A Ganglioside-induced Toxic Soluble Aβ Assembly
2006; Elsevier BV; Volume: 282; Issue: 4 Linguagem: Inglês
10.1074/jbc.m606202200
ISSN1083-351X
AutoresNaoki Yamamoto, Etsuro Matsubara, Sumihiro Maeda, Hirohisa Minagawa, Akihiko Takashima, Wakako Maruyama, Makoto Michikawa, Katsuhiko Yanagisawa,
Tópico(s)Machine Learning in Bioinformatics
ResumoThe mechanism underlying plaque-independent neuronal death in Alzheimer disease (AD), which is probably responsible for early cognitive decline in AD patients, remains unclarified. Here, we show that a toxic soluble Aβ assembly (TAβ) is formed in the presence of liposomes containing GM1 ganglioside more rapidly and to a greater extent from a hereditary variant-type ("Arctic") Aβ than from wild-type Aβ.TAβ is also formed from soluble Aβ through incubation with natural neuronal membranes prepared from aged mouse brains in a GM1 ganglioside-dependent manner. An oligomer-specific antibody (anti-Oligo) significantly suppresses TAβ toxicity. Biophysical and structural analyses by atomic force microscopy and size exclusion chromatography revealed that TAβ is spherical with diameters of 10–20 nm and molecular masses of 200–300 kDa. TAβ induces neuronal death, which is abrogated by the small interfering RNA-mediated knockdown of nerve growth factor receptors, including TrkA and p75 neurotrophin receptor. Our results suggest that soluble Aβ assemblies, such as TAβ, can cause plaque-independent neuronal death that favorably occurs in nerve growth factor-dependent neurons in the cholinergic basal forebrain in AD. The mechanism underlying plaque-independent neuronal death in Alzheimer disease (AD), which is probably responsible for early cognitive decline in AD patients, remains unclarified. Here, we show that a toxic soluble Aβ assembly (TAβ) is formed in the presence of liposomes containing GM1 ganglioside more rapidly and to a greater extent from a hereditary variant-type ("Arctic") Aβ than from wild-type Aβ.TAβ is also formed from soluble Aβ through incubation with natural neuronal membranes prepared from aged mouse brains in a GM1 ganglioside-dependent manner. An oligomer-specific antibody (anti-Oligo) significantly suppresses TAβ toxicity. Biophysical and structural analyses by atomic force microscopy and size exclusion chromatography revealed that TAβ is spherical with diameters of 10–20 nm and molecular masses of 200–300 kDa. TAβ induces neuronal death, which is abrogated by the small interfering RNA-mediated knockdown of nerve growth factor receptors, including TrkA and p75 neurotrophin receptor. Our results suggest that soluble Aβ assemblies, such as TAβ, can cause plaque-independent neuronal death that favorably occurs in nerve growth factor-dependent neurons in the cholinergic basal forebrain in AD. The poor correlation between amyloid load in the brain and the degree of neurological deficits in patients with Alzheimer disease (AD) 2The abbreviations used are: AD, Alzheimer disease; TAβ, toxic soluble Aβ assembly; NGF, nerve growth factor; LDH, lactate dehydrogenase; siRNA, small interfering RNA; AFM, atomic force microscopy; GM1, Galβ1,3GalNAcβ1,4(Neu5Ac-α2,3)Galβ1,4Glcβ1,1-ceramide; ThT, thioflavin-T; NTR, neurotrophin receptor. (1Terry R.D. Masliah E. Hansen L. Terry R.D. Katzman R. Bick K.L. Sisodia S.S. Alzheimer Disease. Lippincott Williams and Wilkins, Philadelphia, PA1999: 187-206Google Scholar) or animal models of AD (2Hsia A.Y. Masliah E. McConlogue L. Yu G.Q. Tatsuno G. Hu K. Kholodenko D. Malenka R.C. Nicoll R.A. Mucke L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3228-3233Crossref PubMed Scopus (994) Google Scholar, 3Mucke L. Masliah E. Yu G.Q. Mallory M. Rockenstein E.M. Tatsuno G. Hu K. Kholodenko D. Johnson-Wood K. McConlogue L. J. 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Dellheden B. Mansson J.E. Fredman P. J. Neurochem. 2005; 92: 171-182Crossref PubMed Scopus (193) Google Scholar), and 2) GM1 ganglioside level also increases in amyloid-positive nerve terminals obtained from the AD cortex (39Gylys K.H. Fein J.A. Yang F. Miller C.A. Cole G.M. Neurobiol. Aging. 2007; 28: 8-17Crossref PubMed Scopus (88) Google Scholar). In this study, we aimed to characterize the toxicity of assemblies formed from Arctic-type Aβ in the presence of GM1 ganglioside. We found that a toxic soluble Aβ assembly (TAβ) is formed more rapidly and to a greater extent from Arctic-type Aβ in the presence of GM1 ganglioside than from wild-type Aβ. Furthermore, our results suggest that TAβ induces nerve growth factor (NGF) receptor-mediated neuronal death. Thus, we propose that soluble Aβ assemblies, such as TAβ, are responsible for plaque-independent neuronal death that favorably occurs in NGF-dependent neurons in AD. Preparation of Seed-free Aβ Solutions and Liposomes—Synthetic wild-type Aβ (Aβ40) and Arctic-type Aβ (Aβ40) (Peptide Institute, Osaka, Japan) were dissolved in 0.02% ammonia solution at 500 μm. To obtain seed-free Aβ solutions, the prepared solutions were centrifuged at 540,000 × g for 3 h using an Optima TL ultracentrifuge (Beckman) to remove undissolved peptides that can act as preexisting seeds. The supernatant was collected and stored in aliquots at -80 °C until use. Immediately before use, the aliquots were thawed and diluted with Tris-buffered saline (150 mm NaCl and 10 mm Tris-HCl, pH 7.4). To prepare liposomes, cholesterol (Sigma), sphingomyelin (Sigma), and GM1 ganglioside (Matreya LLC) were dissolved in chloroform/methanol at a molar lipid ratio of 50:50:0, 45:45:10, 42.5:42.5:15, or 40:40:20. The mixtures were stored at -80 °C until use. Immediately before use, the lipids were resuspended in Tris-buffered saline at a ganglioside concentration of 2.5 mm, and the suspension was subjected to freezing and thawing and sonication. Cell Culture—Cerebral cortical neurons were prepared from embryonic day 17 Sprague-Dawley rats and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium nutrient mixture and N2 supplement. Rat pheochromocytoma PC12 (PC12) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated horse serum (Invitrogen) and 5% fetal bovine serum (Invitrogen). For their differentiation, PC12 cells were plated on 2-cm2 poly-l-lysine-coated (10 mg/ml) dishes at a density of 20,000 cells/cm2 and cultured for 6 days in Dulbecco's modified Eagle's medium supplemented with 100 ng/ml NGF (PC12N) (Alomone Laboratories, Jerusalem, Israel). Human neuroblastoma SH-SY5Y (SY5Y) cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% fetal bovine serum. All of the cells were cultured in humidified 5% CO2 at 37 °C. Aβ Incubation in the Presence of GM1 Ganglioside—A seed-free Aβ solution was incubated at 37 °C and 50 μm, unless otherwise indicated, in the presence or absence of GM1 ganglioside-containing liposomes, as previously reported (40Hayashi H. Kimura N. Yamaguchi H. Hasegawa K. Yokoseki T. Shibata M. Yamamoto N. Michikawa M. Yoshikawa Y. Terao K. Matsuzaki K. Lemere C.A. Selkoe D.J. Naiki H. Yanagisawa K. J. Neurosci. 2004; 24: 4894-4902Crossref PubMed Scopus (217) Google Scholar). The concentration of GM1 ganglioside in the incubation mixtures was 500 μm, and the molar ratio of GM1 ganglioside in the liposomes varied, as indicated in each figure. ThT Assay—Aβ solutions were incubated in the presence of liposomes at 50 μm and 37 °C for various durations. The ThT fluorescence intensity of the incubation mixtures was determined using a spectrofluorophotometer (RF-5300PC) (Shimadzu Co., Kyoto, Japan). The optimum fluorescence intensity of amyloid fibrils was measured at excitation and emission wavelengths of 446 and 490 nm, respectively, with the reaction mixture (1.0 ml) containing 5 μm ThT and 50 mm glycine-NaOH at pH 8.5. The fluorescence intensity was measured immediately after preparing the mixture. LDH Release Assay—The LDH assay was performed on medium using an LDH assay toxicity kit (Promega, Madison, WI). The degree of LDH release in each sample was assessed by measuring absorbance at 490 nm using an Emax precision microplate reader (Molecular Devices Corp., Sunnyvale, CA). Background absorbances, as assessed using cell-free wells, were subtracted from the absorbances of each test sample. Absorbances measured from three wells were averaged, and the percentage degree of LDH release was calculated by dividing the absorbance measured from each test sample following treatment with 1% Triton X-100 to induce the release of intracellular LDH according to instructions provided by the manufacturer. Electron and Atomic Force Microscopies—For electron microscopy, the samples were diluted with distilled water and spread onto carbon-coated grids. The grids were negatively stained with 2% uranyl acetate and examined under a JEM-2000EX transmission electron microscope (Tokyo, Japan) with an acceleration voltage of 100 kV. Atomic force microscopy (AFM) assessment was performed as described elsewhere (41Maeda S. Sahara N. Saito Y. Murayama S. Ikai A. Takashima A. Neurosci. Res. 2006; 54: 197-201Crossref PubMed Scopus (234) Google Scholar). Briefly, the samples were dropped onto a freshly cleaved mica. After leaving them to stand for 3 min and then washing with water, the samples were assessed in a solution using a Nanoscope IIIa (Digital Instruments, Santa Barbara, CA) set in the tapping mode (42Hansma H.G. Laney D.E. Bezanilla M. Sinsheimer R.L. Hansma P.K. Biophys. J. 1995; 68: 1672-1677Abstract Full Text PDF PubMed Scopus (231) Google Scholar). OMCL-TR400PSA (Olympus, Japan) was used as a cantilever. The resonant frequency was ∼9 kHz. Size Exclusion Chromatography—The molecular mass of TAβ was determined using a Superose 12 size exclusion column (1 × 30 cm; GE Healthcare) equilibrated with phosphate-buffered saline (pH 7.4) at a flow rate of 0.5 ml/min. Thirty-five fractions were collected and analyzed by dot blotting using anti-Oligo. Preparation of Synaptosomes—Synaptosomes were prepared as previously described (43Schroeder F. Morrison W.J. Gorka C. Wood W.G. Biochim. Biophys. Acta. 1988; 946: 85-94Crossref PubMed Scopus (83) Google Scholar). A hippocampus or a whole brain minus the hippocampus was homogenized in 0.32 m sucrose buffer containing 0.25 mm EDTA. The homogenate was centrifuged at 580 × g for 8 min. The supernatant was centrifuged at 145,000 × g for 20 min. The resulting pellet was suspended in 0.32 m sucrose buffer without EDTA and layered over Ficoll in sucrose buffer. Following centrifugation at 87,000 × g for 30 min, the synaptosome-rich interface was removed and recentrifuged to remove any remaining Ficoll. RNA Interference—Stealth™ small interfering RNA (siRNA) duplex oligoribonucleotides against PC12 cell TrkA (GenBank™ number NM_021589) and the p75 neurotrophin receptor (p75NTR) (GenBank™ number NM_012610) were synthesized by Invitrogen. The siRNA sequences used were as follows: rTrkA-siRNA (position 1370) sense (5′-GCCCUCCUCCUAGUGCUCAACAAAU-3′) and antisense (5′-AUUUGUUGAGCACUAGGAGGAGGGC-3′); rTrkA-siRNA-control sense (5′-GCCCUCCGAUCUCGUCAACAUCAAU-3′) and antisense (5′-AUUGAUGUUGACGAGAUCGGAGGGC-3′); rp75-siRNA (position 1212) sense (5′-CAGCCUGAACAUAUAGACUCCUUUA-3′) and antisense (5′-UAAAGGAGUCUUAUAUGUUCAGGCUG-3′); rp75-siRNA-control sense (5′-CAGGUAAACAUAUAGUCCCUCCUUA-3′) and antisense (5′-UAAGGAGGGACUAUAUGUUUACCUG-3′). The control siRNA had a random sequence. siRNA oligonucleotides were transfected into PC12 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Toxicity of Aβ Assembly Formed from Arctic-type Aβ—We treated primary neurons with seed-free wild- or Arctic-type Aβ, which had been preincubated for 2 h in the absence or presence of GM1 ganglioside (10 or 20% molar ratio in the lipids composing liposomes). Unexpectedly, extensive neuronal death was observed in the culture treated with Arctic-type Aβ, which had been preincubated for 2 h in the presence of GM1 ganglioside at a 10% molar ratio in liposomes (Fig. 1A). The extent of neuronal death under this condition was greater than that under any other conditions examined in this study (Fig. 1, A and B). To quantitatively characterize the toxic Aβ assembly, we examined its toxicity against NGF-treated PC12 cells (PC12N cells). We found that PC12N cells are also sensitive to the toxic Aβ assembly formed from Arctic-type Aβ (Fig. 1C). We performed an LDH release assay of cultures of PC12N cells under various conditions. The level of LDH released from the PC12N cells, which were treated with the toxic Aβ assembly, increased depending on Aβ dose (Fig. 1D), GM1 ganglioside dose (Fig. 1E), and the duration of the exposure of the cells to the toxic Aβ assembly (Fig. 1F). In regard to the time course of Aβ preincubation with GM1 ganglioside, the level of released LDH increased with peak value at 2 h and then decreased in conjunction with an increase in the ThT fluorescence intensity of the incubation mixtures (Fig. 1G). The Toxic Aβ Assembly Is Soluble—Importantly, the toxicity of the Aβ incubated in the presence of GM1 ganglioside was observed exclusively in the supernatant obtained by ultracentrifuging the incubation mixture (Fig. 2A), suggesting that the toxic Aβ assembly is soluble. To examine the possibility that a TAβ is formed in the presence GM1 ganglioside, we performed dot blotting using an oligomer-specific antibody (anti-Oligo) (23Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Science. 2003; 300: 486-489Crossref PubMed Scopus (3470) Google Scholar). TAβ in the incubation mixtures was readily recognized by anti-Oligo (Fig. 2B). The specificity of TAβ recognition by anti-Oligo was confirmed by the finding that TAβ toxicity was significantly neutralized by coincubating the mixtures with anti-Oligo in the cultures of PC12N cells and primary neurons (Fig. 2C). However, coincubation with a monoclonal antibody (4396C), which inhibits Aβ fibrillogenesis through binding to GM1 ganglioside-bound Aβ as a seed (40Hayashi H. Kimura N. Yamaguchi H. Hasegawa K. Yokoseki T. Shibata M. Yamamoto N. Michikawa M. Yoshikawa Y. Terao K. Matsuzaki K. Lemere C.A. Selkoe D.J. Naiki H. Yanagisawa K. J. Neurosci. 2004; 24: 4894-4902Crossref PubMed Scopus (217) Google Scholar), failed to inhibit the induction of TAβ toxicity (Fig. 2D). TAβ Formation from Wild-type Aβ—We then examined whether TAβ is also formed from wild-type Aβ (Aβ40). We first investigated how TAβ is formed from wild-type Aβ in the presence of liposomes containing GM1 ganglioside. Interestingly, TAβ is favorably formed from wild-type Aβ in the presence of GM1 ganglioside at a 15% molar ratio in liposomes (Fig. 3A). TAβ toxicity was not significant in the nanomolar range of Aβ (Fig. 3B). Biophysical and Structural Features of TAβ—To determine the biophysical and structural features of TAβ, we performed SDS-PAGE of the incubation mixtures containing TAβ. However, no high molecular weight bands corresponding to possible Aβ assemblies were detected. Bands were observed only after cross-linking pretreatment with glutaraldehyde (Fig. 4A), consistent with previous findings showing that soluble Aβ assemblies are probably degraded by denaturing gel electrophoresis (6Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar) unless they are cross-linked (44Atwood C.S. Scarpa R.C. Huang X. Moir R.D. Jones W.D. Fairlie D.P. Tanzi R.E. Bush A.I. J. Neurochem. 2000; 75: 1219-1233Crossref PubMed Scopus (585) Google Scholar, 45Bitan G. Lomakin A. Teplow D.B. J. Biol. Chem. 2001; 276: 35176-35184Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). A morphological analysis of TAβ by electron microscopy failed to detect any definite structure under conditions in which protofibrils, which had been prepared as previously reported (30Nilsberth C. Westlind-Danielsson A. Eckman C.B. Condron M.M. Axelman K. Forsell C. Stenh C. Luthman J. Teplow D.B. Younkin S.G. Naslund J. Lannfelt L. Nat. Neurosci. 2001; 4: 887-893Crossref PubMed Scopus (918) Google Scholar), were readily detectable (Fig. 4B). In contrast, spherical particles with diameters of 10–20 nm, along with rod-shaped structures, were observed by AFM in the supernatant obtained by ultracentrifuging the incubation mixtures containing TAβ (Fig. 4C). We then determined the molecular mass of TAβ by size exclusion chromatography, which was followed by dot blotting using anti-Oligo. The immunoreactivity was recovered as a single peak with relative molecular masses of 200–300 kDa (Fig. 4D). The recovery of TAβ immunoreactivity in the same fraction was also observed in the incubation mixture containing wild-type Aβ (Aβ40) and GM1 ganglioside at a 15% molar ratio in liposomes (Fig. 4D). Furthermore, the collected peak showed a significant toxicity against PC12N cells (Fig. 4E). TAβ Formation in the Presence of Natural Neuronal Membranes—Next, we tested whether TAβ can be formed in the presence of natural neuronal membranes. We incubated Arctic-type Aβ in the presence of synaptosomes prepared from brains of mice from three different age groups. The degree of TAβ formation was significantly higher in the incubation mixture containing synaptosomes prepared from the hippocampus of aged (2-year-old) mouse brains than in any other incubation mixtures, including those containing synaptosomes from the hippocampus or the whole brain minus the hippocampus from younger (1-month-old and 1-year-old) mouse brains (Fig. 5A). To determine the possibility that an alteration in the lipid composition of neuronal membranes, particularly GM1 ganglioside, underlies the acceleration of TAβ formation, we determined the levels of GM1 ganglioside, cholesterol, and phospholipids in synaptosomes prepared from hippocampi of young (1-month-old) and aged (2-year-old) mouse brains. Notably, the GM1 ganglioside level significantly increased, whereas cholesterol level significantly decreased with age (Fig. 5B). Putative Mechanism Underlying TAβ-induced Neuronal Death—To characterize cell death induced by TAβ, we performed nuclear staining with a membrane-permeable dye, Hoechst 33258. PC12N cells, which were treated with incubation mixtures containing TAβ for 12 h, showed characteristics of apoptotic changes, including retracted neurites, shrunken cell bodies, and the condensation and fragmentation of nuclei in conjunction with an increase in the level of LDH released from TAβ-treated PC12N cells (data not shown). To determine if TAβ toxicity is mediated by NGF receptors, we first treated PC12N cells, native PC12 cells, and primary neurons with TAβ in the presence of exogenous NGF. In these cultures, cell death was markedly prevented (Fig. 6). We then knocked down the NGF receptors, including TrkA
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