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Apolipoprotein E/Amyloid-β Complex Accumulates in Alzheimer Disease Cortical Synapses via Apolipoprotein E Receptors and Is Enhanced by APOE4

2019; Elsevier BV; Volume: 189; Issue: 8 Linguagem: Inglês

10.1016/j.ajpath.2019.04.010

ISSN

1525-2191

Autores

Tina Bilousova, Mikhail Melnik, Emily Miyoshi, Bianca Gonzalez, Wayne W. Poon, Harry V. Vinters, Carol A. Miller, María M. Corrada, Claudia H. Kawas, Asa Hatami, Ricardo Albay, Charles Glabe, Karen H. Gylys,

Tópico(s)

Cellular transport and secretion

Resumo

Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)–positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses. Apolipoprotein E (apoE) colocalizes with amyloid-β (Aβ) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aβ are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aβ complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aβ was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)–positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aβ concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aβ was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aβ complex and associated lipids into synaptic terminals, with subsequent Aβ clearance in control synapses and accumulation in AD synapses. Apolipoprotein E4 (APOE4) is the major genetic risk factor for late-onset Alzheimer disease (AD), and early observations that apoE colocalizes with plaque amyloid-β (Aβ) in human AD and in mouse models resulted in designation of apoE as a pathologic chaperone that facilitates Aβ deposition.1Dickson T.C. Saunders H.L. Vickers J.C. Relationship between apolipoprotein E and the amyloid deposits and dystrophic neurites of Alzheimer's disease.Neuropathol Appl Neurobiol. 1997; 23: 483-491Crossref PubMed Scopus (32) Google Scholar, 2Burns M.P. Noble W.J. Olm V. Gaynor K. Casey E. LaFrancois J. Wang L. 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ApoE promotes the proteolytic degradation of Abeta.Neuron. 2008; 58: 681-693Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 9Deane R. Sagare A. Hamm K. Parisi M. Lane S. Finn M.B. Holtzman D.M. Zlokovic B.V. apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain.J Clin Invest. 2008; 118: 4002-4013Crossref PubMed Scopus (522) Google Scholar, 10Bell R.D. Sagare A.P. Friedman A.E. Bedi G.S. Holtzman D.M. Deane R. Zlokovic B.V. Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system.J Cereb Blood Flow Metab. 2007; 27: 909-918Crossref PubMed Scopus (500) Google Scholar, 11DeMattos R.B. Cirrito J.R. Parsadanian M. May P.C. O'Dell M.A. Taylor J.W. Harmony J.A. Aronow B.J. Bales K.R. Paul S.M. Holtzman D.M. ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo.Neuron. 2004; 41: 193-202Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar Uptake by the apoE receptors, low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1), makes an important contribution to Aβ clearance, and in the brain these pathways operate in neurons, brain microvasculature, astrocytes, and microglia.12Kanekiyo T. Xu H. Bu G. ApoE and Abeta in Alzheimer's disease: accidental encounters or partners?.Neuron. 2014; 81: 740-754Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar For example, overexpression of LDLR reduces Aβ deposition without affecting amyloid precursor protein expression in multiple mouse strains,13Kim J. Castellano J.M. Jiang H. Basak J.M. Parsadanian M. Pham V. Mason S.M. Paul S.M. Holtzman D.M. Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular A beta clearance.Neuron. 2009; 64: 632-644Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar and reduction of neuronal LRP1 in amyloid precursor protein/presenilin 1 (PS1) mice increases brain Aβ deposition.14Kanekiyo T. Cirrito J.R. Liu C.C. Shinohara M. Li J. Schuler D.R. Shinohara M. Holtzman D.M. Bu G. Neuronal clearance of amyloid-beta by endocytic receptor LRP1.J Neurosci. 2013; 33: 19276-19283Crossref PubMed Scopus (163) Google Scholar On the other hand, receptor-mediated clearance of Aβ into neurons may lead to toxic accumulations,15Billings L.M. Oddo S. Green K.N. McGaugh J.L. LaFerla F.M. Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice.Neuron. 2005; 45: 675-688Abstract Full Text Full Text PDF PubMed Scopus (1001) Google Scholar, 16Shinohara M. Tachibana M. Kanekiyo T. Bu G. Role of LRP1 in the pathogenesis of Alzheimer's disease: evidence from clinical and preclinical studies.J Lipid Res. 2017; 58: 1267-1281Crossref PubMed Scopus (92) Google Scholar whereas internalization by microglia,17Mandrekar S. Jiang Q. Lee C.Y. Koenigsknecht-Talboo J. Holtzman D.M. Landreth G.E. Microglia mediate the clearance of soluble Abeta through fluid phase macropinocytosis.J Neurosci. 2009; 29: 4252-4262Crossref PubMed Scopus (297) Google Scholar endothelial cells,9Deane R. Sagare A. Hamm K. Parisi M. Lane S. Finn M.B. Holtzman D.M. Zlokovic B.V. apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain.J Clin Invest. 2008; 118: 4002-4013Crossref PubMed Scopus (522) Google Scholar or astrocytes18Koistinaho M. Lin S. Wu X. Esterman M. Koger D. Hanson J. Higgs R. Liu F. Malkani S. Bales K.R. Paul S.M. Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides.Nat Med. 2004; 10: 719-726Crossref PubMed Scopus (442) Google Scholar seems a more likely pathway for uptake and degradation of Aβ.19Holtzman D.M. Herz J. Bu G. Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease.Cold Spring Harb Perspect Med. 2012; 2: a006312Crossref PubMed Scopus (483) Google Scholar ApoE/Aβ complexes have been detected in human brain extracts,20Russo C. Angelini G. Dapino D. Piccini A. Piombo G. Schettini G. Chen S. Teller J.K. Zaccheo D. Gambetti P. Tabaton M. Opposite roles of apolipoprotein E in normal brains and in Alzheimer's disease.Proc Natl Acad Sci U S A. 1998; 95: 15598-15602Crossref PubMed Scopus (81) Google Scholar plasma,21Koudinov A.R. Berezov T.T. Kumar A. Koudinova N.V. Alzheimer's amyloid beta interaction with normal human plasma high density lipoprotein: association with apolipoprotein and lipids.Clin Chim Acta. 1998; 270: 75-84Crossref PubMed Scopus (92) Google Scholar and cerebral spinal fluid,22LaDu M.J. Munson G.W. Jungbauer L. Getz G.S. Reardon C.A. Tai L.M. Yu C. Preferential interactions between ApoE-containing lipoproteins and Abeta revealed by a detection method that combines size exclusion chromatography with non-reducing gel-shift.Biochim Biophys Acta. 2012; 1821: 295-302Crossref PubMed Scopus (24) Google Scholar and some key Aβ-dependent apoE effects on AD seem likely to result from direct interactions between apoE and Aβ. However, the variability arising from multiple detection methods has greatly limited understanding of the role of complex formation in disease progression, as documented in recent reviews.12Kanekiyo T. Xu H. Bu G. ApoE and Abeta in Alzheimer's disease: accidental encounters or partners?.Neuron. 2014; 81: 740-754Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 23Tai L.M. Mehra S. Shete V. Estus S. Rebeck G.W. Bu G. LaDu M.J. Soluble apoE/Abeta complex: mechanism and therapeutic target for APOE4-induced AD risk.Mol Neurodegener. 2014; 9: 2Crossref PubMed Scopus (83) Google Scholar An additional level of variability emerges from the disparate factors that affect binding of apoE to Aβ; these include apoE isoform, apoE lipidation state, and aggregation state of Aβ.12Kanekiyo T. Xu H. Bu G. ApoE and Abeta in Alzheimer's disease: accidental encounters or partners?.Neuron. 2014; 81: 740-754Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 23Tai L.M. Mehra S. Shete V. Estus S. Rebeck G.W. Bu G. LaDu M.J. Soluble apoE/Abeta complex: mechanism and therapeutic target for APOE4-induced AD risk.Mol Neurodegener. 2014; 9: 2Crossref PubMed Scopus (83) Google Scholar A reduction in detectable complex with apoE4 has led to one hypothesis that apoE/Aβ complex level modulates Aβ levels and that reduced lipidation of apoE results in reduced complex formation and increased Aβ accumulation.23Tai L.M. Mehra S. Shete V. Estus S. Rebeck G.W. Bu G. LaDu M.J. Soluble apoE/Abeta complex: mechanism and therapeutic target for APOE4-induced AD risk.Mol Neurodegener. 2014; 9: 2Crossref PubMed Scopus (83) Google Scholar, 24Tai L.M. Bilousova T. Jungbauer L. Roeske S.K. Youmans K.L. Yu C. Poon W.W. Cornwell L.B. Miller C.A. Vinters H.V. Van Eldik L.J. Fardo D.W. Estus S. Bu G. Gylys K.H. Ladu M.J. Levels of soluble apolipoprotein E/amyloid-beta complex are reduced and oligomeric Abeta increased with APOE4 and Alzheimer disease in a transgenic mouse model and human samples.J Biol Chem. 2013; 288: 5914-5926Crossref PubMed Scopus (108) Google Scholar Lipidation status is difficult to assess in vivo, but apoE4 is generally believed to be less lipidated than apoE3. This hypothesis is supported by experiments in mice expressing five familial AD mutations (5XFAD) plus human apoE isoforms23Tai L.M. Mehra S. Shete V. Estus S. Rebeck G.W. Bu G. LaDu M.J. Soluble apoE/Abeta complex: mechanism and therapeutic target for APOE4-induced AD risk.Mol Neurodegener. 2014; 9: 2Crossref PubMed Scopus (83) Google Scholar and also by recent data showing that apoE4 reduces lipidation and enhances Aβ accumulation, whereas apoE2 has the opposite effect.25Hu J. Liu C.C. Chen X.F. Zhang Y.W. Xu H. Bu G. Opposing effects of viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE lipidation and Abeta metabolism in apoE4-targeted replacement mice.Mol Neurodegener. 2015; 10: 6Crossref PubMed Scopus (79) Google Scholar However, several studies demonstrate that blocking the apoE/Aβ interaction reduces Aβ deposition,26Kim J. Eltorai A.E. Jiang H. Liao F. Verghese P.B. Kim J. Stewart F.R. Basak J.M. Holtzman D.M. Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Abeta amyloidosis.J Exp Med. 2012; 209: 2149-2156Crossref PubMed Scopus (90) Google Scholar, 27Liao F. Hori Y. Hudry E. Bauer A.Q. Jiang H. Mahan T.E. Lefton K.B. Zhang T.J. Dearborn J.T. Kim J. Culver J.P. Betensky R. Wozniak D.F. Hyman B.T. Holtzman D.M. Anti-ApoE antibody given after plaque onset decreases Abeta accumulation and improves brain function in a mouse model of Abeta amyloidosis.J Neurosci. 2014; 34: 7281-7292Crossref PubMed Scopus (88) Google Scholar, 28Liu S. Breitbart A. Sun Y. Mehta P.D. Boutajangout A. Scholtzova H. Wisniewski T. Blocking the apolipoprotein E/amyloid beta interaction in triple transgenic mice ameliorates Alzheimer's disease related amyloid beta and tau pathology.J Neurochem. 2014; 128: 577-591Crossref PubMed Scopus (32) Google Scholar contradicting the role of the complexes in Aβ clearance and suggesting important therapeutic implications of apoE/Aβ complex formation. Given the promise of APOE-related therapeutics for increasing clearance or reducing accumulation of Aβ,3Yu J.T. Tan L. Hardy J. Apolipoprotein E in Alzheimer's disease: an update.Annu Rev Neurosci. 2014; 37: 79-100Crossref PubMed Scopus (275) Google Scholar, 19Holtzman D.M. Herz J. Bu G. Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease.Cold Spring Harb Perspect Med. 2012; 2: a006312Crossref PubMed Scopus (483) Google Scholar it is critical to understand the pathway(s) affected by interactions between apoE and Aβ. On the basis of previous data suggesting that apoE isoform affects apoE/Aβ complexes and Aβ clearance, and on our previous work showing that apoE enhances uptake of Aβ into synaptosomes,29Gylys K.H. Fein J.A. Tan A.M. Cole G.M. Apolipoprotein E enhances uptake of soluble but not aggregated amyloid-beta protein into synaptic terminals.J Neurochem. 2003; 84: 1442-1451Crossref PubMed Scopus (67) Google Scholar the present studies examined the hypothesis that apoE receptors mediate uptake of apoE/Aβ complex and lipids into the synaptic compartment in cortical synapses of aged control and AD samples. Multiple SDS-stable apoE/Aβ complexes were observed in human cortical synaptosomes, and complex level was increased in AD samples. Using flow cytometry analysis, we also demonstrate alterations in synaptic lipids and pronounced accumulation of both apoE and Aβ in LRP1- and LDLR-positive synaptosomes, consistent with uptake of Aβ and apoE into the synaptic compartment by these receptors. Experiments also showed that higher synaptic Aβ was associated with apoE4. The antibodies used in the present work are detailed in Table 1.30Yang F. Mak K. Vinters H.V. Frautschy S.A. Cole G.M. Monoclonal antibody to the C-terminus of beta-amyloid.Neuroreport. 1994; 5: 2117-2120Crossref PubMed Scopus (39) Google Scholar, 31Hatami A. Albay 3rd, R. Monjazeb S. Milton S. Glabe C. Monoclonal antibodies against Abeta42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J Biol Chem. 2014; 289: 32131-32143Crossref PubMed Scopus (77) Google Scholar The dyes filipin and Nile red [5H-benzo(a)phenoxazin-5-one, 9-(diethylamino)-7385-67-3] were purchased from Sigma-Aldrich (St. Louis, MO).Table 1ReagentsAntibodyAntigen/epitopeSupplierHostReactivity10G4Aβ peptide: N-terminal residues 5 to 17Kind gift of Greg Cole (Veterans Affairs Medical Center; University of California)30Yang F. Mak K. Vinters H.V. Frautschy S.A. Cole G.M. Monoclonal antibody to the C-terminus of beta-amyloid.Neuroreport. 1994; 5: 2117-2120Crossref PubMed Scopus (39) Google ScholarMouseHuman, mouse6E10N-terminal residues 1 to 16 of Aβ peptideBioLegend (San Diego, CA)MouseHumanmOC1Fibrillar aggregates of amyloid-βKind gift of Charles Glabe (University of California)31Hatami A. Albay 3rd, R. Monjazeb S. Milton S. Glabe C. Monoclonal antibodies against Abeta42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J Biol Chem. 2014; 289: 32131-32143Crossref PubMed Scopus (77) Google ScholarRabbitHumanmOC16Fibrillar aggregates of amyloid-βKind gift of Charles Glabe31Hatami A. Albay 3rd, R. Monjazeb S. Milton S. Glabe C. Monoclonal antibodies against Abeta42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J Biol Chem. 2014; 289: 32131-32143Crossref PubMed Scopus (77) Google ScholarRabbitHumanmOC23Fibrillar aggregates of amyloid-βKind gift of Charles Glabe31Hatami A. Albay 3rd, R. Monjazeb S. Milton S. Glabe C. Monoclonal antibodies against Abeta42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J Biol Chem. 2014; 289: 32131-32143Crossref PubMed Scopus (77) Google ScholarRabbitHumanmOC98Fibrillar aggregates of amyloid-βKind gift of Charles Glabe31Hatami A. Albay 3rd, R. Monjazeb S. Milton S. Glabe C. Monoclonal antibodies against Abeta42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J Biol Chem. 2014; 289: 32131-32143Crossref PubMed Scopus (77) Google ScholarRabbitHumanAnti–PSD-95 antibodyPSD-95Millipore (Temecula, CA)MouseHumanSynaptophysin antibodySynaptophysinGene Tex (Irvine, CA)RabbitHuman, mouse, ratSNAP-25 antibody (SP12) sc-20038SNAP-25Santa Cruz Biotechnology (Dallas, TX)MouseHuman, mouseAnti-apoE goat pAb (178479)ApoECalbiochem (Cambridge, MA)GoatHuman, mouse, ratE6D7ApoEAbcam (Cambridge, MA)MouseHuman, mouseEP1553YLDLRAbcamRabbitHuman, mouseEPR3724LRP1AbcamRabbitHuman, mouse, rat, pigFilipin (dye)Free cholesterolSigma-Aldrich (St. Louis, MO)NANANile red (dye)Cholesterol estersSigma-AldrichNANAApoE, apolipoprotein E; NA, not applicable; mOC, OC monoclonal; pAb, polyclonal antibody; postsynaptic density-95, PSD-95; SNAP-25, synaptosomal nerve-associated protein 25. Open table in a new tab ApoE, apolipoprotein E; NA, not applicable; mOC, OC monoclonal; pAb, polyclonal antibody; postsynaptic density-95, PSD-95; SNAP-25, synaptosomal nerve-associated protein 25. Brain samples of parietal cortex (Brodmann areas A7, A39, and A40) were obtained at autopsy from Alzheimer disease research centers at University of California, Los Angeles, University of California, Irvine, and University of Southern California (Table 2). Samples were selected for each experiment on the basis of the design and availability in the bank, and neurologic controls were used interchangeably with the aged cognitively normal controls. Table 2 lists the total of unique cases (28 controls, 80 AD); some cases were used for more than one experiment. As in a recent study,32Bilousova T. Miller C.A. Poon W.W. Vinters H.V. Corrada M. Kawas C. Hayden E.Y. Teplow D.B. Glabe C. Albay 3rd, R. Cole G.M. Teng E. Gylys K.H. Synaptic amyloid-beta oligomers precede p-tau and differentiate high pathology control cases.Am J Pathol. 2016; 186: 185-198Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar cases were stratified into early (Braak stages II–IV) and late (Braak stages V and VI) AD on the basis of Braak stage because of the inherent dynamic range and long general use of this staging system. Immediately on receipt, samples (approximately 0.3 to 5 g) were minced in a 0.32 mol/L sucrose solution with protease inhibitors for cryopreservation of synaptic structure and membranes33Dodd P.R. Hardy J.A. Baig E.B. Kidd A.M. Bird E.D. Watson W.E. Johnston G.A. Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain.Neurochem Pathol. 1986; 4: 177-198Crossref PubMed Scopus (75) Google Scholar (2 mmol/L EDTA, 2 mmol/L EGTA, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na pyrophosphate, 5 mmol/L NaF, and 10 mmol/L Tris), then stored at −70°C until homogenization. The P-2 (crude synaptosome; synaptosome-enriched fraction) was prepared as previously described29Gylys K.H. Fein J.A. Tan A.M. Cole G.M. Apolipoprotein E enhances uptake of soluble but not aggregated amyloid-beta protein into synaptic terminals.J Neurochem. 2003; 84: 1442-1451Crossref PubMed Scopus (67) Google Scholar; briefly, tissue was homogenized in ice-cold buffer [0.32 mol/L sucrose; 10 mmol/L Tris, pH 7.5; plus protease inhibitors pepstatin (4 μg/mL), aprotinin (5 μg/mL), trypsin inhibitor (20 μg/mL), EDTA (2 mmol/L), EGTA (2 mmol/L), phenylmethylsulfonyl fluoride (0.2 mmol/L), and Leu-peptin (4 μg/mL)]. The homogenate was first centrifuged at 1000 × g for 10 minutes; the resulting supernatant was centrifuged at 10,000 × g for 20 minutes to obtain the crude synaptosomal pellet. Aliquots of P-2 are routinely cryopreserved in 0.32 mol/L sucrose and banked at −70°C until the day of the experiment,33Dodd P.R. Hardy J.A. Baig E.B. Kidd A.M. Bird E.D. Watson W.E. Johnston G.A. Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain.Neurochem Pathol. 1986; 4: 177-198Crossref PubMed Scopus (75) Google Scholar at which time they were defrosted at 37°C, resuspended in phosphate-buffered saline (PBS) with protease inhibitors, sonicated, and centrifuged for 4 minutes at 3380 × g. Supernatant was collected, and total protein concentration was defined using the bicinchoninic acid protein assay (Pierce, Waltham, MA).Table 2Case Information for Human SamplesVariableAged controls∗No dementia.Neurologic controls†Without AD pathology (spinocerebellar ataxia, Pick disease, vascular dementia, and Parkinson disease with dementia).Early AD (Braak stage ≤ IV)Late AD (Braak stage V or VI)Cases, n2172357Age, y‡Data are given as means ± SD.85.3 ± 7.366.5 ± 7.385 ± 8.981.2 ± 12.3PMI, hours‡Data are given as means ± SD.6.9 ± 3.78.1 ± 4.57.6 ± 3.06.2 ± 2.0Female sex, %48755738APOE4 positive, n52828PMI, post-mortem interval.∗ No dementia.† Without AD pathology (spinocerebellar ataxia, Pick disease, vascular dementia, and Parkinson disease with dementia).‡ Data are given as means ± SD. Open table in a new tab PMI, post-mortem interval. Human P-2 samples were separated by nonreducing gel electrophoresis on 10% to 20% Tris-glycine gradient gels either with or without reducing agent dithiothreitol. After transferring to Immobilon-P membrane (Millipore, Burlington, MA), Western blot analysis with primary anti-apoE (E6D7) or anti-Aβ primary antibody (10G4 or 6E10) and secondary anti-mouse horseradish peroxidase–conjugated IgG (Jackson Immunoresearch, West Grove, PA) was performed. Before immunolabeling, membranes were labeled with Ponceau S (0.1% w/v in acetic acid) to verify equal loading; only membranes with equal loading were used for analysis. Immunolabeled proteins were visualized by SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific, Rockford, IL). Resulting films were scanned and quantified on a UVP 600 imaging system (BioSpectrum, Jena, Germany) using VisionWorks software version 6.6A (VisionWorks, Upland, CA). For dot blotting, 1 μL of each sample was pipetted onto a Whatman nitrocellulose membrane (GE Healthcare, Chicago, IL). Membranes were allowed to air dry and were subsequently blocked in 10% nonfat dry milk in Tris-buffered saline (TBS) containing 0.01% Tween 20 (TBS-T) for 1 hour at room temperature. Membranes were then incubated 1:100 with OC monoclonal (mOC) antibodies in 5% nonfat dried milk in TBS-T overnight at 4°C. After three 5-minute washes in TBS-T, membranes were incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (1:10,000 in 5% nonfat dried milk in TBS-T). Membranes were then washed three times for 5 minutes in TBS-T and visualized using enhanced chemiluminescence (GE Healthcare). Images were obtained using a Nikon D700 (Nikon Inc., Melville, NY) camera, as described previously.31Hatami A. Albay 3rd, R. Monjazeb S. Milton S. Glabe C. Monoclonal antibodies against Abeta42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain.J Biol Chem. 2014; 289: 32131-32143Crossref PubMed Scopus (77) Google Scholar Antibodies were covalently coupled to M-270 Epoxi Dynabeads using a conjugation kit (14311D; Invitrogen, Carlsbad, CA), according to manufacturer's instructions. P-2 samples were quickly thawed, centrifuged at 10,000 × g for 10 minutes at 4°C to remove sucrose, homogenized in PBS with protease and phosphatase inhibitor cocktail (Thermo Scientific; v/w 1:9) using a pestle motor mixer (30 seconds on ice), and then run through three freeze-thaw cycles (3 minutes liquid nitrogen, 30 minutes thawing at room temperature) with additional 30 seconds' homogenization after each cycle. Samples were centrifuged at 840 × g for 2 minutes at 4°C, and supernatants (P2-H) were collected. Small volumes of each P2-H sample were set aside for further Western blot analysis; the rest were equally divided for immunoprecipitation (IP) with E6D7, 6E10, and corresponding isotype control antibody conjugated Dynabeads. Specificity for the E6D7 antibody has been demonstrated in apoE knockout mice.34Pang J. Wu Y. Peng J. Yang P. Kuai L. Qin X. Cao F. Sun X. Chen L. Vitek M.P. Jiang Y. Potential implications of apolipoprotein E in early brain injury after experimental subarachnoid hemorrhage: involvement in the modulation of blood-brain barrier integrity.Oncotarget. 2016; 7: 56030-56044Crossref PubMed Scopus (18) Google Scholar Samples were rotated with the beads for 1 hour at 4°C, then placed on magnet, and unbound flow throughs were collected; beads were washed three times with PBS using magnet. P2-H, flow-through samples, and beads were mixed with Tris-glycine sample buffer with DDT and boiled for 10 minutes. Beads were placed on magnet, and the fractions containing apoE/Aβ complexes (IP) were collected. Samples were run using 10% to 20% Tris-glycine SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. Membranes were blocked with 3% milk and 5% bovine serum albumin for 1 hour at room temperature and probed with either goat anti-apoE (178479; Calbiochem, Cambridge, MA) or mouse anti-Aβ antibodies (BioLegend, San Diego, CA) at 4°C. After washes, membranes were incubated with corresponding horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature, SuperSignal West Femto maximum sensitivity substrate was applied for 5 minutes, and images were taken using a UVP reading system (BioSpectrum). P-2 fractions were prepared from cryopreserved brain tissue, as described previously35Gylys K.H. Fein J.A. Yang F. Cole G.M. Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex.Cytometry A. 2004; 60: 90-96Crossref PubMed Scopus (38) Google Scholar; cryopreserved in 0.32 mol/L buffered sucrose solution; and stored at −80°C as aliquots. On

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