFP is necessary for vocal learning. These pathways consist of a set of brain areas called song nuclei. In these song nuclei, many genes are known to be induced by singing. Several researches showed that some of these singing-induced genes are differentially regulated during the critical period of vocal learning, especially in the forebrain nucleus RA. We found an epigenetic change of one of these immediate-early genes, activity-regulated cytoskeleton-associated (Arc) that interacts with endocytosis-related proteins and facilitates the removal of AMPA receptors from the plasma membran. The DNA methylation in the upstream genome region of Arc was differentially regulated through the critical period in zebra finch, a closed-ended learner. This data suggests that epigenetic mechanisms involve in regulation of induction of singing-induced genes in the critical period. To further clarify the relationship between gene expression and epigenetics, we examined expression patterns of epigenetics-related genes, histone H3 variants (H3.3) and Growth arrest and DNA-damage inducible gene 45 (Gadd45) families. Both H3.3B and Gadd45b genes were up-regulated in song nuclei of both VMP and AFP by singing. The induction of gene expressions was differentially regulated during the critical period of song learning in zebra finch. To clarify whether these regulations depend on age or sensorimotor learning itself, we examined gene expression of these two genes after seasonally singing in the canary, an open-ended learner, which has ability to re-learn new songs even after sexually matured as adult. As the result, gene induction of H3.3B and Gadd45b was higher in RA of the canaries singing variable and plastic songs than the birds singing crystallized songs. These data suggest that histone H3.3B and Gadd45b may control duration of the critical period by epigenetic regulation for several gene expressions that are related with vocal learning. Keywords: critical period, DNA Methylation, epigenetics, Immediate early Gene, sensory motor learning, Song learning, songbird, vocalization Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster Presentation (see alternatives below as well) Topic: Genes and Behavior Citation: Wada K, Kobayashi M and Liu W (2012). Epigenetic gene expression dynamics induced by singing in a critical period of vocal learning. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00099 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Apr 2012; Published Online: 07 Jul 2012. * Correspondence: Dr. Kazuhiro Wada, Hokkaido Universituy, Department of Biological Sciences, Sapporo, Hokkaido, 060-0810, Japan, wada@sci.hokudai.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu Google Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu Google Scholar Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu PubMed Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells

Cystatin C (CysC), an endogenous cysteine protease inhibitor, has been implicated in the apoptosis and differentiation processes of neuronal cells. In this study, we have investigated the pathway involved in the process. A human neuronal hybridoma cell line (A1 cell) was treated with CysC in both undifferentiated and retinoic acid (RA)-induced differentiated conditions, which decreased overall process length in both conditions. Also, CysC increased apoptotic cell number time-dependently, as revealed by TUNEL assay. Western blot analysis demonstrated that in differentiated A1 cells, CysC treatment decreased Bcl-2 and increased active caspase-9 protein level time-dependently. Immunocytochemistry results revealed that, CysC treatment significantly increased active form of Bax expressing cell number, which co-localized with mitochondria. Mitogen activated protein (MAP) kinase inhibition experiments showed that Bax mRNA induction and Bcl-2 mRNA inhibition by CysC treatment were c-Jun N-terminal kinase (JNK)-dependent. After RA-induced differentiation, choline acetyltransferase (ChAT) and neurofilament (NF) mRNA levels were increased in A1 cells. CysC treatment inhibited NF mRNA level in both undifferentiated and RA-differentiated, and increased TH mRNA in differentiated A1 neurons. Analysis of signal transduction pathway demonstrated that TH gene induction was also JNK-dependent. Thus, our results demonstrated the significance of JNK-dependent pathways on CysC-induced apoptosis and TH gene expression in neuronal cells, which might be an important target in the management of CysC dependent neurodegenerative processes.

Apigenin isolated from the seeds of Perilla frutescens britton var crispa (Benth.) inhibits food intake in C57BL/6J mice

Energy balance is monitored by the hypothalamus, which responds to peripheral signals by releasing neuropeptides that regulate energy intake and expenditure. In this study, we constructed pro-opiomelanocortin (POMC) and "cocaine and amphetamine-related transcript" (CART) promoter-driven luciferase plasmids and transformed them permanently into both N29-2 neuronal cells and human SHSY5Y cells. Using reporter gene assays, we identified apigenin from the seeds of Perilla frutescens Britton var crispa (Benth.) using activity-guided fractionation. The 50% promoting concentrations (EC₅₀) of apigenin on POMC and CART were 0.93 μM and 0.67 μM, respectively, in N29-2 cells, without significant cytotoxic effects. Shortterm food intake was decreased in C57BL/6J mice after an intraperitoneal injection of apigenin (10 mg/kg; p < 0.05). Food intake and body weight gain for 30 days were also reduced slightly in mice fed a high-fat diet containing apigenin (0.05%, w/w; p < 0.05). These results indicate that apigenin increased POMC and CART gene expression in neuronal cells and significantly reduced food intake in C57BL/6 mice, which may be related to the anorexigenic neuropeptides POMC and CART.

Knockdown of transmembrane protein 132A by RNA interference facilitates serum starvation-induced cell death in Neuro2a cells

Transmembrane protein 132A (TMEM132A) is a novel GRP78 binding protein that we recently discovered. However, the biological functions of TMEM132A are merely characterized because it does not encode any known structural domains. In this study, we down regulated intrinsic TMEM132A by RNA interference and identified a variety of genes that fluctuated during TMEM132A gene silencing using microarray analysis. TMEM132A-knockdown in Neuro2a cells caused neurite-like projection without any stimuli and enhanced the expression of ATF6 mRNA, an ER stress transducer, and GADD153 mRNA, a stress inducible gene. Under serum-deprived condition, TMEM132A-knockdown cells gradually retarded neurite-like projection and decreased cell viability. Moreover, TMEM132A knockdown markedly induced GADD153 expression due to serum starvation without affecting the level of cleaved caspase-3. Our data suggest that TMEM132A is an important factor of cell survival in regulating certain ER stress-related gene expression in neuronal cells.

Zinc as a translation regulator in neurons: implications for P-body aggregation

Post-transcriptional mechanisms of gene expression in neuronal cells include mRNA transport and local protein synthesis, which play a vital role in the control of polarity, synaptic plasticity and growth cone motility. RNA-binding proteins, which form the transported ribonucleoparticle (RNP), control mRNA stability and local translation. Recently, the existence of processing bodies (P-bodies), in which mRNA decapping and degradation take place, was revealed in neurons. It was suggested that P-bodies serve as a transient storage compartment for mRNAs, which can be released and, upon stimulation, resume translation. In this study, we focused on the localization of the Dcp1a protein, which serves as a P-body marker, in PC12 growth cones and P19 neuronal cells and its association with the tau mRNA-binding protein HuD. We found that stimulation of neurons by zinc, which is stored and released from synaptic vesicles, caused a disruption of polysomes into monosomes, whereas HuD protein distribution in sucrose gradient fractions remained unaffected. In addition, zinc application caused an aggregation of Dcp1a protein in an RNA-dependent manner. These findings suggest a role for zinc in translation regulation via disruption of polysomes, aggregation of P-bodies in neurons and impairment of the RNP-polysome interaction.

Retinoic Acid‐Induced Human Secretin Gene Expression in Neuronal Cells Is Mediated by Cyclin‐Dependent Kinase 1

Previously, we found that secretin transcript levels were induced by all-trans retinoic acid (RA) in a neuroblastoma cell model, SH-SY5Y. In this article, this RA-dependent upregulation process was further investigated. In the cyclin-dependent kinase 1 (Cdk1) inhibitor-treated cells, the RA-dependent induction of secretin gene expression was inhibited. Together with our previous works, we propose here that the RA responsiveness of the secretin promotor is mediated by two different pathways. The first pathway is by changing the expression levels of NFI-C and Sp proteins while the second pathway is by modifying the phosphorylation status of both NFI-C and Sp proteins via Cdk1.

A novel adenoviral vector which mediates hypoxia-inducible gene expression selectively in neurons

Selective gene expression in neurons is still a challenge. We have developed several expression vectors using a combination of neuron restrictive silencer elements (NRSEs), hypoxia responsive elements (HREs) and CMV minimal promoter (CMVmp). These elements were packaged into replication defective adenovirus to target gene expression selectively in neurons in a hypoxia-regulated manner. Neuronal selectivity and responsiveness to hypoxia of these novel constructs were determined empirically in both neural cell lines and primary cerebellar granule neurons (CGNs). The construct p5HRE-3NRSE exhibited not only the highest level of reporter gene expression in neuronal cells but also in an oxygen concentration-dependent manner when compared with all other constructs. As expected, this construct did not elicit reporter gene expression in non-neuronal cells including human HEK293A and HT29 cells, rat NRK cells, mouse 3T6 cells and 3T3 L1 cells. This construct was packaged into a replication defective adenoviral vector (Ad/5HRE-3NRSE) to determine neuron-selective and hypoxia-inducible gene expression in cultured mouse postmitotic primary CGNs and differentiated human NT2 neurons (NT2/Ns). Remarkably, in response to hypoxia, Ad/5HRE-3NRSE showed strong hypoxia-inducible gene expression selectively in neurons (12-fold induction in CGNs and 22-fold in NT2/Ns), but not in glial cells. Taken together, this vector with restricted gene expression to neurons under the regulation of hypoxia will be a useful tool for investigations of mechanisms of neuronal damage caused by ischemic insult.

Novel Nuclear Shuttle Proteins, HDBP1 and HDBP2, Bind to Neuronal Cell-specific cis-Regulatory Element in the Promoter for the Human Huntington's Disease Gene

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the HD gene, and the expression level of either normal or mutant huntingtin is implicated in the pathogenesis of HD. However, a molecular base of the HD gene transcription has not been elucidated as yet. In this study, we identified two proteins, HDBP1 and HDBP2, which bind to the promoter region for the HD gene using a yeast one-hybrid system. Amino acid sequence analysis of the proteins deduced the presence of nuclear localization signal, nuclear export signal, zinc finger, serine/proline-rich region, and highly conserved C-terminal region. In vitro DNA binding assay indicated that the C-terminal conserved regions of the proteins were responsible for binding to the unique promoter DNA sequences of the HD gene. The DNA sequence protected from DNase I digestion was a 7-bp consensus sequence (GCCGGCG), which resides in triplicate at intervals of 13 bp within and proximal to the 20-bp direct repeat sequences of the HD promoter region. The mutation of 7-bp consensus sequence abolishes the HD promoter function in a neuronal cell line (IMR32). In human cultured cells, ectopically expressed green fluorescent protein-fused HDBP1 and HDBP2 localized in the cytoplasm, but both proteins totally shift from cytoplasm to nucleus by the treatment with an inhibitor of the nuclear export, leptomycin B, and mutagenesis of the putative nuclear export signals. Taken together, HDBP1 and HDBP2 are novel transcription factors shuttling between nucleus and cytoplasm and bind to the specific GCCGGCG, which is an essential cis-element for HD gene expression in neuronal cells.

Neuroprotective effects of L-carnitine in induced mitochondrial dysfunction.

The neuroprotective action of l-carnitine (LC) in the rat model of 3-nitropropionic acid (3-NPA)-induced mitochondrial dysfunction was examined. 3-NPA is known to produce decreases in neuronal ATP levels via inhibition of the succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain. SDH is involved in reactions of the Krebs cycle and oxidative phosphorylation, and its inhibition leads to both necrosis and apoptosis. LC enhances mitochondrial metabolism and, together with its acetylated form, acetyl-l-carnitine (ALC), via the LC-ALC-mediated transfer of acetyl groups, plays an important modulatory role in neurotransmitter signal transduction pathways and gene expression in neuronal cells. In the study described here, adult male Sprague-Dawley rats were injected with 3-NPA alone or treated with LC prior to 3-NPA administration. Pretreatment with LC totally prevented the 3-NPA-induced decrease in brain temperature measured using temperature probes implanted intracranially. It appears that the protective effects of LC against 3-NPA-induced neurotoxicity are achieved via compensatory enhancement of several pathways of mitochondrial energy metabolism. The results of this and previous studies conducted by our division in the 3-NPA model of mitochondrial dysfunction demonstrate that 3-NPA may be employed in vivo to evaluate enhancers of mitochondrial function that might exert neuroprotective effects.

Estrogen Receptor and Gene Silencing

A complex picture is emerging of the many ways by which nuclear receptors for the hormone estrogen regulate gene expression. Estrogen receptors alpha and beta (ERα and ERβ) can act through either a ligand-dependent or -independent mechanism, and receptor activity is further influenced by various coactivators and corepressors. The expression of caveolin-1, a protein best known for its role in caveolae development at the cell surface, is regulated in some tissues by estrogen. Because caveolin-1 can itself bind to ERα and potentiate gene transcription, the functional relationship between estrogen, ERs, and caveolin in regulating cellular processes is of interest. Zschocke et al. report that in a neuronal cell line, ectopic expression of ERα inhibited caveolin expression in an estrogen-independent manner. The process involved methylation of specific CpG islands in the caveolin-1 promoter region, a modification that is a hallmark of stable gene silencing. ERβ counteracted the effect of ERα on caveolin expression. Although it remains unclear whether ERα directly suppresses caveolin expression, the inverse relationship of ERα and caveolin expression observed in some cancer cell lines may be attributed to this epigenetic effect. Furthermore, exposure of embryonic mice to estrogen resulted in decreased caveolin-1 expression in certain brain regions. The authors propose that persistent down-regulation of caveolin expression by estrogen would not only decrease the availability of this ER coregulator but might also prevent formation of caveolae, thus impairing the integration and transduction of signals from the cell surface.J. Zschocke, D. Manthey, N. Bayatti, B. van der Burg, S. Goodenough, C. Behl, Estrogen receptor α-mediated silencing of caveolin gene expression in neuronal cells. J. Biol. Chem. 277, 38772-38780 (2002). [Abstract] [Full Text]

Estrogen Receptor and Gene Silencing

A complex picture is emerging of the many ways by which nuclear receptors for the hormone estrogen regulate gene expression. Estrogen receptors alpha and beta (ERα and ERβ) can act through either a ligand-dependent or -independent mechanism, and receptor activity is further influenced by various coactivators and corepressors. The expression of caveolin-1, a protein best known for its role in caveolae development at the cell surface, is regulated in some tissues by estrogen. Because caveolin-1 can itself bind to ERα and potentiate gene transcription, the functional relationship between estrogen, ERs, and caveolin in regulating cellular processes is of interest. Zschocke et al. report that in a neuronal cell line, ectopic expression of ERα inhibited caveolin expression in an estrogen-independent manner. The process involved methylation of specific CpG islands in the caveolin-1 promoter region, a modification that is a hallmark of stable gene silencing. ERβ counteracted the effect of ERα on caveolin expression. Although it remains unclear whether ERα directly suppresses caveolin expression, the inverse relationship of ERα and caveolin expression observed in some cancer cell lines may be attributed to this epigenetic effect. Furthermore, exposure of embryonic mice to estrogen resulted in decreased caveolin-1 expression in certain brain regions. The authors propose that persistent down-regulation of caveolin expression by estrogen would not only decrease the availability of this ER coregulator but might also prevent formation of caveolae, thus impairing the integration and transduction of signals from the cell surface. J. Zschocke, D. Manthey, N. Bayatti, B. van der Burg, S. Goodenough, C. Behl, Estrogen receptor α-mediated silencing of caveolin gene expression in neuronal cells. J. Biol. Chem. 277 , 38772-38780 (2002). [Abstract] [Full Text]