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• W2097520077 abstract "In the non-amyloidogenic pathway, amyloid precursor protein (APP) is cleaved by α-secretases to produce α-secretase-cleaved soluble APP (sAPPα) with neuroprotective and neurotrophic properties; therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of Alzheimer's disease. Here, we demonstrate the effects of type III secretory phospholipase A2 (sPLA2-III) on sAPPα secretion. Exposing differentiated neuronal cells (SH-SY5Y cells and primary rat neurons) to sPLA2-III for 24 h increased sAPPα secretion and decreased levels of Aβ1−42 in SH-SY5Y cells, and these changes were accompanied by increased membrane fluidity. We further tested whether sPLA2-III-enhanced sAPPα release is due in part to the production of its hydrolyzed products, including arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC). Addition of AA but neither PA nor LPC mimicked sPLA2-III-induced increases in sAPPα secretion and membrane fluidity. Treatment with sPLA2-III and AA increased accumulation of APP at the cell surface but did not alter total expressions of APP, α-secretases, and β-site APP cleaving enzyme. Taken together, these results support the hypothesis that sPLA2-III enhances sAPPα secretion through its action to increase membrane fluidity and recruitment of APP at the cell surface. In the non-amyloidogenic pathway, amyloid precursor protein (APP) is cleaved by α-secretases to produce α-secretase-cleaved soluble APP (sAPPα) with neuroprotective and neurotrophic properties; therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of Alzheimer's disease. Here, we demonstrate the effects of type III secretory phospholipase A2 (sPLA2-III) on sAPPα secretion. Exposing differentiated neuronal cells (SH-SY5Y cells and primary rat neurons) to sPLA2-III for 24 h increased sAPPα secretion and decreased levels of Aβ1−42 in SH-SY5Y cells, and these changes were accompanied by increased membrane fluidity. We further tested whether sPLA2-III-enhanced sAPPα release is due in part to the production of its hydrolyzed products, including arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC). Addition of AA but neither PA nor LPC mimicked sPLA2-III-induced increases in sAPPα secretion and membrane fluidity. Treatment with sPLA2-III and AA increased accumulation of APP at the cell surface but did not alter total expressions of APP, α-secretases, and β-site APP cleaving enzyme. Taken together, these results support the hypothesis that sPLA2-III enhances sAPPα secretion through its action to increase membrane fluidity and recruitment of APP at the cell surface. The senile plaque composed of neurotoxic amyloid-β peptide (Aβ) is a pathologic characteristic of Alzheimer's disease (AD) (1Dickson D.W. Microglia in Alzheimer's disease and transgenic models. How close the fit?.Am. J. Pathol. 1999; 154: 1627-1631Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 2Frautschy S.A. Yang F. Irrizarry M. Hyman B. Saido T.C. Hsiao K. Cole G.M. Microglial response to amyloid plaques in APPsw transgenic mice.Am. J. Pathol. 1998; 152: 307-317PubMed Google Scholar, 3McGeer P.L. Itagaki S. Tago H. McGeer E.G. Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR.Neurosci. Lett. 1987; 79: 195-200Crossref PubMed Scopus (719) Google Scholar, 4Perlmutter L.S. Barron E. Chui H.C. Morphologic association between microglia and senile plaque amyloid in Alzheimer's disease.Neurosci. Lett. 1990; 119: 32-36Crossref PubMed Scopus (200) Google Scholar, 5Selkoe D.J. The origins of Alzheimer disease: A is for amyloid.JAMA. 2000; 283: 1615-1617Crossref PubMed Scopus (269) Google Scholar, 6Stalder M. Phinney A. Probst A. Sommer B. Staufenbiel M. Jucker M. Association of microglia with amyloid plaques in brains of APP23 transgenic mice.Am. J. Pathol. 1999; 154: 1673-1684Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In the amyloidogenic pathway, Aβ is derived from a proteolytic process of amyloid precursor protein (APP), in which APP is cleaved sequentially by β- and γ-secretases (7Vassar R. BACE1: the beta-secretase enzyme in Alzheimer's disease.J. Mol. Neurosci. 2004; 23: 105-114Crossref PubMed Scopus (306) Google Scholar). Alternatively, the non-amyloidogenic pathway is mediated by α-secretase, which cleaves between amino acids 16 and 17 within the Aβ domain. This secretase is a member of the ADAM (a disintegrin and metalloprotease) family and produces a soluble fragment of APP generally regarded as α-secretase-cleaved soluble APP (sAPPα) (8Allinson T.M. Parkin E.T. Turner A.J. Hooper N.M. ADAMs family members as amyloid precursor protein alpha-secretases.J. Neurosci. Res. 2003; 74: 342-352Crossref PubMed Scopus (381) Google Scholar, 9Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Cleavage of amyloid beta peptide during constitutive processing of its precursor.Science. 1990; 248: 1122-1124Crossref PubMed Scopus (1208) Google Scholar). Due to the neurotrophic and neuroprotective properties of sAPPα (10Thornton E. Vink R. Blumbergs P.C. Van Den Heuvel C. Soluble amyloid precursor protein alpha reduces neuronal injury and improves functional outcome following diffuse traumatic brain injury in rats.Brain Res. 2006; 1094: 38-46Crossref PubMed Scopus (144) Google Scholar), increasing the APP processing by α-secretase has been suggested as a new strategy for the treatment of AD (11Cheng H. Vetrivel K.S. Gong P. Meckler X. Parent A. Thinakaran G. Mechanisms of disease: new therapeutic strategies for Alzheimer's disease–targeting APP processing in lipid rafts.Nat. Clin. Pract. Neurol. 2007; 3: 374-382Crossref PubMed Scopus (84) Google Scholar). APP is a transmembrane protein, and recent studies show that APP processing can be affected by the local membrane environment. The activity of β-site APP cleaving enzyme (BACE) to produce neurotoxic Aβ is favorable in lipid rafts, which are highly ordered membrane microdomains enriched in cholesterol, sphingolipids, and saturated phospholipids (12Kaether C. Haass C. A lipid boundary separates APP and secretases and limits amyloid beta-peptide generation.J. Cell Biol. 2004; 167: 809-812Crossref PubMed Scopus (67) Google Scholar, 13Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.J. Biol. Chem. 2004; 279: 44945-44954Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 14Cordy J.M. Hussain I. Dingwall C. Hooper N.M. Turner A.J. Exclusively targeting beta-secretase to lipid rafts by GPI-anchor addition up-regulates beta-site processing of the amyloid precursor protein.Proc. Natl. Acad. Sci. USA. 2003; 100: 11735-11740Crossref PubMed Scopus (312) Google Scholar, 15Marlow L. Cain M. Pappolla M.A. Sambamurti K. Beta-secretase processing of the Alzheimer's amyloid protein precursor (APP).J. Mol. Neurosci. 2003; 20: 233-239Crossref PubMed Scopus (81) Google Scholar, 16Ehehalt R. Keller P. Haass C. Thiele C. Simons K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (929) Google Scholar, 17Tun H. Marlow L. Pinnix I. Kinsey R. Sambamurti K. Lipid rafts play an important role in A beta biogenesis by regulating the beta-secretase pathway.J. Mol. Neurosci. 2002; 19: 31-35Crossref PubMed Scopus (79) Google Scholar). On the other hand, cleavage of APP by α-secretases is known to occur mainly in nonraft domains (18Reid P.C. Urano Y. Kodama T. Hamakubo T. Alzheimer's disease: cholesterol, membrane rafts, isoprenoids and statins.J. Cell. Mol. Med. 2007; 11: 383-392Crossref PubMed Scopus (113) Google Scholar). Therefore, APP processing can be altered by manipulating membrane lipid composition, such as cholesterol and sphingolipids removals (19Sawamura N. Ko M. Yu W. Zou K. Hanada K. Suzuki T. Gong J.S. Yanagisawa K. Michikawa M. Modulation of amyloid precursor protein cleavage by cellular sphingolipids.J. Biol. Chem. 2004; 279: 11984-11991Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Simons 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 (1085) Google Scholar, 21Kojro E. Gimpl G. Lammich S. Marz W. Fahrenholz F. Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha-secretase ADAM 10.Proc. Natl. Acad. Sci. USA. 2001; 98: 5815-5820Crossref PubMed Scopus (729) Google Scholar, 22von Arnim C.A. von Einem B. Weber P. Wagner M. Schwanzar D. Spoelgen R. Strauss W.L. Schneckenburger H. Impact of cholesterol level upon APP and BACE proximity and APP cleavage.Biochem. Biophys. Res. Commun. 2008; 370: 207-212Crossref PubMed Scopus (54) Google Scholar). Phospholipases A2 (PLA2s) are ubiquitous enzymes responsible for maintenance of phospholipid homeostasis in cell membranes. Aberrant PLA2 activity has been im­plicated in neurodegenerative diseases, including AD, Parkinson's disease, ischemia, spinal cord trauma, and head injury (23Farooqui A.A. Horrocks L.A. Phospholipase A2-generated lipid mediators in the brain: the good, the bad, and the ugly.Neuroscientist. 2006; 12: 245-260Crossref PubMed Scopus (207) Google Scholar, 24Sun G.Y. Xu J. Jensen M.D. Simonyi A. Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases.J. Lipid Res. 2004; 45: 205-213Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 25Muralikrishna Adibhatla R. Hatcher J.F. Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia.Free Radic. Biol. Med. 2006; 40: 376-387Crossref PubMed Scopus (316) Google Scholar, 26Moses G.S. Jensen M.D. Lue L.F. Walker D.G. Sun A.Y. Simonyi A. Sun G.Y. Secretory PLA2-IIA: a new inflammatory factor for Alzheimer's disease.J. Neuroinflammation. 2006; 3: 28Crossref PubMed Scopus (120) Google Scholar). Among many types of secretory PLA2s, secretory phospholipase A2 type III (sPLA2-III) has been found to express in human neuronal cells and contribute to neuronal differentiation (27Masuda S. Yamamoto K. Hirabayashi T. Ishikawa Y. Ishii T. Kudo I. Murakami M. Human group III secreted phospholipase A2 promotes neuronal outgrowth and survival.Biochem. J. 2008; 409: 429-438Crossref PubMed Scopus (49) Google Scholar). sPLA2-III from bee venom is highly homologous to the enzymatic-active central s-domain of human sPLA2-IIIs (28Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme.J. Biol. Chem. 2000; 275: 7492-7496Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). This protein has been reported to alter cellular membrane properties (29Best K.B. Ohran A.J. Hawes A.C. Hazlett T.L. Gratton E. Judd A.M. Bell J.D. Relationship between erythrocyte membrane phase properties and susceptibility to secretory phospholipase A2.Biochemistry. 2002; 41: 13982-13988Crossref PubMed Scopus (27) Google Scholar). In this study, we investigate whether sPLA2-III alters sAPPα in differentiated neuronal cells, including SH-SY5Y cells and primary rat neurons, and Aβ secretion. In addition, we also examine the effects of its hydrolyzed products, i.e., arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC), on sAPPα secretion, membrane fluidity, recruitment of APP to the cell surface, as well as the expressions of α-secretases and BACE. DMEM with high glucose, DMEM/F12 medium (1:1), Ham's F-12 medium, FBS, penicillin, and streptomycin (pen/strep), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Invitrogen (Carlsbad, CA). Neurobasal medium, B27, and trypsin-EDTA were obtained from Gibco (Carlsbad, CA). Bee venom sPLA2-III was from Cayman Chemical (Ann Arbor, MI). AA, LPC, PA, phorbol 12-myristate 13-acetate (PMA), DMSO, all-trans retinoic acid, and poly-l-lysine were from Sigma-Aldrich (St. Louis, MO). Farnesyl-(2-carboxy-2-cyanovinyl)-julolidine (FCVJ) was from Dr. Haidekker's Laboratory (Univerisity of Georgia) (30Nipper M.E. Majd S. Mayer M. Lee J.C. Theodorakis E.A. Haidekker M.A. Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ.Biochim. Biophys. Acta. 2008; 1778: 1148-1153Crossref PubMed Scopus (53) Google Scholar). Human neuroblastoma SH-SY5Y cells (1.0 × 105 cells/well) were seeded into 12-well plates or 1.0 ×106 cells/dish into 60 mm dishes and were cultured in DMEM/F12 medium (1:1) containing 10% FBS. For differentiation, SH-SY5Y cells were exposed to 10 μM all-trans retinoic acid for 6 days with change of fresh culture medium every 2 days. Primary cortical neurons were prepared from embryonic day 17 Sprague-Dawley rats as described previously (31Satoh T. Kosaka K. Itoh K. Kobayashi A. Yamamoto M. Shimojo Y. Kitajima C. Cui J. Kamins J. Okamoto S. et al.Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1.J. Neurochem. 2008; 104: 1116-1131Crossref PubMed Scopus (326) Google Scholar) with slightly modification. In brief, cortical neurons were enzymatically dissociated (0.05% trypsin with EDTA) and dispersed into a single-cell suspension with pasture pipette and seeded onto glass growth chambers and 6-well dishes coated with 50 mg/l poly-l-lysine. The cells were maintained in neural basal medium with 2% B27, 2 mM glutamine, and 1% pen/strep for 7 days before experiments. All cells were maintained at 37°C in a 5% CO2 humidified incubator. Cell viability was determined by MTT reduction. Briefly, differentiated SH-SY5Y cells or primary neurons cultured in 12-well plates were treated with different compounds, e.g., sPLA2-III, AA, LPC, and PA. After treatment, medium was removed and 1 ml of MTT reagent (0.5 mg/ml) in DMEM was added into each well. Cells were incubated for 4 h at 37°C, and after dissolving formazan crystals with DMSO, absorption at 540 nm was measured. A fluorescent molecular rotor, FCVJ was used to measure the relative membrane fluidity in SH-SY5Y cells. FCVJ was designed to be a more membrane-compatible fluorescent molecular rotor (32Haidekker M.A. Ling T. Anglo M. Stevens H.Y. Frangos J.A. Theodorakis E.A. New fluorescent probes for the measurement of cell membrane viscosity.Chem. Biol. 2001; 8: 123-131Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) with the quantum yield strongly dependent on the local free volume. A higher fluorescent intensity of FCVJ reflects the intramolecular rotational motions being restricted by a smaller local free volume, indicating a more viscous membrane. Previously, we verified the application of FCVJ for measuring membrane viscosity by comparing the results obtained using FCVJ with those from the technique of fluorescence recovery after photobleaching (30Nipper M.E. Majd S. Mayer M. Lee J.C. Theodorakis E.A. Haidekker M.A. Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ.Biochim. Biophys. Acta. 2008; 1778: 1148-1153Crossref PubMed Scopus (53) Google Scholar). In this study, we adapted the protocol from Haidekker et al. (32Haidekker M.A. Ling T. Anglo M. Stevens H.Y. Frangos J.A. Theodorakis E.A. New fluorescent probes for the measurement of cell membrane viscosity.Chem. Biol. 2001; 8: 123-131Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) to fluorescently label cells with FCVJ. Briefly, after undergoing different treatment protocols, e.g., sPLA2-III, AA, PA, and LPC, SH-SY5Y cells or primary neurons were washed with PBS and incubated in DMEM containing 20% FBS and 1 μM FCVJ for 20 min. Excess FCVJ was removed by washing cells with PBS three times. Fluorescence intensity measurements were performed at room temperature using a Nikon TE-2000 U fluorescence microscope with an oil immersion 60× objective lens. Images were acquired using a CCD camera controlled by a computer running MetaVue imaging software (Universal Imaging, PA). The fluorescence intensities of FCVJ per cell were measured. Background subtraction was done for all images prior to data analysis. After treating cells with sPLA2-III or lipid metabolites for 24 h, culture medium was collected and the same volume of the cell lysate from each sample was used for Western blot analysis using β-actin as internal standard. The culture medium was centrifuged at 12,000 g for 5 min to remove cell debris, and the same volume of medium from each sample (e.g., 40 μl) was diluted with Laemmli buffer, boiled for 5 min, subjected to electrophoresis in 7.5% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% (w/v) nonfat dry milk in TBS containing 0.1% (v/v) Tween 20 (TBST) and incubated overnight at 4°C in 3% (w/v) BSA with 0.02% (w/v) sodium azide in TBST with a 6E10 monoclonal antibody (1:1,000 dilution; Millipore, Billerica, MA) that recognizes residues 1–17 of the Aβ domain of human sAPPα or with a rodent specific polyclonal antibody (1:1,000 dilution; Covance, Dedham, MA). Membranes were washed three times during a 15 min period with TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:2,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 5% (w/v) nonfat dry milk in TBST at room temperature for 1 h. After washing with TBST for three times, the membrane was subjected to SuperSignal West Pico Chemiluminescent detection reagents from Pierce (Rockford, IL) to visualize bands. The protein bands detected on X-ray film were quantified using a computer-driven scanner and Quantity One software (Bio-Rad). After treatments, the protein concentration of the cell lysate was determined by BCA protein assay kit (Pierce Biotechnology) according to the manufacturer's instruction. Equivalent amounts of protein from each sample (e.g., 30 μg) was diluted with Laemmli buffer, boiled for 5 min, subjected to electrophoresis in 7.5% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% (w/v) nonfat dry milk in TBST and incubated overnight at 4°C in 3% (w/v) BSA with 0.02% (w/v) sodium azide in TBST with 6E10 monoclonal antibody, anti-ADAM9 antibody (1:1,000 dilution; Abcam, Cambridge, MA), anti-ADAM10 antibody (1:1,000 dilution; Millipore), anti-ADAM17 antibody (1:1,000 dilution; Santa Cruz Biotechnology) or anti-BACE1 antibody (1:1,000 dilution; Sigma-Aldrich). Membranes were washed three times during a 15 min period with TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibody (1:2,000 dilution; Santa Cruz Biotechnology) in 5% (w/v) nonfat dry milk in TBST at room temperature for 1 h. After washing with TBST for three times, the membrane was subjected to SuperSignal West Pico Chemiluminescent detection reagents from Pierce to visualize bands. The protein bands detected on X-ray film were quantified using a computer-driven scanner and Quantity One software (Bio-Rad). SH-SY5Y cells were plated onto cover slips. After differentiation and treatments, cells were fixed in PBS containing 4% paraformaldehyde without prior permeablization with detergent. After washing three times with PBS, nonspecific binding of antibodies was blocked by 5% goat serum for 1 h at room temperature. Cells were then incubated overnight at 4°C in 3% goat serum with anti-APP mouse antibody (1:200 dilution; Assay Designs, Ann Arbor, MI) that recognizes the N terminus of APP. The cover slips were washed with PBS and incubated for 1 h at room temperature with FITC-labeled goat anti-mouse secondary antibody (1:400) and washed with PBS. Cover slips were then mounted, and fluorescent intensity measurements were performed at room temperature using the Nikon TE-2000 U fluorescence microscope and oil immersion 60× objective lens. Images were acquired using a CCD camera controlled by a computer running MetaVue imaging software (Universal Imaging). The fluorescent intensities per cell area were measured. Background subtraction was done for all images prior to data analysis. After differentiation and treatments, SH-SY5Y cells were detached with nonenzymatic cell dissociation solution (Gibco, Carlsbad, CA). The cells were fixed in PBS containing 4% paraformaldehyde without permeablization. After washing three times with PBS, nonspecific binding of antibodies was blocked by 5% goat serum for 1 h at room temperature. The cells were then incubated for 2 h at room temperature in 3% goat serum with anti-APP mouse antibody (1:200 dilution; Assay Designs) that recognizes the N terminus of APP. After washing with PBS, cells were then incubated for 1 h at room temperature with FITC-­labeled goat anti-mouse secondary antibody (1:400) and washed with PBS. Background fluorescence intensity was assessed in the absence of primary antibody. All measurements were performed on a FACScan flow cytometry system (BD Biosciences, San Jose, CA) equipped with an argon laser. The excitation wavelength was 488 nm and emission intensity was detected with a FITC 525/30 nm filter set. A total of 10,000 cells were analyzed from each sample. Curves were generated with CellQuest software (BD Biosciences), and the median values of intensity were measured for data analysis. After treatments, culture medium was collected, supplemented with protease inhibitor cocktail, and centrifuged at 12,000 g for 5 min at 4°C to remove cell debris. An aliquot (100 μl) of supernatant was used for Aβ1-42 quantification using an ELISA kit (Invitrogen) following the manufacturer's recommendation. According to the instruction manual, substances including Aβ1−12, Aβ1−20, Aβ12−28, Aβ22−35, Aβ1−40, Aβ1−43, Aβ42−1, and APP have no cross-reactivity. The minimum detectable dose of Aβ1-42 is <1.0 pg/ml. The level of Aβ1-42 in each sample was measured in duplicates and expressed in pg/ml. Data are presented as mean ± SD from at least three independent experiments. Comparison between two groups was made with a Student's t-test. Comparisons of more than two groups were made with one-way ANOVA, followed by Bonferroni's post hoc tests. Values of P < 0.05 are considered to be statistically significant. sPLA2-III hydrolyzes sn-2 fatty acids of phospholipids in cell membranes, resulting in release of PUFAs and lysophospholipids. To test whether fatty acids or lysophospholipids are responsible for the increase in sAPPα secretion and alteration of membrane fluidity, we used AA and LPC as representative polyunsaturated fatty acids and lysophospholipids, respectively. For a negative control, PA, a saturated fatty acid and not likely a hydrolyzed product of sPLA2-III, was also applied. Since sPLA2-III from bee venom is highly homologous to the sPLA2-III in human (28Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme.J. Biol. Chem. 2000; 275: 7492-7496Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), sPLA2-III from bee venom was used to investigate the effect of sPLA2-III on sAPPα secretion in neuronal cells in relation to membrane fluidity. We first examined the viability of SH-SY5Y cells and primary rat neurons in response to different doses of sPLA2-III using the MTT test. As shown in Fig. 1, there is a dose-dependent decrease in cell viability upon exposing SH-SY5Y cells and primary neurons to sPLA2-III for 24 h. Based on these results, subsequent studies used 100 and 500 ng/ml of sPLA2-III for treating SH-SY5Y cells and 50 and 100 ng for treating primary neurons. Similar approaches were applied to determine the concentrations of AA (Fig. 1B), PA, and LPC (data not shown) for this study. In this study, 1 and 10 μM of AA (Fig. 1B), 10 and 100 μM of PA, and 1 and 10 μM of LPC were used. Western blot analysis showed that sPLA2-III and AA increased sAPPα secretion in SH-SY5Y cells in a dose-­dependent manner (Fig. 2A). Since it has been reported that PMA, a protein kinase C agonist, increases sAPPα secretion (33Caporaso G.L. Gandy S.E. Buxbaum J.D. Ramabhadran T.V. Greengard P. Protein phosphorylation regulates secretion of Alzheimer beta/A4 amyloid precursor protein.Proc. Natl. Acad. Sci. USA. 1992; 89: 3055-3059Crossref PubMed Scopus (326) Google Scholar, 34Slack B.E. Nitsch R.M. Livneh E. Kunz Jr., G.M. Eldar H. Wurtman R.J. Regulation of amyloid precursor protein release by protein kinase C in Swiss 3T3 fibroblasts.Ann. N. Y. Acad. Sci. 1993; 695: 128-131Crossref PubMed Scopus (19) Google Scholar, 35Camden J.M. Schrader A.M. Camden R.E. Gonzalez F.A. Erb L. Seye C.I. Weisman G.A. P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing.J. Biol. Chem. 2005; 280: 18696-18702Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), treatment with PMA (10 nM) was used as a positive control. However, PA and LPC did not alter sAPPα secretion (Fig. 2A). The increase in sAPPα secretion induced by sPLA2-III and AA was not due to the change of APP content in cells as shown in Fig. 2B; exposing cells to sPLA2-III, AA, LPC, and PA for 24 h did not alter total APP expression in SH-SY5Y cells (Fig. 2B). Consistent with the results from SH-SY5Y cells, sPLA2-III also increased sAPPα secretion in primary rat neurons (Fig. 2C). To study the effects of sPLA2-III and its hydrolyzed products on membrane fluidity, we applied a fluorescent molecular rotor, FCVJ. As explained in Materials and Methods, FCVJ integrated into a highly fluidized membrane exhibits lower quantum yield, as reflected by a lower fluorescent intensity. To validate the application of this technique for the measurement of membrane fluidity in neuronal cells, we exposed cells to ethanol, a compound known to increase membrane fluidity, and measured the fluorescent intensity of FCVJ integrated in cell membranes. Consistent with the notion that ethanol makes phospholipid bilayer membranes become more fluidized, ethanol caused a decrease in fluorescent intensity of FCVJ in SH-SY5Y cell and primary rat neuron membranes (data not shown). After treatment with sPLA2-III and AA, cells exhibited a lower fluorescent intensity of FCVJ compared with control (Fig. 3A, B), indicating that sPLA2-III and AA increased membrane fluidity in SH-SY5Y cells. These results are in agreement with the ability for sPLA2-III to increase membrane fluidity. However, PA and LPC were not capable of increasing membrane fluidity (Fig. 3B). Consistent with the results from SH-SY5Y cells, sPLA2-III was capable of increasing membrane fluidity in primary rat neurons. Together with the results for sAPPα secretion, these data suggest that sPLA2-III and AA increased sAPPα through their actions to increase membrane fluidity. There is strong evidence suggesting that the amyloidogenic pathway to generate Aβ occurs preferentially in the intracellular compartments, whereas the non-amyloidogenic pathway for production of sAPPα preferentially occurs at the plasma membranes (22von Arnim C.A. von Einem B. Weber P. Wagner M. Schwanzar D. Spoelgen R. Strauss W.L. Schneckenburger H. Impact of cholesterol level upon APP and BACE proximity and APP cleavage.Biochem. Biophys. Res. Commun. 2008; 370: 207-212Crossref PubMed Scopus (54) Google Scholar, 36Haass C. Hung A.Y. Schlossmacher M.G. Teplow D.B. Selkoe D.J. beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms.J. Biol. Chem. 1993; 268: 3021-3024Abstract Full Text PDF PubMed Google Scholar, 37Koo E.H. Squazzo S.L. 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To test this hypothesis, we fluorescently labeled the extracellular domain of APP without invoking the procedure for membrane permeabilization. Immunofluorescence microscopy of APP at the cell surfa" @default.
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• W2097520077 modified "2023-10-16" @default.
• W2097520077 title "Secretory phospholipase A2 type III enhances α-secretase-dependent amyloid precursor protein processing through alterations in membrane fluidity" @default.
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• W2097520077 doi "https://doi.org/10.1194/jlr.m002287" @default.
• W2097520077 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2853463" @default.
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