The Synaptic Accumulation of Hyperphosphorylated Tau Oligomers in Alzheimer Disease Is Associated With Dysfunction of the Ubiquitin-Proteasome System

• DOI: 10.1016/j.ajpath.2012.06.033
• Publication Date: 2012-08-04T06:31:04Z
• Authors: Hwan-Ching Tai; Alberto Serrano-Pozo; Tadafumi Hashimoto; Matthew P. Frosch; Tara L. Spires-Jones; Bradley T. Hyman
• Keywords: Neocortex; Proteotoxicity; Tau protein

In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss synapses in the neocortex limbic system each correlate strongly with cognitive impairment. Tangles are composed misfolded hyperphosphorylated tau proteins; however, link between abnormalities synaptic dysfunction remains unclear. We examined location control AD cortices using biochemical morphologic methods. found that, addition to its well-described axonal localization, normal is present at both presynaptic postsynaptic terminals human brains. AD, becomes terminals, this abnormally posttranslationally modified enriched synaptoneurosomal fractions. Synaptic seems be ubiquitinated, forms stable oligomers resistant SDS denaturation. The accumulation associated increased ubiquitinated substrates proteasome components, consistent ubiquitin-proteasome system. Our findings suggest that may an important mediator proteotoxicity disrupts AD. 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Activity-dependent dynamics sequestration proteasomes spines.Nature. 441: 1144-1148Crossref (275) 17Yi Ehlers M.D. Emerging roles for function.Pharmacol 2007; 59: 14-39Crossref (177) By isolating we observed brains, terminals. contrast, synaptoneurosomes isolated from brains p-tau form SDS-resistant on sides synapse, showing enrichment when compared cytoplasm. mirrors same fraction, well chaperones, which suggests aggregates impaired proteolysis mediated by (UPS).18Keck Nitsch Grune Ullrich O. Proteasome inhibition filament-tau patients disease.J Neurochem. 85: 115-122Crossref (405) Protease inhibitor (cOmplete tablet) was purchased Roche Applied Science (Roche Diagnostics Corp., Indianapolis, IN). Phosphatase cocktails 3 were Sigma-Aldrich Corp. (St. Louis, MO) 1:1 combination. Mouse monoclonal antibodies PHF1 (pS396/pS404 tau), CP13 (pS202 DA9 (total tau) gifts Peter (Albert Einstein College Medicine, Bronx, NY). Rabbit anti–total (A20024) Dako Denmark A/S (Glostrup, Denmark); rabbit anti-PSD95 (No. 2507) Cell Signaling Technology, Inc. (Danvers, MA); anti-MBP (SMI-99P) Covance, (Princeton, NJ); anti-actin (A4700), (A5060), anti-tubulin β3 (T8660) Sigma-Aldrich; anti-synaptophysin (AB8049), anti-VCP (AB19444), anti-VDAC (AB34726) Abcam (Cambridge, anti-GAPDH (MAB374), anti-histone H3 (05-928), anti-Myc (06-549) Millipore (Billerica, anti-ubiquitin conjugates (UG9510), anti-α7 (20S subunit, PW8110), anti-Rpt1 (26S PW8852) Enzo Clinical Laboratories, (Farmingdale, Brains subjects diagnosis no deficits obtained Massachusetts Disease Research Center General Hospital Department. All donor tissue accord local national institutional review board regulations. Characteristics controls quantitative analyses given Table 1.Table 1Characteristics Control AD-Affected Used Quantitative StudiesCase No.Age (years)SexClinical DiagnosisDisease Duration (years)ApoE GenotypePMI (hours)Braak StageExperimentC189FControlNA2/3132Figure 4C291FControlNA3/3192Figure 4C371MControlNANA50Figure 4C487MControlNANA361Figure 4C580FControlNA2/4541Figure 7C676MControlNA3/4481Figure 7C785MControlNA3/3242Figure 7C857FControlNA3/3130Figure 7C974FControlNA3/3241Figure 7C1088FControlNA3/3202Figure 7AD183FAD133/4125Figure 4AD282MAD63/476Figure 4AD391FAD143/495Figure 4AD495MADNA3/3116Figure 4AD585FAD43/4105Figure 5AD673FAD193/3145Figure 5AD784FAD163/4125Figure 5AD865MAD83/3215Figures 7, 8AD984FAD73/376Figures 8AD1075FAD53/3266Figures 8AD1192FAD93/346Figures 8AD1293MAD173/365Figures 8AD1374MAD113/3155Figures 8AD1492MAD224/4125Figures 5, 8AD1568FAD114/4206Figures 8AD1680FAD124/4116Figures 8AD1771FAD174/4146Figures 8AD1874MAD174/4205Figures 8AD1989MAD104/4105Figures 8F = female; M male; disease; NA applicable available; PMI postmortem interval. Open table tab F Cortical gray matter (200 300 mg) taken frozen gently ground Potter-Elvehjem homogenizer 1.5 mL ice-cold buffer A (25 mmol/L HEPES [pH 7.5], 120 NaCl, 5 KCl, 1 MgCl2, CaCl2) supplemented dithiothreitol (DTT), protease inhibitors, phosphatase inhibitors. homogenate passed two layers 80-μm nylon filters (Millipore) remove debris, 200-μL aliquot saved. saved mixed 200 μL water 70 10% SDS, 27-gauge needle, boiled minutes prepare total extract. To filtered Hollingsworth et al,19Hollingsworth E.B. McNeal Burton J.L. Williams R.J. Daly J.W. Creveling C.R. Biochemical characterization synaptoneurosome preparation guinea pig cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, enzymes.J 1985; 5: 2240-2253Crossref remainder 5-μm Supor membrane filter (Pall Port Washington, NY) large organelles nuclei, centrifuged 1000 × g minutes. pellet washed once again, yielding pellet. Supernatant first centrifugation clarified via 100,000 hour obtain cytosol fraction. Cytosolic extract prepared adding 1.5% boiling Synaptoneurosome pellets extracted 0.5 B (50 Tris DTT), Flotation sucrose gradient previously published procedures.20Lathia Wesemann W. Serotonin uptake release biochemically characterized nerve endings concomitant flotation sedimentation centrifugation.J Neural Transm. 1975; 37: 111-126Crossref (4) Ice-cold solutions 0.3, 0.93, 1.2 mol/L 10 (pH 7.5). resuspended transferred centrifuge tube, then overlaid 1.2, 0.3 three-layer discontinuous gradient. After 60,000 hours, collected 0.93/1.2-mol/L interface, myelin 0.3/0.93-mol/L diluted about final concentration. sucrose. three fractions 20,000 20 minutes, C [50 7.5), 150 2% Triton X-100, inhibitors]. incubation rotating mixture hour. supernatant 1% SDS-denatured extracts 15 followed bicinchoninic acid assays (Pierce Protein Biology, Fisher Scientific, Inc., Rockford, IL) determine concentrations. Extracts again after 5X sample (250 5% 400 DTT, 50% glycerol, 0.2% Orange G). Samples resolved SDS-PAGE Bis-Tris 4% 12% gels (Invitrogen Carlsbad, CA), low-fluorescence polyvinylidene difluoride nitrocellulose membranes immunoblotting, detected Odyssey laser scanner (LI-COR Biosciences, Lincoln, NE). Blocking buffer, stripping secondary LI-COR, according manufacturer's protocols. D (20 4 cleared 15,000 equal volume E sodium deoxycholate) neutralize SDS. pre-cleared G Sepharose (GE Healthcare, Pittsburg, PA) 4°C hour, overnight S5a UIM agarose conjugate (UW9820; Laboratories) sepharose (control). flow-through resin times cold X-100) 1.5X elute captured proteins. immunoprecipitation experiments, incubated (control), hours. Western blots enhanced chemiluminescence TrueBlot Ultra anti-mouse horseradish peroxidase (eBioscience, San Diego, CA) Clean-Blot (against primary antibodies; Pierce) minimize signals denatured IgG. A, needles, paraformaldehyde PBS-MC (1 MgCl2 Lab-Tek II CC2 pre-coated chamber slides (Nunc, Rochester, settling 4°C, became fixed attached glass surface, (room temperature point on). Synaptoneurosomes permeabilized 0.05% X-100 3% bovine serum albumen (BSA), times. Slides blocked goat BSA 30 washes. Secondary slide mounted No. coverslip Prolong Antifade reagent (Invitrogen). Primary immunostaining included anti-vGlut1 (Millipore AB590, 1:150), chicken anti-MAP2 (Abcam AB5392, 100), AB12093, 1:100), (1:150), (1:80). Fluorescent donkey Jackson ImmunoResearch (West Grove, 1:100 dilutions (anti-guinea DyLight 649, anti-chicken Cy3, anti-goat Alexa 488, 488). brightfield images immunostained acquired Axio Imager Z epifluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) equipped 63X oil immersion objective (numerical aperture, 1.40). Images deconvolved Iterative Deconvolution plug-in (by Bob Dougherty, OptiNav, Inc.) ImageJ software (version 1.44). co-localization determined manual analysis randomly selected areas wide-field images. glutaraldehyde PBS rinsed, post-fixed osmium tetroxide, embedded LR White (Electron Microscopy Sciences, Hatfield, PA). transmission electron ATM digital camera (JEOL1011; JEOL USA, Peabody, MA). quantified densitometry gel Statistical tests (paired t-test, Mann-Whitney test, two-way variance, linear regression) performed statistical (GraphPad version 5.03; Prism Software La Jolla, CA). Proteins cortical homogenates analyzed SDS-PAGE. extracts, exhibited only monomeric nonhyperphosphorylated 65 kDa). AD-affected contained species (reactive against CP1321Duff Knight Refolo Sanders Yu X. Picciano Malester Hutton Adamson Goedert Burki Characterization pathology transgenic mice over-expressing genomic cDNA transgenes.Neurobiol Dis. 2000; 7: 87-98Crossref (251) PHF122Greenberg S.G. Schein J.D. Hydrofluoric acid-treated PHF display properties tau.J 267: 564-569Abstract Scholar) migrated smear (Figure 1). Fast-migrating (15 50 kDa) represented truncated p-tau; slow-migrating (65 several hundred oligomers,23Watanabe Deamidation isoaspartate smeared filaments Unusual microtubule-binding domain 274: 7368-7378Abstract (106) stably denaturation reducing agents. understand subcellular localization separated into cytosolic (synaptoneurosome) under microscopy, showed well-preserved structures. synaptoneurosomes, fibrillar microscopy 2A). samples nonphosphorylated than expected revealed accumulated high levels remained surprisingly absent 2B). Considered together, our data nonfibrillar pellet, containing oligomers. examine rather originating other contaminants inclusions, further purify 3A). One potential concern could co-precipitated during low-speed (1000 g). Because assemblies dense lipid-rich vesicles, chose gradient–purified great reductions organelle nuclei 3B). they low clearly detectable tau, suggesting confirming role synapse.13Ittner 24Roberson E.D. Scearce-Levie Palop Yan Cheng I.H. Wu Gerstein G.Q. Reducing endogenous ameliorates beta-induced model.Science. 316: 750-754Crossref (1545) including aggregate 3C), hyperphosphorylation oligomerization specific synapses. did demonstrate terminals; developed protocol this. visualize spread dilute solution onto lightly place (see Materials Methods). vesicular glutamate transporter (vGlut1) marker, (MAP2) marker 4A). MAP2 lower inside spines PSD.25Morales Fifkova colocalization actin: immunogold electron-microscope study.Cell Tissue Res. 1989; 256: 447-456Crossref (90) 26Caceres Payne M.R. Bender Rebhun Steward Differential tubulin immunocytochemistry hybridoma antibodies.J 1984; 4: 394-410Crossref confirmed co-staining PSD95 4D), noted vGlut1 often seemed so close almost overlap, whereas frequently adjacent distinct puncta; we, use mark enable straightforward identification tau. Using antibody (DA9) recognizes phosphorylated vGlut1-positive puncta (55.3%) MAP2-positive (70.2%) 63.3% 70.1% 4, C). With detect significant difference Under approximately 80% contain visible microtubules.27Gordon-Weeks Burgoyne Gray E.G. Presynaptic microtubules: organisation assembly/disassembly.Neuroscience. 1982; 739-749Abstract (46) Inasmuch 55.3% presynapses reasonable, possibly underestimate. criteria, neighboring structures exhibit 70.2% labeling, indicated localization.13Ittner Immunocytochemistry labeled substantial percentage synapses: 20.8% 32.0% 5). These support hypothesis already progression misfolding occur synapse. oligomers, solubilize free cytoplasmic proteins, remaining insoluble material crude PSD retained nearly all actin microtubule cytoskeleton 6). Most extractable Triton, small amount co-immunoprecipitated PSD95.13Ittner essentially Triton-insoluble indicating supports spines. ubiquitin-