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

2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês

10.1016/j.ajpath.2012.06.033

1525-2191

Hwan‐Ching Tai, Alberto Serrano‐Pozo, Tadafumi Hashimoto, Matthew P. Frosch, Tara L. Spires‐Jones, Bradley T. Hyman,

Cholinesterase and Neurodegenerative Diseases

In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD. In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD. Alzheimer disease (AD) is the most common neurodegenerative disorder in the elderly, and affects primarily the neocortex and the limbic system, with complex pathophysiologic features that include tau inclusions (neurofibrillary tangles, neuropil threads, and dystrophic neurites), β-amyloid inclusions (plaques and cerebral amyloid angiopathy), loss of neurons and synapses, astrogliosis, microglial activation, and inflammation.1Serrano-Pozo A. Frosch M.P. Masliah E. Hyman B.T. Neuropathological alterations in Alzheimer disease.Cold Spring Harb Perspect Med. 2011; 1: a006189Crossref PubMed Scopus (2195) Google Scholar, 2Braak E. Griffing K. Arai K. Bohl J. Bratzke H. Braak H. Neuropathology of Alzheimer's disease: what is new since A. Alzheimer?.Eur Arch Psychiatry Clin Neurosci. 1999; 249: 14-22Crossref PubMed Google Scholar Among these features, synaptic loss3Terry R.D. Masliah E. Salmon D.P. Butters N. DeTeresa R. Hill R. Hansen L.A. Katzman R. Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment.Ann Neurol. 1991; 30: 572-580Crossref PubMed Scopus (3511) Google Scholar, 4DeKosky S.T. Scheff S.W. Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity.Ann Neurol. 1990; 27: 457-464Crossref PubMed Scopus (1764) Google Scholar and neurofibrillary tangle deposition5Arriagada P.V. Marzloff K. Hyman B.T. Distribution of Alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer's disease.Neurology. 1992; 42: 1681-1688Crossref PubMed Google Scholar, 6Giannakopoulos P. Herrmann F.R. Bussiere T. Bouras C. Kovari E. Perl D.P. Morrison J.H. Gold G. Hof P.R. Tangle and neuron numbers, but not amyloid load, predict cognitive status in Alzheimer's disease.Neurology. 2003; 60: 1495-1500Crossref PubMed Scopus (828) Google Scholar seem to best correlate with cognitive decline. Neurofibrillary and synaptic loss are correlated in clinicopathologic studies of AD7Ingelsson M. Fukumoto H. Newell K.L. Growdon J.H. Hedley-Whyte E.T. Frosch M.P. Albert M.S. Hyman B.T. Irizarry M.C. Early Abeta accumulation and progressive synaptic loss, gliosis, and tangle formation in AD brain.Neurology. 2004; 62: 925-931Crossref PubMed Scopus (542) Google Scholar; however, whether this is a co-occurrence of parallel pathologic processes or synaptic loss is more directly related to alterations in tau biology is uncertain.8Morris M. Maeda S. Vossel K. Mucke L. The many faces of tau.Neuron. 2011; 70: 410-426Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, 9Spires-Jones T.L. Stoothoff W.H. de Calignon A. Jones P.B. Hyman B.T. Tau pathophysiology in neurodegeneration: a tangled issue.Trends Neurosci. 2009; 32: 150-159Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar We, therefore, used new biochemical and morphologic techniques to address the issue of tau protein accumulation in synapses in the adult human brain and in AD. Normal tau is an abundant microtubule-associated protein that has been described as predominantly localized in axons in mature neurons.10Dotti C.G. Banker G.A. Binder L.I. The expression and distribution of the microtubule-associated proteins tau and microtubule-associated protein 2 in hippocampal neurons in the rat in situ and in cell culture.Neuroscience. 1987; 23: 121-130Abstract Full Text PDF PubMed Scopus (173) Google Scholar In AD, abnormally folded and hyperphosphorylated tau (p-tau) accumulates in axons, dendrites, and somas.11Avila J. Lucas J.J. Perez M. Hernandez F. Role of tau protein in both physiological and pathological conditions.Physiol Rev. 2004; 84: 361-384Crossref PubMed Scopus (721) Google Scholar, 12Brandt R. Hundelt M. Shahani N. Tau alteration and neuronal degeneration in tauopathies: mechanisms and models.Biochim Biophys Acta. 2005; 1739: 331-354Crossref PubMed Scopus (205) Google Scholar In contrast to these long-held generalizations, recent reports have suggested that tau is also normally present in dendritic spines, where it interacts with postsynaptic density (PSD) proteins such as Fyn kinase.13Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wolfing H. Chieng B.C. Christie M.J. Napier I.A. Eckert A. Staufenbiel M. Hardeman E. Gotz J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar We hypothesized that tau may pathologically accumulate at synaptic sites in AD because it has been recently suggested that tau can be present in postsynaptic locales in normal mice,13Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wolfing H. Chieng B.C. Christie M.J. Napier I.A. Eckert A. Staufenbiel M. Hardeman E. Gotz J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar ubiquitinated tau accumulates in the brain in AD,14Cripps D. Thomas S.N. Jeng Y. Yang F. Davies P. Yang A.J. Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation.J Biol Chem. 2006; 281: 10825-10838Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 15Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Titani K. Ihara Y. Ubiquitin is conjugated with amino-terminally processed tau in paired helical filaments.Neuron. 1993; 10: 1151-1160Abstract Full Text PDF PubMed Scopus (302) Google Scholar and a major site of protein ubiquitination and proteasome-mediated degradation is at presynaptic and postsynaptic structures.16Bingol B. Schuman E.M. Activity-dependent dynamics and sequestration of proteasomes in dendritic spines.Nature. 2006; 441: 1144-1148Crossref PubMed Scopus (275) Google Scholar, 17Yi J.J. Ehlers M.D. Emerging roles for ubiquitin and protein degradation in neuronal function.Pharmacol Rev. 2007; 59: 14-39Crossref PubMed Scopus (177) Google Scholar By isolating synaptic terminals, we observed that, in control brains, tau is present at both presynaptic and postsynaptic terminals. In contrast, in synaptoneurosomes isolated from brains in AD, p-tau can form stable SDS-resistant oligomers that accumulate on both sides of the synapse, showing synaptic enrichment when compared with the cytoplasm. The accumulation of p-tau at the synapse mirrors the accumulation of ubiquitinated proteins in the same fraction, as well as the accumulation of proteasomes and related chaperones, which suggests that tau aggregates are associated with impaired proteolysis mediated by the ubiquitin-proteasome system (UPS).18Keck S. Nitsch R. Grune T. Ullrich O. Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease.J Neurochem. 2003; 85: 115-122Crossref PubMed Scopus (405) Google Scholar Protease inhibitor (cOmplete tablet) was purchased from Roche Applied Science (Roche Diagnostics Corp., Indianapolis, IN). Phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma-Aldrich Corp. (St. Louis, MO) and used in a 1:1 combination. Mouse monoclonal antibodies PHF1 (pS396/pS404 tau), CP13 (pS202 tau), and DA9 (total tau) were gifts of Peter Davies (Albert Einstein College of Medicine, Bronx, NY). Rabbit anti–total tau (A20024) was purchased from Dako Denmark A/S (Glostrup, Denmark); rabbit anti-PSD95 (No. 2507) from Cell Signaling Technology, Inc. (Danvers, MA); mouse anti-MBP (SMI-99P) from Covance, Inc. (Princeton, NJ); mouse anti-actin (A4700), rabbit anti-actin (A5060), and mouse anti-tubulin β3 (T8660) from Sigma-Aldrich; mouse anti-synaptophysin (AB8049), mouse anti-VCP (AB19444), and rabbit anti-VDAC (AB34726) from Abcam Inc. (Cambridge, MA); mouse anti-GAPDH (MAB374), rabbit anti-histone H3 (05-928), and rabbit anti-Myc (06-549) from Millipore Corp. (Billerica, MA); and rabbit anti-ubiquitin conjugates (UG9510), mouse anti-α7 (20S subunit, PW8110), and mouse anti-Rpt1 (26S subunit, PW8852) from Enzo Clinical Laboratories, Inc. (Farmingdale, NY). Brains from human subjects with a diagnosis of AD or no cognitive deficits were obtained through the Massachusetts Alzheimer's Disease Research Center and Massachusetts General Hospital Neuropathology Department. All donor tissue was obtained in accord with local and national institutional review board regulations. Characteristics of controls and subjects with AD used for quantitative analyses are given in Table 1.Table 1Characteristics of Control and AD-Affected Brains Used in 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 7, 8AD1075FAD53/3266Figures 7, 8AD1192FAD93/346Figures 7, 8AD1293MAD173/365Figures 7, 8AD1374MAD113/3155Figures 7, 8AD1492MAD224/4125Figures 5, 7, 8AD1568FAD114/4206Figures 7, 8AD1680FAD124/4116Figures 7, 8AD1771FAD174/4146Figures 7, 8AD1874MAD174/4205Figures 7, 8AD1989MAD104/4105Figures 7, 8F = female; M = male; AD = Alzheimer disease; NA = not applicable or not available; PMI = postmortem interval. Open table in a new tab F = female; M = male; AD = Alzheimer disease; NA = not applicable or not available; PMI = postmortem interval. Cortical gray matter (200 to 300 mg) taken from frozen human brains was gently ground in a Potter-Elvehjem homogenizer with 1.5 mL ice-cold buffer A (25 mmol/L HEPES [pH 7.5], 120 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, and 2 mmol/L CaCl2) supplemented with 2 mmol/L dithiothreitol (DTT), protease inhibitors, and phosphatase inhibitors. The homogenate was passed through two layers of 80-μm nylon filters (Millipore) to remove tissue debris, and a 200-μL aliquot was saved. The saved aliquot was mixed with 200 μL water and 70 μL 10% SDS, passed through a 27-gauge needle, and boiled for 5 minutes to prepare the total extract. To prepare filtered synaptoneurosomes as described by Hollingsworth et al,19Hollingsworth E.B. McNeal E.T. Burton J.L. Williams R.J. Daly J.W. Creveling C.R. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, and enzymes.J Neurosci. 1985; 5: 2240-2253Crossref PubMed Google Scholar the remainder of the homogenate was passed through a 5-μm Supor membrane filter (Pall Corp., Port Washington, NY) to remove large organelles and nuclei, and centrifuged at 1000 × g for 5 minutes. The pellet was washed once with buffer A and centrifuged again, yielding the synaptoneurosome pellet. Supernatant from the first centrifugation was clarified via centrifugation at 100,000 × g for 1 hour to obtain the cytosol fraction. Cytosolic extract was prepared by adding 1.5% SDS and boiling for 5 minutes. Synaptoneurosome pellets were extracted using 0.5 mL buffer B (50 mmol/L Tris [pH 7.5], 1.5% SDS, and 2 mmol/L DTT), and were boiled for 5 minutes. Flotation sucrose gradient for isolating synaptic terminals was modified from previously published procedures.20Lathia D. Wesemann W. Serotonin uptake and release by biochemically characterized nerve endings isolated from rat brain by concomitant flotation and sedimentation centrifugation.J Neural Transm. 1975; 37: 111-126Crossref PubMed Scopus (4) Google Scholar Ice-cold sucrose solutions of 0.3, 0.93, and 1.2 mol/L were prepared using 10 mmol/L HEPES (pH 7.5). The synaptoneurosome pellet was resuspended in 1.2 mmol/L sucrose and transferred to a centrifuge tube, and then overlaid with 1.2, 0.93, and 0.3 mol/L sucrose to form a three-layer discontinuous gradient. After centrifugation at 60,000 × g for 2 hours, synaptic terminals were collected from the 0.93/1.2-mol/L interface, and myelin from the 0.3/0.93-mol/L interface, and both were diluted with water to about 0.3 mol/L final sucrose concentration. The pellet was also resuspended in 0.3 mol/L sucrose. All three fractions were centrifuged at 20,000 × g for 20 minutes, and the pellets were extracted using buffer B and boiled for 5 minutes. The synaptoneurosome pellet was resuspended in ice-cold buffer C [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 2% Triton X-100, and protease inhibitors]. After 20 minutes of incubation in a rotating tube, the mixture was centrifuged at 100,000 × g for 1 hour. The collected supernatant was supplemented with 1% SDS and boiled for 5 minutes. The pellet was extracted using buffer B and boiled for 5 minutes. SDS-denatured protein extracts were clarified via centrifugation at 20,000 × g for 15 minutes, followed by bicinchoninic acid assays (Pierce Protein Biology, Fisher Scientific, Inc., Rockford, IL) to determine protein concentrations. Extracts were boiled again for 3 minutes after adding 5X sample buffer (250 mmol/L Tris [pH 7.5], 5% SDS, 400 mmol/L DTT, 50% glycerol, and 0.2% Orange G). Samples were resolved via SDS-PAGE using Bis-Tris 4% to 12% gels (Invitrogen Corp., Carlsbad, CA), and were transferred to low-fluorescence polyvinylidene difluoride or nitrocellulose membranes for immunoblotting, detected using an Odyssey laser scanner (LI-COR Biosciences, Inc., Lincoln, NE). Blocking buffer, stripping buffer, and secondary antibodies were purchased from LI-COR, and were used according to the manufacturer's protocols. Synaptoneurosome pellets were resuspended in buffer D (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 0.2% SDS, and 4 mmol/L DTT), boiled for 5 minutes, and cleared via centrifugation at 15,000 × g for 10 minutes. The supernatant was mixed with an equal volume of buffer E (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 2% Triton X-100, and 1% sodium deoxycholate) to neutralize SDS. The mixture was pre-cleared via incubation using Protein G Sepharose (GE Healthcare, Pittsburg, PA) at 4°C for 1 hour, followed by overnight incubation with S5a UIM agarose conjugate (UW9820; Enzo Clinical Laboratories) or protein G sepharose (control). After centrifugation at 1000 × g for 2 minutes, the supernatant was collected as the flow-through fraction. The resin was washed three times with cold buffer F (20 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, and 1% Triton X-100) and boiled for 5 minutes with 1.5X SDS-PAGE sample buffer to elute captured ubiquitinated proteins. In immunoprecipitation experiments, the pre-cleared extract was incubated overnight at 4°C with rabbit anti–total tau or anti-Myc (control), followed by incubation with protein G sepharose for 2 hours. After centrifugation at 1000 × g for 2 minutes, the supernatant was collected as the flow-through fraction. The resin was washed three times with cold buffer F and boiled for 5 minutes with 1.5X SDS-PAGE sample buffer to elute captured proteins. Western blots of immunoprecipitation proteins were detected via enhanced chemiluminescence using TrueBlot Ultra anti-mouse horseradish peroxidase conjugate (eBioscience, Inc., San Diego, CA) or Clean-Blot horseradish peroxidase conjugate (against rabbit primary antibodies; Pierce) to minimize signals from denatured IgG. Synaptoneurosome pellets were resuspended in ice-cold buffer A, passed through 27-gauge needles, and mixed with an equal volume of 2% paraformaldehyde in PBS-MC (1 mmol/L MgCl2 and 1 mmol/L CaCl2) in Lab-Tek II CC2 pre-coated chamber slides (Nunc, Rochester, NY). After 10 minutes of settling at 4°C, synaptoneurosomes became fixed and attached to the glass surface, and were washed three times using PBS-MC (room temperature from this point on). Synaptoneurosomes were permeabilized using 0.05% Triton X-100 in PBS-MC with 3% bovine serum albumen (BSA), and washed three times. Slides were blocked using 4% normal goat serum and 3% BSA in PBS-MC for 30 minutes and then incubated with primary antibodies diluted in PBS-MC with 3% BSA for 2 hours, followed by three washes. Secondary antibodies diluted in PBS-MC with 3% BSA were incubated for 1 hour, followed by three washes. The slide was mounted with a No. 1.5 glass coverslip and Prolong Gold Antifade reagent (Invitrogen). Primary antibodies for immunostaining included guinea pig anti-vGlut1 (Millipore AB590, 1:150), chicken anti-MAP2 (Abcam AB5392, 1: 100), goat anti-PSD95 (Abcam AB12093, 1:100), DA9 (1:150), and PHF1 (1:80). Fluorescent secondary donkey antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and used at 1:100 dilutions (anti-guinea pig DyLight 649, anti-chicken Cy3, anti-goat Alexa 488, and anti-mouse Alexa 488). Fluorescent and brightfield images of immunostained synaptoneurosomes were acquired using an Axio Imager Z epifluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a 63X oil immersion objective (numerical aperture, 1.40). Images were deconvolved using the Iterative Deconvolution plug-in (by Bob Dougherty, OptiNav, Inc.) in ImageJ software (version 1.44). Synaptic protein co-localization was determined via manual analysis on randomly selected areas from wide-field images. Synaptoneurosome pellets were fixed in 2% glutaraldehyde and 2% paraformaldehyde in PBS overnight at 4°C, rinsed, post-fixed in 1% osmium tetroxide, and embedded in LR White resin (Electron Microscopy Sciences, Hatfield, PA). Images were acquired using a transmission electron microscope equipped with an ATM digital camera (JEOL1011; JEOL USA, Inc., Peabody, MA). Western blots were quantified via densitometry using the gel analysis function in ImageJ software (version 1.44). Statistical tests (paired t-test, Mann-Whitney test, two-way analysis of variance, and linear regression) were performed using statistical software (GraphPad version 5.03; Prism Software Corp., La Jolla, CA). Proteins from control and AD cortical homogenates were extracted using 1.5% SDS and analyzed using SDS-PAGE. In total protein extracts, control brains exhibited only monomeric nonhyperphosphorylated tau (50 to 65 kDa). AD-affected brains contained tau species that were hyperphosphorylated (reactive against p-tau antibodies CP1321Duff K. Knight H. Refolo L.M. Sanders S. Yu X. Picciano M. Malester B. Hutton M. Adamson J. Goedert M. Burki K. Davies P. Characterization of pathology in transgenic mice over-expressing human genomic and cDNA tau transgenes.Neurobiol Dis. 2000; 7: 87-98Crossref PubMed Scopus (251) Google Scholar and PHF122Greenberg S.G. Davies P. Schein J.D. Binder L.I. Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.J Biol Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar) and migrated as a smear (Figure 1). Fast-migrating species (15 to 50 kDa) represented truncated p-tau; slow-migrating species (65 to several hundred kDa) represented p-tau oligomers,23Watanabe A. Takio K. Ihara Y. Deamidation and isoaspartate formation in smeared tau in paired helical filaments Unusual properties of the microtubule-binding domain of tau.J Biol Chem. 1999; 274: 7368-7378Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar which were stably misfolded and resistant to SDS denaturation and reducing agents. To understand the subcellular localization of tau proteins in AD, we separated cortical tissue homogenates into cytosolic and synaptic (synaptoneurosome) fractions. The synaptoneurosome fraction, when examined under electron microscopy, showed well-preserved synaptic structures. In both control and AD synaptoneurosomes, no fibrillar protein aggregates were detected under electron microscopy (Figure 2A). Biochemical analysis of tau in control samples showed that nonphosphorylated tau was more enriched in the cytosol than in synaptoneurosomes, consistent with its expected primary distribution in the axonal cytoplasm. In contrast, analysis of AD samples revealed that p-tau accumulated to high levels in AD synaptoneurosomes, but remained surprisingly absent in cytosolic extracts (Figure 2B). Considered together, our data suggest that p-tau can form nonfibrillar aggregates associated with the synaptoneurosomal pellet, containing SDS-resistant oligomers. To examine whether the p-tau oligomers observed in AD synaptoneurosomes were associated with synapses rather than originating from other contaminants such as tau inclusions, we used sucrose gradient to further purify synaptic terminals (Figure 3A). One potential concern was that large nonfibrillar tau aggregates could have co-precipitated with synaptoneurosomes during low-speed centrifugation (1000 × g). Because large protein assemblies are more dense than lipid-rich vesicles, we chose a flotation gradient to purify synaptic terminals. Flotation gradient–purified synaptic terminals showed great reductions in organelle contaminants such as nuclei and myelin (Figure 3B). In control brains, they contained low but clearly detectable levels of nonphosphorylated monomeric tau, suggesting a synaptic localization of tau and confirming recent reports of a normal role for tau at the synapse.13Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wolfing H. Chieng B.C. Christie M.J. Napier I.A. Eckert A. Staufenbiel M. Hardeman E. Gotz J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar, 24Roberson E.D. Scearce-Levie K. Palop J.J. Yan F. Cheng I.H. Wu T. Gerstein H. Yu G.Q. Mucke L. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model.Science. 2007; 316: 750-754Crossref PubMed Scopus (1545) Google Scholar In AD-affected brains, they contained high levels of p-tau including aggregate species (Figure 3C), which suggested that hyperphosphorylation and oligomerization are specific to AD synapses. Biochemical analysis did not demonstrate whether tau and p-tau were localized to presynaptic or postsynaptic terminals; therefore, we developed a new immunostaining protocol to determine this. To visualize isolated synaptic terminals, we spread a dilute solution of synaptoneurosomes onto a glass slide and fixed it lightly in place (see Materials and Methods). The fixed permeabilized synaptoneurosomes were immunostained for vesicular glutamate transporter 1 (vGlut1) as a presynaptic marker, and microtubule-associated protein 2 (MAP2) as a postsynaptic marker (Figure 4A). MAP2 is a major microtubule-binding protein in dendrites, but is also present at lower levels inside dendritic spines and at the PSD.25Morales M. Fifkova E. Distribution of MAP2 in dendritic spines and its colocalization with actin: an immunogold electron-microscope study.Cell Tissue Res. 1989; 256: 447-456Crossref PubMed Scopus (90) Google Scholar, 26Caceres A. Binder L.I. Payne M.R. Bender P. Rebhun L. Steward O. Differential subcellular localization of tubulin and the microtubule-associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies.J Neurosci. 1984; 4: 394-410Crossref PubMed Google Scholar We confirmed the postsynaptic localization of MAP2 by co-staining with PSD95 (Figure 4D), and noted that PSD95 and vGlut1 often seemed to be so close as to almost overlap, whereas MAP2 and vGlut1 seemed most frequently to be adjacent but distinct puncta; we, therefore, chose to use MAP2 to mark postsynaptic sites to enable more straightforward identification of the presynaptic or postsynaptic localization of other proteins including tau. Using an antibody (DA9) that recognizes both phosphorylated and nonphosphorylated forms of tau, we detected tau in many vGlut1-positive presynaptic puncta (55.3%) and also in most MAP2-positive postsynaptic sites (70.2%) in control human brains. In AD-affected brains, we detected tau at 63.3% of presynaptic sites and 70.1% of postsynaptic sites (Figure 4, B and C). With two-way analysis of variance, we did not detect a significant difference in tau distribution between presynaptic and postsynaptic sites or between control and AD-affected brains. Under electron microscopy, approximately 80% of the presynaptic terminals isolated from rat brains contain visible microtubules.27Gordon-Weeks P.R. Burgoyne R.D. Gray E.G. Presynaptic microtubules: organisation and assembly/disassembly.Neuroscience. 1982; 7: 739-749Abstract Full Text PDF PubMed Scopus (46) Google Scholar Inasmuch as tau is an abundant microtubule-binding protein in axons, our identification of tau in 55.3% of the presynapses is reasonable, and possibly an underestimate. With use of the same criteria, neighboring postsynaptic structures from control brains exhibit 70.2% tau labeling, which is consistent with recent reports that indicated which tau may also have a postsynaptic localization.13Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wolfing H. Chieng B.C. Christie M.J. Napier I.A. Eckert A. Staufenbiel M. Hardeman E. Gotz J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar Immunocytochemistry using PHF1 antibody did not detect p-tau in control synapses, but labeled a substantial percentage of AD synapses: 20.8% of vGlut1-positive presynaptic puncta and 32.0% of MAP2-positive postsynaptic puncta (Figure 5). These data support the hypothesis that normal tau is already present at both presynaptic and postsynaptic terminals in normal brains and that, during the pathologic progression of AD, tau misfolding and hyperphosphorylation occur on both sides of the synapse. To understand the properties of synaptic p-tau oligomers, we extracted synaptoneurosomes using 2% Triton X-100 to solubilize membrane and free cytoplasmic proteins, and the remaining insoluble material was collected as the crude PSD pellet, which retained nearly all of the PSD95 protein and most of the actin and microtubule cytoskeleton (Figure 6). Most of the normal tau protein in control human synapses was extractable using Triton, and the small amount of tau retained in the crude PSD pellet was consistent with mouse studies that co-immunoprecipitated tau and PSD95.13Ittner L.M. Ke Y.D. Delerue F. Bi M. Gladbach A. van Eersel J. Wolfing H. Chieng B.C. Christie M.J. Napier I.A. Eckert A. Staufenbiel M. Hardeman E. Gotz J. Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models.Cell. 2010; 142: 387-397Abstract Full Text Full Text PDF PubMed Scopus (1418) Google Scholar In contrast, in AD synaptoneurosomes, essentially all p-tau species remained in the Triton-insoluble pellet, indicating that they were associated with the PSD and the cytoskeleton fraction, which supports our immunostaining data that they accumulate in dendritic spines. The crude PSD pellet also exhibited an enrichment of ubiquitinated proteins and ubiquitin-