Ethanol increases fetal human neurosphere size and alters adhesion molecule gene expression.

BACKGROUND Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC). METHODS Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways. RESULTS Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of alpha and beta Laminins 1, beta Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan epsilon. No change in the expression of beta Actin was observed while the expression of beta Integrin 2 was significantly suppressed. CONCLUSIONS ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD.

[1]  L. Wickstrom,et al.  SALMONELLAS IN MEAT. , 1964, Lancet.

[2]  A. Streissguth,et al.  Pattern of Malformation in Offspring of Chronic Alcoholic Mothers , 1995, Alcohol health and research world.

[3]  David W. Smith,et al.  Recognition of the fetal alcohol syndrome in early infancy. , 1973, Lancet.

[4]  K. Jones,et al.  A pattern of craniofacial and limb defects secondary to aberrant tissue bands. , 1974, The Journal of pediatrics.

[5]  A. Streissguth,et al.  Outcome in offspring of chronic alcoholic women. , 1974, Lancet.

[6]  S. Hockfield,et al.  Identification of major cell classes in the developing mammalian nervous system , 1985, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[7]  J. Perper,et al.  Tolerance at high blood alcohol concentrations: a study of 110 cases and review of the literature. , 1986, Journal of forensic sciences.

[8]  M. Miller,et al.  Effect of prenatal exposure to ethanol on the development of cerebral cortex: I. Neuronal generation. , 1988, Alcoholism, clinical and experimental research.

[9]  S. Al-Rabiai,et al.  Effect of prenatal exposure to ethanol on the ultrastructure of layer V of mature rat somatosensory cortex , 1989, Journal of neurocytology.

[10]  R. McKay,et al.  CNS stem cells express a new class of intermediate filament protein , 1990, Cell.

[11]  Michael W. Miller,et al.  Numbers of neurons and glia in mature rat somatosensory cortex: Effects of prenatal exposure to ethanol , 1990, The Journal of comparative neurology.

[12]  Y. Mizoi,et al.  Degrees of alcohol intoxication in 117 hospitalized cases. , 1991, Journal of studies on alcohol.

[13]  C. Alling,et al.  A method for maintaining constant ethanol concentrations in cell culture media. , 1992, Alcohol and alcoholism.

[14]  E P Riley,et al.  Verbal learning and memory in children with fetal alcohol syndrome. , 1996, Alcoholism, clinical and experimental research.

[15]  J. V. van Dongen,et al.  Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. , 1996, Leukemia.

[16]  A. Falek,et al.  A comparison of children affected by prenatal alcohol exposure and attention deficit, hyperactivity disorder. , 1997, Alcoholism, clinical and experimental research.

[17]  P. Hoffman,et al.  Ethanol Promotes Apoptosis in Cerebellar Granule Cells by Inhibiting the Trophic Effect of NMDA , 1997, Journal of neurochemistry.

[18]  E P Riley,et al.  A review of the neurobehavioral deficits in children with fetal alcohol syndrome or prenatal exposure to alcohol. , 1998, Alcoholism, clinical and experimental research.

[19]  E. Riley,et al.  Comparison of social abilities of children with fetal alcohol syndrome to those of children with similar IQ scores and normal controls. , 1998, Alcoholism, clinical and experimental research.

[20]  S. Smith,et al.  Ethanol-induced neural crest apoptosis is coincident with their endogenous death, but is mechanistically distinct. , 1998, Alcoholism, clinical and experimental research.

[21]  L. Ghoda,et al.  Brain-Derived Neurotrophic Factor Mediates the Anti-Apoptotic Effect of NMDA in Cerebellar Granule Neurons: Signal Transduction Cascades and Site of Ethanol Action , 1999, The Journal of Neuroscience.

[22]  G. Cheng,et al.  Ethanol Inhibits L1-mediated Neurite Outgrowth in Postnatal Rat Cerebellar Granule Cells* , 1999, The Journal of Biological Chemistry.

[23]  J. West,et al.  Ethanol induces Fas/Apo [apoptosis]-1 mRNA and cell suicide in the developing cerebral cortex. , 2000, Alcoholism, clinical and experimental research.

[24]  I. Weissman,et al.  Direct isolation of human central nervous system stem cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. Renau‐Piqueras,et al.  Alcohol Exposure Alters the Expression Pattern of Neural Cell Adhesion Molecules During Brain Development , 2000, Journal of neurochemistry.

[26]  P. May,et al.  Emotion-related learning in individuals prenatally exposed to alcohol: an investigation of the relation between set shifting, extinction of responses, and behavior , 2001, Neuropsychologia.

[27]  D. Delis,et al.  Verbal and nonverbal fluency in children with heavy prenatal alcohol exposure. , 2001, Journal of studies on alcohol.

[28]  F. Diaz,et al.  Transplantation of human fetal brain cells into ischemic lesions of adult gerbil hippocampus. , 2001, Journal of neurosurgery.

[29]  S. Goldman,et al.  Identification and characterization of neuronal precursors and their progeny from human fetal tissue , 2001, Journal of neuroscience research.

[30]  J. Hobbins,et al.  The impact of prenatal alcohol exposure on frontal cortex development in utero. , 2001, American journal of obstetrics and gynecology.

[31]  Michael W. Miller,et al.  Effects of Ethanol and Transforming Growth Factor β (TGFβ) on Neuronal Proliferation and nCAM Expression , 2002 .

[32]  C. Bearer,et al.  Ethanol and membrane protein trafficking: diverse mechanisms of ethanol action. , 2002, Alcoholism, clinical and experimental research.

[33]  Kazuhiko Sawada,et al.  Adverse effects of maternal ethanol consumption on development of dorsal hippocampus in rat offspring , 2002, Acta Neuropathologica.

[34]  I. Weissman,et al.  Engraftment of sorted/expanded human central nervous system stem cells from fetal brain , 2002, Journal of neuroscience research.

[35]  N. Spear,et al.  Neonatal activation of alcohol-related prenatal memories: impact on the first suckling response. , 2002, Alcoholism, clinical and experimental research.

[36]  W. Buczko,et al.  Ethanol-induced neurotoxicity is counterbalanced by increased cell proliferation in mouse dentate gyrus , 2002, Neuroscience Letters.

[37]  A. Kaasik,et al.  Neurodegeneration and production of the new cells in the dentate gyrus of juvenile rat hippocampus after a single administration of ethanol , 2003, Brain Research.

[38]  K. Barami,et al.  Differential responses of human neural and hematopoietic stem cells to ethanol exposure. , 2003, Journal of hematotherapy & stem cell research.

[39]  S. Maffi,et al.  Ethanol‐induced oxidative stress precedes mitochondrially mediated apoptotic death of cultured fetal cortical neurons , 2003, Journal of neuroscience research.

[40]  K. Barami,et al.  Human neural stem cells are more sensitive than astrocytes to ethanol exposure. , 2003, Alcoholism, clinical and experimental research.

[41]  A. Villa,et al.  Expression of Laminin Receptors in Schwann Cell Differentiation: Evidence for Distinct Roles , 2003, The Journal of Neuroscience.

[42]  Dominique Muller,et al.  Integrins are involved in synaptogenesis, cell spreading, and adhesion in the postnatal brain. , 2003, Brain research. Developmental brain research.

[43]  D. Holtzman,et al.  Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain , 2003, Cell Death and Differentiation.

[44]  Lia S. Campos,et al.  β1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance , 2004, Development.

[45]  Michael W. Miller,et al.  Transforming growth factor beta1 modulates cell migration in rat cortex: effects of ethanol. , 2004, Cerebral cortex.

[46]  E. Sowell,et al.  Teratogenic effects of alcohol: A decade of brain imaging , 2004, American journal of medical genetics. Part C, Seminars in medical genetics.

[47]  M. Tateno,et al.  Ethanol inhibition of neural stem cell differentiation is reduced by neurotrophic factors. , 2004, Alcoholism, clinical and experimental research.

[48]  J. Takahashi,et al.  Neural precursor cells derived from human embryonic brain retain regional specificity , 2004, Journal of neuroscience research.

[49]  Zbigniew Darzynkiewicz,et al.  Analysis of cell cycle by flow cytometry. , 2004, Methods in molecular biology.

[50]  Rajesh C Miranda,et al.  Ethanol induces cell-cycle activity and reduces stem cell diversity to alter both regenerative capacity and differentiation potential of cerebral cortical neuroepithelial precursors , 2005, BMC Neuroscience.

[51]  M. Götz,et al.  Radial Glial Cells Defined and MajorIntermediates between EmbryonicStem Cells and CNS Neurons , 2005, Neuron.

[52]  C. ffrench-Constant,et al.  Human diseases reveal novel roles for neural laminins , 2005, Trends in Neurosciences.

[53]  M. Vemuri,et al.  Alcohol impairs astrogliogenesis by stem cells in rodent neurospheres , 2005, Neurochemistry International.

[54]  M. Tateno,et al.  The effect of ethanol on cell fate determination of neural stem cells. , 2005, Alcoholism, clinical and experimental research.

[55]  N. Spear,et al.  Fetal or infantile exposure to ethanol promotes ethanol ingestion in adolescence and adulthood: a theoretical review. , 2005, Alcoholism, clinical and experimental research.

[56]  L. Olson,et al.  Moderate ethanol consumption increases hippocampal cell proliferation and neurogenesis in the adult mouse. , 2005, The international journal of neuropsychopharmacology.

[57]  K. Roth,et al.  Molecular Regulation of Acute Ethanol-Induced Neuron Apoptosis , 2005, Journal of neuropathology and experimental neurology.

[58]  M. Frotscher,et al.  Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology , 2006, Nature Methods.

[59]  E. Snyder,et al.  Adhesive Interactions Between Human Neural Stem Cells and Inflamed Human Vascular Endothelium Are Mediated by Integrins , 2006, Stem cells.

[60]  E. Snyder,et al.  Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed , 2006, Experimental Neurology.

[61]  Gail M Williams,et al.  In utero alcohol exposure and prediction of alcohol disorders in early adulthood: a birth cohort study. , 2006, Archives of general psychiatry.

[62]  Michael W. Miller,et al.  Transforming growth factor β1 and ethanol affect transcription and translation of genes and proteins for cell adhesion molecules in B104 neuroblastoma cells , 2006, Journal of neurochemistry.

[63]  M. S. Kallos,et al.  Large-scale expansion of mammalian neural stem cells: a review , 2003, Medical and Biological Engineering and Computing.

[64]  Yuqing Li,et al.  Myoclonus, Motor Deficits, Alterations in Emotional Responses and Monoamine Metabolism in ε-Sarcoglycan Deficient Mice , 2006 .

[65]  R. Miranda,et al.  Embryonic cerebral cortical progenitors are resistant to apoptosis, but increase expression of suicide receptor DISC-complex genes and suppress autophagy following ethanol exposure. , 2007, Alcoholism, clinical and experimental research.

[66]  C. Esapa,et al.  SGCE missense mutations that cause myoclonus-dystonia syndrome impair epsilon-sarcoglycan trafficking to the plasma membrane: modulation by ubiquitination and torsinA. , 2007, Human molecular genetics.

[67]  S. Bressman,et al.  Myoclonus-dystonia, obsessive-compulsive disorder, and alcohol dependence in SGCE mutation carriers , 2007, Neurology.

[68]  H. Komuro,et al.  How does alcohol impair neuronal migration? , 2007, Journal of neuroscience research.