The inhibition of cellular toxicity of amyloid-β by dissociated transthyretin

Abstract

The protective effect of transthyretin (TTR) on cellular toxicity of β-amyloid (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aβ, a protein associated with Alzheimer's disease. TTR binds Aβ, alters its aggregation, and inhibits its toxicity both in vitro and in vivo. In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aβ oligomers. These findings suggest that the Aβ-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aβ aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aβ oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aβ catalyzed by Aβ fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease. The protective effect of transthyretin (TTR) on cellular toxicity of β-amyloid (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aβ, a protein associated with Alzheimer's disease. TTR binds Aβ, alters its aggregation, and inhibits its toxicity both in vitro and in vivo. In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aβ oligomers. These findings suggest that the Aβ-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aβ aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aβ oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aβ catalyzed by Aβ fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease. The physiological importance of transthyretin in Alzheimer's disease was first reported by Schwarzman et al. in 1994 (1Schwarzman A.L. Gregori L. Vitek M.P. Lyubski S. Strittmatter W.J. Enghilde J.J. Bhasin R. Silverman J. Weisgraber K.H. Coyle P.K. Transthyretin sequesters amyloid β protein and prevents amyloid formation.Proc. Natl. Acad. Sci. U.S.A. 1994; 91 (8078889): 8368-837210.1073/pnas.91.18.8368Crossref PubMed Scopus (329) Google Scholar). One of the hallmarks of Alzheimer's disease (AD) is the formation of brain plaques composed of β-amyloid peptide (Aβ). A 42-residue-long β-amyloid peptide (Aβ42) is the predominant variant in neuritic plaques of AD patients, with higher amyloidogenicity and cellular toxicity in vitro (2Roher A.E. Lowenson J.D. Clarke S. Woods A.S. Cotter R.J. Gowing E. Ball M.J. β-Amyloid-(1–42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease.Proc. Natl. Acad. Sci. U.S.A. 1993; 90 (8248178): 10836-1084010.1073/pnas.90.22.10836Crossref PubMed Scopus (591) Google Scholar, 3Burdick D. Soreghan B. Kwon M. Kosmoski J. Knauer M. Henschen A. Yates J. Cotman C. Glabe C. Assembly and aggregation properties of synthetic Alzheimer's A4/β amyloid peptide analogs.J. Biol. Chem. 1992; 267 (1730616): 546-554Abstract Full Text PDF PubMed Google Scholar). Many studies have shown that transthyretin (TTR) binds to Aβ, alters its aggregation, and inhibits its toxicity both in vitro and in vivo (4Stein T.D. Anders N.J. DeCarli C. Chan S.L. Mattson M.P. Johnson J.A. Neutralization of transthyretin reverses the neuroprotective effects of secreted amyloid precursor protein (APP) in APPSW mice resulting in tau phosphorylation and loss of hippocampal neurons: support for the amyloid hypothesis.J. Neurosci. 2004; 24 (15342738): 7707-771710.1523/JNEUROSCI.2211-04.2004Crossref PubMed Scopus (215) Google Scholar, 5Choi S.H. Leight S.N. Lee V.M. Li T. Wong P.C. Johnson J.A. Saraiva M.J. Sisodia S.S. Accelerated Aβ deposition in APPswe/PS1deltaE9 mice with hemizygous deletions of TTR (transthyretin).J. Neurosci. 2007; 27 (17596449): 7006-701010.1523/JNEUROSCI.1919-07.2007Crossref PubMed Scopus (109) Google Scholar, 6Buxbaum J.N. Ye Z. Reixach N. Friske L. Levy C. Das P. Golde T. Masliah E. Roberts A.R. Bartfai T. Transthyretin protects Alzheimer's mice from the behavioral and biochemical effects of Aβ toxicity.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18272491): 2681-268610.1073/pnas.0712197105Crossref PubMed Scopus (210) Google Scholar, 7Costa R. Gonçalves A. Saraiva M.J. Cardoso I. Transthyretin binding to A-β peptide: impact on A-β fibrillogenesis and toxicity.FEBS Lett. 2008; 582 (18295603): 936-94210.1016/j.febslet.2008.02.034Crossref PubMed Scopus (107) Google Scholar, 8Du J. Cho P.Y. Yang D.T. Murphy R.M. Identification of β-amyloid–binding sites on transthyretin.Protein Eng. Des. Sel. 2012; 25 (22670059): 337-34510.1093/protein/gzs026Crossref PubMed Scopus (43) Google Scholar). In vitro, TTR co-aggregates with Aβ oligomers into large nontoxic assemblies, thereby inhibiting cellular toxicity (9Cascella R. Conti S. Mannini B. Li X. Buxbaum J.N. Tiribilli B. Chiti F. Cecchi C. Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.Biochim. Biophys. Acta. 2013; 1832 (24075940): 2302-231410.1016/j.bbadis.2013.09.011Crossref PubMed Scopus (53) Google Scholar, 10Garai K. Posey A.E. Li X. Buxbaum J.N. Pappu R.V. Inhibition of amyloid β fibril formation by monomeric human transthyretin.Protein Sci. 2018; 27 (29498118): 1252-126110.1002/pro.3396Crossref PubMed Scopus (22) Google Scholar). In vivo, TTR sequesters Aβ and facilitates its clearance in the brain (1Schwarzman A.L. Gregori L. Vitek M.P. Lyubski S. Strittmatter W.J. Enghilde J.J. Bhasin R. Silverman J. Weisgraber K.H. Coyle P.K. Transthyretin sequesters amyloid β protein and prevents amyloid formation.Proc. Natl. Acad. Sci. U.S.A. 1994; 91 (8078889): 8368-837210.1073/pnas.91.18.8368Crossref PubMed Scopus (329) Google Scholar). More recently, the Buxbaum laboratory (6Buxbaum J.N. Ye Z. Reixach N. Friske L. Levy C. Das P. Golde T. Masliah E. Roberts A.R. Bartfai T. Transthyretin protects Alzheimer's mice from the behavioral and biochemical effects of Aβ toxicity.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18272491): 2681-268610.1073/pnas.0712197105Crossref PubMed Scopus (210) Google Scholar) showed that overexpression of WT human TTR suppressed disease progression in the APP23 transgenic AD mouse model. They also showed that silencing the endogenous TTR gene in AD transgenic mice accelerated Aβ42 deposition (5Choi S.H. Leight S.N. Lee V.M. Li T. Wong P.C. Johnson J.A. Saraiva M.J. Sisodia S.S. Accelerated Aβ deposition in APPswe/PS1deltaE9 mice with hemizygous deletions of TTR (transthyretin).J. Neurosci. 2007; 27 (17596449): 7006-701010.1523/JNEUROSCI.1919-07.2007Crossref PubMed Scopus (109) Google Scholar). What makes the pair of TTR and Aβ42 particularly interesting is the amyloid nature of the two elements. In health, TTR functions as a transporter of retinol and thyroxine in blood, cerebrospinal fluid, and the eye and is secreted by the liver, choroid plexus, and retinal epithelium, respectively. However, dissociation of tetrameric TTR leads to amyloid fibril formation and systemic TTR amyloid deposition in patients of transthyretin amyloidosis (11Westermark P. Sletten K. Johansson B. Cornwell G.G. Fibril in senile systemic amyloidosis is derived from normal transthyretin.Proc. Natl. Acad. Sci. U.S.A. 1990; 87 (2320592): 2843-284510.1073/pnas.87.7.2843Crossref PubMed Scopus (585) Google Scholar, 12Colon W. Kelly J.W. Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.Biochemistry. 1992; 31 (1390650): 8654-866010.1021/bi00151a036Crossref PubMed Scopus (456) Google Scholar, 13Saelices L. Chung K. Lee J.H. Cohn W. Whitelegge J.P. Benson M.D. Eisenberg D.S. Amyloid seeding of transthyretin by ex vivo cardiac fibrils and its inhibition.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (29954863): E6741-E675010.1073/pnas.1805131115Crossref PubMed Scopus (35) Google Scholar). Whether the amyloidogenicity of TTR is linked to its interaction to Aβ42 is still under debate. Previous work showed that different aggregation propensities of TTR result in distinct interaction capabilities: fewer amyloidogenic variants had increased affinity for Aβ (7Costa R. Gonçalves A. Saraiva M.J. Cardoso I. Transthyretin binding to A-β peptide: impact on A-β fibrillogenesis and toxicity.FEBS Lett. 2008; 582 (18295603): 936-94210.1016/j.febslet.2008.02.034Crossref PubMed Scopus (107) Google Scholar). However, this did not impact on the levels of inhibition of Aβ aggregation, and cytotoxicity protection was not assessed. Here we attempt to fill the experimental gap by studying the protective effect of TTR variants at different aggregated states over Aβ42 cellular toxicity. In addition, we show the identification and characterization of a segment of TTR that binds Aβ42 triggering the formation of nonamyloid amorphous aggregates that are more sensitive to protease digestion, therefore mimicking TTR inhibitory effect over Aβ42. We first evaluated the protection against Aβ42 cytotoxicity by nine TTR variants with distinct aggregation propensities in several aggregated states (Table 1). The amyloidogenic behavior of three representative TTR variants is shown in Fig. 1 (A and B). For this assay, recombinant transthyretin was incubated at 37 °C, pH 4.3; protein aggregation was followed by immunodot blot of the insoluble fractions collected by centrifugation at several time points (Fig. 1A); and EM was performed after 4 days of incubation (Fig. 1B). We found that M-TTR, a of transthyretin that is at physiological pH X. P. Kelly J.W. transthyretin that is is PubMed Scopus Google aggregation after 1 of incubation at pH (Fig. is an variant of TTR the tetrameric in (Fig. a aggregation (Fig. A and B). is a that did not show of aggregation in 4 days (Fig. in a of the of TTR amyloidosis in patients both and T. M. Saraiva M.J. I. A of in an found to be a for two TTR TTR and TTR 1993; Scholar). We incubated Aβ42 with the from Fig. for and evaluated its cytotoxicity by of TTR variants evaluated in this in in a The first is that TTR not prevent Aβ42 toxicity, for of the variants (Fig. In addition, the variant which aggregates at a to be (Fig. The is that which not and not prevent toxicity (Fig. We the the TTR variants evaluated (Fig. variants are monomeric in the protection at is not that are tetrameric in (Fig. and Fig. that the to from Aβ42 toxicity not with the of variants (Fig. and Fig. with the dissociation at the of the with Aβ42 (Fig. A and and Fig. A that the TTR variants in this not in the of with the of M-TTR, which in a of (Fig. that dissociation of TTR is required for the inhibition of Aβ In addition, the suggest that a more TTR more Aβ42 inhibition a more amyloidogenic These findings to whether a segment of TTR that is not in the be capable of altering Aβ toxicity. of the interaction of a segment of Aβ42 and the TTR a of the and the (Fig. have shown that the amyloidogenic segment is TTR is to Aβ X. X. A.R. D. Kelly J.W. Buxbaum J.N. of transthyretin inhibition of β-amyloid aggregation in Neurosci. 2013; PubMed Scopus Google Scholar). In the Eisenberg laboratory the of three of by J.P. A. M. M. L. D. D. Eisenberg D. for Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). We these three as as the monomeric of WT TTR L. Johnson D. P. Whitelegge J. L. R. Eisenberg D.S. the of aggregation of human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to computational docking (Fig. For the segment was the TTR The segment is in the monomeric of We whether this segment TTR protective effects The peptide was found to be We first the amyloidogenicity of TTR by which the of segment to amyloid fibrils L. P.K. R. Eisenberg D. the proteins capable of Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, M.J. J. D. Eisenberg D. The for of Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) (Fig. TTR show that the that this segment is to amyloid In we found the segment and the peptide to be amyloidogenic in (Fig. which may the of inhibitory effect in studies P.Y. Johnson J.A. Murphy R.M. as β-amyloid Chem. Neurosci. PubMed Scopus Google Scholar). In to the amorphous aggregates by recombinant these fibrils (Fig. We several to by the first a to the The and of the are in We found that amyloidogenicity was by the of a (Fig. of evaluated in this in a The of the segment Aβ42 fibril formation (Fig. and and We first evaluated the inhibitory effect of over Aβ42 fibril formation in (Fig. and Fig. the that we the Aβ42 inhibitory effect was with which a followed by (Fig. an incubation of we the by EM and found that the of the formation of amorphous aggregates (Fig. that not with (Fig. we found that also the formation of amorphous incubated with Aβ42 fibrils (Fig. These aggregates also found to be (Fig. and with (Fig. The characterization of Aβ42 amorphous aggregates by showed that the of to fibrils in a from β to (Fig. we found that this in cytotoxicity protection (Fig. and to Aβ42 in the and of at different and was followed by (Fig. We found that the of in a of Aβ42 cellular toxicity. protective effect may be by the inhibition of the formation of oligomers as a result of the incubation with (Fig. is that the aggregates did not in the inhibition of Aβ42 followed by in which and with Aβ42 in the and the of of of Aβ42 and are a cytotoxicity of was also and for toxicity in the of is with a and are not followed by of Aβ42 and after of incubation at 37 and with to Aβ42 and after incubation and are shown in The by blot with the oligomer These suggest that the inhibition of Aβ42 cytotoxicity by from the of we found that the binding of to and Aβ42 alters protease have shown that TTR Aβ42 clearance in vivo (1Schwarzman A.L. Gregori L. Vitek M.P. Lyubski S. Strittmatter W.J. Enghilde J.J. Bhasin R. Silverman J. Weisgraber K.H. Coyle P.K. Transthyretin sequesters amyloid β protein and prevents amyloid formation.Proc. Natl. Acad. Sci. U.S.A. 1994; 91 (8078889): 8368-837210.1073/pnas.91.18.8368Crossref PubMed Scopus (329) Google Scholar). We that the large that binding to TTR be more sensitive to amyloid thereby also the protective effect found in vivo (6Buxbaum J.N. Ye Z. Reixach N. Friske L. Levy C. Das P. Golde T. Masliah E. Roberts A.R. Bartfai T. Transthyretin protects Alzheimer's mice from the behavioral and biochemical effects of Aβ toxicity.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18272491): 2681-268610.1073/pnas.0712197105Crossref PubMed Scopus (210) Google Scholar). We this by of amorphous aggregates (Fig. We incubated and Aβ42 with and collected the insoluble fractions by The immunodot blot of insoluble fractions showed that amorphous aggregates that result from the incubation of with both and Aβ42 by after 1 (Fig. A and B). In Aβ42 fibrils showed a higher to digestion. We Aβ42 in the as a of Finally, we evaluated ability to amyloid seeding by fibrils extracted from the brain of an AD (Fig. The of Aβ fibrils was performed by several of and as by R. and for in amyloid Sci. PubMed Scopus Google Scholar). Amyloid seeding was by we found that the of fibrils to Aβ42 in the of fibril In incubation with in inhibition of Aβ42 fibril formation in the of Aβ that the dissociation of the TTR is required to prevent cytotoxicity from Aβ oligomers. These are with the by Buxbaum and X. X. A.R. D. Kelly J.W. Buxbaum J.N. of transthyretin inhibition of β-amyloid aggregation in Neurosci. 2013; PubMed Scopus Google in which TTR Aβ which are to be for cellular toxicity D. M. The amyloid of proteins in human 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). They also found that tetrameric TTR binds to monomeric Aβ, thereby aggregation in vitro X. X. A.R. D. Kelly J.W. Buxbaum J.N. of transthyretin inhibition of β-amyloid aggregation in Neurosci. 2013; PubMed Scopus Google Scholar). have also found an of transthyretin and of cerebrovascular disease R. A. of cerebrovascular and Biol. 2013; PubMed Scopus Google Scholar). an additional found that the of the tetrameric of TTR promotes Aβ clearance in a mouse of Alzheimer's disease A. Saraiva M.J. Cardoso I. Transthyretin by promotes peptide its and in an Alzheimer's disease mouse PubMed Scopus Google Scholar). In study, we did not effect of tetrameric TTR on Aβ42 For the tetrameric variant did not Aβ42 cytotoxicity (Fig. 1). the variant did not the amyloidogenic variant did (Fig. studies and suggest that tetrameric TTR may be protective in vivo not by by and clearance of Aβ In TTR to Aβ cytotoxicity by binding to triggering the formation of large nontoxic Using computational we a peptide that inhibits Aβ42 fibril formation and cellular toxicity (Fig. the amyloidogenic segment followed by a (Fig. are with studies that the interaction of TTR and with Aβ to the inhibition of Aβ fibril formation in vitro X. X. A.R. D. Kelly J.W. Buxbaum J.N. of transthyretin inhibition of β-amyloid aggregation in Neurosci. 2013; PubMed Scopus Google Scholar). In addition, have shown that the segment was capable of binding Aβ on a J. Cho P.Y. Yang D.T. Murphy R.M. Identification of β-amyloid–binding sites on transthyretin.Protein Eng. Des. Sel. 2012; 25 (22670059): 337-34510.1093/protein/gzs026Crossref PubMed Scopus (43) Google Scholar). However, the peptide was not inhibiting Aβ aggregation in P.Y. Johnson J.A. Murphy R.M. as β-amyloid Chem. Neurosci. PubMed Scopus Google the peptide was therefore not amyloidogenic P.Y. M.D. Johnson J.A. Murphy R.M. A peptide of the β-amyloid binding on Chem. Neurosci. PubMed Scopus Google of the of this segment (Fig. In study, we by a which higher and also (Fig. The of a was sufficient to a amyloidogenic peptide into an of Aβ42 cellular toxicity at (Fig. inhibits Aβ42 oligomer formation the formation of nontoxic, amorphous Previous studies of the inhibition of Aβ cytotoxicity by TTR have revealed that TTR co-aggregates with Aβ oligomers into large nontoxic assemblies, thereby inhibiting cellular toxicity in vitro (9Cascella R. Conti S. Mannini B. Li X. Buxbaum J.N. Tiribilli B. Chiti F. Cecchi C. Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.Biochim. Biophys. Acta. 2013; 1832 (24075940): 2302-231410.1016/j.bbadis.2013.09.011Crossref PubMed Scopus (53) Google Scholar). this effect and promotes the formation of large aggregates (Fig. that not (Fig. not show with (Fig. and a to (Fig. These amorphous with the large aggregates found after the of to Aβ and amyloid proteins Lee J. Kelly J.W. the by which treatment amyloid Chem. 2013; PubMed Scopus Google Scholar). to Aβ aggregates, aggregates a that in and a of binding to (Fig. A and Aβ42 amorphous aggregates binding to are more sensitive to protease TTR inhibits Aβ oligomer toxicity by into large nontoxic (9Cascella R. Conti S. Mannini B. Li X. Buxbaum J.N. Tiribilli B. Chiti F. Cecchi C. Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro.Biochim. Biophys. Acta. 2013; 1832 (24075940): 2302-231410.1016/j.bbadis.2013.09.011Crossref PubMed Scopus (53) Google Scholar). However, studies did not protease of Aβ We that aggregates, from both and Aβ, are more sensitive to Aβ We that TTR may Aβ clearance in vivo by a this the formation of large that are more to and Finally, we evaluated the inhibitory effect of on Aβ amyloid shown we found that the of ex vivo fibril to Aβ fibril formation The of the β amyloid protein is for the seeding of amyloid implications for the of Alzheimer's 1993; PubMed Scopus Google Scholar, R. for in Alzheimer's β-amyloid Natl. Acad. Sci. U.S.A. 2008; 105 PubMed Scopus Google Scholar). In we found that inhibits amyloid seeding catalyzed by Aβ extracted from the brain of an AD (Fig. In suggest that the dissociation of the TTR into is required to prevent cytotoxicity from Aβ oligomers. In addition, we found that a segment derived from antiamyloid thereby inhibiting Aβ42 cytotoxicity and amyloid We also found that binds to and a that leads to an of protease Finally, we found that inhibits amyloid seeding catalyzed by Aβ fibrils extracted from the brain of an AD patient. of the inhibitory effect of by additional may to cytotoxicity and of the that result from the inhibition of by ex vivo fibrils as EM and protease The represents an of the on the of protection of TTR over Aβ42 cellular toxicity and a brain from the of an of Alzheimer's disease was from at the of the of The was previously evaluated for the of amyloid plaques by and for Alzheimer's disease. The of of the from the was TTR and as previously L. Johnson D. P. Whitelegge J. L. R. Eisenberg D.S. the of aggregation of human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with 1 of for TTR by followed by a on an peptide (Aβ42) was E. recombinant and was as reported previously I. R.M. R. The recombinant peptide aggregates and is more synthetic Biol. PubMed Scopus Google Scholar). The for Aβ42 an followed by of protease and the human Aβ42 the was into in E. was to the proteins followed by Aβ42 peptide was from the by followed by aggregation, Aβ42 was in which was by of protein by a with The 1 which was on an to pH with and to in a The was was The by at and of and with from the and the and from the of TTR aggregation performed as previously L. Chung K. A. Lee J.H. T. J. Benson M.D. Eisenberg D.S. A pair of inhibits seeding of the transporter transthyretin into amyloid Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 1 TTR in pH and was incubated at a of EM after days of fractions and to TTR aggregation by immunodot EM was performed to TTR aggregation and the of Aβ42 in the of TTR of was EM The three with of after of followed by with for A at an of was to the The by a and and for the toxicity of Aβ42. in with in with and We performed to the cytotoxicity of Aβ42 with the of TTR peptide A was to toxicity and at and in The in for at 37 in For Aβ42 and Aβ42 was in at the of followed by the of TTR at The with a and incubated for at 37 for the assay, of was to 90 24 of incubation at 37 in of was into incubation for 4 at 37 °C, of was to of incubation at the was at with at for of the The the of the treatment of the of Aβ42 and The after treatment with Aβ42 with and peptide was by the the as and as docking monomeric TTR and Aβ42 was performed the D. B. D. C. D. S. The for PubMed Scopus Google Scholar). TTR was by of the of L. Johnson D. P. Whitelegge J. L. R. Eisenberg D.S. the of aggregation of human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the Aβ42 segment and J.P. A. M. M. L. D. D. Eisenberg D. for Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). binding of was in the Aβ42 was in at the of Aβ42 was into at the of and was with and different of TTR peptide The was into and in a with The was the with and of and at 37 The aggregation of TTR was followed by immunodot blot as by et al. L. Johnson D. P. Whitelegge J. L. R. Eisenberg D.S. the of aggregation of human Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the of was at for and the was in the of and The was in and We a of the of The aggregation of Aβ42 was followed by and immunodot For the of by and a by For the immunodot blot analysis, of a We of of T. amyloid in the cerebrospinal from patients with Alzheimer's and Sci. 1994; Full Text PDF PubMed Scopus Google of and of and F. T. R. R. M. C. S. I. E. D. C. Glabe C. E. A a of Aβ plaques in Alzheimer and transgenic mouse PubMed Scopus Google Scholar). The was as at a of and was to and at a of The was the the of Aβ42 by The into a A was The in a of with a time of a of and a of was the of of the at a of in The at C. The are as the is the in is the in is the in is the and is in the of in of the The of the of Aβ after of was by the of as a The of the was by the which three to the and N. of protein from of and with an PubMed Scopus Google Scholar). The are as the and of the three Aβ42 was in and Aβ42 fibrils after of incubation in at of was to Aβ42 and incubated for and insoluble fractions extracted by centrifugation of of the to was performed in a of of Aβ42 and of to in pH for 1 at 37 The with 1 and the blot we a with The was of was performed with for an in the are as as of at three The and the are from the on are the We Eisenberg and for We also for the AD brain and the with Alzheimer's disease transthyretin peptide peptide monomeric TTR variant TTR variant TTR segment with a

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