Interactions between neuroactive steroids and reelin haploinsufficiency in Purkinje cell survival

We determined total Purkinje cell (PC) numbers in cerebella of wild-type (+/+) and heterozygous (rl/+) reeler mice of either sex during early postnatal development; in parallel, we quantified levels of neuroactive steroids in the cerebellum with mass spectrometry. We also quantified reelin mRNA and protein expression with RT-PCR and Western blotting. PC numbers are selectively reduced at postnatal day 15 (P15) in rl/+ males in comparison to +/+ males, +/+ females, and rl/+ females. Administration of 17beta-estradiol (17beta-E) into the cisterna magna at P5 increases PC numbers in rl/+ males, but not in the other groups; conversely, estrogen antagonists 4-OH-tamoxifen or ICI 182,780 reduce PC numbers in +/+ and rl/+ females, but have no effect in males. Testosterone (T) levels at P5 are much higher in males than in females, reflecting the perinatal testosterone surge in males. In addition, rl/+ male cerebella at P5 show a peculiar hormonal profile in comparison with the other groups, consisting of increased levels of T and 17beta-E, and decreased levels of dihydrotestosterone. RT-PCR analysis indicated that heterozygosity leads to a 50% reduction of reelin mRNA in the cerebellum in both sexes, as expected, and that 17beta-E upregulates reelin mRNA, particularly in rl/+ males; reelin mRNA upregulation is associated with an increase of all major reelin isoforms. These effects may represent a novel model of how reelin deficiency interacts with variable perinatal levels of neuroactive steroids, leading to gender-dependent differences in genetic vulnerability.

[1]  G. Laviola,et al.  Gene–environment interaction during early development in the heterozygous reeler mouse: Clues for modelling of major neurobehavioral syndromes , 2009, Neuroscience & Biobehavioral Reviews.

[2]  N. Maidment,et al.  Forebrain steroid levels fluctuate rapidly during social interactions , 2008, Nature Neuroscience.

[3]  Patrick R Hof,et al.  Neuropathological findings in autism. , 2004, Brain : a journal of neurology.

[4]  Philippe Absil,et al.  Effects of testosterone on Reelin expression in the brain of male European starlings , 2003, Cell and Tissue Research.

[5]  K. Ukena,et al.  Novel brain function: biosynthesis and actions of neurosteroids in neurons , 2000, Neuroscience Research.

[6]  C Martin-Ruiz,et al.  Nicotinic receptor abnormalities in the cerebellar cortex in autism. , 2002, Brain : a journal of neurology.

[7]  E. Weeber,et al.  The role of reelin in adult synaptic function and the genetic and epigenetic regulation of the reelin gene. , 2008, Biochimica et biophysica acta.

[8]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[9]  Chris I De Zeeuw,et al.  Estradiol Improves Cerebellar Memory Formation by Activating Estrogen Receptor β , 2007, The Journal of Neuroscience.

[10]  S. Prasad,et al.  Age- and sex-related analysis of methylation of 5′-upstream sequences of Fmr-1 gene in mouse brain and modulation by sex steroid hormones , 2008, Biogerontology.

[11]  Ramin Homayouni,et al.  Reelin Is a Ligand for Lipoprotein Receptors , 1999, Neuron.

[12]  K. Ukena,et al.  Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. , 2003, Endocrinology.

[13]  S. Furukawa,et al.  Reduction of nerve growth factor level in the brain of genetically ataxic mice (weaver, reeler) , 1990, FEBS letters.

[14]  T. Kemper,et al.  Neuropathology of infantile autism. , 1998, Journal of neuropathology and experimental neurology.

[15]  Joachim Herz,et al.  Direct Binding of Reelin to VLDL Receptor and ApoE Receptor 2 Induces Tyrosine Phosphorylation of Disabled-1 and Modulates Tau Phosphorylation , 1999, Neuron.

[16]  H. Zoghbi,et al.  Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2 , 1999, Nature Genetics.

[17]  D. Pfaff,et al.  Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha. , 2003, Endocrinology.

[18]  A. Arnold,et al.  Expression of androgen receptor mRNA in zebra finch song system: Developmental regulation by estrogen , 2004, The Journal of comparative neurology.

[19]  L. Garcia-Segura,et al.  Neuroactive steroids: State of the art and new perspectives , 2008, Cellular and Molecular Life Sciences.

[20]  T. Curran,et al.  Detection of the reelin breakpoint in reeler mice. , 1996, Brain research. Molecular brain research.

[21]  L. Garcia-Segura,et al.  Aromatase: a neuroprotective enzyme , 2003, Progress in Neurobiology.

[22]  D. Grayson,et al.  DNA Methyltransferase Inhibitors Coordinately Induce Expression of the Human Reelin and Glutamic Acid Decarboxylase 67 Genes , 2007, Molecular Pharmacology.

[23]  S. Handler,et al.  Diurnal incorporation of 3H‐leucine into liver protein , 1970, FEBS letters.

[24]  K. Tsutsui.  Biosynthesis and organizing action of neurosteroids in the developing Purkinje cell , 2006, The Cerebellum.

[25]  R. Jakab,et al.  Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum. , 2001, The Journal of comparative neurology.

[26]  N. Delhaye-bouchaud,et al.  Gender effect on Purkinje cell loss in the cerebellum of the heterozygous reeler mouse. , 1996, Journal of neurogenetics.

[27]  Tao Li,et al.  Genome-Wide Association Identifies a Common Variant in the Reelin Gene That Increases the Risk of Schizophrenia Only in Women , 2008, PLoS genetics.

[28]  A. Guidotti,et al.  DNA-methyltransferase 1 mRNA is selectively overexpressed in telencephalic GABAergic interneurons of schizophrenia brains , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Guidotti,et al.  The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes , 2009, Molecular Pharmacology.

[30]  S. Mellon.  Neurosteroids: biochemistry, modes of action, and clinical relevance. , 1994, The Journal of clinical endocrinology and metabolism.

[31]  L. Garcia-Segura,et al.  Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair , 1999, Neuroscience.

[32]  A. Bailey,et al.  Autism as a strongly genetic disorder: evidence from a British twin study , 1995, Psychological Medicine.

[33]  L. Eisenman,et al.  Purkinje cell reduction in the reeler mutant mouse: A quantitative immunohistochemical study , 1989, The Journal of comparative neurology.

[34]  P. Strata,et al.  A change in the pattern of activity affects the developmental regression of the purkinje cell polyinnervation by climbing fibers in the rat cerebellum , 2003, Neuroscience.

[35]  A. Zimmerman,et al.  Autism : current theories and evidence , 2008 .

[36]  J. DeFelipe,et al.  Aromatase expression in the human temporal cortex , 2006, Neuroscience.

[37]  F. Labrie,et al.  Androgen receptor antagonists (antiandrogens): structure-activity relationships. , 2000, Current medicinal chemistry.

[38]  L. Doncarlos,et al.  Neuroprotection by estradiol , 2001, Progress in Neurobiology.

[39]  D. A. Edwards,et al.  The neonatal testosterone surge: a comparative study. , 1992, Archives internationales de physiologie, de biochimie et de biophysique.

[40]  D. Geschwind,et al.  Advances in autism genetics: on the threshold of a new neurobiology , 2008, Nature Reviews Genetics.

[41]  N. Harada,et al.  Mode of Action and Functional Significance of Estrogen-Inducing Dendritic Growth, Spinogenesis, and Synaptogenesis in the Developing Purkinje Cell , 2007, The Journal of Neuroscience.

[42]  K. Herrup,et al.  Numerical matching during cerebellar development: quantitative analysis of granule cell death in staggerer mouse chimeras , 1987, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[43]  H. Kölsch,et al.  Effects of estrogen on brain development and neuroprotection—implications for negative symptoms in schizophrenia 1 1 Part of this review was presented at the Seventh International Congress of Biological Psychiatry in Berlin, Germany, 1–7 July 2001. , 2003, Psychoneuroendocrinology.

[44]  T. Curran,et al.  A protein related to extracellular matrix proteins deleted in the mouse mutant reeler , 1995, Nature.

[45]  R. V. Rajala,et al.  Involvement of Insulin/Phosphoinositide 3-Kinase/Akt Signal Pathway in 17β-Estradiol-mediated Neuroprotection* , 2004, Journal of Biological Chemistry.

[46]  L. Garcia-Segura,et al.  Evaluation of neuroactive steroid levels by liquid chromatography–tandem mass spectrometry in central and peripheral nervous system: Effect of diabetes , 2008, Neurochemistry International.

[47]  M. McCarthy,et al.  Cellular mechanisms of estradiol-mediated masculinization of the brain , 2008, The Journal of Steroid Biochemistry and Molecular Biology.

[48]  Flavio Keller,et al.  The neurobiological context of autism , 2003, Molecular Neurobiology.

[49]  A. Goffinet,et al.  Reelin and brain development , 2003, Nature Reviews Neuroscience.

[50]  J. Schmahmann,et al.  The neuropsychiatry of the cerebellum — insights from the clinic , 2008, The Cerebellum.

[51]  E. London,et al.  The Neuropathology of Autism: A Review , 2005, Journal of neuropathology and experimental neurology.

[52]  F. Tempia,et al.  Selection of Reference Genes for Quantitative Real-time RT-PCR Studies in Mouse Brain , 2009, Journal of Molecular Neuroscience.

[53]  A. Sinclair,et al.  Rapid DNA extraction and PCR‐sexing of mouse embryos , 2001, Molecular reproduction and development.

[54]  Erminio Costa,et al.  Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis , 2006, Proceedings of the National Academy of Sciences.

[55]  Paul J. Harrison,et al.  Cellular basis of reduced cortical reelin expression in schizophrenia. , 2006, The American journal of psychiatry.

[56]  A. Sandler.  The 2nd to 4th digit ratio and autism. , 2001 .

[57]  N. Andreasen,et al.  The Role of the Cerebellum in Schizophrenia , 2008, Biological Psychiatry.

[58]  P. Grant,et al.  Sex differences in histone modifications in the neonatal mouse brain , 2009, Epigenetics.

[59]  T. Kemper,et al.  Histoanatomic observations of the brain in early infantile autism , 1985, Neurology.

[60]  L. Swanson,et al.  Distribution of androgen and estrogen receptor mRNA‐containing cells in the rat brain: An in situ hybridization study , 1990, The Journal of comparative neurology.

[61]  H. Gundersen,et al.  Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator , 1991, The Anatomical record.

[62]  D. Pfaff,et al.  Immunolocalization of estrogen receptor β in the mouse brain: Comparison with estrogen receptor α , 2003 .

[63]  Rebecca C. Knickmeyer,et al.  Edinburgh Research Explorer Fetal testosterone and autistic traits , 2014 .

[64]  G. Blatt,et al.  A qualitative and quantitative light microscopic study of the inferior olivary complex of normal, reeler, and weaver mutant mice , 1985, The Journal of comparative neurology.

[65]  K. Goto,et al.  Human Apolipoprotein E Receptor 2 , 1996, The Journal of Biological Chemistry.

[66]  D. Woodruff-Pak,et al.  Stereological estimation of Purkinje neuron number in C57BL/6 mice and its relation to associative learning , 2006, Neuroscience.

[67]  Rebecca C. Knickmeyer,et al.  Androgens and autistic traits: A study of individuals with congenital adrenal hyperplasia , 2006, Hormones and Behavior.

[68]  A. Goffinet,et al.  Reelin receptors in developing laminated brain structures of mouse and human , 2004, The European journal of neuroscience.

[69]  R. Handa,et al.  Expression of estrogen receptor-beta protein and mRNA in the cerebellum of the rat , 2000, Neuroscience Letters.

[70]  Rebecca C. Knickmeyer,et al.  Topical Review: Fetal Testosterone and Sex Differences in Typical Social Development and in Autism , 2006, Journal of child neurology.

[71]  F. Biamonte,et al.  Interaction between Genetic Vulnerability and Neurosteroids in Purkinje cells as a Possible Neurobiological Mechanism in Autism Spectrum Disorders , 2008 .

[72]  R. Jakab,et al.  Estrogen receptor β immunoreactivity in differentiating cells of the developing rat cerebellum , 2001 .

[73]  K. Kranda,et al.  Translocation of cytochrome c during cerebellar degeneration in Lurcher and weaver mutant mice , 2006, Brain Research Bulletin.

[74]  J. Sutcliffe,et al.  DNA methylation represses FMR-1 transcription in fragile X syndrome. , 1992, Human molecular genetics.

[75]  David A. Pearce,et al.  Reelin signaling is impaired in autism , 2005, Biological Psychiatry.

[76]  Daniel R. Weinberger,et al.  Variants in the estrogen receptor alpha gene and its mRNA contribute to risk for schizophrenia , 2008, Human molecular genetics.

[77]  L. Galea,et al.  Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats , 2007, Developmental neurobiology.