Statins Reduce Amyloid-β Production through Inhibition of Protein Isoprenylation
2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês
10.1074/jbc.m702640200
ISSN1083-351X
AutoresStephen M. Ostrowski, Brandy Wilkinson, Todd E. Golde, Gary E. Landreth,
Tópico(s)Cellular transport and secretion
ResumoEpidemiological evidence suggests that long term treatment with hydroxymethylglutaryl-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins have pleiotropic actions and act to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. In neuroblastoma cell lines, treatment with statins inhibits the membrane localization of Rho and Rab proteins at statin doses as low as 200 nm, without affecting cellular cholesterol levels. In addition, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of a subset of GTPases. The amyloid precursor protein (APP) is proteolytically cleaved to generate β-amyloid (Aβ), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of Rab family proteins involved in vesicular trafficking. Moreover, inhibition of Rho family protein function reduces levels of APP C-terminal fragments due to enhanced lysosomal dependent degradation. Statin inhibition of protein isoprenylation results in decreased Aβ secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Aβ production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis. Epidemiological evidence suggests that long term treatment with hydroxymethylglutaryl-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins have pleiotropic actions and act to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. In neuroblastoma cell lines, treatment with statins inhibits the membrane localization of Rho and Rab proteins at statin doses as low as 200 nm, without affecting cellular cholesterol levels. In addition, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of a subset of GTPases. The amyloid precursor protein (APP) is proteolytically cleaved to generate β-amyloid (Aβ), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of Rab family proteins involved in vesicular trafficking. Moreover, inhibition of Rho family protein function reduces levels of APP C-terminal fragments due to enhanced lysosomal dependent degradation. Statin inhibition of protein isoprenylation results in decreased Aβ secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Aβ production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis. Alzheimer disease (AD) 3The abbreviations used are:ADAlzheimer diseaseAPPamyloid precursor proteinCTFC-terminal fragmentAβamyloid-βFPPfarnesylpyrophosphateGGPPgeranylgeranylpyrophosphateERKextracellular signal-regulated kinaseHMG-CoA3-hydroxy-3-methylglutarylcoenzyme AGAPDHglyceraldehyde-3-phosphate dehydrogenaseELISAenzyme-linked immunosorbent assayBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPIPES1,4-piperazinediethanesulfonic acid 3The abbreviations used are:ADAlzheimer diseaseAPPamyloid precursor proteinCTFC-terminal fragmentAβamyloid-βFPPfarnesylpyrophosphateGGPPgeranylgeranylpyrophosphateERKextracellular signal-regulated kinaseHMG-CoA3-hydroxy-3-methylglutarylcoenzyme AGAPDHglyceraldehyde-3-phosphate dehydrogenaseELISAenzyme-linked immunosorbent assayBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPIPES1,4-piperazinediethanesulfonic acid is a progressive neurodegenerative disease and the most common cause of dementia in the elderly. The pathologic hallmarks of AD are extraneuronal senile plaques composed of β-amyloid (Aβ) fibrils and intraneuronal accumulations of hyperphosphorylated Tau (1Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3594) Google Scholar, 2Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4142) Google Scholar). Aβ is generated by sequential proteolytic processing of the type I trans-membrane protein, amyloid precursor protein (APP), by β- and γ-secretases (3Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3252) Google Scholar, 4Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. 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Cholesterol-independent actions of the statins have already been demonstrated to be important for the clinical benefits of these drugs on cardiovascular disease (35Bellosta S. Ferri N. Arnaboldi L. Bernini F. Paoletti R. Corsini A. Diabetes Care. 2000; 23: B72-B78PubMed Google Scholar, 36Bellosta S. Ferri N. Bernini F. Paoletti R. Corsini A. Ann. Med. 2000; 32: 164-176Crossref PubMed Scopus (320) Google Scholar, 37Takemoto M. Liao J.K. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1712-1719Crossref PubMed Scopus (1217) Google Scholar), as well as in animal models of central nervous system diseases with an inflammatory component, including multiple sclerosis and ischemic stroke (38Youssef S. Stuve O. Patarroyo J.C. Ruiz P.J. Radosevich J.L. Hur E.M. Bravo M. Mitchell D.J. Sobel R.A. Steinman L. Zamvil S.S. Nature. 2002; 420: 78-84Crossref PubMed Scopus (987) Google Scholar, 39Zamvil S.S. Steinman L. Neurology. 2002; 59: 970-971Crossref PubMed Scopus (52) Google Scholar, 40Kwak B. Mulhaupt F. Myit S. Mach F. Nat. Med. 2000; 6: 1399-1402Crossref PubMed Scopus (1226) Google Scholar).In cell culture, it has been shown that statin inhibition of GTPase isoprenylation causes these proteins to lose their normal membrane association and function (41Cordle A. Koenigsknecht-Talboo J. Wilkinson B. Limpert A. Landreth G. J. Biol. Chem. 2005; 280: 34202-34209Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, the effects of statins on protein isoprenylation have not been well studied in neurons. In addition, reports of statin effects on Rab family proteins have been quite limited, although statins have been shown to modulate protein trafficking through inhibition of Rab protein function (42Overmeyer J.H. Maltese W.A. J. Biol. Chem. 1992; 267: 22686-22692Abstract Full Text PDF PubMed Google Scholar, 43Ivessa N.E. Gravotta D. De Lemos-Chiarandini C. Kreibich G. J. Biol. Chem. 1997; 272: 20828-20834Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). As APP is trafficked by Rab-dependent mechanisms and perturbation of Rab function is associated with suppression of APP processing and Aβ generation (9Dugan J.M. deWit C. McConlogue L. Maltese W.A. J. Biol. Chem. 1995; 270: 10982-10989Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10McConlogue L. Castellano F. deWit C. Schenk D. Maltese W.A. J. Biol. Chem. 1996; 271: 1343-1348Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 11Maltese W.A. Wilson S. Tan Y. Suomensaari S. Sinha S. Barbour R. McConlogue L. J. Biol. Chem. 2001; 276: 20267-20279Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), we thought it important to examine the effects of statins on Rab isoprenylation, and whether modulation of Rab function by statins may perturb Aβ production.The physiological levels of statins in the brain have only recently been determined. Johnson-Anuna et al. (44Johnson-Anuna L.N. Eckert G.P. Keller J.H. Igbavboa U. Franke C. Fechner T. Schubert-Zsilavecz M. Karas M. Muller W.E. Wood W.G. J. Pharmacol. Exp. Ther. 2005; 312: 786-793Crossref PubMed Scopus (159) Google Scholar) reported simvastatin reaches concentrations of 300–500 nm in the brains of mice. Effects of statins at these lower, more clinically relevant, doses are not well documented. We report that, in neuronal cell types, statins inhibit the isoprenylation and membrane association of GTPases of the Rho and Rab family at doses of statins as low as 200 nm. Importantly, we show that while at high doses statins universally impair GTPase function, at low doses statins preferentially impair the isoprenylation and membrane localization of only a subset of GTPases. These GTPases may represent specific, clinically relevant targets of statin action. We also show that statins impact APP metabolism through Rab- and Rho-dependent mechanisms, leading to reduced Aβ production. In summary, we show that statins can selectively inhibit the isoprenylation of GTPases at physiologically relevant doses, and suggest that statins may act by cholesterol-independent mechanisms to lower Aβ production and limit AD pathogenesis.EXPERIMENTAL PROCEDURESMaterials and Reagents—Simvastatin and lovastatin were purchased from Calbiochem (La Jolla, CA) and prepared following the manufacturer's instructions. The statins were converted to the active form by dissolving them in absolute EtOH, followed by the addition of 1 m NaOH to a final concentration of 100 mm. This solution was stored at–20 °C until use. Immediately before use, the statin solution was neutralized with 1 m HCl, and diluted in vehicle (50% EtOH, 5 mm HEPES, pH 7.2). Geranylgeranyl pyrophosphate triammonium salt and farnesyl pyrophosphate triammonium salt were purchased from Biomol (Plymouth Meeting, PA). Mevalonic acid was purchased from Sigma and reconstituted in 100% ethanol. Clostridium difficile Toxin A was purchased from List Labs (Campbell, CA). Cell-permeable C3 exoenzyme was obtained from Cytoskeleton (Denver, CO). The Amplex Red Cholesterol Assay kit was purchased from Molecular Probes (Eugene, OR). Antibodies to APP (22c11) and the APP C-terminal fragment were purchased from Chemicon (Temecula, CA). Antibodies to Rac and Rab4 were obtained from Upstate (Waltham, MA). Antibodies to flotillin and calnexin were obtained from BD Bioscience. The antibody to GAPDH was obtained from Trevigen (Gaithersburg, MD). Antibodies to ERK2, β-tubulin, RhoA, Cdc42, Rab1b, Rab5b, and Rab6 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies 6E10 and 4G8 were purchased from Covance (Cumberland, VA). Peroxidase-conjugated secondary antibodies were purchased from GE Healthcare. Cell culture reagents were purchased from Invitrogen.Cell Culture—Mouse N2a (parental) neuroblastoma cells were obtained from American Type Culture Collection (Manassas, VA). N2a.Swe cells were obtained from Dr. Gopal Thinakaran (University of Chicago). APPsw-293 cells were obtained from Dr. Robert Vassar (Northwestern University). N2a and APPsw-293 cells were cultured in 50% Opti-MEM, 50% Dulbecco's modified Eagle's medium, 5% fetal bovine serum (Hyclone, Logan, UT), and 1% penicillin/streptomycin.H4.APPWT and H4.HPLAP neuroglioma cells were maintained in Opti-MEM plus 4% fetal bovine serum, 1% penicillin/streptomycin, and 1% Zeocin. H4.APPWT cells overexpress wild type human APP under an actin promoter (45Murphy M.P. Uljon S.N. Fraser P.E. Fauq A. Lookingbill H.A. Findlay K.A. Smith T.E. Lewis P.A. McLendon D.C. Wang R. Golde T.E. J. Biol. Chem. 2000; 275: 26277-26284Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). H4.HPLAP express endogenous levels of APP, but overexpress the human APP C-terminal fragment fused to human placental alkaline phosphatase. All cells were cultured at 37 °C and 5% CO2.Neuron Culture—Primary cultures of cortical neurons were prepared from embryonic day 15–16 C57BL/6 mouse embryos as described (50Brown M.S. Goldstein J.L. J. Lipid Res. 1980; 21: 505-517Abstract Full Text PDF PubMed Google Scholar). Briefly, embryo cortices were dissected, and meninges were removed. Tissue was digested, mechanically dissociated, and suspended in neurobasal medium (B27 supplement, 100 μg/ml penicillin/streptomycin, 0.5 mm glutamine, and 25 μm glutamate), and plated densely onto poly-d-lysinecoated 6-well plates (1 × 106 cells/well). Neurons were maintained under serum-free conditions in neurobasal medium with B27 supplement prior to drug treatment.Drug Treatments—Cells were plated at 5 × 105 cells per well in 6-well plates, and allowed to grow for 1 (for 48 h treatment) or 2 days (for 12–24 h treatment) before drug treatment. H4 cells were plated on poly-L-lysine-coated 6-well plates. Cells were then treated with the indicated compounds for 12–48 h.Western Blotting—Cells were collected and lysed with radio-immuno precipitation assay (RIPA) buffer (1% Triton X-100, 20 mm Tris, pH 7.5, 100 mm NaCl, 40 mm NaF, 0.2% SDS, 0.5% deoxycholate, 1 mm EDTA, 1 mm EGTA, and 1 mm Na3VO4). Lysates were sonicated for 2 × 10 s on ice and cleared by centrifugation (16,000 × g, 15 min, 4 °C). Protein concentration was determined by the Bradford method (46Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213161) Google Scholar). The samples were boiled under reducing conditions then resolved on 9% SDS-PAGE gels or NuPage 4–12% BisTris gels (Invitrogen) and transferred to polyvinylidene fluoride membranes. After blocking in a 5% milk or 5% normal goat serum solution, blots were incubated overnight at 4 °C with the indicated antibodies. Bands were visualized by incubation of blots with anti-mouse, rabbit, or goat horseradish peroxidase-conjugated secondary antibodies (1:1000; 90 min at room temperature) and visualized by enhanced chemiluminescence (Pierce). Protein loading was evaluated by probing with anti-ERK2 (1:3000) or anti-GAPDH (1:5000) antibodies. Images were scanned using Adobe Photoshop and band intensities quantified using Image-Pro Plus software package (Media Cybernetics, Inc., Silver Springs, MD). Band densities were normalized for protein loading by comparison with ERK2 or GAPDH band densities. Mean ± S.E. were calculated. Pairwise comparisons were determined using the Tukey-Kramer post hoc test.Quantification of Secreted Aβ Peptide Levels by ELISA—Following drug treatments, the culture medium was collected, a protease inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride was added, and medium was centrifuged at 16,000 × g for 15 min at 4 °C. Media from H4.APPWT cells were diluted 1:5 and assayed by ELISA specific for Aβ1–40 from BIOSOURCE/Invitrogen (Carlsbad, CA). H4.APPWT cells do not produce detectable levels of Aβ1–42.Membrane Localization and Western Blotting for GTPases— N2a cells were plated into 6-well plates and 24 h later were treated with simvastatin for 24 or 48 h. Cellular fractionation was carried out as described previously (47Zhao X. Bey E.A. Wientjes F.B. Cathcart M.K. J. Biol. Chem. 2002; 277: 25385-25392Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Briefly, following statin treatment the cells were lysed by incubation in relaxation buffer (100 mm KCl, 3 mm NaCl, 3.5 mm MgCl2, 1.25 mm EGTA, and 10 mm PIPES, pH 7.3) on ice for 15 min followed by a 10-s sonication. Cells were cleared by centrifugation at 500 × g for 5 min at 4 °C. The resulting supernatant was centrifuged for 1 h at 110,000 × g at 4 °C in a Beckman-Coulter ultracentrifuge (SW50.1 rotor). The resulting supernatant was removed (cytosolic fraction), and the membrane pellet was then resuspended in relaxation buffer (membrane fraction). The protein concentration from each fraction was measured using the BCA protein assay from Pierce. Standard Western blotting procedures were used to separate the fractions and transfer them to polyvinylidene difluoride membranes. Blots were probed with antibodies for the individual GTPases, as well as markers for cytosolic (GAPDH, ERK2) and membrane (flotillin, calnexin) fractions.RESULTSSelective Effects of Statin Treatment on G-protein Isoprenylation and Abundance—We investigated whether statins act uniformly to inhibit the isoprenylation of members of the Rho and Rab families of G-proteins in N2a neuroblastoma cells. We initially monitored statin-mediated effects by decreased electrophoretic mobility of non-prenylated versus prenylated forms of GTPases as examined by SDS-PAGE. It has been reported previously that Ras and Rab (42Overmeyer J.H. Maltese W.A. J. Biol. Chem. 1992; 267: 22686-22692Abstract Full Text PDF PubMed Google Scholar, 43Ivessa N.E. Gravotta D. De Lemos-Chiarandini C. Kreibich G. J. Biol. Chem. 1997; 272: 20828-20834Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 48Sinensky M. Beck L.A. Leonard S. Evans R. J. Biol. Chem. 1990; 265: 19937-19941Abstract Full Text PDF PubMed Google Scholar), but not the Rho family proteins (49Cicha I. Schneiderhan-Marra N. Yilmaz A. Garlichs C.D. Goppelt-Struebe M. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2046-2050Crossref PubMed Scopus (32) Google Scholar) exhibit altered electrophoretic mobility depending upon protein prenylation status, although we have reported altered isoprenoid-dependent Rac electrophoretic mobility in some cell types (41Cordle A. Koenigsknecht-Talboo J. Wilkinson B. Limpert A. Landreth G. J. Biol. Chem. 2005; 280: 34202-34209Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In statin-treated N2a cells we observed lowered mobility of Rab family proteins after statin treatment but not of the Rho family proteins Rac, Rho, or Cdc42 (Fig. 1A). The statin-dependent change in electrophoretic mobility of these protein families likely reflects the fact that Rab family proteins have two geranylgeranyl moieties added to them, whereas Rho family proteins possess only one lipid moiety. We find that after exposure of N2a cells to high doses of statins (10 μm), the Rab GTPases Rab1b, Rab4, Rab5, and Rab6 are converted entirely into the lower mobility, non-prenylated species (Fig. 1A). Statins reduced the levels of Rab6, requiring overexposure of the blots to visualize the unprenylated Rab6 band. Similar results were observed in H4 neuroglioma cells (data not shown). Provision of mevalonate allows restoration of the isoprenyl intermediate pools, without significant effects on cholesterol synthesis (20Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6460-6464Crossref PubMed Scopus (1075) Google Scholar, 21Kojro E. Gimpl G. Lammich S. Marz W. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5815-5820Crossref PubMed Scopus (718) Google Scholar, 25Fassbender K. Simons M. Bergmann C. Stroick M. Lutjohann D. Keller P. Runz H. Kuhl S. Bertsch T. von Bergmann K. Hennerici M. Beyreuther K. Hartmann T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5856-5861Crossref PubMed Scopus (1023) Google Scholar, 50Brown M.S. Goldstein J.L. J. Lipid Res. 1980; 21: 505-517Abstract Full Text PDF PubMed Google Scholar, 51Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4498) Google Scholar). Consistent with previous reports that show electrophoretic shifts are a result of loss of protein isoprenylation, we observe that changes in electrophoretic protein mobility caused by statin treatment were reversed upon provision of exogenous mevalonate, demonstrating that these effects are dependent upon protein isoprenylation (Fig. 1A). Statins do not alter cellular cholesterol content when serum is present in the media, as has been reported previously (data not shown) (52Cole S.L. Grudzien A. Manhart I.O. Kelly B.L. Oakley H. Vassar R. J. Biol. Chem. 2005; 280: 18755-18770Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 53Cordle A. Landreth G. J. Neurosci. 2005; 25: 299-307Crossref PubMed Scopus (156) Google Scholar).One remarkable outcome of these experiments was the effect of the statins on Cdc42. We find that in N2a cells, statins cause a dramatic increase in Cdc42 levels (Fig. 1A, B). Similar results were found in H4 cells (data not shown). Elevated Cdc42 levels were seen in N2a cells at doses of statins as low as 50 nm (Fig. 1B). Cdc42 mRNA levels were not increased after statin treatment (data not shown), suggesting that isoprenylation may be required for the normal turnover of this protein.Statins Block the Membrane Association of Ras Superfamily GTPases—The isoprenoid modification of the small GTPases is esse
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