Dynamics of membrane lipid domains in neuronal cells differentiated in culture
2003; Elsevier BV; Volume: 44; Issue: 11 Linguagem: Inglês
10.1194/jlr.m300247-jlr200
ISSN1539-7262
AutoresElena Ottico, Alessandro Prinetti, Simona Prioni, Claudia Giannotta, Luisa Basso, Vanna Chigorno, Sandro Sonnino,
Tópico(s)Cellular transport and secretion
ResumoTreatment with methyl-β-cyclodextrin (MCD) induced a time- and dose-dependent efflux of cholesterol, sphingolipids, and phosphatidylcholine (PC) from cerebellar neurons differentiated in culture. With a "mild" treatment, the loss of cell lipids induced a deep reorganization of the remaining membrane lipids. In fact, the amount of PC associated with a Triton X-100-insoluble membrane fraction (highly enriched in sphingolipids and cholesterol in nontreated cells) was lowered by the treatment. This suggested a reduction of the lipid domain area. However, the cholesterol and sphingolipid enrichment of this fraction remained substantially unchanged, suggesting the existence of dynamic processes aimed at preserving the segregation of cholesterol and sphingolipids in membrane domains. Under these conditions, the lipid membrane domains retained the ability to sort signaling proteins, such as Lyn and c-Src, but cells displayed deep alterations in their membrane permeability. However, normal membrane permeability was restored by loading cells with cholesterol. When MCD treatment was more stringent, a large loss of cell lipids occurred, and the lipid domains were much less enriched in cholesterol and lost the ability to sort specific proteins.The loss of the integrity and properties of lipid domains was accompanied by severe changes in the membrane permeability, distress, and eventually cell death. Treatment with methyl-β-cyclodextrin (MCD) induced a time- and dose-dependent efflux of cholesterol, sphingolipids, and phosphatidylcholine (PC) from cerebellar neurons differentiated in culture. With a "mild" treatment, the loss of cell lipids induced a deep reorganization of the remaining membrane lipids. In fact, the amount of PC associated with a Triton X-100-insoluble membrane fraction (highly enriched in sphingolipids and cholesterol in nontreated cells) was lowered by the treatment. This suggested a reduction of the lipid domain area. However, the cholesterol and sphingolipid enrichment of this fraction remained substantially unchanged, suggesting the existence of dynamic processes aimed at preserving the segregation of cholesterol and sphingolipids in membrane domains. Under these conditions, the lipid membrane domains retained the ability to sort signaling proteins, such as Lyn and c-Src, but cells displayed deep alterations in their membrane permeability. However, normal membrane permeability was restored by loading cells with cholesterol. When MCD treatment was more stringent, a large loss of cell lipids occurred, and the lipid domains were much less enriched in cholesterol and lost the ability to sort specific proteins. The loss of the integrity and properties of lipid domains was accompanied by severe changes in the membrane permeability, distress, and eventually cell death. A number of data deriving from very heterogeneous research areas and experimental approaches, including biochemical analysis, optical and electron immunomicroscopy, atom force microscopy, and single particle tracking [as reviewed in ref. (1Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5112) Google Scholar)], led to the notion that membrane complex lipids are not randomly distributed within a cell membrane but form lipid domains, where sphingolipids and cholesterol are segregated in dipalmitoylphosphatidylcholine-rich membrane areas. These domains are involved in the trafficking and sorting of specific proteins, as well as in signal transduction processes (2Hakomori S. Handa K. Iwabuchi K. Yamamura S. Prinetti A. New insights in glycosphingolipid function: "glycosignaling domain," a cell surface assembly of glycosphingolipids with signal transducer molecules, involved in cell adhesion coupled with signaling.Glycobiology. 1998; 8: xi-xixCrossref PubMed Scopus (186) Google Scholar, 3Brown D.A. London E. Functions of lipid rafts in biological membranes.Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2542) Google Scholar). The lipid domain composition and the machinery of signal transduction were extensively studied in neurons (4Prinetti A. Iwabuchi K. Hakomori S. Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.J. Biol. Chem. 1999; 274: 20916-20924Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 5Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. Functionally different GPI proteins are organized in different domains on the neuronal surface.EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (331) Google Scholar, 6Bilderback T.R. Gazula V.R. Lisanti M.P. Dobrowsky R.T. Caveolin interacts with Trk A and p75NTR and regulates neurotrophin signaling pathways.J. Biol. Chem. 1999; 274: 257-263Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Prinetti A. Marano N. Prioni S. Chigorno V. Mauri L. Casellato R. Tettamanti G. Sonnino S. Association of Src-family protein tyrosine kinases with sphingolipids in rat cerebellar granule cells differentiated in culture.Glycoconj. J. 2000; 17: 223-232Crossref PubMed Scopus (40) Google Scholar, 8Prinetti A. Chigorno V. Tettamanti G. Sonnino S. Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 9Prinetti A. Chigorno V. Prioni S. Loberto N. Marano N. Tettamanti G. Sonnino S. Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.J. Biol. Chem. 2001; 276: 21136-21145Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 10Prinetti A. Prioni S. Chigorno S. Karagogeos D. Tettamanti G. Sonnino S. Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.J. Neurochem. 2001; 78: 1162-1167Crossref PubMed Scopus (67) Google Scholar, 11Loberto N. Prioni S. Prinetti A. Ottico E. Chigorno S. Karagogeos D. Sonnino S. The adhesion protein TAG-1 has a ganglioside environment in the sphingolipid-enriched membrane domains of neuronal cells in culture.J. Neurochem. 2003; 85: 224-233Crossref PubMed Scopus (33) Google Scholar, 12Prioni S. Loberto N. Prinetti A. Chigorno S. Guzzi F. Maggi R. Parenti M. Sonnino S. Sphingolipid metabolism and caveolin expression in gonadotropin-releasing hormone-expressing GN11 and gonadotropin-releasing hormone-secreting GT1–7 neuronal cells.Neurochem. Res. 2002; 27: 831-840Crossref PubMed Scopus (27) Google Scholar, 13Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. Caveolin interacts with Trk A and p75NTR and regulates neurotrophin signaling pathways.J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 14Kasahara K. Watanabe Y. Takeuchi K. Kaneko H. Ooohira A. Yamamoto T. Sanai Y. Association of Src family tyrosine kinase Lyn with ganglioside GD3 in rat brain. Possible regulation of Lyn by glycosphingolipid in caveolae-like domains.J. Biol. Chem. 2000; 275: 34701-34709Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In neurons and neuronal cell lines, many lines of evidence suggest that lipid membrane domains or multimolecular complexes localized within such domains are directly involved in the process of neuronal differentiation. As a well-documented example, in cerebellar neurons spontaneously differentiating in culture, neuronal differentiation is accompanied by a marked increase in the membrane area occupied by these domains and by deep changes in their lipid composition (8Prinetti A. Chigorno V. Tettamanti G. Sonnino S. Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 9Prinetti A. Chigorno V. Prioni S. Loberto N. Marano N. Tettamanti G. Sonnino S. Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.J. Biol. Chem. 2001; 276: 21136-21145Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In particular, lipid membrane domains from fully differentiated neurons bear the highest sphingolipid density and content with respect to undifferentiated and senescent cells, while cholesterol relative content steadily and markedly decreases with in vitro development (9Prinetti A. Chigorno V. Prioni S. Loberto N. Marano N. Tettamanti G. Sonnino S. Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.J. Biol. Chem. 2001; 276: 21136-21145Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Particularly significant seem to be the interactions of sphingolipids with protein kinases of the Src family, probably the most typical protein family localized in lipid domains, as well as with other proteins implicated in the Src signaling cassette (4Prinetti A. Iwabuchi K. Hakomori S. Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.J. Biol. Chem. 1999; 274: 20916-20924Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Prinetti A. Marano N. Prioni S. Chigorno V. Mauri L. Casellato R. Tettamanti G. Sonnino S. Association of Src-family protein tyrosine kinases with sphingolipids in rat cerebellar granule cells differentiated in culture.Glycoconj. J. 2000; 17: 223-232Crossref PubMed Scopus (40) Google Scholar, 8Prinetti A. Chigorno V. Tettamanti G. Sonnino S. Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 9Prinetti A. Chigorno V. Prioni S. Loberto N. Marano N. Tettamanti G. Sonnino S. Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.J. Biol. Chem. 2001; 276: 21136-21145Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 10Prinetti A. Prioni S. Chigorno S. Karagogeos D. Tettamanti G. Sonnino S. Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.J. Neurochem. 2001; 78: 1162-1167Crossref PubMed Scopus (67) Google Scholar, 11Loberto N. Prioni S. Prinetti A. Ottico E. Chigorno S. Karagogeos D. Sonnino S. The adhesion protein TAG-1 has a ganglioside environment in the sphingolipid-enriched membrane domains of neuronal cells in culture.J. Neurochem. 2003; 85: 224-233Crossref PubMed Scopus (33) Google Scholar, 12Prioni S. Loberto N. Prinetti A. Chigorno S. Guzzi F. Maggi R. Parenti M. Sonnino S. Sphingolipid metabolism and caveolin expression in gonadotropin-releasing hormone-expressing GN11 and gonadotropin-releasing hormone-secreting GT1–7 neuronal cells.Neurochem. Res. 2002; 27: 831-840Crossref PubMed Scopus (27) Google Scholar, 13Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. Caveolin interacts with Trk A and p75NTR and regulates neurotrophin signaling pathways.J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 14Kasahara K. Watanabe Y. Takeuchi K. Kaneko H. Ooohira A. Yamamoto T. Sanai Y. Association of Src family tyrosine kinase Lyn with ganglioside GD3 in rat brain. Possible regulation of Lyn by glycosphingolipid in caveolae-like domains.J. Biol. Chem. 2000; 275: 34701-34709Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Interestingly, the lipid composition changes were accompanied by changes in the association of signaling molecules with the lipid membrane domain during neuronal cell differentiation (9Prinetti A. Chigorno V. Prioni S. Loberto N. Marano N. Tettamanti G. Sonnino S. Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.J. Biol. Chem. 2001; 276: 21136-21145Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Similarly, in the case of Neuro2a, neuroblastoma cell differentiation induced by treatment with exogenous gangliosides (likely able to artificially increase the ganglioside content of lipid membrane domains), activation of c-Src kinase, and dissociation of Csk from the lipid membrane domain were observed (4Prinetti A. Iwabuchi K. Hakomori S. Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.J. Biol. Chem. 1999; 274: 20916-20924Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). These findings suggest a direct link between the lipid composition of lipid membrane domains and their biological and functional properties as signaling units. However, the molecular basis of this link is poorly understood.A very promising experimental approach to a better understanding of the involvement of lipids in the structural and functional properties of membrane domains is represented by the artificial manipulation of lipid composition and/or organization within these structures. To this purpose, a wide variety of experimental approaches allowing the downregulation of the biosynthesis of membrane lipids (15Stevens V.L. Tang J. Fumonisin B1-induced sphingolipid depletion inhibits vitamin uptake via the glycosylphosphatidylinositol-anchored folate receptor.J. Biol. Chem. 1997; 272: 18020-18024Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 16Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. Sphingolipid depletion increases formation of the scrapie prion protein in neuroblastoma cells infected with prions.J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 17Sheets E.D. Lee G.M. Simson R. Jacobson K. Transient confinement of a glycosylphosphatidylinositol-anchored protein in the plasma membrane.Biochemistry. 1997; 36: 12449-12458Crossref PubMed Scopus (279) Google Scholar, 18Inokuchi J. Uemura S. Kabayama K. Igarashi Y. Glycosphingolipid deficiency affects functional microdomain formation in Lewis lung carcinoma cells.Glycoconj. J. 2000; 17: 239-246Crossref PubMed Scopus (30) Google Scholar, 19Taraboulos A. Scott M. Semenov A. Avraham D. Laszlo L. Prusiner S.B. Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform.J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (512) Google Scholar, 20Ledesma M.D. Simons K. Dotti C.G. Neuronal polarity: essential role of protein-lipid complexes in axonal sorting.Proc. Natl. Acad. Sci. USA. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar, 21Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons.Proc. Natl. Acad. Sci. USA. 1998; 95: 6460-6464Crossref PubMed Scopus (1076) Google Scholar) or the lowering of cholesterol levels in the plasma membrane by external ligands (20Ledesma M.D. Simons K. Dotti C.G. Neuronal polarity: essential role of protein-lipid complexes in axonal sorting.Proc. Natl. Acad. Sci. USA. 1998; 95: 3966-3971Crossref PubMed Scopus (199) Google Scholar, 21Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons.Proc. Natl. Acad. Sci. USA. 1998; 95: 6460-6464Crossref PubMed Scopus (1076) Google Scholar, 22Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. Intracellular trafficking of cholesterol monitored with a cyclodextrin.J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 23Furuchi T. Anderson R.G.W. Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase ERK.J. Biol. Chem. 1998; 273: 21099-21104Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 24Ilangumaran S. Hoessli D.C. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.Biochem. J. 1998; 335: 433-440Crossref PubMed Scopus (398) Google Scholar, 25Sheets E.D. Holowka D. Baird B. Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes.J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (286) Google Scholar, 26Fukasawa M. Nishijima M. Itabe H. Takano T. Hanada K. Reduction of sphingomyelin level without accumulation of ceramide in Chinese hamster ovary cells affects detergent-resistant membrane domains and enhances cellular cholesterol efflux to methyl-beta -cyclodextrin.J. Biol. Chem. 2000; 275: 34028-34034Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27Shogomori H. Futerman A.H. Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism.J. Biol. Chem. 2001; 276: 9182-9188Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 28Mendez A.J. Lin G. Wade D.P. Lawn R.M. Oram J.F. Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway.J. Biol. Chem. 2001; 276: 3158-3166Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) has been introduced. Among these tools, the extraction of cholesterol from cells by treatment with cyclodextrins is certainly the most widely used, as evidenced by the hundreds of papers found combining the two key words "cyclodextrin" and "cholesterol" in a Medline search from 1996 to the present. However, although the efflux of cholesterol mediated by cyclodextrin has been studied in detail, only limited information can be found regarding the effect of cyclodextrins on the possible removal of other complex lipids from living cells (24Ilangumaran S. Hoessli D.C. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.Biochem. J. 1998; 335: 433-440Crossref PubMed Scopus (398) Google Scholar, 25Sheets E.D. Holowka D. Baird B. Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes.J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (286) Google Scholar, 29Kilsdonk E.P.C. Yancey P.G. Stoudt G.W. Bangerter F.W. Johnson W.J. Phillips M.C. Rothblat G.H. Cellular cholesterol efflux mediated by cyclodextrins.J. Biol. Chem. 1995; 270: 17250-17256Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, 30Hansen G.H. Immerdal L. Thorsen E. Niels-Christiansen L-L. Nystr⊘m B.T. Demant E.J.F. Danielsen E.M. Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes.J. Biol. Chem. 2001; 276: 32338-32344Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). This is quite surprising, insofar as it is known that cyclodextrins are able to form complexes with sphingolipid monomers (31Singh I. Kishimoto Y. Effect of cyclodextrins on the solubilization of lignoceric acid, ceramide, and cerebroside, and on the enzymatic reactions involving these compounds.J. Lipid Res. 1983; 24: 662-665Abstract Full Text PDF PubMed Google Scholar, 32Casu B. Grenni A. Naggi A. Torri G. Virtuani M. Focher B. Interaction of cyclodextrinscyclomalto-oligosaccharides with glycolipids: N.M.R. studies of aqueous system of cyclomaltohexaose and alkyl glycosides.Carbohydr. Res. 1990; 200: 101-109Crossref Scopus (21) Google Scholar, 33Shiraishi T. Hiraiwa M. Uda Y. Effects of cyclodextrins on the hydrolysis of ganglioside GM1 by acid beta-galactosidases.Glycoconj. J. 1993; 10: 170-174Crossref PubMed Scopus (12) Google Scholar) and to disrupt ganglioside aggregates (32Casu B. Grenni A. Naggi A. Torri G. Virtuani M. Focher B. Interaction of cyclodextrinscyclomalto-oligosaccharides with glycolipids: N.M.R. studies of aqueous system of cyclomaltohexaose and alkyl glycosides.Carbohydr. Res. 1990; 200: 101-109Crossref Scopus (21) Google Scholar).In the present study, we analyzed the efflux of cholesterol, sphingolipids, and glycerophospholipids from cells and the changes in the composition of membrane lipid domains after treating rat cerebellar granule cells differentiated in culture with methyl-β-cyclodextrin (MCD). Our data provide a new scenario, in which the membrane lipid domains appear as dynamic structures whose existence strongly influences cell membrane properties.EXPERIMENTAL PROCEDURESMaterialsCommercial chemicals were the purest available, common solvents were distilled before use, and water was doubly distilled in a glass apparatus. MCD, trypsin, crystalline BSA, and several reagents for cell culture were purchased from Sigma Chemicals. Basal modified Eagle's medium (BME) and fetal calf serum were purchased from EuroClone. Sphingosine was prepared from cerebroside (34Carter H.E. Rothfus J.A. Gigg R. Biochemistry of the sphingolipids: XII. Conversion of cerebrosides to ceramides and sphingosine; structure of Gaucher cerebroside.J. Lipid Res. 1961; 2: 228-234Abstract Full Text PDF Google Scholar). Standard sphingolipids and glycerophospholipids were extracted from rat brain, purified, and characterized (35Tettamanti G. Bonali F. Marchesini S. Zambotti V. Parallelism of subcellular location of major particulate neuraminidase and gangliosides in rabbit brain cortex.Biochim. Biophys. Acta. 1973; 296: 160-170Crossref PubMed Scopus (510) Google Scholar). [1-3H]sphingosine was prepared by specific chemical oxidation of the primary hydroxyl group of sphingosine, followed by reduction with sodium boro[3H]hydride (36Toyokuni T. Nisar M. Dean B. Hakomori S. A facile and regiospecific titration of sphingosine: synthesis of 2S,3R,4E.-2-amino-4-octadecene-1,3-diol-1-3H.J. Label. Compd. Radiopharm. 1991; 29: 567-574Crossref Scopus (54) Google Scholar) (radiochemical purity over 98%; specific radioactivity 2 Ci/mmol). 3H-labeled lipids were extracted from [1-3H]sphingosine-fed cells, purified, characterized, and used as chromatographic standards. [9,10(n)-3H]palmitic acid, 55.0 Ci/mmol, was from Amersham Pharmacia Biotech. Anti-Lyn, anti-c-Src, anti-Akt rabbit polyclonal IgG, and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology.Cell culturesGranule cells obtained from the cerebellum of 8-day-old Harlan Sprague-Dawley rats were prepared and cultured as described (37Gallo V. Ciotti M. Coletti A. Aloisi F. Levi G. Selective release of glutamate from cerebellar granule cells differentiating in culture.Proc. Natl. Acad. Sci. USA. 1982; 79: 7919-7923Crossref PubMed Scopus (486) Google Scholar, 38Thangnipon W. Kingsbury A. Webb M. Balazs R. Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurones and astrocytes.Brain Res. 1983; 11: 177-189Crossref Scopus (177) Google Scholar, 39Kingsbury A.E. Gallo V. Woodhams P.L. Balazs R. Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium.Brain Res. 1985; 17: 17-25Crossref Scopus (141) Google Scholar). The cells were seeded in 60 mm or 100 mm dishes at a density of 0.155 × 106 cells/cm2 and cultured with BME containing 10% fetal calf serum. Primary cultures from 8-day-old rat cerebellum are highly enriched in neurons. In these cultures, about 90% of the cells are immature excitatory granule cells. The replication of non-neuronal cells was prevented by adding 10 μM cytosine arabinoside to the culture medium. Rat cerebellar granule cells in culture spontaneously undergo a developmental pattern that resembles that of cerebellar neurons in vivo, reaching a mature state after 8 days. The eighth day in culture corresponds to morphologically and biochemically fully differentiated neurons; the cells are mostly grouped in large aggregates, connected by a complex net of fasciculate fibers characterized by the presence of axo-axonic synapses. From the biochemical point of view, granule cells at this stage of in vitro development are characterized by the expression of voltage-dependent sodium channels, neurotransmitter receptors, neuronal surface sialoglycoproteins, and by the ability to synthesize glutamate and release this neurotransmitter under depolarizing conditions (37Gallo V. Ciotti M. Coletti A. Aloisi F. Levi G. Selective release of glutamate from cerebellar granule cells differentiating in culture.Proc. Natl. Acad. Sci. USA. 1982; 79: 7919-7923Crossref PubMed Scopus (486) Google Scholar, 40Balasz R. Gallo V. Kingsbury A. Effect of depolarization on the maturation of cerebellar granule cells in culture.Brain Res. 1988; 468: 269-276PubMed Google Scholar). Typical protein content was 700 μg of protein/dish. Cell integrity was assessed by the Trypan blue exclusion method, and cell mitochondrial metabolic activity was determined with the 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) test (41Rizzardini M. Lupi M. Bernasconi S. Mangolini A. Cantoni L. Mitochondrial dysfunction and death in motor neurons exposed to the glutathione-depleting agent ethacrynic acid.J. Neurol. Sci. 2002; 207: 51-58Abstract Full Text Full Text PDF Scopus (75) Google Scholar).Treatment of cell cultures with [1-3H]sphingosineCells at the sixth day in culture were incubated in the presence of 3 × 10−8 M [1-3H]sphingosine (2 ml/dish, or 5 ml/dish for 60 mm or 100 mm dishes, respectively) in cell-conditioned medium for a 2 h pulse followed by a 48 h chase. Under these conditions, free radioactive sphingosine was barely detectable in the cells, and all cell sphingolipids and phosphatidylethanolamine (obtained by recycling of radioactive ethanolamine formed in the catabolism of [1-3H]sphingosine) were metabolically radiolabeled (8Prinetti A. Chigorno V. Tettamanti G. Sonnino S. Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.J. Biol. Chem. 2000; 275: 11658-11665Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar).Treatment of cell cultures with [9,10(n)-3H]palmitic acidCells at the fifth day in culture were incubated in the presence of 4,5 × 10−7 M [9,10(n)-3H]palmitic acid (2 ml/dish) in cell-conditioned medium for a 24 h pulse followed by a 48 h chase. Under these conditions, all cell sphingolipids and glycerolipids were metabolically radiolabeled (42van Echten G. Iber H. Stotz H. Takatsuki A. Sandhoff K. Uncoupling of ganglioside biosynthesis by Brefeldin A.Eur. J. Cell Biol. 1990; 51: 135-139PubMed Google Scholar).Treatment of cell cultures with MCDAfter sphingolipid metabolic labeling as described in the previous section, cells at the eighth day in culture were treated with MCD as follows. At the time of the experiments, the chase medium was discarded and dishes (60 mm dishes) were washed three times with 1 ml Locke's balanced saline solution [156 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 5 mM HEPES (pH 7.4)] prewarmed to 37°C. Cells were incubated in the presence of 2 ml Locke's solution (incubation buffer) containing different concentrations of MCD (1–10 mM) or not (control) at 37°C for different times (5–120 min). Incubation buffer was removed, centrifuged at 1,000 g for 10 min to remove detached cells and cell debris, and stored at −80°C until lipid analysis. The number of cells remaining after each treatment was determined by counting the cells in 4–5 random fields in each dish using a phase-contrast microscope. Cells were quickly harvested in ice-cold water, snap frozen, lyophilized, and stored at −80°C until lipid analysis.Cholesterol repletionCells at the eighth day in culture were treated with 1–5 mM MCD or with incubation buffer for 30 min as described in the previous section. The incubation buffer was removed, and cells were further incubated for 1 h in BME or in BME containing 16 μg/ml cholesterol and 0.4% MCD complex. A stock solution of 0.4 mg/ml cholesterol and 10% MCD was prepared by adding 200 μl cholesterol solution (20 mg/ml in ethanol) to 10 ml of 10% MCD in BME and vortexing at ∼40°C (23Furuchi T. Anderson R.G.W. Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase ERK.J. Biol. Chem. 1998; 273: 21099-21104Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar).Sucrose gradient centrifugationAfter metabolic radiolabeling with [1-3H]sphingosine or [9,10(n)-3H]palmitic acid, cells were treated with 5 mM or 10 mM MCD in Locke's solution or Locke's alone (control) for 30 min at 37°C as described above. The number of cells remaining after each treatment was determined as described above. Cells were subjected to ultracentrifugation on discontinuous sucrose gradients as previously described (4Prinetti A. Iwabuchi K. Hakomori S. Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.J. Biol. Chem. 1999; 274: 20916-20924Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 8Prinetti A. Chigorno V. Tettamanti G. Sonnino S. Sphingolipid-enriched membrane domains from rat cerebellar granule cells differen
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