- Email: [email protected]
Bcl-2 overexpression protects the neonatal cerebellum from ethanol neurotoxicity
Brain Research 817 Ž1999. 13–18
Research report
Bcl-2 overexpression protects the neonatal cerebellum from ethanol neurotoxicity Marieta Barrow Heaton a
a, )
, D. Blaine Moore a , Michael Paiva a , Theresa Gibbs b , Ora Bernard
b
Department of Neuroscience, UniÕersity of Florida Brain Institute, Center for Alcohol Research, UniÕersity of Florida College of Medicine, GainesÕille, FL 32610-0244, USA b The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia Accepted 27 October 1998
Abstract The developing nervous system is extremely sensitive to ethanol, and exposure often produces a condition known as the fetal alcohol syndrome. Although mechanisms underlying developmental ethanol toxicity have long been sought, they remain poorly understood. In this study, we examined the ability of the cell death repressor gene bcl-2 to protect against ethanol neurotoxicity. Transgenic mice overexpressing bcl-2 in neurons were exposed to ethanol vapor on postnatal days 4 and 5, which is the peak period of vulnerability of cerebellar Purkinje cells to ethanol. While exposure of wild-type animals to ethanol resulted in significant loss of Purkinje cells by P5, similar exposure of homozygous and heterozygous transgenics had no effect on the number of these neurons. This study suggests that bcl-2 can protect neurons from ethanol neurotoxicity and that modulation of cell death effector or repressor gene products may play a significant role in developmental ethanol neurotoxicity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bcl-2; Apoptosis; Fetal alcohol syndrome; Neuroprotection; Ethanol
1. Introduction Developmental exposure to ethanol leads to a wide range of structural and functional anomalies, often resulting in the condition known as fetal alcohol syndrome ŽFAS, Ref. w21x.. The most devastating deficits associated with FAS are those involving the central nervous system ŽCNS., producing an array of dysfunctions, including hyperactivity, attention deficits, difficulties with learning and memory, impaired motor abilities, and lowered IQ w39x. Significant and permanent alterations have been well documented in regions such as the cerebral cortex w28x, hippocampus w4x and cerebellum w7x. This sensitivity appears to be most acute during the so-called brain growth spurt ŽBGS., a dynamic period of differentiation and synaptoge-
)
Corresponding author. Department of Neuroscience, University of Florida College of Medicine, Health Science Center, PO Box 100244, Gainesville, FL 32610-0244, USA. Fax: q1-352-392-8347; E-mail: [email protected]
nesis, which occurs during the third trimester in humans and in the early postnatal period Žpostnatal days 4–10. in the rodent model of FAS, in which many of the behavioral and neuroanatomical abnormalities seen in FAS children have been duplicated w11x. The developing cerebellum is exquisitely vulnerable to ethanol during a very brief period. In the rat model, exposure to even a single bolus of ethanol on postnatal day 4 ŽP4. results in loss of Purkinje cells comparable to that seen with exposure throughout the BGS w14x. Administration of ethanol solely during the prenatal period or slightly later in the postnatal period ŽP7-8. does not induce neuronal loss in this region w17,23x. In human FAS children, cerebellar abnormalities have also been described, and a number of behavioral deficits such as impairment in the development of motor competence, problems in balance, gait coordination and fine motor control reflect this disruption w34x. These deficits correlate with cell loss and reduced size of the anterior vermal cerebellar region, particularly lobules I to V, which is also the region shown to be particularly sensitive to ethanol in the animal studies w17x.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 7 3 - 1
14
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
The mechanisms whereby ethanol exerts its neurotoxicity in the developing nervous system have long been sought and a number of contributing factors have been proposed. These include hypoxiarischemia, hypoglycemia w43x, alterations in calcium homeostasis w42x, production of reactive oxygen species or depletion of protective antioxidants w35x, and deceases in neurotrophic support or responsiveness w10,18,19x. Since ethanol neurotoxicity has been demonstrated both in vivo and in vitro to be an apoptotic process w6,9x, an intriguing possibility is that the gene products which regulate cell death may be involved in ethanol-induced neuronal loss. The bcl-2 gene family, the mammalian homologs of the C. elegans ced-9 gene, has been well characterized in recent years. This family consists of cell death repressors Že.g., bcl-2, bcl-xl . and effectors Že.g., bax, bad, bcl-xs .. bcl-2 expression has been shown to protect against a number of adverse conditions or substances, including growth factor or serum withdrawal, high levels of intracellular calcium, glutamate toxicity, ischemia, hypoglycemia, oxidative stress, glutamate toxicity, 6-hydroxydopamine and MPTP neurotoxicity, and axotomy w12,31,47x. In addition, high levels of expression of the Bcl-2a protein have been found to confer increased resistance to several stressors in an Epstein Barr virus-infected human B lymphoblastoid cell line, including ethanol w40x. The present study represents the first investigation of the potential of Bcl-2 overexpression to afford similar protection against ethanol neurotoxicity in the developing CNS. For this study we chose to view neonatal cerebellar Purkinje cells following ethanol exposure during the extremely vulnerable early postnatal period, in wild-type animals, and in transgenic animals overexpressing Bcl-2 in neurons.
2. Materials and methods 2.1. Transgenic mice The NSE–bcl-2 transgenic mice used in this study were generated by microinjection of the human bcl-2 cDNA fused to the neuron-specific enolase ŽNSE. promoter into the pronuclei of fertilized C57BLr6J ova, as described earlier w12x. Integration of the transgene was detected by dot hybridization of DNA obtained from tail biopsies to the 750-bp EcoRIrBamHI simian virus 40 fragment from pNSE-CAT as a probe. The homozygous transgene offspring, and to a lesser extent the heterozygous offspring, express high levels of Bcl-2 protein in neurons of the CNS and PNS. Expression of the transgene was confirmed by Western blot analysis of whole neonatal brain lysates. For this confirmation, brains were homogenized in lysis buffer containing 1% Nonidet-P-40 and protease inhibitors. Samples were subjected to SDSrPAGE followed by electroblotting onto Hybond C-extra nitrocellulose membranes.
Filters were probed with a monoclonal anti-human Bcl-2 antibody, Bcl-2-100, followed by HRP-coupled rabbit anti-mouse immunoglobulin antibody and visualized by enhanced chemiluminescence. We have found that expression of the transgene is detected in embryos as early as 10.5 days post-coitus w5x. The transgene is expressed exclusively in neurons w12x, and in immunohistochemical studies, it was found to be highly expressed in cerebellar Purkinje cells, but not in granule cells w5x. The mice used in the present study were descended from the same transgenic animals which were characterized in our previous studies w5,12x. The desired genotypes were obtained by mating homozygotes= homozygotes to yield offspring homozygous for the bcl-2 allele, and homozygotes = C57BLr6J animals to yield offspring heterozygotes for the bcl-2 allele. 2.2. Ethanol Õapor inhalation exposure paradigm Neonatal mice were exposed to ethanol via the vapor inhalation procedure, using the modification of Pal and Alkana w32x. For this procedure, pups in their home cage along with the nursing dam were placed in an inhalation apparatus, thus eliminating the stress of maternal separation. This procedure is preferable to the usual inhalation protocol, and the pups resume feeding more quickly than when they are separated from the dam during exposure, which minimizes differences in weight gain between groups. Wild-type, heterozygous and homozygous pups were assigned to one of two groups: Ethanol vapor-exposed or control vapor-exposed. The inhalation procedure was carried out on P4 and P5, the peak period of Purkinje cell vulnerability to ethanol w14x. The ethanol or control exposure period was 2 h per day. The home cage containing the dam and the pups was placed in a large Plexiglas inhalation chamber which was fitted with intake and exhaust hoses. Prior to vapor exposure, the ethanol litters were culled to 5 pups on P4, while control litters were culled to 7 pups. The decreased number of pups in the Ethanol group reduced the possibility of nutritional differences arising between ethanol-exposed and control pups w36x. Air flow into the intake hose was provided by an air pump set to deliver approximately 0.8–1.0 lrmin, and it flowed into a 1 l Erlenmeyer vacuum flask containing 520 ml 95% ethanol. As the air was forced into the flask it passed through a 3.5-cm air stone submerged in the ethanol. The ethanol-laden vapor was then carried to the chamber. The exhaust hose led ethanol vapor from the chamber to a fume hood. Two hours following termination of ethanol exposure, blood ethanol concentrations were determined by the Sigma microenzymatic assay. This procedure was carried out in compliance with established guidelines of the Walter and Eliza Hall Institute, and was approved by the Royal Melbourne Hospital Campus Animal Ethics Committee ŽHCAEC Project 008r971..
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
2.3. Histological procedures On the day of sacrifice ŽP5, 2 h after the final ethanol exposure., the animals were sacrificed by methoxyflurane overdose, and the cerebellum was dissected out and placed in Bouin’s fixative for 24 h. Following fixation, specimens were dehydrated, cleared, and embedded in paraffin. Serial sagittal 6 mm sections were then cut through the vermis, mounted onto slides and stained with hematoxylin and eosin. 2.4. Purkinje cell counts In order to assess ethanol effects on developing cerebellum, Purkinje cells were counted in Nissl-stained sections from the midline vermis. Only cells in lobule I were counted, since this early maturing region has been shown to be maximally sensitive to ethanol and experiences the greatest Purkinje cell loss w14x. In addition to cell counts, the length of the lobules was measured in order to correct for any possible size differential between groups. The cell counts were then converted to cellsrmm. Prior to Purkinje cell counting, slides were coded and randomized with an identifying number. Cell counts were performed in the manner of Hamre and West w17x on three sections separated by 24 mm to insure that the same cells were not counted twice. Purkinje cells were identified by size and location, and were counted if they contained a well-delineated nuclear membrane, distinct nucleolus, and darkly stained cytoplasm. The quantification was carried out using a Zeiss Axioplan photomicroscope, with a drawing tube attachment, at a magnification of 400 = . As has recently been discussed, there are various advantages and disadvantages to all of the currently practiced cell enumeration protocols w15x. For this study, we used a basic manual sampling technique, which is appropriate since we intended to compare mean cells mmy1 sectiony1 within a circumscribed cerebellar region between groups, but did not attempt to estimate population totals w16x. The same procedure using widely spaced sections was employed for all conditions and all counts were done ‘blind’. Also, vermal lobule area was measured to determine whether ethanol treatment affected volume or area, and corrections for volumetric changes were made. It should be noted that Purkinje cell loss following developmental ethanol exposure has been determined in previous studies using both manual counting w17x and stereological procedures w30x with comparable results. Counts were performed on 4–9 animals from each group Ži.e., wild-type, homozygous and heterozygous in ethanol and control groups.. The average number of Purkinje cellsrmm was determined for each section from each animal. Statistical comparisons were performed via the analysis of variance ŽANOVA. using the StatView Program on a Macintosh computer. When appropriate, individual differences between groups were as-
15
sessed using the Fisher’s protected least significant difference ŽPLSD. post-hoc test.
3. Results The inhalation paradigm of ethanol exposure as described above resulted in peak blood ethanol concentrations averaging 232 mgrdl in the pups, with relatively low blood ethanol readings in the dam Žaveraging 25 mgrdl.. The pups were weighed on each exposure day, just prior to being placed in the inhalation chamber. The ANOVA indicated that the ethanol-exposed pups’ weight gain was comparable to their control counterparts Ž F s 0.965, df s 5, 27, p s 0.4565.. When the ANOVA was applied to the Purkinje cell counts, a significant effect of treatment was revealed Ž F s 7.96, df s 5, 31, p - 0.0001.. The PLSD post-hoc test indicated that in the wild-type mice, ethanol exposure during this period produced a significant loss of Purkinje cells Žy24%., compared to control vapor-exposed animals Ž p s 0.0007.. The wild-type ethanol-exposed animals also differed significantly from the heterozygous ethanol-exposed Ž p s 0.0021., heterozygous control Ž p s 0.0031., homozygous ethanol-exposed Ž p - 0.0001., and homozygous control animals Ž p - 0.0001.. Neither the heterozygous nor the homozygous ethanol-exposed neonates differed from their genotypic controls. The only other difference detected was a small but significant increase in
Fig. 1. Purkinje cell counts, expressed as cellsrmm, in lobule I of P5 mouse cerebellum following 2-h exposure to ethanol vapors or control conditions on P4–5, in wild-type, and heterozygous and homozygous transgenic mice overexpressing bcl-2 in the nervous system. While there is a significant reduction of neurons in this population in the wild-type ethanol-exposed animals, this reduction is completely blocked in both the heterozygous and homozygous transgenics. ConsControl groups; Etoh s Ethanol groups. Ža. significantly different from wild-type controls Ž ps 0.0007., heterozygous controls Ž ps 0.0031., heterozygous ethanols Ž p s 0.0021., homozygous controls Ž p - 0.0001. and homozygous ethanols Ž p- 0.0001.. Žb. significantly different from wild-type controls Ž ps 0.0477..
16
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
Purkinje cells Žq14%. in the homozygous controls compared to the wild-type controls Ž p s 0.0477.. These results are depicted in Fig. 1. Photomicrographs of the Purkinje cell layer in ethanol-exposed animals from the three genotypes are shown in Fig. 2.
Fig. 2. Photomicrographs of Purkinje cell layer in lobule I of P5 wild-type and bcl-2 transgenic mouse cerebellum following 2-h exposure to ethanol vapors on P4–5. Ža. Wild-type, Žb. heterozygous bcl-2 transgenic, and Žc. homozygous bcl-2 transgenic animals. A number of Purkinje cells are delineated by arrows in each micrograph. Note the widely spaced Purkinje cells in the wild-type ethanol-exposed animals compared to both the heterozygous and homozygous transgenics. Bar s 50 mm.
4. Discussion This study has provided the first demonstration that the apoptosis repressor, Bcl-2, can protect a vulnerable neuronal population against ethanol toxicity during the sensitive early postnatal period of CNS development. The protection was complete, with no Purkinje cell loss detected in the transgenic animals overexpressing Bcl-2. This cell loss was prevented in both the homozygous and the heterozygous animals, suggesting that even moderate increases in the levels of this cell survival-promoting protein are sufficient to counter ethanol toxicity within this neuronal population. These findings are consistent with several previous studies in which Bcl-2 overexpression was shown to protect against a variety of adverse conditions or substances, including ischemia, hypoglycemia, oxidative stress, neurotrophic factor withdrawal and high levels of intracellular calcium w12,47x. It may be significant that these same conditions have often been hypothesized to be mechanisms contributing to the detrimental effects of ethanol on nervous system development w43x. Our results are also consistent with an earlier report in which Bcl-2a overexpression was shown to produce increased resistance to ethanol in cultured B lymphoblastoid cells w40x. The current results serve as an extension of and a complement to a recent study in our laboratory, using the RNase protection assay ŽRPA., in which we found marked upregulation of mRNA for the pro-apoptotic molecules bax and bcl-xs in the same region viewed in the present study Žcerebellar vermis. shortly after ethanol exposure on P4. The increased expression of these cell death effectors correlated with loss of Purkinje cells in parallel studies Žas determined on P21; Moore et al., unpublished observations.. No change in the level of these molecules was seen following exposure on P7, however, and, correspondingly, no loss of Purkinje cells was found after exposure at that time. Although the level of bcl-2 mRNA was not significantly altered in that study, the implication that ethanol can promote neuronal loss through regulation of expression within this gene family is strong. Bcl-2 protein is highly expressed during embryonic development, particularly in proliferating and early differentiating cells, with highest expression at E15 in the developing mouse brain, approximately 2–3 times higher than in the adult brain w1,26x. Bcl-2 expression declines as the brain matures w26x, except in regions with postnatal neurogenesis such as cerebellar cortex, in which granule cell proliferation is still underway Žgeneration of Purkinje cell is completed prenatally, from gestation day 13 wG13x to G16 w2x.. Bcl-2 expression is relatively high in the neonatal mouse cerebellum during the first postnatal month, and has been localized to Purkinje cells, and granule cells of both the external and internal granule cell layers w27x. Cerebellar Bcl-2 distribution declines during later postnatal periods, however w41x. Particularly compelling evidence for the importance of cell death repressors to Purkinje cell
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
survival was provided in a study by Gillardon et al. w13x, which examined the expression of bcl-2 and bax mRNA in Purkinje cell degeneration Ž pcd . mutant mice in which Purkinje cells die between P22 and P28. They found that at P22, at the onset of Purkinje cell loss, the level of bcl-2 mRNA decreased by 35% in the pcd mutants, while bax transcripts remained unchanged. In a previous study using bcl-2 transgenic mice which overexpressed Bcl-2 either embryonically or postnatally, increased numbers of Purkinje cells were seen w24,46x. The Purkinje cell rescue from programmed cell death was 43% in strains with embryonic overexpression, and 27% in those with postnatal overexpression. Only a relatively modest Ž14%. increase in Purkinje cells was seen in the homozygous transgenic animals in the present study, probably owing to the fact that the cell death period, which extends from G20 to P14 w8x, was not yet completed. Ethanol has been found to trigger neuronal apoptosis both in vivo and in vitro, as determined by DNA fragmentation analyses w6,9,47x. Recent studies have provided considerable new information concerning the mechanisms of Bcl-2 activity within the apoptosis network. The Bcl-2 family members are characterized by their ability to form complex combinations of homo- and heterodimers, the relative abundance of which appears to determine cellular sensitivity to apoptotic signals w25,33,44x. Bcl-2, for example, heterodimerizes with Bax and Bcl-xs Žamong others., which serves to inhibit their pro-apoptotic functions. Bcl-2 can also act as an adaptor or docking protein, sequestering pro-apoptotic proteins from the cytosol, either localizing them to the membrane, or tethering them for interaction and inactivation by other membrane-associated proteins. In addition, Bcl-2, Bcl-xl and Bax can form selective pores in lipid membranes w3,29,37x and can in this way insert into the mitochondrial membrane and regulate the mitochondrial membrane potential w20,45x. Loss of this membrane potential has been correlated with induction of cell death. Bax, in its pore-forming capacity, promotes loss of the mitochondrial membrane potential, while Bcl-2 and Bcl-xl block this pore-forming activity w3,38x. If the mitochondrial membrane potential is sufficiently disrupted, cytochrome-c is released into the cytosol w22x, binding to apoptosis protease-activating factor-1 ŽApaf-1., which mediates activation of the cysteine protease caspase-3, initiating the effector phase of apoptosis w48x. The increased expression of Bcl-2 in the transgenic animals, as in the present study, could clearly abrogate the pro-apoptotic functions of Bax and Bcl-xs, shown to be upregulated in our RPA study, either by forming heterodimers and altering the ratio of pro- to anti-apoptotic molecules, or by interfering with the disruption of the mitochondrial membrane potential by the cell death effectors, thereby preventing cytochrome-c release and subsequent caspase activation. The present investigation, then, has provided the first clear evidence that Bcl-2 can rescue neurons from
17
ethanol-induced death. Together with our previous studies in which increases in the levels of the cell death effector molecules Bax and Bcl-xs were seen to accompany neuronal loss as a function of ethanol exposure, this study suggests that modulation of gene expression within this family represents an important mechanism contributing to neuronal death in the nervous system following developmental ethanol exposure.
Acknowledgements This study was supported by NIAAA Grant AA09128 to MBH; DBM was supported by NIAAA Grants T32AA07561 and AA05502; OB was supported by the National Health and Medical Research Council of Australia and the National Multiple Sclerosis Society of Australia. We thank Lisa Dube´ for excellent technical assistance.
References w1x S. Abe-Dohmae, N. Harada, K. Yamada, R. Tanaka, Bcl-2 gene is highly expressed during neurogenesis in the central nervous system, Biochem. Biophys. Res. Commun. 191 Ž1993. 915–921. w2x J. Altman, S. Bayer, Embryonic development of the rat cerebellum: III. Regional differences in the time of origin, migration, and settling of Purkinje cells, J. Comp. Neurol. 231 Ž1985. 42–65. w3x B. Antonsson, F. Conti, A. Ciavatta, S. Montssuit, S. Lewis, I. Martinou, L. Bernasconi, A. Bernard, J.J. Mermod, G. Mazzei, K. Maundrell, F. Gambale, R. Sadoul, J.-C. Martinou, Inhibition of bax channel-forming activity by bcl-2, Science 277 Ž1977. 370–372. w4x D.E. Barnes, D.W. Walker, Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus, Dev. Brain Res. 1 Ž1981. 333–340. w5x R. Bernard, P. Farlie, O. Bernard, NSE– bcl-2 transgenic mice, a model system for studying neuronal death and survival, Dev. Neurosci. 19 Ž1997. 79–85. w6x S.V. Bhave, P.L. Hoffman, Ethanol promotes apoptosis in cerebellar granule cells by inhibiting the trophic effect of NMDA, J. Neurochem. 68 Ž1997. 578–586. w7x D.J. Bonthius, J.R. West, Alcohol-induced neuronal loss in developing rats: Increased brain damage with binge exposure, Alcohol. Clin. Exp. Res. 14 Ž1990. 107–118. w8x B. Cragg, S. Phillips, Natural loss of Purkinje cells during development and increased loss with alcohol, Brain Res. 325 Ž1985. 151– 160. w9x A. De, N.I. Boyadjieva, M. Pastorcic, B.V. Reddy, D.K. Sarkar, Cyclic AMP and ethanol interact to control apoptosis and differentiation in hypothalamic b-endorphin neurons, J. Biol. Chem. 269 Ž1994. 26697–26705. w10x D.P. Dohrman, J.R. West, N.J. Pantazis, Ethanol reduces expression of the nerve growth factor receptor, but not nerve growth factor protein levels in the neonatal rat cerebellum, Alcohol. Clin. Exp. Res. 21 Ž1997. 882–893. w11x C.D. Driscoll, A.P. Streissguth, E.P. Riley, Prenatal alcohol exposure: comparability of effects in humans and animal models, Neurotoxicol. Teratol. 12 Ž1990. 231–237. w12x P.G. Farlie, R. Dringen, S.M. Rees, G. Kannourakis, O. Bernard, Bcl-2 transgene expression can protect neurons against developmental and induced cell death, P.N.A.S. 92 Ž1995. 4397–4401. w13x F. Gillardon, J. Baurle, H. Wickert, U. Grusser-Cornehls, M. Zim-
18
w14x
w15x
w16x
w17x
w18x
w19x
w20x
w21x w22x
w23x
w24x
w25x
w26x w27x
w28x w29x
w30x
M.B. Heaton et al.r Brain Research 817 (1999) 13–18 merman, Differential regulation of bcl-2, bax, c-fos, junB, and krox-24 expression in the cerebellum of Purkinje cell degeneration mutant mice, J. Neurosci. Res. 41 Ž1995. 708–715. C.R. Goodlett, A.T. Eilers, Alcohol-induced Purkinje cell loss with a single binge exposure in neonatal rats: a stereological study of temporal windows of vulnerability, Alcohol. Clin. Exp. Res. 21 Ž1997. 738–744. R.W. Guillery, K. Herrup, Quantification without pontification: choosing a method for counting objects in sectioned tissues, J. Comp. Neurol. 386 Ž1997. 2–7. T. Hagg, C.E.M. Van der Zee, G.M. Ross, R.J. Riopelle, Basal forebrain neuronal loss in mice lacking neurotrophin receptor p75. Technical comment, response, Science 277 Ž1997. 838–839. K.M. Hamre, J.R. West, The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles, Alcohol. Clin. Exp. Res. 17 Ž1993. 610–622. M.B. Heaton, M. Paiva, D.J. Swanson, D.W. Walker, Modulation of ethanol neurotoxicity by nerve growth factor, Brain Res. 620 Ž1993. 78–85. M.B. Heaton, M. Paiva, D.J. Swanson, D.W. Walker, Alterations in responsiveness to ethanol and neurotrophic substances in fetal septohippocampal neurons following chronic prenatal ethanol exposure, Dev. Brain Res. 85 Ž1995. 1–13. M.G.V. Heiden, N.S. Chandel, E.K. Williamson, P.T. Schumacker, C.B. Thompson, Bcl-xL regulates the membrane potential and volume of homeostasis of mitochondria, Cell 91 Ž1997. 627–637. K.L. Jones, D.W. Smith, Recognition of the fetal alcohol syndrome in early infancy, Lancet 2 Ž1973. 999–1001. R.M. Kluck, E. Bossy-Wetzel, D.R. Green, D.D. Newmeyer, The release of cytochrome c from mitochondria: a primary site for bcl-2 regulation of apoptosis, Science 275 Ž1997. 1132–1136. S.E. Maier, W.J.A. Chen, J.A. Miller, J.R. West, Fetal alcohol exposure and temporal vulnerability: regional differences in alcohol-induced microencephaly as a function of the timing of binge-like alcohol exposure during rat brain development, Alcohol. Clin. Exp. Res. 21 Ž1997. 1418–1428. J. Martinou, M. Dubois-Dauphin, J. Staple, I. Rodriguez, H. Frankowski, M. Missotten, P. Albertini, D. Talabot, S. Catsicas, C. Pietra, Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia, Neuron 13 Ž1994. 1017–1030. K. Maundrell, B. Antonsson, E. Magnenant, M. Camps, M. Muda, C. Chabert, C. Gillieron, U. Boschert, E. Vial-Knecht, J.-C. Martinou, S. Arkinstall, Bcl-2 undergoes phosphorylation by c-Jun Nterminal kinaserstress-activated protein kinases in the presence of the constitutively active GTP-binding protein Raf1, J. Biol. Chem. 272 Ž1997. 25238–25242. D.E. Merry, S.J. Korsmeyer, Bcl-2 gene family in the nervous system, Annu. Rev. Neurosci. 20 Ž1997. 245–267. D. Merry, D. Veis, W. Hickey, J. Korsmeyer, Bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS, Development 120 Ž1994. 301–311. M.W. Miller, Effects of alcohol on the generation and migration of cerebral cortical neurons, Science 233 Ž1986. 1308–1311. A.J. Minn, P. Velez, S.L. Schendel, H. Liang, S.W. Muchmore, S.W. Fesik, M. Fill, C.B. Thompson, Bcl-xL forms an ion channel in synthetic lipid membranes, Nature 385 Ž1997. 353–357. R.M.A. Napper, J.R. West, Permanent neuronal cell loss in the cerebellum of rats exposed to continuous low blood alcohol levels during the brain growth spurt: a stereological investigation, J. Comp. Neurol. 362 Ž1995. 283–292.
w31x D. Offen, P.M. Beart, N.S. Cheung, C.J. Pascoe, A. Hochman, S. Gorodin, E. Melamed, R. Bernard, O. Bernard, Transgenic mice expressing Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, P.N.A.S. 95 Ž1998. 5789–5794. w32x N. Pal, R.L. Alkana, Use of inhalation to study the effect of ethanol and ethanol dependence on neonatal mouse development without maternal separation: a preliminary study, Life Science 61 Ž1997. 1269–1281. w33x J.C. Reed, Double identity for proteins of the bcl-2 family, Nature 387 Ž1997. 773–776. w34x T.M. Roebuck, M.N. Mattson, E.P. Riley, A review of the neuronatomical findings in children with fetal alcohol syndrome or prenatal exposure to alcohol, Alcohol. Clin. Exp. Res. 22 Ž1998. 339–344. w35x H. Rouach, P. Houze, M. Gentil, M.-T. Orfanelli, R. Nordmann, Changes in some pro- and antioxidants in rat cerebellum after chronic alcohol intake, Biochem. Pharmacol. 53 Ž1997. 539–545. w36x A.E. Ryabinin, M. Cole, F.E. Bloom, M.C. Wilson, Exposure of neonatal rats to alcohol by vapor inhalation demonstrates specificity of microcephaly and Purkinje cell loss but not astrogliosis, Alcohol. Clin. Exp. Res. 19 Ž1995. 784–791. w37x S.L. Schendel, Z. Xie, M.O. Montal, S. Matsuyama, M. Montal, J. Reed, Channel formation by antiapoptotic protein bcl-2, P.N.A.S. 94 Ž1997. 5113–5118. w38x V.P. Skulachev, Cytochrome c in the apoptotic antioxidant cascades, FEBS Lett. 423 Ž1998. 275–280. w39x A.P. Streissguth, J.M. Aase, S.K. Clarren, S.P. Randels, R.A. LaDue, D.F. Smith, Fetal alcohol syndrome in adolescents and adults, J.A.M.A. 265 Ž1991. 1961–1967. w40x Y. Tsujimoto, Stress-resistance conferred by high level of bcl-2a protein in human B lymphoblastoid cell, Oncogene 4 Ž1989. 1331– 1336. w41x K. Vekrellis, M. McCarthy, A. Watson, J. Whitfield, L. Rubin, J. Ham, Bax promotes neuronal cell death and is downregulated during the development of the nervous system, Development 124 Ž1997. 1239–1249. w42x B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization, Brain Res. 701 Ž1995. 61–74. w43x J.R. West, W.J.A. Chen, N.J. Pantazis, Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage, Metabol. Brain Dis. 9 Ž1994. 291–323. w44x E. Yang, J. Zha, J. Jockel, L. Boise, C. Thompson, S. Korsmeyer, Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death, Cell 80 Ž1995. 285–291. w45x J. Yang, X. Liu, K.C.N. Bhalla, A.M. Ibrado, J. Cai, T.-I. Peng, D.P. Jones, X. Wang, Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked, Science 275 Ž1997. 1129–1132. w46x H.S. Zanjani, M.W. Vogel, N. Delhaye-Bouchaud, J.C. Martinou, J. Mariani, Increased cerebellar Purkinje cell numbers in mice overexpressing a human bcl-2 transgene, J. Comp. Neurol. 374 Ž1996. 332–341. w47x L.T. Zhong, T. Sarafian, D.J. Kane, A.C. Charles, S.P. Mah, R.H. Edwards, D.E. Bredesen, Bcl-2 inhibits death of central neural cells induced by multiple agents, P.N.A.S. 90 Ž1993. 4533–4537. w48x H. Zou, W.J. Henzel, X. Liu, A. Lutsch, X. Wang, Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3, Cell 90 Ž1997. 405– 413.
Research report
Bcl-2 overexpression protects the neonatal cerebellum from ethanol neurotoxicity Marieta Barrow Heaton a
a, )
, D. Blaine Moore a , Michael Paiva a , Theresa Gibbs b , Ora Bernard
b
Department of Neuroscience, UniÕersity of Florida Brain Institute, Center for Alcohol Research, UniÕersity of Florida College of Medicine, GainesÕille, FL 32610-0244, USA b The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia Accepted 27 October 1998
Abstract The developing nervous system is extremely sensitive to ethanol, and exposure often produces a condition known as the fetal alcohol syndrome. Although mechanisms underlying developmental ethanol toxicity have long been sought, they remain poorly understood. In this study, we examined the ability of the cell death repressor gene bcl-2 to protect against ethanol neurotoxicity. Transgenic mice overexpressing bcl-2 in neurons were exposed to ethanol vapor on postnatal days 4 and 5, which is the peak period of vulnerability of cerebellar Purkinje cells to ethanol. While exposure of wild-type animals to ethanol resulted in significant loss of Purkinje cells by P5, similar exposure of homozygous and heterozygous transgenics had no effect on the number of these neurons. This study suggests that bcl-2 can protect neurons from ethanol neurotoxicity and that modulation of cell death effector or repressor gene products may play a significant role in developmental ethanol neurotoxicity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bcl-2; Apoptosis; Fetal alcohol syndrome; Neuroprotection; Ethanol
1. Introduction Developmental exposure to ethanol leads to a wide range of structural and functional anomalies, often resulting in the condition known as fetal alcohol syndrome ŽFAS, Ref. w21x.. The most devastating deficits associated with FAS are those involving the central nervous system ŽCNS., producing an array of dysfunctions, including hyperactivity, attention deficits, difficulties with learning and memory, impaired motor abilities, and lowered IQ w39x. Significant and permanent alterations have been well documented in regions such as the cerebral cortex w28x, hippocampus w4x and cerebellum w7x. This sensitivity appears to be most acute during the so-called brain growth spurt ŽBGS., a dynamic period of differentiation and synaptoge-
)
Corresponding author. Department of Neuroscience, University of Florida College of Medicine, Health Science Center, PO Box 100244, Gainesville, FL 32610-0244, USA. Fax: q1-352-392-8347; E-mail: [email protected]
nesis, which occurs during the third trimester in humans and in the early postnatal period Žpostnatal days 4–10. in the rodent model of FAS, in which many of the behavioral and neuroanatomical abnormalities seen in FAS children have been duplicated w11x. The developing cerebellum is exquisitely vulnerable to ethanol during a very brief period. In the rat model, exposure to even a single bolus of ethanol on postnatal day 4 ŽP4. results in loss of Purkinje cells comparable to that seen with exposure throughout the BGS w14x. Administration of ethanol solely during the prenatal period or slightly later in the postnatal period ŽP7-8. does not induce neuronal loss in this region w17,23x. In human FAS children, cerebellar abnormalities have also been described, and a number of behavioral deficits such as impairment in the development of motor competence, problems in balance, gait coordination and fine motor control reflect this disruption w34x. These deficits correlate with cell loss and reduced size of the anterior vermal cerebellar region, particularly lobules I to V, which is also the region shown to be particularly sensitive to ethanol in the animal studies w17x.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 7 3 - 1
14
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
The mechanisms whereby ethanol exerts its neurotoxicity in the developing nervous system have long been sought and a number of contributing factors have been proposed. These include hypoxiarischemia, hypoglycemia w43x, alterations in calcium homeostasis w42x, production of reactive oxygen species or depletion of protective antioxidants w35x, and deceases in neurotrophic support or responsiveness w10,18,19x. Since ethanol neurotoxicity has been demonstrated both in vivo and in vitro to be an apoptotic process w6,9x, an intriguing possibility is that the gene products which regulate cell death may be involved in ethanol-induced neuronal loss. The bcl-2 gene family, the mammalian homologs of the C. elegans ced-9 gene, has been well characterized in recent years. This family consists of cell death repressors Že.g., bcl-2, bcl-xl . and effectors Že.g., bax, bad, bcl-xs .. bcl-2 expression has been shown to protect against a number of adverse conditions or substances, including growth factor or serum withdrawal, high levels of intracellular calcium, glutamate toxicity, ischemia, hypoglycemia, oxidative stress, glutamate toxicity, 6-hydroxydopamine and MPTP neurotoxicity, and axotomy w12,31,47x. In addition, high levels of expression of the Bcl-2a protein have been found to confer increased resistance to several stressors in an Epstein Barr virus-infected human B lymphoblastoid cell line, including ethanol w40x. The present study represents the first investigation of the potential of Bcl-2 overexpression to afford similar protection against ethanol neurotoxicity in the developing CNS. For this study we chose to view neonatal cerebellar Purkinje cells following ethanol exposure during the extremely vulnerable early postnatal period, in wild-type animals, and in transgenic animals overexpressing Bcl-2 in neurons.
2. Materials and methods 2.1. Transgenic mice The NSE–bcl-2 transgenic mice used in this study were generated by microinjection of the human bcl-2 cDNA fused to the neuron-specific enolase ŽNSE. promoter into the pronuclei of fertilized C57BLr6J ova, as described earlier w12x. Integration of the transgene was detected by dot hybridization of DNA obtained from tail biopsies to the 750-bp EcoRIrBamHI simian virus 40 fragment from pNSE-CAT as a probe. The homozygous transgene offspring, and to a lesser extent the heterozygous offspring, express high levels of Bcl-2 protein in neurons of the CNS and PNS. Expression of the transgene was confirmed by Western blot analysis of whole neonatal brain lysates. For this confirmation, brains were homogenized in lysis buffer containing 1% Nonidet-P-40 and protease inhibitors. Samples were subjected to SDSrPAGE followed by electroblotting onto Hybond C-extra nitrocellulose membranes.
Filters were probed with a monoclonal anti-human Bcl-2 antibody, Bcl-2-100, followed by HRP-coupled rabbit anti-mouse immunoglobulin antibody and visualized by enhanced chemiluminescence. We have found that expression of the transgene is detected in embryos as early as 10.5 days post-coitus w5x. The transgene is expressed exclusively in neurons w12x, and in immunohistochemical studies, it was found to be highly expressed in cerebellar Purkinje cells, but not in granule cells w5x. The mice used in the present study were descended from the same transgenic animals which were characterized in our previous studies w5,12x. The desired genotypes were obtained by mating homozygotes= homozygotes to yield offspring homozygous for the bcl-2 allele, and homozygotes = C57BLr6J animals to yield offspring heterozygotes for the bcl-2 allele. 2.2. Ethanol Õapor inhalation exposure paradigm Neonatal mice were exposed to ethanol via the vapor inhalation procedure, using the modification of Pal and Alkana w32x. For this procedure, pups in their home cage along with the nursing dam were placed in an inhalation apparatus, thus eliminating the stress of maternal separation. This procedure is preferable to the usual inhalation protocol, and the pups resume feeding more quickly than when they are separated from the dam during exposure, which minimizes differences in weight gain between groups. Wild-type, heterozygous and homozygous pups were assigned to one of two groups: Ethanol vapor-exposed or control vapor-exposed. The inhalation procedure was carried out on P4 and P5, the peak period of Purkinje cell vulnerability to ethanol w14x. The ethanol or control exposure period was 2 h per day. The home cage containing the dam and the pups was placed in a large Plexiglas inhalation chamber which was fitted with intake and exhaust hoses. Prior to vapor exposure, the ethanol litters were culled to 5 pups on P4, while control litters were culled to 7 pups. The decreased number of pups in the Ethanol group reduced the possibility of nutritional differences arising between ethanol-exposed and control pups w36x. Air flow into the intake hose was provided by an air pump set to deliver approximately 0.8–1.0 lrmin, and it flowed into a 1 l Erlenmeyer vacuum flask containing 520 ml 95% ethanol. As the air was forced into the flask it passed through a 3.5-cm air stone submerged in the ethanol. The ethanol-laden vapor was then carried to the chamber. The exhaust hose led ethanol vapor from the chamber to a fume hood. Two hours following termination of ethanol exposure, blood ethanol concentrations were determined by the Sigma microenzymatic assay. This procedure was carried out in compliance with established guidelines of the Walter and Eliza Hall Institute, and was approved by the Royal Melbourne Hospital Campus Animal Ethics Committee ŽHCAEC Project 008r971..
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
2.3. Histological procedures On the day of sacrifice ŽP5, 2 h after the final ethanol exposure., the animals were sacrificed by methoxyflurane overdose, and the cerebellum was dissected out and placed in Bouin’s fixative for 24 h. Following fixation, specimens were dehydrated, cleared, and embedded in paraffin. Serial sagittal 6 mm sections were then cut through the vermis, mounted onto slides and stained with hematoxylin and eosin. 2.4. Purkinje cell counts In order to assess ethanol effects on developing cerebellum, Purkinje cells were counted in Nissl-stained sections from the midline vermis. Only cells in lobule I were counted, since this early maturing region has been shown to be maximally sensitive to ethanol and experiences the greatest Purkinje cell loss w14x. In addition to cell counts, the length of the lobules was measured in order to correct for any possible size differential between groups. The cell counts were then converted to cellsrmm. Prior to Purkinje cell counting, slides were coded and randomized with an identifying number. Cell counts were performed in the manner of Hamre and West w17x on three sections separated by 24 mm to insure that the same cells were not counted twice. Purkinje cells were identified by size and location, and were counted if they contained a well-delineated nuclear membrane, distinct nucleolus, and darkly stained cytoplasm. The quantification was carried out using a Zeiss Axioplan photomicroscope, with a drawing tube attachment, at a magnification of 400 = . As has recently been discussed, there are various advantages and disadvantages to all of the currently practiced cell enumeration protocols w15x. For this study, we used a basic manual sampling technique, which is appropriate since we intended to compare mean cells mmy1 sectiony1 within a circumscribed cerebellar region between groups, but did not attempt to estimate population totals w16x. The same procedure using widely spaced sections was employed for all conditions and all counts were done ‘blind’. Also, vermal lobule area was measured to determine whether ethanol treatment affected volume or area, and corrections for volumetric changes were made. It should be noted that Purkinje cell loss following developmental ethanol exposure has been determined in previous studies using both manual counting w17x and stereological procedures w30x with comparable results. Counts were performed on 4–9 animals from each group Ži.e., wild-type, homozygous and heterozygous in ethanol and control groups.. The average number of Purkinje cellsrmm was determined for each section from each animal. Statistical comparisons were performed via the analysis of variance ŽANOVA. using the StatView Program on a Macintosh computer. When appropriate, individual differences between groups were as-
15
sessed using the Fisher’s protected least significant difference ŽPLSD. post-hoc test.
3. Results The inhalation paradigm of ethanol exposure as described above resulted in peak blood ethanol concentrations averaging 232 mgrdl in the pups, with relatively low blood ethanol readings in the dam Žaveraging 25 mgrdl.. The pups were weighed on each exposure day, just prior to being placed in the inhalation chamber. The ANOVA indicated that the ethanol-exposed pups’ weight gain was comparable to their control counterparts Ž F s 0.965, df s 5, 27, p s 0.4565.. When the ANOVA was applied to the Purkinje cell counts, a significant effect of treatment was revealed Ž F s 7.96, df s 5, 31, p - 0.0001.. The PLSD post-hoc test indicated that in the wild-type mice, ethanol exposure during this period produced a significant loss of Purkinje cells Žy24%., compared to control vapor-exposed animals Ž p s 0.0007.. The wild-type ethanol-exposed animals also differed significantly from the heterozygous ethanol-exposed Ž p s 0.0021., heterozygous control Ž p s 0.0031., homozygous ethanol-exposed Ž p - 0.0001., and homozygous control animals Ž p - 0.0001.. Neither the heterozygous nor the homozygous ethanol-exposed neonates differed from their genotypic controls. The only other difference detected was a small but significant increase in
Fig. 1. Purkinje cell counts, expressed as cellsrmm, in lobule I of P5 mouse cerebellum following 2-h exposure to ethanol vapors or control conditions on P4–5, in wild-type, and heterozygous and homozygous transgenic mice overexpressing bcl-2 in the nervous system. While there is a significant reduction of neurons in this population in the wild-type ethanol-exposed animals, this reduction is completely blocked in both the heterozygous and homozygous transgenics. ConsControl groups; Etoh s Ethanol groups. Ža. significantly different from wild-type controls Ž ps 0.0007., heterozygous controls Ž ps 0.0031., heterozygous ethanols Ž p s 0.0021., homozygous controls Ž p - 0.0001. and homozygous ethanols Ž p- 0.0001.. Žb. significantly different from wild-type controls Ž ps 0.0477..
16
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
Purkinje cells Žq14%. in the homozygous controls compared to the wild-type controls Ž p s 0.0477.. These results are depicted in Fig. 1. Photomicrographs of the Purkinje cell layer in ethanol-exposed animals from the three genotypes are shown in Fig. 2.
Fig. 2. Photomicrographs of Purkinje cell layer in lobule I of P5 wild-type and bcl-2 transgenic mouse cerebellum following 2-h exposure to ethanol vapors on P4–5. Ža. Wild-type, Žb. heterozygous bcl-2 transgenic, and Žc. homozygous bcl-2 transgenic animals. A number of Purkinje cells are delineated by arrows in each micrograph. Note the widely spaced Purkinje cells in the wild-type ethanol-exposed animals compared to both the heterozygous and homozygous transgenics. Bar s 50 mm.
4. Discussion This study has provided the first demonstration that the apoptosis repressor, Bcl-2, can protect a vulnerable neuronal population against ethanol toxicity during the sensitive early postnatal period of CNS development. The protection was complete, with no Purkinje cell loss detected in the transgenic animals overexpressing Bcl-2. This cell loss was prevented in both the homozygous and the heterozygous animals, suggesting that even moderate increases in the levels of this cell survival-promoting protein are sufficient to counter ethanol toxicity within this neuronal population. These findings are consistent with several previous studies in which Bcl-2 overexpression was shown to protect against a variety of adverse conditions or substances, including ischemia, hypoglycemia, oxidative stress, neurotrophic factor withdrawal and high levels of intracellular calcium w12,47x. It may be significant that these same conditions have often been hypothesized to be mechanisms contributing to the detrimental effects of ethanol on nervous system development w43x. Our results are also consistent with an earlier report in which Bcl-2a overexpression was shown to produce increased resistance to ethanol in cultured B lymphoblastoid cells w40x. The current results serve as an extension of and a complement to a recent study in our laboratory, using the RNase protection assay ŽRPA., in which we found marked upregulation of mRNA for the pro-apoptotic molecules bax and bcl-xs in the same region viewed in the present study Žcerebellar vermis. shortly after ethanol exposure on P4. The increased expression of these cell death effectors correlated with loss of Purkinje cells in parallel studies Žas determined on P21; Moore et al., unpublished observations.. No change in the level of these molecules was seen following exposure on P7, however, and, correspondingly, no loss of Purkinje cells was found after exposure at that time. Although the level of bcl-2 mRNA was not significantly altered in that study, the implication that ethanol can promote neuronal loss through regulation of expression within this gene family is strong. Bcl-2 protein is highly expressed during embryonic development, particularly in proliferating and early differentiating cells, with highest expression at E15 in the developing mouse brain, approximately 2–3 times higher than in the adult brain w1,26x. Bcl-2 expression declines as the brain matures w26x, except in regions with postnatal neurogenesis such as cerebellar cortex, in which granule cell proliferation is still underway Žgeneration of Purkinje cell is completed prenatally, from gestation day 13 wG13x to G16 w2x.. Bcl-2 expression is relatively high in the neonatal mouse cerebellum during the first postnatal month, and has been localized to Purkinje cells, and granule cells of both the external and internal granule cell layers w27x. Cerebellar Bcl-2 distribution declines during later postnatal periods, however w41x. Particularly compelling evidence for the importance of cell death repressors to Purkinje cell
M.B. Heaton et al.r Brain Research 817 (1999) 13–18
survival was provided in a study by Gillardon et al. w13x, which examined the expression of bcl-2 and bax mRNA in Purkinje cell degeneration Ž pcd . mutant mice in which Purkinje cells die between P22 and P28. They found that at P22, at the onset of Purkinje cell loss, the level of bcl-2 mRNA decreased by 35% in the pcd mutants, while bax transcripts remained unchanged. In a previous study using bcl-2 transgenic mice which overexpressed Bcl-2 either embryonically or postnatally, increased numbers of Purkinje cells were seen w24,46x. The Purkinje cell rescue from programmed cell death was 43% in strains with embryonic overexpression, and 27% in those with postnatal overexpression. Only a relatively modest Ž14%. increase in Purkinje cells was seen in the homozygous transgenic animals in the present study, probably owing to the fact that the cell death period, which extends from G20 to P14 w8x, was not yet completed. Ethanol has been found to trigger neuronal apoptosis both in vivo and in vitro, as determined by DNA fragmentation analyses w6,9,47x. Recent studies have provided considerable new information concerning the mechanisms of Bcl-2 activity within the apoptosis network. The Bcl-2 family members are characterized by their ability to form complex combinations of homo- and heterodimers, the relative abundance of which appears to determine cellular sensitivity to apoptotic signals w25,33,44x. Bcl-2, for example, heterodimerizes with Bax and Bcl-xs Žamong others., which serves to inhibit their pro-apoptotic functions. Bcl-2 can also act as an adaptor or docking protein, sequestering pro-apoptotic proteins from the cytosol, either localizing them to the membrane, or tethering them for interaction and inactivation by other membrane-associated proteins. In addition, Bcl-2, Bcl-xl and Bax can form selective pores in lipid membranes w3,29,37x and can in this way insert into the mitochondrial membrane and regulate the mitochondrial membrane potential w20,45x. Loss of this membrane potential has been correlated with induction of cell death. Bax, in its pore-forming capacity, promotes loss of the mitochondrial membrane potential, while Bcl-2 and Bcl-xl block this pore-forming activity w3,38x. If the mitochondrial membrane potential is sufficiently disrupted, cytochrome-c is released into the cytosol w22x, binding to apoptosis protease-activating factor-1 ŽApaf-1., which mediates activation of the cysteine protease caspase-3, initiating the effector phase of apoptosis w48x. The increased expression of Bcl-2 in the transgenic animals, as in the present study, could clearly abrogate the pro-apoptotic functions of Bax and Bcl-xs, shown to be upregulated in our RPA study, either by forming heterodimers and altering the ratio of pro- to anti-apoptotic molecules, or by interfering with the disruption of the mitochondrial membrane potential by the cell death effectors, thereby preventing cytochrome-c release and subsequent caspase activation. The present investigation, then, has provided the first clear evidence that Bcl-2 can rescue neurons from
17
ethanol-induced death. Together with our previous studies in which increases in the levels of the cell death effector molecules Bax and Bcl-xs were seen to accompany neuronal loss as a function of ethanol exposure, this study suggests that modulation of gene expression within this family represents an important mechanism contributing to neuronal death in the nervous system following developmental ethanol exposure.
Acknowledgements This study was supported by NIAAA Grant AA09128 to MBH; DBM was supported by NIAAA Grants T32AA07561 and AA05502; OB was supported by the National Health and Medical Research Council of Australia and the National Multiple Sclerosis Society of Australia. We thank Lisa Dube´ for excellent technical assistance.
References w1x S. Abe-Dohmae, N. Harada, K. Yamada, R. Tanaka, Bcl-2 gene is highly expressed during neurogenesis in the central nervous system, Biochem. Biophys. Res. Commun. 191 Ž1993. 915–921. w2x J. Altman, S. Bayer, Embryonic development of the rat cerebellum: III. Regional differences in the time of origin, migration, and settling of Purkinje cells, J. Comp. Neurol. 231 Ž1985. 42–65. w3x B. Antonsson, F. Conti, A. Ciavatta, S. Montssuit, S. Lewis, I. Martinou, L. Bernasconi, A. Bernard, J.J. Mermod, G. Mazzei, K. Maundrell, F. Gambale, R. Sadoul, J.-C. Martinou, Inhibition of bax channel-forming activity by bcl-2, Science 277 Ž1977. 370–372. w4x D.E. Barnes, D.W. Walker, Prenatal ethanol exposure permanently reduces the number of pyramidal neurons in rat hippocampus, Dev. Brain Res. 1 Ž1981. 333–340. w5x R. Bernard, P. Farlie, O. Bernard, NSE– bcl-2 transgenic mice, a model system for studying neuronal death and survival, Dev. Neurosci. 19 Ž1997. 79–85. w6x S.V. Bhave, P.L. Hoffman, Ethanol promotes apoptosis in cerebellar granule cells by inhibiting the trophic effect of NMDA, J. Neurochem. 68 Ž1997. 578–586. w7x D.J. Bonthius, J.R. West, Alcohol-induced neuronal loss in developing rats: Increased brain damage with binge exposure, Alcohol. Clin. Exp. Res. 14 Ž1990. 107–118. w8x B. Cragg, S. Phillips, Natural loss of Purkinje cells during development and increased loss with alcohol, Brain Res. 325 Ž1985. 151– 160. w9x A. De, N.I. Boyadjieva, M. Pastorcic, B.V. Reddy, D.K. Sarkar, Cyclic AMP and ethanol interact to control apoptosis and differentiation in hypothalamic b-endorphin neurons, J. Biol. Chem. 269 Ž1994. 26697–26705. w10x D.P. Dohrman, J.R. West, N.J. Pantazis, Ethanol reduces expression of the nerve growth factor receptor, but not nerve growth factor protein levels in the neonatal rat cerebellum, Alcohol. Clin. Exp. Res. 21 Ž1997. 882–893. w11x C.D. Driscoll, A.P. Streissguth, E.P. Riley, Prenatal alcohol exposure: comparability of effects in humans and animal models, Neurotoxicol. Teratol. 12 Ž1990. 231–237. w12x P.G. Farlie, R. Dringen, S.M. Rees, G. Kannourakis, O. Bernard, Bcl-2 transgene expression can protect neurons against developmental and induced cell death, P.N.A.S. 92 Ž1995. 4397–4401. w13x F. Gillardon, J. Baurle, H. Wickert, U. Grusser-Cornehls, M. Zim-
18
w14x
w15x
w16x
w17x
w18x
w19x
w20x
w21x w22x
w23x
w24x
w25x
w26x w27x
w28x w29x
w30x
M.B. Heaton et al.r Brain Research 817 (1999) 13–18 merman, Differential regulation of bcl-2, bax, c-fos, junB, and krox-24 expression in the cerebellum of Purkinje cell degeneration mutant mice, J. Neurosci. Res. 41 Ž1995. 708–715. C.R. Goodlett, A.T. Eilers, Alcohol-induced Purkinje cell loss with a single binge exposure in neonatal rats: a stereological study of temporal windows of vulnerability, Alcohol. Clin. Exp. Res. 21 Ž1997. 738–744. R.W. Guillery, K. Herrup, Quantification without pontification: choosing a method for counting objects in sectioned tissues, J. Comp. Neurol. 386 Ž1997. 2–7. T. Hagg, C.E.M. Van der Zee, G.M. Ross, R.J. Riopelle, Basal forebrain neuronal loss in mice lacking neurotrophin receptor p75. Technical comment, response, Science 277 Ž1997. 838–839. K.M. Hamre, J.R. West, The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles, Alcohol. Clin. Exp. Res. 17 Ž1993. 610–622. M.B. Heaton, M. Paiva, D.J. Swanson, D.W. Walker, Modulation of ethanol neurotoxicity by nerve growth factor, Brain Res. 620 Ž1993. 78–85. M.B. Heaton, M. Paiva, D.J. Swanson, D.W. Walker, Alterations in responsiveness to ethanol and neurotrophic substances in fetal septohippocampal neurons following chronic prenatal ethanol exposure, Dev. Brain Res. 85 Ž1995. 1–13. M.G.V. Heiden, N.S. Chandel, E.K. Williamson, P.T. Schumacker, C.B. Thompson, Bcl-xL regulates the membrane potential and volume of homeostasis of mitochondria, Cell 91 Ž1997. 627–637. K.L. Jones, D.W. Smith, Recognition of the fetal alcohol syndrome in early infancy, Lancet 2 Ž1973. 999–1001. R.M. Kluck, E. Bossy-Wetzel, D.R. Green, D.D. Newmeyer, The release of cytochrome c from mitochondria: a primary site for bcl-2 regulation of apoptosis, Science 275 Ž1997. 1132–1136. S.E. Maier, W.J.A. Chen, J.A. Miller, J.R. West, Fetal alcohol exposure and temporal vulnerability: regional differences in alcohol-induced microencephaly as a function of the timing of binge-like alcohol exposure during rat brain development, Alcohol. Clin. Exp. Res. 21 Ž1997. 1418–1428. J. Martinou, M. Dubois-Dauphin, J. Staple, I. Rodriguez, H. Frankowski, M. Missotten, P. Albertini, D. Talabot, S. Catsicas, C. Pietra, Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia, Neuron 13 Ž1994. 1017–1030. K. Maundrell, B. Antonsson, E. Magnenant, M. Camps, M. Muda, C. Chabert, C. Gillieron, U. Boschert, E. Vial-Knecht, J.-C. Martinou, S. Arkinstall, Bcl-2 undergoes phosphorylation by c-Jun Nterminal kinaserstress-activated protein kinases in the presence of the constitutively active GTP-binding protein Raf1, J. Biol. Chem. 272 Ž1997. 25238–25242. D.E. Merry, S.J. Korsmeyer, Bcl-2 gene family in the nervous system, Annu. Rev. Neurosci. 20 Ž1997. 245–267. D. Merry, D. Veis, W. Hickey, J. Korsmeyer, Bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS, Development 120 Ž1994. 301–311. M.W. Miller, Effects of alcohol on the generation and migration of cerebral cortical neurons, Science 233 Ž1986. 1308–1311. A.J. Minn, P. Velez, S.L. Schendel, H. Liang, S.W. Muchmore, S.W. Fesik, M. Fill, C.B. Thompson, Bcl-xL forms an ion channel in synthetic lipid membranes, Nature 385 Ž1997. 353–357. R.M.A. Napper, J.R. West, Permanent neuronal cell loss in the cerebellum of rats exposed to continuous low blood alcohol levels during the brain growth spurt: a stereological investigation, J. Comp. Neurol. 362 Ž1995. 283–292.
w31x D. Offen, P.M. Beart, N.S. Cheung, C.J. Pascoe, A. Hochman, S. Gorodin, E. Melamed, R. Bernard, O. Bernard, Transgenic mice expressing Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, P.N.A.S. 95 Ž1998. 5789–5794. w32x N. Pal, R.L. Alkana, Use of inhalation to study the effect of ethanol and ethanol dependence on neonatal mouse development without maternal separation: a preliminary study, Life Science 61 Ž1997. 1269–1281. w33x J.C. Reed, Double identity for proteins of the bcl-2 family, Nature 387 Ž1997. 773–776. w34x T.M. Roebuck, M.N. Mattson, E.P. Riley, A review of the neuronatomical findings in children with fetal alcohol syndrome or prenatal exposure to alcohol, Alcohol. Clin. Exp. Res. 22 Ž1998. 339–344. w35x H. Rouach, P. Houze, M. Gentil, M.-T. Orfanelli, R. Nordmann, Changes in some pro- and antioxidants in rat cerebellum after chronic alcohol intake, Biochem. Pharmacol. 53 Ž1997. 539–545. w36x A.E. Ryabinin, M. Cole, F.E. Bloom, M.C. Wilson, Exposure of neonatal rats to alcohol by vapor inhalation demonstrates specificity of microcephaly and Purkinje cell loss but not astrogliosis, Alcohol. Clin. Exp. Res. 19 Ž1995. 784–791. w37x S.L. Schendel, Z. Xie, M.O. Montal, S. Matsuyama, M. Montal, J. Reed, Channel formation by antiapoptotic protein bcl-2, P.N.A.S. 94 Ž1997. 5113–5118. w38x V.P. Skulachev, Cytochrome c in the apoptotic antioxidant cascades, FEBS Lett. 423 Ž1998. 275–280. w39x A.P. Streissguth, J.M. Aase, S.K. Clarren, S.P. Randels, R.A. LaDue, D.F. Smith, Fetal alcohol syndrome in adolescents and adults, J.A.M.A. 265 Ž1991. 1961–1967. w40x Y. Tsujimoto, Stress-resistance conferred by high level of bcl-2a protein in human B lymphoblastoid cell, Oncogene 4 Ž1989. 1331– 1336. w41x K. Vekrellis, M. McCarthy, A. Watson, J. Whitfield, L. Rubin, J. Ham, Bax promotes neuronal cell death and is downregulated during the development of the nervous system, Development 124 Ž1997. 1239–1249. w42x B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Ethanol and nerve growth factor effects on calcium homeostasis in cultured embryonic rat medial septal neurons before and during depolarization, Brain Res. 701 Ž1995. 61–74. w43x J.R. West, W.J.A. Chen, N.J. Pantazis, Fetal alcohol syndrome: the vulnerability of the developing brain and possible mechanisms of damage, Metabol. Brain Dis. 9 Ž1994. 291–323. w44x E. Yang, J. Zha, J. Jockel, L. Boise, C. Thompson, S. Korsmeyer, Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death, Cell 80 Ž1995. 285–291. w45x J. Yang, X. Liu, K.C.N. Bhalla, A.M. Ibrado, J. Cai, T.-I. Peng, D.P. Jones, X. Wang, Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked, Science 275 Ž1997. 1129–1132. w46x H.S. Zanjani, M.W. Vogel, N. Delhaye-Bouchaud, J.C. Martinou, J. Mariani, Increased cerebellar Purkinje cell numbers in mice overexpressing a human bcl-2 transgene, J. Comp. Neurol. 374 Ž1996. 332–341. w47x L.T. Zhong, T. Sarafian, D.J. Kane, A.C. Charles, S.P. Mah, R.H. Edwards, D.E. Bredesen, Bcl-2 inhibits death of central neural cells induced by multiple agents, P.N.A.S. 90 Ž1993. 4533–4537. w48x H. Zou, W.J. Henzel, X. Liu, A. Lutsch, X. Wang, Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3, Cell 90 Ž1997. 405– 413.