Title: Ethanol Impairs Insulin-stimulated Neuronal Survival in the Developing Brain
Abstract: Gestational exposure to ethanol causes fetal alcohol syndrome, which is associated with cerebellar hypoplasia. Previous in vitro studies demonstrated ethanol-impaired neuronal survival with reduced signaling through the insulin receptor (IRβ). We examined insulin signaling in an experimental rat model of chronic gestational exposure to ethanol in which the pups exhibited striking cerebellar hypoplasia with increased apoptosis. Immunoprecipitation and Western blot analyses detected reduced levels of tyrosyl-phosphorylated IRβ, tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1), and p85-associated IRS-1 but no alterations in IRβ, IRS-1, or p85 protein expression in cerebellar tissue from ethanol-exposed pups. In addition, ethanol exposure significantly reduced the levels of total phosphoinositol 3-kinase, Akt kinase, phospho-BAD (inactive), and glyceraldehyde-3-phosphate dehydrogenase and increased the levels of glycogen synthase kinase-3 activity, activated BAD, phosphatase and tensin homolog deleted in chromosome 10 (PTEN) protein, and PTEN phosphatase activity in cerebellar tissue. Cerebellar neurons isolated from ethanol-exposed pups had reduced levels of insulin-stimulated phosphoinositol 3-kinase and Akt kinase activities and reduced insulin inhibition of PTEN and glycogen synthase kinase-3 activity. The results demonstrate that cerebellar hypoplasia produced by chronic gestational exposure to ethanol is associated with impaired survival signaling through insulin-regulated pathways, including failure to suppress PTEN function. Gestational exposure to ethanol causes fetal alcohol syndrome, which is associated with cerebellar hypoplasia. Previous in vitro studies demonstrated ethanol-impaired neuronal survival with reduced signaling through the insulin receptor (IRβ). We examined insulin signaling in an experimental rat model of chronic gestational exposure to ethanol in which the pups exhibited striking cerebellar hypoplasia with increased apoptosis. Immunoprecipitation and Western blot analyses detected reduced levels of tyrosyl-phosphorylated IRβ, tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1), and p85-associated IRS-1 but no alterations in IRβ, IRS-1, or p85 protein expression in cerebellar tissue from ethanol-exposed pups. In addition, ethanol exposure significantly reduced the levels of total phosphoinositol 3-kinase, Akt kinase, phospho-BAD (inactive), and glyceraldehyde-3-phosphate dehydrogenase and increased the levels of glycogen synthase kinase-3 activity, activated BAD, phosphatase and tensin homolog deleted in chromosome 10 (PTEN) protein, and PTEN phosphatase activity in cerebellar tissue. Cerebellar neurons isolated from ethanol-exposed pups had reduced levels of insulin-stimulated phosphoinositol 3-kinase and Akt kinase activities and reduced insulin inhibition of PTEN and glycogen synthase kinase-3 activity. The results demonstrate that cerebellar hypoplasia produced by chronic gestational exposure to ethanol is associated with impaired survival signaling through insulin-regulated pathways, including failure to suppress PTEN function. Ethanol exposure during development is one of the leading causes of mental retardation in Europe and North America. Heavy gestational exposure to ethanol can cause fetal alcohol syndrome, which encompasses a broad array of neurologic and systemic lesions including central nervous system (CNS) malformations such as microencephaly, reduced cerebral white matter volume, ventriculomegaly, cerebellar hypoplasia, and disorders of neuronal migration (1Clarren S.K. Alvord E.J. Sumi S.M. Streissguth A.P. Smith D.W. J. Pediatr. 1978; 92: 64-67Abstract Full Text PDF PubMed Scopus (538) Google Scholar). Experimental models of fetal alcohol syndrome have demonstrated that the accompanying CNS abnormalities are associated with impaired neuronal survival, growth, synaptogenesis, maturation, neurotransmitter function, and intracellular adhesion (2Maier S.E. West J.R. Alcohol. 2001; 23: 49-57Crossref PubMed Scopus (139) Google Scholar, 3Minana R. Climent E. Barettino D. Segui J.M. Renau-Piqueras J. Guerri C. J. Neurochem. 2000; 75: 954-964Crossref PubMed Scopus (92) Google Scholar, 4Olney J.W. Ishimaru M.J. Bittigau P. Ikonomidou C. Apoptosis. 2000; 5: 515-521Crossref PubMed Scopus (113) Google Scholar, 5Swanson D.J. King M.A. Walker D.W. Heaton M.B. Alcohol Clin. Exp. Res. 1995; 19: 1252-1260Crossref PubMed Scopus (42) Google Scholar, 6Yanni P.A. Lindsley T.A. Brain Res. Dev. Brain Res. 2000; 120: 233-243Crossref PubMed Scopus (63) Google Scholar, 7Liesi P. J. Neurosci. Res. 1997; 48: 439-448Crossref PubMed Scopus (92) Google Scholar). Even with shorter durations and lower levels of exposure, ethanol can be neurotoxic during development and substantially reduce the populations of CNS neurons (2Maier S.E. West J.R. Alcohol. 2001; 23: 49-57Crossref PubMed Scopus (139) Google Scholar). Previous experiments demonstrated that neuronal loss following ethanol exposure was mediated by apoptosis (8Ikonomidou C. Bittigau P. Ishimaru M.J. Wozniak D.F. Koch C. Genz K. Price M.T. Stefovska V. Horster F. Tenkova T. Dikranian K. Olney J.W. Science. 2000; 287: 1056-1060Crossref PubMed Scopus (1203) Google Scholar, 9Zhang F.X. Rubin R. Rooney T.A. J. Neurochem. 1998; 71: 196-204Crossref PubMed Scopus (108) Google Scholar, 10de La Monte S.M. Wands J.R. Alcohol Clin. Exp. Res. 2001; 25: 898-906Crossref PubMed Google Scholar) or mitochondrial dysfunction (10de La Monte S.M. Wands J.R. Alcohol Clin. Exp. Res. 2001; 25: 898-906Crossref PubMed Google Scholar, 11de la Monte S.M. Wands J.R. Cell. Mol. Life Sci. 2002; 59: 882-893Crossref PubMed Scopus (106) Google Scholar, 12Ramachandran V. Perez A. Chen J. Senthil D. Schenker S. Henderson G.I. Alcohol Clin. Exp. Res. 2001; 25: 862-871Crossref PubMed Scopus (148) Google Scholar), and recent studies correlated these adverse effects of ethanol to inhibition of growth factor-stimulated survival signaling (9Zhang F.X. Rubin R. Rooney T.A. J. Neurochem. 1998; 71: 196-204Crossref PubMed Scopus (108) Google Scholar, 11de la Monte S.M. Wands J.R. Cell. Mol. Life Sci. 2002; 59: 882-893Crossref PubMed Scopus (106) Google Scholar, 12Ramachandran V. Perez A. Chen J. Senthil D. Schenker S. Henderson G.I. Alcohol Clin. Exp. Res. 2001; 25: 862-871Crossref PubMed Scopus (148) Google Scholar, 13de la Monte S.M. Neely T.R. Cannon J. Wands J.R. Cell. Mol. Life Sci. 2001; 58: 1950-1960Crossref PubMed Scopus (66) Google Scholar). In the developing CNS, insulin and insulin-like growth factor type 1 (IGF-1) 1The abbreviations used are: IGF-1, insulin-like growth factor type 1; IRS-1, insulin receptor substrate-1; PY, tyrosyl-phosphorylated; PI3 kinase, phosphatidylinositol 3-kinase; GSK-3, glycogen synthase kinase-3; P, postnatal day; TUNEL, terminal transferase dUTP endlabeling; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; MOPS, 4-morpholinepropanesulfonic acid; RT, reversetranscribed; IRβ, insulin receptor β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p, phospho.1The abbreviations used are: IGF-1, insulin-like growth factor type 1; IRS-1, insulin receptor substrate-1; PY, tyrosyl-phosphorylated; PI3 kinase, phosphatidylinositol 3-kinase; GSK-3, glycogen synthase kinase-3; P, postnatal day; TUNEL, terminal transferase dUTP endlabeling; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; MOPS, 4-morpholinepropanesulfonic acid; RT, reversetranscribed; IRβ, insulin receptor β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p, phospho. receptors are abundantly expressed (14Goodyer C.G. De S.L. Lai W.H. Guyda H.J. Posner B.I. Endocrinology. 1984; 114: 1187-1195Crossref PubMed Scopus (169) Google Scholar, 15Gammeltoft S. Fehlmann M. Van O.E. Biochimie (Paris). 1985; 67: 1147-1153Crossref PubMed Scopus (40) Google Scholar, 16Hill J.M. Lesniak M.A. Pert C.B. Roth J. Neuroscience. 1986; 17: 1127-1138Crossref PubMed Scopus (328) Google Scholar), and the corresponding growth factor-stimulated responses are critical mediators of neuronal growth, viability, energy metabolism, and synapse formation. Because insulin and IGF-1 signaling pathways are among the important targets of ethanol neurotoxicity in immature nervous system (9Zhang F.X. Rubin R. Rooney T.A. J. Neurochem. 1998; 71: 196-204Crossref PubMed Scopus (108) Google Scholar, 13de la Monte S.M. Neely T.R. Cannon J. Wands J.R. Cell. Mol. Life Sci. 2001; 58: 1950-1960Crossref PubMed Scopus (66) Google Scholar, 17de la Monte S.M. Ganju N. Banerjee K. Brown N.V. Luong T. Wands J.R. Alcohol Clin. Exp. Res. 2000; 24: 716-726Crossref PubMed Google Scholar, 18Hallak H. Seiler A.E. Green J.S. Henderson A. Ross B.N. Rubin R. Alcohol Clin. Exp. Res. 2001; 25: 1058-1064Crossref PubMed Scopus (37) Google Scholar), neuronal loss associated with microencephaly in ethanol-exposed fetuses may be caused, in part, by ethanol inhibition of insulin/IGF-1stimulated survival mechanisms. The stimulatory effects of insulin and IGF-1 are mediated through complex pathways, beginning with ligand binding and activation of intrinsic receptor tyrosine kinases (19Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokawa M. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1512) Google Scholar, 20O'Hare T. Pilch P.F. Int. J. Biochem. 1990; 22: 315-324Crossref PubMed Scopus (24) Google Scholar), which phosphorylate specific cytosolic molecules, including two of their major substrates, the insulin receptor substrate types 1 (IRS-1) and 2 (IRS-2) (21Myers M.G. Sun X.J. White M.F. Trends Biochem. Sci. 1994; 19: 289-293Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 22Shpakov A.O. Pertseva M.N. Membr. Cell Biol. 2000; 13: 455-484PubMed Google Scholar). Tyrosyl-phosphorylated IRS-1 (PY-IRS-1) transmits intracellular signals that mediate growth, metabolic functions, and viability by interacting with downstream Src homology 2-containing molecules through specific motifs located in the C-terminal region of IRS-1 (21Myers M.G. Sun X.J. White M.F. Trends Biochem. Sci. 1994; 19: 289-293Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 22Shpakov A.O. Pertseva M.N. Membr. Cell Biol. 2000; 13: 455-484PubMed Google Scholar). The 897YVNI motif of IRS-1 binds to the Grb2 (growth factor receptor-bound protein 2) adapter molecule (23Skolnik E.Y. Batzer A. Li N. Lee C.H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (503) Google Scholar, 24Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J.K. Chawla A. Banerjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (232) Google Scholar). The 1180YIDL motif binds to Syp protein tyrosine phosphatase, and the 613YMPM and 942YMKM motifs bind to the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) (25Sun X.J. Crimmins D.L. Myers M.J. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). Binding of PY-IRS-1 to p85 stimulates glucose transport (26Lam K. Carpenter C.L. Ruderman N.B. Friel J.C. Kelly K.L. J. Biol. Chem. 1994; 269: 20648-20652Abstract Full Text PDF PubMed Google Scholar) and inhibits apoptosis by activating Akt/protein kinase B (27Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (792) Google Scholar, 28Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1875) Google Scholar) or inhibiting glycogen synthase kinase-3 (GSK-3) (29Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). Akt kinase inhibits apoptosis by phosphorylating GSK-3 (29Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar, 30van Weeren P.C. de Bruyn K.M. de Vries-Smits A.M. van Lint J. Burgering B.M. J. Biol. Chem. 1998; 273: 13150-13156Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and BAD (31Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4920) Google Scholar), rendering them inactive. Low levels of Akt kinase and high levels of GSK-3 activity or activated BAD are associated with increased neuronal death (32Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar, 33Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2215) Google Scholar, 34Eves E.M. Xiong W. Bellacosa A. Kennedy S.G. Tsichlis P.N. Rosner M.R. Hay N. Mol. Cell. Biol. 1998; 18: 2143-2152Crossref PubMed Scopus (175) Google Scholar). BAD inactivates anti-apoptotic Bcl family proteins, rendering the mitochondrial membrane more susceptible to pro-apoptotic molecules that promote membrane permeabilization, cytochrome c release, and caspase activation (35Condorelli F. Salomoni P. Cotteret S. Cesi V. Srinivasula S.M. Alnemri E.S. Calabretta B. Mol. Cell. Biol. 2001; 21: 3025-3036Crossref PubMed Scopus (109) Google Scholar). Perturbations in mitochondrial membrane permeability increase cellular free radicals that cause mitochondrial DNA damage, impair mitochondrial function, and activate pro-apoptosis cascades (36Halestrap A.P. Doran E. Gillespie J.P. O'Toole A. Biochem. Soc. Trans. 2000; 28: 170-177Crossref PubMed Scopus (283) Google Scholar, 37Hirsch T. Susin S.A. Marzo I. Marchetti P. Zamzami N. Kroemer G. Cell Biol. Toxicol. 1998; 14: 141-145Crossref PubMed Scopus (120) Google Scholar). Our previous studies (11de la Monte S.M. Wands J.R. Cell. Mol. Life Sci. 2002; 59: 882-893Crossref PubMed Scopus (106) Google Scholar, 13de la Monte S.M. Neely T.R. Cannon J. Wands J.R. Cell. Mol. Life Sci. 2001; 58: 1950-1960Crossref PubMed Scopus (66) Google Scholar) using in vitro or in vivo models demonstrated that ethanol profoundly inhibits insulin-stimulated survival and mitochondrial function in cultured neuronal cells. The present study was designed to examine the effects of chronic gestational exposure to ethanol on insulin-, IRS-1-, and PI3 kinase-mediated signaling in the intact brain to determine the extent to which abnormalities in these pathways correlate with ethanol-induced developmental defects in the CNS. PTEN expression and activity were also examined, because PTEN dephosphorylates and negatively regulates PI3 kinase function (38Dahia P.L. Aguiar R.C. Alberta J. Kum J.B. Caron S. Sill H. Marsh D.J. Ritz J. Freedman A. Stiles C. Eng C. Hum. Mol. Genet. 1999; 8: 185-193Crossref PubMed Scopus (266) Google Scholar), and the effects of ethanol on phosphatase and tensin homolog deleted in chromosome 10 (PTEN) were unknown. Cerebellar tissue was studied, because it represents a major in vivo target of ethanol neurotoxicity (2Maier S.E. West J.R. Alcohol. 2001; 23: 49-57Crossref PubMed Scopus (139) Google Scholar, 39Mohamed S.A. Nathaniel E.J. Nathaniel D.R. Snell L. Exp. Neurol. 1987; 97: 35-52Crossref PubMed Scopus (28) Google Scholar). In Vivo Model of Chronic Ethanol Exposure—Long-Evans female rats were adapted to an ethanol-containing or isocaloric control liquid diet (BioServ, Frenchtown, NJ) over a 3-week interval, after which they were mated with normal males. Ethanol comprised 11.8, 23.6, and 35.4% of the caloric content of the feedings during the first, second, and third weeks of adaptation. The 35.4% ethanol-containing or control diets were maintained throughout pregnancy. Using this protocol, the serum ethanol concentrations in the rats ranged from 25 to 43 mm, which is within the range observed in human disease states (40Urso T. Gavaler J.S. Van T.D. Life Sci. 1981; 28: 1053-1056Crossref PubMed Scopus (210) Google Scholar). Rats were monitored daily to ensure equivalent caloric consumption and maintenance of body weight. Typically, in the ethanol-fed group, the litter sizes were reduced by 20%, and pup mean body weight was reduced by 10 to 15%. Studies were conducted with cerebella harvested from control and ethanol-exposed postnatal day 2 (P2) pups to evaluate the effects of ethanol in the early postnatal period and prior to the occurrence of any major compensatory developmental responses. Cerebellar tissue was fixed in Histochoice fixative (Amresco, Solon, OH) and embedded in paraffin. Histological sections were stained with hematoxylin and eosin to detect morphological abnormalities. Fresh cerebellar tissue was snap-frozen in liquid nitrogen and stored at –80 °C for use in protein studies, assays of kinase or phosphatase activity, and measurement of PTEN mRNA levels. In Situ Assays for Apoptosis—The terminal transferase dUTP end-labeling (TUNEL) assay was used to detect nicked or fragmented DNA in cryostat sections of cerebella. TUNEL assays were performed using fluorescein-labeled dUTP ([Fl]dUTP; Invitrogen) and terminal deoxynucleotide transferase (17de la Monte S.M. Ganju N. Banerjee K. Brown N.V. Luong T. Wands J.R. Alcohol Clin. Exp. Res. 2000; 24: 716-726Crossref PubMed Google Scholar). The labeled DNA was detected with biotinylated secondary antibody, alkaline phosphatase-conjugated Streptavidin, and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as the substrate. To detect nuclear pyknosis and fragmentation characteristic of apoptosis, adjacent sections were stained with Hoechst H33258 (1 μg/ml in phosphate-buffered saline) for 2 min at room temperature. The slides were rinsed thoroughly in phosphate-buffered saline, cover-slipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and examined by fluorescence microscopy. Adjacent sections were immunostained with polyclonal antibodies to activated (cleaved) caspase-3 (Cell Signaling, Beverly, MA). Histological sections were prepared for immunostaining according to the manufacturer's protocol. Immunoreactivity was detected with biotinylated secondary antibody, alkaline phosphatase-conjugated Streptavidin, and the BCIP/NBT substrate. Western Blot Analysis and Immunoprecipitation Studies—For Western blot analysis, tissue from individual cerebella were polytron homogenized in radioimmunoprecipitation assay buffer (50 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 2 mm EGTA) containing protease and phosphatase inhibitors (1 mm NaF, 1 mm Na4P2O7, 2 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, pepstatin A, and leupeptin) (41Bonifacino J.S. Dell'Angelica E.C. Springer T.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, New York1988: 10.16.1-10.16.29Google Scholar). For immunoprecipitations, homogenates were prepared in Triton lysis buffer (50 mm Tris-HCl, pH 7.5, 10 mm EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors as indicated. Cellular debris was pelleted by centrifuging the samples at 14,000 × g for 15 min at 4 °C, and supernatant fractions were used in the studies. Protein concentration was measured with the bis-chloracetate (BCA) assay (Pierce). 60- or 100-μg protein aliquots were used for Western blot analysis (17de la Monte S.M. Ganju N. Banerjee K. Brown N.V. Luong T. Wands J.R. Alcohol Clin. Exp. Res. 2000; 24: 716-726Crossref PubMed Google Scholar, 42Banerjee K. Mohr L. Wands J.R. de la Monte S.M. Alcohol Clin. Exp. Res. 1998; 22: 2093-2101Crossref PubMed Google Scholar, 43de la Monte S.M. Ganju N. Tanaka S. Banerjee K. Karl P.J. Brown N.V. Wands J.R. Alcohol Clin. Exp. Res. 1999; 23: 770-777Crossref PubMed Google Scholar), and 500-μg samples were used for immunoprecipitation/Western immunoblotting or kinase assays (11de la Monte S.M. Wands J.R. Cell. Mol. Life Sci. 2002; 59: 882-893Crossref PubMed Scopus (106) Google Scholar, 17de la Monte S.M. Ganju N. Banerjee K. Brown N.V. Luong T. Wands J.R. Alcohol Clin. Exp. Res. 2000; 24: 716-726Crossref PubMed Google Scholar). Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibody and SuperSignal enhanced chemiluminescence reagents (Pierce). Immunoreactivity was quantified using the Eastman Kodak Co. Digital Science Imaging Station (PerkinElmer Life Sciences). Kinase Assays—PI3 kinase activity was measured in p85 immunoprecipitates (42Banerjee K. Mohr L. Wands J.R. de la Monte S.M. Alcohol Clin. Exp. Res. 1998; 22: 2093-2101Crossref PubMed Google Scholar) obtained from individual 500-μg protein samples using rabbit polyclonal anti-p85 (1 μg/ml) and protein A-Sepharose (Amersham Biosciences). Immunoprecipitates complexed with protein A-Sepharose were suspended in 50 μl of TNE buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA), and reactions were initiated by sequentially adding 20 μg of sonicated phosphatidylinositol (10 μl), 10 μl of 100 mm MgCl2, and 5 μl of [γ-32P]ATP working solution composed of 0.88 mm [γ-32P]ATP (30 μCi of [γ-32P]ATP/3000 Ci/mmol), 20 mm MgCl2, and 150 mm cold ATP. Reactions were incubated for 10 min at 37 °C and 300 rpm and stopped by adding 15 μl of 6 n HCl. Phospholipids extracted with chloroform/methanol were analyzed by thin layer chromatography using plates pre-coated with 1% potassium oxalate (Merck). PI3 kinase activity was detected by film autoradiography and quantified using the Kodak Digital Science Imaging Station. To measure Akt and GSK-3β kinase activities, corresponding immunoprecipitates captured onto protein A-Sepharose beads (41Bonifacino J.S. Dell'Angelica E.C. Springer T.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, New York1988: 10.16.1-10.16.29Google Scholar) were suspended in 10 μl of 5× assay dilution buffer (ADB; 100 mm MOPS, pH 7.2, 125 mm β-glycerol phosphate, 5 mm EGTA, 5 mm sodium orthovanadate, and 5 mm dithiothreitol). Reactions were performed by sequentially adding 10 μl each of Mg2+/ATP mixture (100 mm non-radioactive ATP, 75 mm MgCl2 in ADB), [γ-32P]ATP (diluted to a final concentration of 1 μCi/μl using Mg2+/ATP mixture), and 10 nm synthetic peptide substrate. Crosstide (Upstate Biotechnology, Inc., Lake Placid, NY) was used to measure Akt activity, and cAMP-response element-binding protein (New England Biolabs, Beverly, MA) was used as the substrate for GSK-3β (Upstate Biotechnology). Reactions were incubated for 10 (GSK-3) or 15 (Akt) min at 30 °C and 300 rpm and then terminated by adding 5 μl of 0.5 m EDTA. 10 μl of each reaction were spotted in duplicate onto P81 phosphocellulose. Nonspecific counts were removed by washing the P81 three times in 0.85% phosphoric acid (41Bonifacino J.S. Dell'Angelica E.C. Springer T.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley, New York1988: 10.16.1-10.16.29Google Scholar) followed by 95% ethanol. [γ-32P]ATP incorporation was measured in a TopCount machine (Packard Instrument Co., Meriden, CT). PTEN Studies—PTEN expression was measured by Western blot analysis and real-time quantitative reverse-transcribed (RT)-PCR assays. In addition, PTEN phosphatase activity was measured in PTEN immunoprecipitates using the Biomol Green Reagent according to the manufacturer's protocol. Phosphatase inhibitors were omitted from the lysis buffer. For the real-time RT-PCR studies, total RNA was isolated from cerebellar tissue homogenates using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Samples containing 2 μg of RNA were reverse-transcribed using the AMV First Strand cDNA synthesis kit (Roche Molecular Biochemicals) and random oligodeoxynucleotide primers. Highly conserved regions of PTEN and 18 S cDNAs isolated from rat cerebellar tissue by RT-PCR were cloned into the PCRII vector (Invitrogen) and used to generate standard curves for determining transcript abundance. PCR amplifications were performed using 25-μl reaction volumes containing 20 ng of RT product, 0.4 μm each of forward and reverse primers (Table I), and SYBR Green I PCR reagent (Applied Biosystems, Foster, CA). The amplified signals were detected continuously with the Bio-Rad iCycler and iCycler iQ MultiColor Real Time PCR Detection System (Hercules, CA). The following real-time PCR amplification protocol was used: 1) initial denaturation, 95 °C for 10 min; 2) a three-segment amplification and quantification program consisting of 40 cycles of 95 °C × 60 s, 60 °C × 45 s and 72 °C for 30 s; and 3) a cooling step down to 4 °C.Table IForward (for) and reverse (rev) primers used for PTEN and 18 S RT-PCR amplificationPrimerSequence (5′ → 3′)Position (nucleotide)PTEN forGGA AAG GAC GGA CTG GTG TA380PTEN revTGC CAC TGG TCT GTA ATC CA55718 S forGGA CCA GAG CGA AAG CAT98518 S revTCA ATC TCG GGT GGC TGA A1462 Open table in a new tab In Vitro Experiments—In vitro experiments were used to examine the effects of ethanol on PTEN expression, phosphorylation, and phosphatase activity in CNS neurons and to determine whether the responses observed in vivo were mediated by impaired insulin or IGF-1 signaling. Primary neuronal cultures were generated with cerebellar tissue harvested from P2 pups (44Nikolic M. Dudek H. Kwon Y.T. Ramos Y.F. Tsai L.H. Genes Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar). Fluorescence-activated cell sorting demonstrated that greater than 95% of cells isolated from control or ethanol-exposed pup cerebella were neuronal as evidenced by the immunoreactivity with antibodies to HuC/HuD neuron-specific RNA binding protein (45Szabo A. Dalmau J. Manley G. Rosenfeld M. Wong E. Henson J. Posner J.B. Furneaux H.M. Cell. 1991; 67: 325-333Abstract Full Text PDF PubMed Scopus (515) Google Scholar) (Molecular Probes, Eugene, OR). Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 2 mm glutamine, 10 mm non-essential amino acid mixture (Invitrogen), 25 mm KCl, and 9 g/liter glucose. After overnight seeding, cells were treated with 6 μm cytosine arabinoside to inhibit DNA synthesis. Cultures were exposed to 50 mm ethanol or nothing for 2 days using sealed humidified chambers (17de la Monte S.M. Ganju N. Banerjee K. Brown N.V. Luong T. Wands J.R. Alcohol Clin. Exp. Res. 2000; 24: 716-726Crossref PubMed Google Scholar, 42Banerjee K. Mohr L. Wands J.R. de la Monte S.M. Alcohol Clin. Exp. Res. 1998; 22: 2093-2101Crossref PubMed Google Scholar), after which they were serum-starved for 12 h and then stimulated with 50 nm insulin (Humulin; Eli Lilly & Co., Indianapolis, IN) or 25 nm IGF-1 for 0, 15, 30, 60, or 120 min in the presence or absence of 50 mm ethanol. PTEN protein was detected by Western blot analysis. PTEN phosphorylation was evaluated by PTEN Western blot analysis of anti-phospho-serine/phospho-threonine immunoprecipitates. PTEN phosphatase activity was measured in PTEN immunoprecipitates using the Biomol Green Reagent (Cell Signaling, Beverly, MA). To examine the effects of ethanol on insulin-stimulated viability and kinase activity, primary neuronal cultures were generated with cerebella harvested from control or ethanol-exposed P1 pups. However, to mimic the in vivo model in which the ethanol exposure was discontinued after birth, the cultured cells were not further exposed to ethanol. Micro-cultures (96-well plates) in which 5 × 104 viable cells (determined by Trypan Blue exclusion) were seeded per well were used for viability assays, and 60-mm Petri dish cultures were used for kinase or PTEN phosphatase assays. To measure insulin-stimulated responses, after 1 day in culture, the cells were serum-starved for 12 h and then stimulated with 50 nm insulin (Humulin; Eli Lilly & Co., Indianapolis, IN). Viability was measured after 24 h of insulin stimulation using the crystal violet assay (13de la Monte S.M. Neely T.R. Cannon J. Wands J.R. Cell. Mol. Life Sci. 2001; 58: 1950-1960Crossref PubMed Scopus (66) Google Scholar, 17de la Monte S.M. Ganju N. Banerjee K. Brown N.V. Luong T. Wands J.R. Alcohol Clin. Exp. Res. 2000; 24: 716-726Crossref PubMed Google Scholar, 42Banerjee K. Mohr L. Wands J.R. de la Monte S.M. Alcohol Clin. Exp. Res. 1998; 22: 2093-2101Crossref PubMed Google Scholar). IRS-1-associated PI3 kinase, total PI3 kinase, Akt kinase, GSK-3 kinase, and PTEN phosphatase activities were measured in corresponding immunoprecipitates from cells stimulated with insulin for 0, 5, 15, or 30 min. Source of Reagents—Monoclonal antibodies to phospho-tyrosine (PY20) and phospho-serine were purchased from Transduction Laboratories (Lexington, KY). Polyclonal antibodies to p85, IRβ, IRS-1, and PTEN phosphatase were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Phospho-specific antibodies to Akt, GSK-3β, and BAD were obtained from Cell Signaling (Beverly, MA). Protein A-Sepharose was purchased from Amersham Biosciences. Monoclonal antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and GSK-3β were purchased from Chemicon Corp. All other reagents were purchased from CalBiochem or Sigma-Aldrich. Statistical Analysis—Data depicted in the graphs represent the mean ± S.D. of results. Inter-group comparisons were made with the Student's t test or analysis of variance and the Fisher least significance post hoc significance test using Number Cruncher Statistical Systems (Dr. Jerry L. Hintze, Kaysville, UT). Ethanol-induced Cerebellar Hypoplasia—Histological studies of the cerebellar tissue revealed well developed folia and distinct laminar architecture in control pups (Fig. 1A, C, and E) and hypoplasia with marked simplification of the folia and poor lamination of the cortex in ethanol-exposed pups (Fig. 1, B, D, and F). Adjacent sections stained with H33258 revealed uniform nuclear morphology and only occasional apoptotic bodies in contro