Browsing by Author "Zhou, Feng C."

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    Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum
    (Plos, 2016-09) Zhou, Feng C.; Resendiz, Marisol; Lo, Chiao-Ling; Chen, Yuanyuan; Department of Anatomy & Cell Biology, IU School of Medicine
    Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology.
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    Cis‐acting allele specific expression (ASE) differences induced by alcohol and impacted by sex as well as parental genotype of origin
    (Wiley, 2018) Lo, Chiao-Ling; Lumeng, Lawrence; Bell, Richard L.; Liang, Tiebing; Lossie, Amy C.; Muir, Williams M.; Zhou, Feng C.; Anatomy and Cell Biology, School of Medicine
    Background Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high and low alcohol drinking (HAD and LAD) rat lines. Here we report allele specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. Methods The HAD2 and LAD2 rats which have been sequenced were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2; and, for the other 5 litters, the sire was a LAD2. From these 10 litters, two males and two females were picked from each F1 litter (N = 40 total). The F1‐pups were divided, with balancing for sex and direction of cross, into an alcohol (15%) vs a water control group. Alcohol‐drinking started in the middle of adolescence (~PND 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA‐sequencing and ASE analyses. Results Analyses revealed that adolescent ethanol drinking, individual ethanol drinking levels, parentage, and sex‐of‐animal affected ASEs of about 300 genes. The identified genes included those associated with ethanol metabolism (e.g., Aldh2); neuromodulatory function [e.g., Cckbr, Slc6a7, and Slc1a1]; ion channel activity (e.g., Kcnc3); as well as other synaptic and epigenetic function. Conclusion These data indicate that ethanol drinking differentially amplified paternal vs maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to ethanol‐induced changes in cis‐regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene‐by‐environment interactions mediating a genetic predisposition for, and/or the active development of, alcohol use disorders.
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    Constructing a new nigrostriatal pathway in the Parkinsonian model with bridged neural transplantation in substantia nigra
    (Society for Neuroscience, 1996-11-01) Zhou, Feng C.; Chiang, Yung H.; Wang, Yun; Anatomy and Cell Biology, School of Medicine
    The physical repair and restoration of a completely damaged pathway in the brain has not been achieved previously. In a previous study, using excitatory amino acid bridging and fetal neural transplantation, we demonstrated that a bridged mesencephalic transplant in the substantia nigra generated an artificial nerve pathway that reinnervated the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. In the current study, we report that a bridged mesencephalic transplant can anatomically, neurochemically, and functionally reinstate the 6-OHDA-eradicated nigro-striatal pathway. An excitatory amino acid, kainic acid, laid down in a track during the transplant generated a trophic environment that effectively guided the robust growth of transplanted neuronal fibers in a bundle to innervate the distal striatum. Growth occurred at the remarkable speed of approximately 200 microm/d. Two separate and distinct types of dopamine (DA) innervation from the transplant have been achieved for the first time: (1) DA innervation of the striatum, and (2) DA innervation of the pars reticularis of the substantia nigra. In addition, neuronal tracing revealed that reciprocal connections were achieved. The grafted DA neurons in the SNr innervated the host's striatum, whereas the host's striatal neurons, in turn, innervated the graft within 3-8 weeks. Electrochemical volt- ammetry recording revealed the restoration of DA release and clearance in a broad striatal area associated with the DA reinnervation. Furthermore, the amphetamine-induced rotation was attenuated, which indicates that the artificial pathways were motor functional. This study provides additional evidences that our bridged transplantation technique is a potential means for the repair of a completely damaged neuronal pathway.
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    DNA Methylation program in normal and alcohol-induced thinning cortex
    (Elsevier, 2017-05) Öztürk, Nail Can; Resendiz, Marisol; Öztürk, Hakan; Zhou, Feng C.; Anatomy and Cell Biology, School of Medicine
    While cerebral underdevelopment is a hallmark of fetal alcohol spectrum disorders (FASD), the mechanism(s) guiding the broad cortical neurodevelopmental deficits are not clear. DNA methylation is known to regulate early development and tissue specification through gene regulation. Here, we examined DNA methylation in the onset of alcohol-induced cortical thinning in a mouse model of FASD. C57BL/6 (B6) mice were administered a 4% alcohol (v/v) liquid diet from embryonic (E) days 7–16, and their embryos were harvested at E17, along with isocaloric liquid diet and lab chow controls. Cortical neuroanatomy, neural phenotypes, and epigenetic markers of methylation were assessed using immunohistochemistry, Western blot, and methyl-DNA assays. We report that cortical thickness, neuroepithelial proliferation, and neuronal migration and maturity were found to be deterred by alcohol at E17. Simultaneously, DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxcylmethylcytosine (5hmC), which progresses as an intrinsic program guiding normal embryonic cortical development, was severely affected by in utero alcohol exposure. The intricate relationship between cortical thinning and this DNA methylation program disruption is detailed and illustrated. DNA methylation, dynamic across the multiple cortical layers during the late embryonic stage, is highly disrupted by fetal alcohol exposure; this disruption occurs in tandem with characteristic developmental abnormalities, ranging from structural to molecular. Finally, our findings point to a significant question for future exploration: whether epigenetics guides neurodevelopment or whether developmental conditions dictate epigenetic dynamics in the context of alcohol-induced cortical teratogenesis.
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    Effects of Chronic Alcohol and Repeated Deprivations on Dopamine D1 and D2 Receptor Levels in the Extended Amygdala of Inbred Alcohol-Preferring Rats
    (Wiley Blackwell (Blackwell Publishing), 2006-01) Sari, Youssef; Bell, Richard L.; Zhou, Feng C.; Department of Anatomy & Cell Biology, IU School of Medicine
    Background Dopaminergic (DA) activity in the extended amygdala (EA) has been known to play a pivotal role in mediating drug and alcohol addiction. Alterations of DA activity within the EA after chronic exposure to alcohol or substances of abuse are considered a major mechanism for the development of alcoholism and addiction. To date, it is not clear how different patterns of chronic alcohol drinking affect DA receptor levels. Therefore, the current studies investigated the effects of chronic ethanol consumption, with or without deprivations, on D1 and D2 receptor densities within the EA. Methods Inbred alcohol-preferring (iP) rats were divided into 3 groups with the following treatments: (1) water for 14 weeks; (2) continuous alcohol (C-Alc) for 14 weeks [24-hour concurrent access to 15 and 30% (v/v) ethanol]; or (3) repeatedly deprived of alcohol (RD-Alc) (24-hour concurrent access to 15 and 30% ethanol for 6 weeks, followed by 2 cycles of 2 weeks of deprivation of and 2 weeks of reexposure to ethanol access). At the end of 14 weeks, the rats were killed for autoradiographic labeling of D1 and D2 receptors. Results Compared with the water control group, both the C-Alc and the RD-Alc groups displayed increases in D1 receptor binding density in the anterior region of the Acb core, whereas the RD-Alc group displayed additional increases in D1 receptor binding density in anterior regions of the lateral and intercalated nuclei of the amygdala. Additionally, both C-Alc and RD-Alc rats displayed increases in D2 receptor binding density in anterior regions of the Acb shell and core, whereas RDAlc rats displayed additional increases in D2 receptor binding density in the dorsal striatum. Conclusion The results of this study indicate that 14-week extended alcohol drinking with continuous chronic or repeated deprivations increase binding sites of D1 and D2 receptors in specific regions of the EA with greater sensitivity in the anterior regions. The repeated deprivation has greater effect on altering D1 and D2 receptor binding sites in the Acb, dorsal striatum, and subamygdala regions. The current result indicates that the two drinking paradigms may have common as well as differential mechanisms on alteration of dopamine receptor–binding sites in specific regions of the EA.
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    Effects of one- and three-day binge alcohol exposure in neonatal C57BL/6 mice on spatial learning and memory in adolescence and adulthood
    (Elsevier B.V., 2014-03) Wagner, Jennifer L.; Zhou, Feng C.; Goodlett, Charles R.; Department of Anatomy & Cell Biology, IU School of Medicine
    Binge-like alcohol exposure during the early postnatal period in rats and mice causes deficits in spatial learning and memory that persist into adulthood. Wozniak et al. (2004) reported that heavy binge alcohol exposure on postnatal day 7 (PD 7) in C57BL/6 (B6) mice produced profound spatial learning deficits in the Morris water maze when tested in adolescence (P30–39); when tested in adulthood, however, the deficits were greatly attenuated. Using a similar PD 7 binge alcohol exposure paradigm in B6 mice, we tested whether a single-day (PD 7 only) alcohol treatment produced place learning deficits in both adolescence and in adulthood, and further tested whether a more extended (3-day, PD 7–9) alcohol exposure would induce more severe and enduring deficits. B6 mice were given either 2 subcutaneous injections of alcohol (2.5 g/kg each) 2 h apart on PD 7 or on PD 7–9, and compared with controls that received saline vehicle injections and controls that received no injections. The alcohol injections on PD 7 produced average peak blood alcohol concentrations of 472 mg/dL and evoked typical patterns of activated caspase-3-positive neurons in the cortex, hippocampal formation, and striatum 6 h after the last injection. Mice were given standard place training or random location training in the Morris water maze either as adolescents (PD 30–39) or adults (PD 70–79). The adolescents acquired the place learning more slowly than adults, and the alcohol treatments produced only modest place acquisition deficits. In contrast, both the PD7 and the PD 7–9 alcohol treatments resulted in large and significant spatial learning impairments in adults. In contrast to the previous findings of Wozniak et al. (2004), these results indicate that binge alcohol exposure in the 3rd trimester equivalent produces significant and enduring deficits in spatial learning in B6 mice.
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    Epigenetic alteration by prenatal alcohol exposure in developing mouse hippocampus and cortex
    (2014-08) Chen, Yuanyuan; Zhou, Feng C.; Jin, Xiao-Ming; Truitt, William A.; Reiter, Jill L.
    Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.
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    Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics
    (SpringerNature, 2017-02-09) Lo, Chiao-Ling; Choudhury, Samrat Roy; Irudayaraj, Joseph; Zhou, Feng C.; Department of Anatomy & Cell Biology, IU School of Medicine
    Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.
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    Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus
    (Nature Publishing Group, 2016-05-31) Shim, Joon W.; Sandlund, Johanna; Hameed, Mustafa Q.; Blazer-Yost, Bonnie L.; Zhou, Feng C.; Klagsbrun, Michael; Madsen, Joseph R.; Department of Anatomy & Cell Biology, IU School of Medicine
    Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus.
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    Fetal Alcohol Spectrum Disorder (FASD) Associated Neural Defects: Complex Mechanisms and Potential Therapeutic Targets
    (MDPI, 2013-06-19) Muralidharan, Pooja; Sarmah, Swapnalee; Zhou, Feng C.; Marrs, James A.; Biology, School of Science
    Fetal alcohol spectrum disorder (FASD), caused by prenatal alcohol exposure, can result in craniofacial dysmorphism, cognitive impairment, sensory and motor disabilities among other defects. FASD incidences are as high as 2% to 5 % children born in the US, and prevalence is higher in low socioeconomic populations. Despite various mechanisms being proposed to explain the etiology of FASD, the molecular targets of ethanol toxicity during development are unknown. Proposed mechanisms include cell death, cell signaling defects and gene expression changes. More recently, the involvement of several other molecular pathways was explored, including non-coding RNA, epigenetic changes and specific vitamin deficiencies. These various pathways may interact, producing a wide spectrum of consequences. Detailed understanding of these various pathways and their interactions will facilitate the therapeutic target identification, leading to new clinical intervention, which may reduce the incidence and severity of these highly prevalent preventable birth defects. This review discusses manifestations of alcohol exposure on the developing central nervous system, including the neural crest cells and sensory neural placodes, focusing on molecular neurodevelopmental pathways as possible therapeutic targets for prevention or protection.