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Dimeric Amyloid β Protein Rapidly Accumulates in Lipid Rafts followed by Apolipoprotein E and Phosphorylated Tau Accumulation in the Tg2576 Mouse Model of Alzheimer's Disease

Kawarabayashi, Takeshi, Shoji, Mikio, Younkin, Linda H., Wen-Lang, Lin, Dickson, Dennis W., Murakami, Tetsuro, Matsubara, Etsuro, Abe, Koji, Ashe, Karen Hsiao, Younkin, Steven G.·2004·The Journal of Neuroscience
DOI 10.1523/jneurosci.5543-03.2004
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Abstract

To investigate lipid rafts as a site where amyloid β protein (Aβ) oligomers might accumulate and cause toxicity in Alzheimer's disease (AD), we analyzed Aβ in the Tg2576 transgenic mouse model of AD. Aβ was highly concentrated in lipid rafts, which comprise a small fraction of brain volume but contain 27% of brain Aβ42 and 24% of Aβ40 in young mice. In the Tg2576 model, memory impairment begins at 6 months before amyloid plaques are visible. Here we show that Aβ dimers appear in lipid rafts at 6 months and that raft Aβ, which is primarily dimeric, rapidly accumulates reaching levels >500× those in young mice by 24–28 months. A similar large accumulation of dimeric Aβ was observed in lipid rafts from AD brain. In contrast to extracellular amyloid fibrils, which are SDS-insoluble, virtually all Aβ in lipid rafts is SDS soluble. Coupled with recent studies showing that synthetic and naturally occurring Aβ oligomers can inhibit hippocampal long-term potentiation, the in vivo age-dependent accumulation of SDS-soluble Aβ dimers in lipid rafts at the time when memory impairment begins in Tg2576 mice provides strong evidence linking Aβ oligomers to memory impairment. After dimeric Aβ began to accumulate in lipid rafts of the Tg2576 brain, apolipoprotein E (ApoE) and then phosphorylated tau accumulated. A similar increase in ApoE and a large increase in phosphorylated tau was observed in lipid rafts from AD brain. These findings suggest that lipid rafts may be an important site for interaction between dimeric Aβ, ApoE, and tau.

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Figures

Figure1 Characterization of lipid rafts from 3-month-old Tg2576 mouse brains. A, Western blotting of sucrose gradient centrifuge fractions. Lanes 1-11 show the 11 fractions from the sucrosegradient.Thelipidraftmarkers,flotillin and GM1ganglioside,localizedinfraction4.The ER marker, calnexin, localized in fractions 8 -11. APP, CTF of APP, BACE1, PS1-N, PS1-C, neprilysin, and ApoE localized in lipid rafts as well as in fractions 8 -11. B, Fraction 4 was composed of vesicular structures of various sizes by EM. Scale bar, 600 nm. C , D, Levels of A 𝛃 42 SDS ( C ) and A 𝛃 40 SDS ( D ) in fractions 1-11 from 3-month-old Tg2576 brains ( n = 6). E, Immunoblotting of A 𝛃 in lipid rafts from 3-month-old transgenic brains. Synthetic A 𝛃 1-40( a, 2,000 fmol; b, 500 fmol; c, 125 fmol), fraction 4 of 3-month-old transgenic brains ( d, 0.5 ml; e, 2 ml; f, 2 ml) were immunoprecipitated with 3160 (anti-A 𝛃 1-40). Proteins were separated on 16% tricine gels, transferred to polyvinylidene difluoride membrane, and detected with BAN-50 (anti-A 𝛃 1-16, lanes a- e) or 4G8 (anti-A 𝛃 17-24, lane f). The arrowhead identifies CTF 𝛃 , and the arrow identifies A 𝛃 monomers.
Figure1 Characterization of lipid rafts from 3-month-old Tg2576 mouse brains. A, Western blotting of sucrose gradient centrifuge fractions. Lanes 1-11 show the 11 fractions from the sucrosegradient.Thelipidraftmarkers,flotillin and GM1ganglioside,localizedinfraction4.The ER marker, calnexin, localized in fractions 8 -11. APP, CTF of APP, BACE1, PS1-N, PS1-C, neprilysin, and ApoE localized in lipid rafts as well as in fractions 8 -11. B, Fraction 4 was composed of vesicular structures of various sizes by EM. Scale bar, 600 nm. C , D, Levels of A 𝛃 42 SDS ( C ) and A 𝛃 40 SDS ( D ) in fractions 1-11 from 3-month-old Tg2576 brains ( n = 6). E, Immunoblotting of A 𝛃 in lipid rafts from 3-month-old transgenic brains. Synthetic A 𝛃 1-40( a, 2,000 fmol; b, 500 fmol; c, 125 fmol), fraction 4 of 3-month-old transgenic brains ( d, 0.5 ml; e, 2 ml; f, 2 ml) were immunoprecipitated with 3160 (anti-A 𝛃 1-40). Proteins were separated on 16% tricine gels, transferred to polyvinylidene difluoride membrane, and detected with BAN-50 (anti-A 𝛃 1-16, lanes a- e) or 4G8 (anti-A 𝛃 17-24, lane f). The arrowhead identifies CTF 𝛃 , and the arrow identifies A 𝛃 monomers.CC-BY-NC-SA · From the PDF
Image 2
CC-BY-NC-SA · From the PDF
Figure 3 Electron microscopy of A 𝛃 42, A 𝛃 40, and A 𝛃 fibrils in the lipid raft fraction and pellets from 28-month-old Tg2576 brain. a-c , IEM showed that vesicular profiles in fraction 4 werelabeledwithanti-A 𝛃 antibodies ( a, BAN-50; b, BA-27; c, BC-05). No labeling was observed in control experiments in which anti-A 𝛃 antibodies were omitted (data not shown). d, Many amyloid fibrils stained with 3160 coupled with 5 nm gold particles were present in fraction 12 (high speed gradient pellet). e, Amyloid cores were detected in fraction 14 (low-speed Triton pellet). Scale bar: a-d , 150 nm; e , 300.
Figure 3 Electron microscopy of A 𝛃 42, A 𝛃 40, and A 𝛃 fibrils in the lipid raft fraction and pellets from 28-month-old Tg2576 brain. a-c , IEM showed that vesicular profiles in fraction 4 werelabeledwithanti-A 𝛃 antibodies ( a, BAN-50; b, BA-27; c, BC-05). No labeling was observed in control experiments in which anti-A 𝛃 antibodies were omitted (data not shown). d, Many amyloid fibrils stained with 3160 coupled with 5 nm gold particles were present in fraction 12 (high speed gradient pellet). e, Amyloid cores were detected in fraction 14 (low-speed Triton pellet). Scale bar: a-d , 150 nm; e , 300.CC-BY-NC-SA · From the PDF
Figure 4 A 𝛃 42andA 𝛃 40in control, pathological aging (PA), and AD brains. The figures on the left show A 𝛃 42, and those on the right A 𝛃 40 in control ( n = 2), pathologic aging ( n = 2), andAD( n = 6) brains. Fractions shown are lipid raft A 𝛃 (fractions 3-5; A, D ), Triton-soluble A 𝛃 (fractions 8 -11; B, E ), and pellet A 𝛃 (fractions 12 + 14; C, F ). SDS-soluble A 𝛃 (A 𝛃 SDS ) is shown in filled columns; SDS-insoluble A 𝛃 that was solubilized in formic acid (A 𝛃 FA ) is shown in open columns. Standard error bars are not shown for pathologic aging and controls because only two brains were analyzed in each instance.
Figure 4 A 𝛃 42andA 𝛃 40in control, pathological aging (PA), and AD brains. The figures on the left show A 𝛃 42, and those on the right A 𝛃 40 in control ( n = 2), pathologic aging ( n = 2), andAD( n = 6) brains. Fractions shown are lipid raft A 𝛃 (fractions 3-5; A, D ), Triton-soluble A 𝛃 (fractions 8 -11; B, E ), and pellet A 𝛃 (fractions 12 + 14; C, F ). SDS-soluble A 𝛃 (A 𝛃 SDS ) is shown in filled columns; SDS-insoluble A 𝛃 that was solubilized in formic acid (A 𝛃 FA ) is shown in open columns. Standard error bars are not shown for pathologic aging and controls because only two brains were analyzed in each instance.CC-BY-NC-SA · From the PDF
Figure 5 A 𝛃 dimers in lipid rafts from Tg2576 and human brains. A, Fractions 1-11 from 11-month-old Tg2576 brain. Ba, Fraction 4 from 4-, 8-, 10-, and 12-month-old Tg2576 brains. Bb, Fraction 4 from 5-, 6-, 7-, 8-, 10-, and 11-month-old Tg2576 brains labeled with 4G8. The A 𝛃 in A and B was immunoprecipitated with polyclonal antibody 3160 and then immunoblotted with 4G8, a monoclonal antibody that detects both monomers and dimers. Dimers were the predominant form of A 𝛃 detected in lipid rafts. A 𝛃 dimers were detected at 6 months and progressively accumulated thereafter. C, Epitope mapping of fraction 4 from 17-month-old Tg2576 brain using anti-A 𝛃 and anti-APP antibodies. A 𝛃 was immunoprecipitated with 3160 and then immunoblotted with Z31preA ( B ), BAN-50 ( C ), 4G8 ( D ), BA-27 ( E ), BC-05 ( F ), and O443( G ). Lane A is synthetic A 𝛃 40(100pmol)immunoprecipitatedwith3160anddetectedby BAN-50. The 8 kDa A 𝛃 dimer and the 4 kDa A 𝛃 monomer were detected by all anti-A 𝛃 antibodies. BA-27 specifically detects the C terminus of A 𝛃 40 and BC-05 is selective for the C terminus of A 𝛃 42, so the labeling by these antibodies shows that the 8 kDa protein terminates at A 𝛃 40oratA 𝛃 42onthecarboxylside.Z31preA,whichrecognizestheAPPepitopejustamino to A 𝛃 failed to detect the 8 kDa band, showing that the 8 kDa band is dimeric A 𝛃 and not an A 𝛃 -bearing APP fragment that extends past A 𝛃 on the amino side. The 8 kDa A 𝛃 dimers were also not detected by anti-C terminus of APP (O443). Because 3160 recognizes the N-terminal region of A 𝛃 , p3 was not detected with this immunoprecipitation. D, Fractions 1-11 from AD, pathological aging (PA), and control brains. The A 𝛃 in the fractions shown in D was directly immunoblotted with 4G8.
Figure 5 A 𝛃 dimers in lipid rafts from Tg2576 and human brains. A, Fractions 1-11 from 11-month-old Tg2576 brain. Ba, Fraction 4 from 4-, 8-, 10-, and 12-month-old Tg2576 brains. Bb, Fraction 4 from 5-, 6-, 7-, 8-, 10-, and 11-month-old Tg2576 brains labeled with 4G8. The A 𝛃 in A and B was immunoprecipitated with polyclonal antibody 3160 and then immunoblotted with 4G8, a monoclonal antibody that detects both monomers and dimers. Dimers were the predominant form of A 𝛃 detected in lipid rafts. A 𝛃 dimers were detected at 6 months and progressively accumulated thereafter. C, Epitope mapping of fraction 4 from 17-month-old Tg2576 brain using anti-A 𝛃 and anti-APP antibodies. A 𝛃 was immunoprecipitated with 3160 and then immunoblotted with Z31preA ( B ), BAN-50 ( C ), 4G8 ( D ), BA-27 ( E ), BC-05 ( F ), and O443( G ). Lane A is synthetic A 𝛃 40(100pmol)immunoprecipitatedwith3160anddetectedby BAN-50. The 8 kDa A 𝛃 dimer and the 4 kDa A 𝛃 monomer were detected by all anti-A 𝛃 antibodies. BA-27 specifically detects the C terminus of A 𝛃 40 and BC-05 is selective for the C terminus of A 𝛃 42, so the labeling by these antibodies shows that the 8 kDa protein terminates at A 𝛃 40oratA 𝛃 42onthecarboxylside.Z31preA,whichrecognizestheAPPepitopejustamino to A 𝛃 failed to detect the 8 kDa band, showing that the 8 kDa band is dimeric A 𝛃 and not an A 𝛃 -bearing APP fragment that extends past A 𝛃 on the amino side. The 8 kDa A 𝛃 dimers were also not detected by anti-C terminus of APP (O443). Because 3160 recognizes the N-terminal region of A 𝛃 , p3 was not detected with this immunoprecipitation. D, Fractions 1-11 from AD, pathological aging (PA), and control brains. The A 𝛃 in the fractions shown in D was directly immunoblotted with 4G8.CC-BY-NC-SA · From the PDF
Figure6 Accumulation of ApoE and phosphorylated tau in lipid rafts. A, Onemicroliter of fraction 4 at 3, 4, 5, 8, 12, 18, 24, and 28monthswasanalyzedbyimmunoblotwithantibodiestoflotillin,APP,ApoE,WKS46(aphosphorylation-independentantibody against tau 359 -370), and PHF-1 (an antibody to tau phosphorylated at pSer396/pSer404) sites. The amount of flotillin and APP did not change with aging. ApoE increased substantially beginning at 12 months. Total tau detected by WKS46 did not change with aging, whereas phosphorylated-tau stained by PHF-1 increased beginning at 18 months. Sucrose gradient centrifuge fractions (1-11) from AD and control brains were analyzed by immunoblot using anti-flotillin antibody ( B ), anti-human ApoE antibody ( C ), or PHF-1 ( D ). Note the accumulation of ApoE in lipid rafts (fraction 4) from AD as compared with control brains and the intensely labeled smear of phosphorylated tau in lipid rafts from AD but not control brain.
Figure6 Accumulation of ApoE and phosphorylated tau in lipid rafts. A, Onemicroliter of fraction 4 at 3, 4, 5, 8, 12, 18, 24, and 28monthswasanalyzedbyimmunoblotwithantibodiestoflotillin,APP,ApoE,WKS46(aphosphorylation-independentantibody against tau 359 -370), and PHF-1 (an antibody to tau phosphorylated at pSer396/pSer404) sites. The amount of flotillin and APP did not change with aging. ApoE increased substantially beginning at 12 months. Total tau detected by WKS46 did not change with aging, whereas phosphorylated-tau stained by PHF-1 increased beginning at 18 months. Sucrose gradient centrifuge fractions (1-11) from AD and control brains were analyzed by immunoblot using anti-flotillin antibody ( B ), anti-human ApoE antibody ( C ), or PHF-1 ( D ). Note the accumulation of ApoE in lipid rafts (fraction 4) from AD as compared with control brains and the intensely labeled smear of phosphorylated tau in lipid rafts from AD but not control brain.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Kawarabayashi, T., Shoji, M., Younkin, L. H., Wen-Lang, L., Dickson, D. W., Murakami, T., Matsubara, E., Abe, K., Ashe, K. H., & Younkin, S. G.
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2004-04-14
Volume
24
Issue
15
Pages
3801-3809
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.5543-03.2004
Cited by
320
References
11
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 183 d)

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