Inhibition of Neuronal Apoptosis by Docosahexaenoic Acid (22:6n-3)
2000; Elsevier BV; Volume: 275; Issue: 45 Linguagem: Inglês
10.1074/jbc.m004446200
ISSN1083-351X
AutoresHee‐Yong Kim, Mohammed Akbar, Audrey O.T. Lau, Lisa C. Edsall,
Tópico(s)Lipid metabolism and biosynthesis
ResumoEnrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitrobiomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes. Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitrobiomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes. Dulbecco's modified Eagle's medium phosphate-buffered saline docosahexaenoic acid arachidonic acid oleic acid phosphatidylserine phosphatidylethanolamine phosphatidylcholine sphingomyelin liquid chromatography/mass spectrometry phospholipase A2 polymerase chain reaction polyacrylamide gel electrophoresis brain derived neurotrophic factor Ca2+-independent phospholipase A2 Mammalian brain is rich in long chain polyunsaturated fatty acids. Docosahexaenoic acid (22:6n-3), the major n-3 fatty acid found in brain, is highly enriched in neuronal cells (1Salem Jr., N. Omega-3 Fatty Acids: Molecular and Biochemical Aspects. Alan R. Liss, New York1989: 109-228Google Scholar). Growing evidences support the essential role of 22:6n-3 in neuronal function. In animal models n-3 fatty acid deficiency caused memory deficit (2Gamoh S. Hashimoto M. Sugioka K. Shahdat Hossain M. Hata N. Misawa Y. Masumura S. Neuroscience. 1999; 93: 237-241Crossref PubMed Scopus (253) Google Scholar), learning disability (3Yoshida S. Yasuda A. Kawazato H. Sakai K. Shimada T. Takeshita M. Yuasa S. Kobayashi T. Watanabe S. Okuyama H. J. Neurochem. 1997; 68: 1261-1268Crossref PubMed Scopus (101) Google Scholar, 4Carrie I. Clement M. De Javel D. Frances H. Bourre J.M. Neurosci. Lett. 1999; 266: 69-72Crossref PubMed Scopus (33) Google Scholar), and visual acuity loss (5Neuringe M. Am. J. Clin. Nutr. 2000; 71 (suppl.): 256-267Crossref Google Scholar). In humans, various neurological disease states have been shown to be associated with a deficient 22:6n-3 status, implying the influence of this fatty acid in neuronal function (6Hoffman D.R. Birch D.G. World Rev. Nutr. 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Neuronal cell survival is critically dependent on the supply of trophic factors, which influences downstream signaling pathways (17Barde Y.A. Neuron. 1989; 2: 1525-1534Abstract Full Text PDF PubMed Scopus (1442) Google Scholar). For example, in many cells phosphatidylinositol 3-kinase-dependent Akt serine/threonine kinase transduces a survival signal through phosphorylating proapoptotic protein BAD, which in turn associates with 14-3-3, preventing the interaction of BAD with Bcl-2 and Bcl-XL (18Zha J. Harda H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2233) Google Scholar, 19Datta 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 (4895) Google Scholar, 20Dudek H. Datta S.R. Franke T. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2212) Google Scholar). Deprivation of trophic factors inhibits phosphatidylinositol 3-kinase/Akt and subsequently BAD phosphorylation, which enables binding of BAD to Bcl-XL, resulting in mitochondrial damage. Subsequent release of cytochromec activates caspases, ultimately leading to apoptotic cell death (21Green D. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Growing evidence indicates that Raf-1 activation, which is known to be essential for transducing signals of many growth factors, can play an important role in the regulation of apoptotic processes (22Hoyle P.E. Moye P.W. Steelman L.S. Blalock W.L. Franklin R.A. Pearce M. Cherwinski H. Bosch E. McMahon M. McCubrey J.A. Leukemia (Baltimore ). 2000; 14: 642-656Crossref PubMed Scopus (87) Google Scholar, 23Neshat M.S. Raitano A.B. Wang H.G. Reed J.C. Sawyers C.L. Mol. Cell. Biol. 2000; 20: 1179-1186Crossref PubMed Scopus (162) Google Scholar, 24Salomoni P. Wasik M.A. Riedel R.F. Reiss K. Choi J.K. Skorski T. Calabretta B. J. Exp. Med. 1998; 187: 1995-2007Crossref PubMed Scopus (101) Google Scholar). Activation of Raf-1 kinase has been shown to prevent apoptosis in hematopoietic cells (22Hoyle P.E. Moye P.W. Steelman L.S. Blalock W.L. Franklin R.A. Pearce M. Cherwinski H. Bosch E. McMahon M. McCubrey J.A. Leukemia (Baltimore ). 2000; 14: 642-656Crossref PubMed Scopus (87) Google Scholar). It has been also shown that inhibition of Raf-1 in cells expressing BCR/ABL, which protects these cells from apoptosis induced by growth factor deprivation, can induce apoptosis (23Neshat M.S. Raitano A.B. Wang H.G. Reed J.C. Sawyers C.L. Mol. Cell. Biol. 2000; 20: 1179-1186Crossref PubMed Scopus (162) Google Scholar). In addition, expression of constitutively active mitochondrial Raf-1 has been shown to restore antiapoptotic potential of a transformation-deficient BCR/ABL mutant (24Salomoni P. Wasik M.A. Riedel R.F. Reiss K. Choi J.K. Skorski T. Calabretta B. J. Exp. Med. 1998; 187: 1995-2007Crossref PubMed Scopus (101) Google Scholar). Recently, it has been reported that activation of mitochondrial Raf-1 is involved in the antiapoptotic effect of Akt (25Majewski M. Nieborowska-Skorska M. Salomoni P. Slupianek A. Reiss K. Trotta R. Calabretta B. Skorski T. Cancer Res. 1999; 59: 2815-2819PubMed Google Scholar). Although mechanisms of Raf-1 activation is complex and still remains controversial, translocation of Raf-1 to the membrane and subsequent phosphorylation are considered to be important steps for its activation (26Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264 (; Correction (1994) Science266, 1792–1793): 1463-1467Crossref PubMed Scopus (832) Google Scholar, 27Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (875) Google Scholar, 28King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-183Crossref PubMed Scopus (380) Google Scholar, 29Morrison D.K. Cutler R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (531) Google Scholar). It has been shown that Raf-1 kinase contains distinct binding domains for acidic phospholipids, phosphatidylserine, and phosphatidic acid (30Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), and therefore the membrane localization of Raf-1 may be dependent on the concentration of these phospholipids. Phosphatidylserine is the major acidic phospholipid in mammalian cell membranes and is particularly enriched with 22:6n-3 fatty acid (1Salem Jr., N. Omega-3 Fatty Acids: Molecular and Biochemical Aspects. Alan R. Liss, New York1989: 109-228Google Scholar). We have previously demonstrated that 22:6n-3, which is abundantly present in neuronal cells, promotes the accumulation of phosphatidylserine in cell membranes (31Garcia M., C. Ward G. Ma Y.C. Salem Jr., N. Kim H.Y. J. Neurochem. 1998; 70: 24-30Crossref PubMed Scopus (83) Google Scholar, 32Kim H.Y. Hamilton J. Lipids. 2000; 35: 187-195Crossref PubMed Scopus (24) Google Scholar). In the present study, we explored the biological significance of 22:6n-3 by examining its effect on apoptotic behavior upon trophic factor removal in relation to its capacity to modulate phosphatidylserine accumulation. We found that enrichment of neuronal cells with 22:6n-3 increased the accumulation of PS and the membrane localization of Raf-1, down-regulated caspase-3 activity, and prevented apoptotic cell death under serum-free conditions. Its protective potential was sensitive to the extent of PS accumulation, suggesting that the observed antiapoptotic effect of 22:6n-3 may be mediated at least in part through the enhanced PS accumulation in neuronal membranes. Dulbecco's modified Eagle's medium (DMEM),1 fetal bovine serum, and other tissue culture reagents were obtained from Life Technologies, Inc. Monoclonal antibodies for Raf-1 and caspase-3 were purchased from Transduction Laboratories (Lexington, KY), and horse radish peroxidase-conjugated secondary antibodies were from Amersham Pharmacia Biotech. Apoptotic DNA ladder kit was purchased from Roche Molecular Biochemicals. Hoechst dye #33258 (bisbenzimide trihydrochloride #33258) was purchased from Sigma. Silica gel 60 plates were obtained from Analtech (Newark, DE). Fatty acids were obtained from Nu-Check (Elysian, MN). [1-14C]Docosahexaenoic acid (50 mCi/mmol) and [3H]thymidine (15 Ci/mmol) were purchased from NEN Life Science Products and Amersham Pharmacia Biotech, respectively. Rat pheochromocytoma PC12 cells were obtained from American Type Cell Culture (ATCC). Cells were maintained in RMPI medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum in a 37 °C incubator containing 5% CO2 water saturated atmosphere. Mouse neuroblastoma Neuro 2A cells (ATCC) were maintained in DMEM (Life Technologies, Inc.) with 5% fetal bovine serum in 75-cm2 Corning culture flasks under a humidified atmosphere of 95% air and 5% CO2 at 37 °C. The medium was changed twice weekly, and cells were subcultured when confluent. For DNA fragmentation assay by sedimentation or Hoechst staining, cells were plated on 6-well plates at a density of 5 × 104/cm2 and 2.5 × 104/cm2, respectively. For DNA or mRNA isolation, cells were cultured in 10 ml of medium in 10-cm culture dishes. For direct exposure of cells to fatty acids, fatty acids were bound to fat-free bovine serum albumin and presented to cells in medium containing 40 μm vitamin E during serum starvation. To test the effect of fatty acid enrichment, Neuro 2A cells were supplemented with fatty acids for 24 or 48 h and then subjected to serum starvation. Fatty acid stock solutions in chloroform or methanol were dried, bound to fetal bovine serum in the presence of 40 μm vitamin E, and diluted in DMEM under the argon atmosphere so that final concentrations of fatty acids and fetal bovine serum became 25 μm and 0.5%, respectively. Non-enriched controls were treated similarly during the enrichment period, but fatty acids were omitted. The DNA fragmentation assay by differential sedimentation was performed as reported earlier (33Duke R.C. Cohen J.J. Current Protocols in Immunology , Suppl. 3. Green/Wiley, New York1992: 3.17.1-3.17.16Google Scholar). Nuclei of PC12 or Neuro 2A cells were labeled with 1 μCi of [3H]thymidine for 24 h. To induce apoptosis, cells were washed gently twice with serum-free medium to remove unincorporated label and then incubated in the serum-free medium for 5–24 h. When cells were enriched with fatty acids before serum starvation, [3H]thymidine was added 24 h before the termination of enrichment. After serum starvation, cells were harvested and centrifuged at 200 × g for 10 min at 4 °C. An aliquot of the supernatant was then precipitated with 25% trichloroacetic acid. This fraction (S) reflects the amount of [3H]thymidine released during apoptosis induced by serum deprivation. The remaining cells were solubilized in a lysis buffer containing 0.2% Triton X-100 in 10 mm Tris/EDTA (TTE). The intact DNA (B) and the fragmented DNA (T) were then separated by centrifugation at 13,000 × g for 10 min at 4 °C. The fragmented DNA was precipitated from the supernatant with 25% trichloroacetic acid. The pellets were resuspended in 1% SDS and subjected to liquid scintillation. The percent DNA fragmentation is expressed as the sum of counts from (S + T)/(B + S + T) × 100. Total genomic DNA was isolated from Neuro 2A cells by using an apoptotic DNA ladder assay kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. Briefly, after 48 h of serum withdrawal as mentioned above, the cells were harvested by trypsinization, suspended in 200 μl of PBS, and lysed with equal amount of lysis buffer and incubated at 70 °C for 10 min. After adding 100 μl of isopropanol, lysates were mixed, and the genomic DNA was sheared by passing a few times through a 25-gage needle attached to a 1-ml disposable syringe. The whole lysate was charged on glass filters and washed, and DNA was isolated. The isolated DNA was precipitated in ethanol and extracted with phenol/chloroform/isoamyl alcohol, air-dried, and suspended in Tris/EDTA buffer. Six to eight μg of total DNA was charged on 2% agarose gel (Bio-Rad) in loading buffer, electrophoresed in Tris-buffered EDTA buffer containing 1 μg/ml ethidium bromide at 75 volts, and photographed under UV illumination. After 48 h of serum deprivation, the medium was centrifuged gently at 100 × g to collect detached cells, which were subsequently fixed in 250 μl of 3.7% formaldehyde. The cells still attached to the plate were fixed directly on the plate with 750 μl of 3.7% formaldehyde for 15 min. Cells were combined and centrifuged, and then 100 μl of Hoechst dye (24 μg/ml) dissolved in 50% glycerol/PBS was added. After incubating for at least 10 min, cells were observed by fluorescence microscopy with a 365-nm filter. Neuro 2A cells were washed twice with cold PBS, and the pellet was suspended in 100 μl of lysis buffer that contained 20% Triton X-100, 50 mm NaCl, 25 mm Tris/HCl, and 1 mm phenylmethylsulfonyl fluoride. The protein concentration was determined by BCA assay using bicinchoninic acid reagent (34Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18349) Google Scholar). Ten micrograms of protein were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed at a constant current of 30 mA, then transferred from the gel to a polyvinylidene difluoride membrane at 45 volts for 1 h. Procaspase-3 (32 kDa) and the 17-kDa fragment were immunoblotted with anti-caspase-3 (Transduction Laboratories) and visualized by enhanced chemoluminescence detection. Caspase-3 activity was measured using a colorimetric assay kit (Biomol, Plymouth Meeting, PA) according to the manufacturer's protocol. Briefly, cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and protein concentrations in the resulting supernatants were determined by BCA assay. Aliquots were incubated with acetyl-DEVD-p-nitroanilide for 2 h at 37 °C, and the absorbance at 405 nm was measured spectrophotometrically. Total RNA was isolated from Neuro 2A cells using TriZOL reagent (Life Technologies, Inc.) according to the manufacturer's protocol and quantified spectrophotometrically. One microgram of isolated RNA was treated with DNase 1 and used for first-strand cDNA synthesis. The treated RNA was incubated with 1 μl (0.5 μg) of oligo(dT)12–18 primer for 10 min at 70 °C and reverse-transcribed by using 1 μl (200 units of Moloney murine leukemia virus reverse transcriptase) of Superscript II RT (Life Technologies, Inc.) in 20 μl of reaction buffer containing 2 μl of 2 × PCR buffer, 25 mm MgCl2, 1 μl of 10 mm dNTP mix, and 2 μl of 0.1 m of dithiothreitol. The mixture was placed in a Perkin-Elmer 2400 Gene Amp PCR system set at 42 °C for a 50-min cycle followed by a 15-min incubation at 70 °C and a 4 °C soak. After reaction, the prepared cDNA was recovered from the mixture after RNA was digested by incubating with 1 μl of RNase H (2 units) for 20 min at 37 °C. To 1–2 μl of template cDNA, 25 μl of PCR reaction buffer (PCR master mix, Roche Molecular Biochemicals) was added along with 1 μl (40 pmol) each of an upstream primer GTC CAG TAG CCC CAA CAA TC-3′ (a 20-mer positioned at 202 -221) and a downstream primer 5′-GCG CAG AAC AGC CAC CTC AT-3′ (a 20-mer positioned at 517–498) obtained from Lofstrand Lab, Ltd (Gaithersburg, MD). PCR was then performed using 35 cycles programmed as follows: initial denaturation for 2 min at 94 °C and 15 s at 94 °C, annealing for 30 s at 55 °C, and primer extension for 1 min at 72 °C. One microliter (20 pmol) of each G3PDH primers (CLONTECH, California, CA) was used as a control providing a 450-base pair band. A band of 316 base pairs was visualized by illuminating under UV light after 4 μl of the PCR product was charged on 2% agarose gel containing 1 μg/ml ethidium bromide (Sigma) and electrophoresed for 1 h at 80 V. Neuro 2A cells were seeded on 6-well plates at a density of 4 × 105/cm in 2 ml of DMEM containing 5% fetal bovine serum. On the next day, 0.5 μCi of [3H]22:6n-3 was added in 2 ml of DMEM containing 0.5% fetal bovine serum and 40 μm vitamin E. The final concentration of the fatty acid was adjusted to 20 μmwith unlabeled fatty acids. After 5, 11, 24, and 48 h of incubation, the medium was removed, and the cells were washed with medium containing 0.2% bovine serum albumin twice. Cells were collected in methanol containing 0.5% (w/v) 2,6-di-tert-butyl-p-cresol (BHT), and lipids were extracted according to the method of Bligh and Dyer (35Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar). The lipid extracts were dried and reconstituted in chloroform, and aliquots were taken for radioactivity counting. The rest of the extracts were mixed with 25 μmol each of standard phospholipids and loaded on the TLC plates. Lipids were separated, and each lipid band was scraped and subjected to liquid scintillation counting as described earlier (36Garcia M.C. Kim H.Y. Brain Res. 1997; 768: 43-48Crossref PubMed Scopus (94) Google Scholar). Separately, cells were enriched with 20 μm nonlabeled 18:1n-9 or 22:6n-3, and lipids were extracted as described above. Phosphatidylserine molecular species were determined by electrospray liquid chromatography/mass spectrometry as described previously (31Garcia M., C. Ward G. Ma Y.C. Salem Jr., N. Kim H.Y. J. Neurochem. 1998; 70: 24-30Crossref PubMed Scopus (83) Google Scholar, 32Kim H.Y. Hamilton J. Lipids. 2000; 35: 187-195Crossref PubMed Scopus (24) Google Scholar, 37Kim H.Y. Wang T.C. Ma Y.C. Anal. Chem. 1994; 6: 3977-3982Crossref Scopus (222) Google Scholar). Liposomes of varying concentrations of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were prepared by the following methods. Desired amounts of 18:0–22:6 PC, PE, and PS (Avanti Polar Lipids, Alabaster, AL) in chloroform were mixed, then dried under argon. Lipids were reconstituted in 2 ml of 75 μm2,6-di-tert-butyl-p-cresol (BHT) in cyclohexane. Samples were frozen on dry ice and then lyophilized under vacuum until only a lipid film remained. The samples were purged under argon before removing to an argon box, whereupon the lipids were reconstituted with an appropriate volume of 50 μm diethylenetriamine pentaacetic acid in PBS. Solutions were mixed with a Vortex until a colloidal suspension formed and then passed through a 0.1-μm polycarbonate filter on a mini-extruder (Avanti Polar Lipids) 11 times to make unilamellar vesicles. Anti-Raf-1 antibody (Transduction Laboratories) was immobilized on a CM5 sensor chip (Biacore, Upsala, Sweden) using Biacore X as directed by manufacturer's instructions. Briefly, upon activating the chip withN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, bovine serum albumin-free anti-Raf was coupled to the sensor chip for a total of approximately 5000 RUs bound. After the coupling was finished, the chip surface was deactivated with ethanolamine/HCl, pH 8.5. The wash buffer used throughout was PBS. Raf-1 was captured on the chip using cell lysate from Neuro-2A cells (ATCC). The cell lysate was collected in radioimmune precipitation buffer (1× PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) containing 100 μg/ml phenylmethylsulfonyl fluoride. One flow cell was kept as the control cell, and no lysate was passed over this cell. The experimental cell had 15–20 RUs of Raf-1 captured on the surface, allowing liposome and Raf-1 interaction at approximately a 1:1.5 ratio. The liposomes were injected into the flow cells, and the association with and dissociation from Raf-1 was measured. Regeneration of the anti-Raf surface was completed in a two-step process. First, 10 mmacetate, pH 4.0, was injected into the cells, followed by an injection of radioimmune precipitation buffer. After allowing the chip surface to equilibrate with PBS, the chip was again ready for use. The recovered protein was analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting using Raf-1 antibody to confirm the identity of the captured protein. After enrichment of Neuro 2A cells with fatty acids for 48 h, Neuro-2A cells were washed and grown overnight in serum-free DMEM at 37 °C. The next day, experimental cells were stimulated with 250 ng of recombinant human BDNF (Promega, Madison, WI) in 5 ml of serum-free DMEM for 5–30 min at 37 °C. Membrane and cytosolic fractions were separated as described earlier (30Ghosh S. Strum J.C. Sciorra V.A. Daniel L. Bell R.M. J. Biol. Chem. 1996; 271: 8472-8480Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar), with slight modifications. Briefly, after the incubation, cells were washed with ice-cold PBS buffer, collected in 10 ml of Buffer A (10 mm Hepes, pH 7.4, 2 mmEDTA, 1 mm Na3VO4, and 1 mm phenylmethylsulfonyl fluoride), pelleted, and lysed by sonication in 40 μl of Buffer B (Buffer A plus 50 mm NaF, 10 μg/ml aprotinin, and 10 mg/ml leupeptin). Unbroken cells and nuclei were removed by centrifuging at 1000 ×g for 5 min at 4 °C. The supernatant was further centrifuged at 100,000 × g for 80 min at 4 °C. The supernatant (cytosol fraction) was collected, and the membrane pellet was solubilized by sonication in 80 μl of Buffer B containing 100 mm NaCl and 1% Triton X-100. The protein content was measured by the BCA protein assay. The Raf-1 protein from cytosol and membrane fractions was detected by SDS-polyacrylamide gel electrophoresis and Western blotting. Statistical analysis was performed using the Student's t test or Bonfferoni/Dunn post hoc analysis. Incubation of PC-12 or Neuro 2A cells under the serum-free conditions for 5 h induced apoptotic cell death, as determined by genomic DNA fragmentation, although Neuro 2A cells yielded much less fragmentation. Although coincubation of cells with 20:4n-6 during the serum deprivation period dose-dependently decreased the genomic DNA fragmentation induced by serum starvation, 22:6n-3 (1–25 μm) was not effective at all (Fig. 1). At a higher concentration (50 μm), both fatty acids were toxic, and DNA fragmentation increased significantly. The protective effect appeared to be 20:4n-6-specific, since 12.5–25 μm18:1n-9 did not have any significant effect as was the case with 22:6n-3. Since 22:6n-3 fatty acid exists mainly as membrane phospholipids in neuronal cells, accumulation of this fatty acid in membrane phospholipids may play an important role rather than the free fatty acid itself. Therefore, Neuro 2A cells were first enriched with 25 μm 22:6n-3, and the DNA fragmentation induced by subsequent serum starvation was examined (Fig.2). Unlike the case with direct exposure of 22:6n-3 during 5-h serum starvation periods, Neuro 2A cells enriched with 22:6n-3 for 24 h before serum starvation showed considerably less DNA fragmentation in comparison with the cells enriched with 18:1n-9 or non-enriched controls. Enrichment of cells with 22:6n-3 for 48 h further protected cells, as was indicated by even less DNA fragmentation. It was observed that the extent of protection or the degree of DNA fragmentation induced by serum starvation differed depending on the cell conditions or lot to lot variations in serum or medium constituents. However, it was consistently observed that the protective effect of 22:6n-3 was improved as the cells were enriched for a longer period up to 48 h. The cells enriched with 20:4n-6 also showed similarly less DNA fragmentation (Fig. 2); however, the protective effect was sensitive to the duration of serum starvation as shown in Fig. 3. The protective effect of 20:4n-6 observed during the 24-h serum-free conditions was abolished when the cells were deprived of serum for 48 h. Only 22:6n-3 remained protective, with up to 48 h of serum starvation. Similar results were obtained for the activity of caspase-3, a member of cysteine protease family that has been shown to mediate apoptosis in mammalian cells. Neuro 2A cells were first enriched with various fatty acids for 48 h and then exposed to the serum-free medium for up to 48 h, during which period caspase-3 activity was followed. Fig.4 shows the increase of caspase-3 activity as a function of the starvation period, with the exception of cells enriched with 22:6n-3. After 24 h of serum starvation, both 20:4n-6- and 22:6n-3-treated cells showed less caspase-3 activity in comparison to non-enriched control or 18:1n-9-enriched cells. Upon prolonged serum starvation, however, the protective effect of 20:4n-6 was no longer observed, and only 22:6n-3-treated cells maintained caspase-3 activity at a level similar to 5% serum control. Neuro 2A cells exhibited a characteristic DNA ladder pattern on an agarose gel after serum starvation, confirming the occurrence of apoptotic cell death. Neuro 2A cells required at least 48 h of serum starvation for DNA ladder formation. Shown in Fig.5 is the DNA ladder observed after the cells were enriched with fatty acids for 48 h and subsequently deprived of serum for 48 h. In agreement with the data shown in Figs. 3 and 4, DNA ladder formation was significantly reduced after enrichment with 22:6n-3, whereas other fatty acids such as 18:1n-9 or 20:4n-6 did not decrea
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