Elsevier

Developmental Brain Research

Volume 139, Issue 2, 15 December 2002, Pages 237-245
Developmental Brain Research

Research report
Effects of prenatal exposure to ethanol on the cyclin-dependent kinase system in the developing rat cerebellum

https://doi.org/10.1016/S0165-3806(02)00573-4Get rights and content

Abstract

Prenatal exposure to ethanol inhibits neurogenesis in the developing cerebellum. Cyclin-dependent kinases (CDKs) are a family of protein kinases that play multiple roles in the regulation of cell proliferation, differentiation and survival. The activity of CDKs is positively regulated by CDK activators, cyclins, and negatively regulated by CDK inhibitors (CDKIs). We hypothesize that impaired cerebellar development induced by gestational ethanol exposure is mediated by disruption of the CDK system. Pregnant rats were fed ad libitum with an ethanol-containing liquid diet (Et) or pair-fed an isocaloric control diet (Ct). Cerebella were collected from pups (postnatal day (P) 0 through P21) and examined for CDK, cyclin, or CDKI expression using a quantitative immunoblotting procedure. In Ct-treated rats, the expression of CDK2 and its activator, cyclin A, paralleled the pattern of granule cell proliferation. Prenatal ethanol exposure produced a significant down-regulation of CDK2/cyclin A expression. Although the amounts of CDK4/CDK6 and their activator, cyclin D2, did not oscillate during postnatal development, their expression in Et-treated pups was significantly (P<0.05) higher than in controls. The expression of a CDK inhibitor, p27Kip, was inversely correlated to proliferation of cerebellar granule progenitors. Prenatal ethanol exposure caused the down-regulation of p27Kip between P0 and P21. Thus, prenatal exposure to ethanol disturbed the expression of cell cycle machineries in the postnatal cerebellum. This may account for the teratogenic effects of ethanol on the developing cerebellum.

Introduction

Prenatal ethanol exposure is deleterious to the developing central nervous system (CNS) [19], [35], [49]. Gestational exposure to ethanol can alter the number of neurons and glia in the mature brain. Such damage varies among CNS structures. Cerebellum is one of the CNS regions that is especially vulnerable to ethanol [21], [40], [41], [42]. A feature of early ethanol exposure is the depletion of cerebellar neurons. Animal studies have consistently shown that ethanol exposure reduces the numbers of both Purkinje cells and granule cells in cerebellum [7], [8], [40], [42], [63]. The number of neurons within cerebellum depends upon the sum of two opposing processes: the additive effect of cell acquisition (neurogenesis) and the subtractive effect of neuronal death. Ethanol affects both processes [6], [8], [10], [37], [55], [75], [78]. The cellular and molecular mechanisms underlying perturbation of these processes are yet to be determined.

The cerebellum is emerging as being more crucial for cognition than previously thought. Damage to the cerebellum results in motor function impairments [30], [33], [38], [60] and cognitive disorders [45], [69] seen in fetal alcohol syndrome. Elucidating cellular and molecular mechanisms of ethanol action would greatly enhance our understanding of how ethanol produces its various detrimental effects, and may provide insight necessary to develop therapies to improve the outcome of maternal ethanol abuse.

Cyclin-dependent kinases (CDKs) are a family of protein kinases that play multiple roles in the regulation of cell proliferation, differentiation and survival [13], [27], [28], [34], [66]. The activity of CDKs is positively regulated by a group of proteins (CDK activators) called cyclins, and negatively regulated by CDK inhibitors (CKDIs) [2]. CDKs have been shown to play a critical role in neurogenesis as well as neuronal differentiation [11], [20], [22], [34], [66], [77]. Ethanol inhibits cell proliferation and induces apoptosis in cultured cerebellar granule progenitors and this damage is accompanied by a severe disruption of CDK system [32]. The present study is designed to determine whether prenatal ethanol exposure affects the developmental expression of CDKs and their activators/inhibitors in the postnatal cerebellum.

Section snippets

Animals and ethanol exposure

Pregnant Long–Evans rats were obtained from Harlan–Sprague–Dawley (Indianapolis, IN, USA) on gestational day (G) 4. The animals were maintained in a temperature/humidity controlled facility with a 12-h light/dark cycle. Beginning on G6, rats were arbitrarily assigned to one of two treatment groups, and fed one of two liquid diets, an ethanol diet (Et), or a control diet (Ct). Both liquid diets were protein-enriched diets that were nutritionally balanced and met the requirements of pregnant rats

Expression of cyclin-dependent kinases

Developmental expression of three cyclin-dependent kinases, CDK2, CDK4 and CDK6 was examined in the cerebellum. In control pups, the expression of CDK2 increased between P0 and P6, and it decreased thereafter (Fig. 1). By P21, the expression of CDK2 was barely detectable. In contrast, the amounts of CDK4 declined from P0 to P3, and then expression stabilized during the rest of developmental period (Fig. 2). CDK6 expression remained quite constant, that is, there was little change of expression

Cyclin-dependent kinase system and the developing cerebellum

The development of rodent cerebellum occurs postnatally [3]. The production of major cerebellar neurons in rats, granule cells, begins at birth and ends after approximately 3 weeks. The cerebellar granule cells, the most abundant cells in the CNS (estimated at 1011), are generated in the proliferative external germinal layer (EGL) and migrate to their final destination in the internal granule layers (IGL) where they differentiate into neurons. From postnatal day 0 (P0) to P6, the cells in the

Acknowledgements

This research was supported by grants from the National Institutes of Health (AA07658, AA12968 and CA 90385).

References (78)

  • J. Luo et al.

    Growth factor-mediated neural proliferation: target of ethanol toxicity

    Brain Res. Rev.

    (1998)
  • S.E. Maier et al.

    Prenatal binge-like alcohol exposure in the rat results in region-specific deficits in brain growth

    Neurotoxicol. Teratol.

    (1999)
  • S.E. Maier et al.

    Regional differences in cell loss associated with binge-like alcohol exposure during the first two trimesters equivalent in the rat

    Alcohol

    (2001)
  • I.L. Miale et al.

    An autoradiographic analysis of histogenesis in the mouse cerebellum

    Exp. Neurol.

    (1961)
  • A. Migheli et al.

    A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum

    Am. J. Pathol.

    (1999)
  • M.W. Miller

    Circadian rhythm of cell proliferation in the telencephalic ventricular zone: effect of in utero exposure to ethanol

    Brain Res.

    (1992)
  • M.W. Miller

    Effect of early exposure to ethanol on the protein and DNA contents of specific brain regions in the rat

    Brain Res.

    (1996)
  • E.J. Nathaniel et al.

    Prenatal ethanol exposure and cerebellar development in rats

    Exp. Neurol.

    (1986)
  • M.J. O’Hare et al.

    Induction and modulation of cerebellar granule neuron death by E2F-1

    J. Biol. Chem.

    (2000)
  • G. Perez-Juste et al.

    The cyclin-dependent kinase inhibitor p27(Kip1) is involved in thyroid hormone-mediated neuronal differentiation

    J. Biol. Chem.

    (1999)
  • E. Perez-Torrero et al.

    Effects of acute prenatal ethanol exposure on Bergmann glia cells early postnatal development

    Brain Res.

    (1997)
  • Y. Rajgopal et al.

    Ethanol induced changes in cyclin-dependent kinase-5 activity and its activators, P35, P67 (Munc-18) in rat brain

    Neurosci. Lett.

    (2001)
  • M.J. Ravitz et al.

    Cyclin-dependent kinase regulation during G1 phase and cell cycle regulation by TGF-beta

    Adv. Cancer Res.

    (1997)
  • J.D. Schmahmann et al.

    The cerebellum and cognition: the cerebrocerebellar system

    Int. Rev. Neurobiol.

    (1997)
  • C.J. Sherr

    G1 phase progression: cycling on cue

    Cell

    (1994)
  • L. Shi et al.

    Chronic ethanol increases lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced tumor necrosis factor alpha production

    J. Biol. Chem.

    (2002)
  • G.K. Acquaah-Mensah et al.

    In utero ethanol suppresses cerebellar activator protein-1 and nuclear factor-kappa B transcriptional activation in a rat fetal alcohol syndrome model

    J. Pharmacol. Exp. Ther.

    (2002)
  • J. Altman et al.

    Development of the Cerebellar System

    (1996)
  • A.M. Beckmann et al.

    AP-1 and Egr DNA-binding activities are increased in rat brain during ethanol withdrawal

    J. Neurochem.

    (1997)
  • S.V. Bhave et al.

    Ethanol promotes apoptosis in cerebellar granule cells by inhibiting the trophic effect of NMDA

    J. Neurochem.

    (1997)
  • D.J. Bonthius et al.

    Permanent neuronal deficits in rats exposed to alcohol during the brain growth spurt

    Teratology

    (1991)
  • S. Borges et al.

    The effect of ethanol on the cellular composition of the cerebellum

    Neuropathol. Appl. Neurobiol.

    (1983)
  • E. Climent et al.

    Ethanol exposure enhances cell death in the developing cerebral cortex: role of brain-derived neurotrophic factor and its signaling pathways

    J. Neurosci. Res.

    (2002)
  • M.J. Courtney et al.

    The mechanism of Ara-C-induced apoptosis of differentiating cerebellar granule neurons

    Eur. J. Neurosci.

    (1999)
  • M.H. Farah et al.

    Generation of neurons by transient expression of neural bHLH proteins in mammalian cells

    Development

    (2000)
  • S.F. Giardina et al.

    Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

    J. Neurochem.

    (1998)
  • C.R. Goodlett et al.

    Mechanisms of alcohol-induced damage to the developing nervous system

    Alcohol Res. Health

    (2001)
  • P. Grant et al.

    Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism

    Eur. J. Biochem.

    (2001)
  • C. Guerri

    Neuroanatomical and neurophysiological mechanisms involved in central nervous system dysfunctions induced by prenatal alcohol exposure

    Alcohol Clin. Exp. Res.

    (1998)
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