Transthyretin binding to A-Beta peptide – Impact on A-Beta fibrillogenesis and toxicity

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Transthyretin binding to A-Beta peptide – Impact on A-Beta fibrillogenesis and toxicity

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  Transthyretin binding to A-Beta peptide – Impact onA-Beta fibrillogenesis and toxicity R. Costa a , A. Gonc¸alves a , M.J. Saraiva a,b , I. Cardoso a,* a Molecular Neurobiology Unit, Instituto de Biologia Molecular e Celular (IBMC), Rua do Campo Alegre, 823, 4150-180 Porto, Portugal  b ICBAS, University of Porto, Porto, Portugal  Received 15 January 2008; revised 31 January 2008; accepted 11 February 2008Available online 22 February 2008Edited by Jesus Avila Abstract It has been suggested that transthyretin (TTR) is in-volved in preventing A-Beta fibrillization in Alzheimer  s disease(AD). Here, we characterized the TTR/A-Beta interaction bycompetition binding assays. TTR binds to different A-Beta pep-tide species: soluble (Kd, 28 nM), oligomers and fibrils; diverseTTR variants bind differentially to A-Beta. Transmission elec-tron microscopy (TEM) analysis demonstrated that TTR iscapable of interfering with A-Beta fibrillization by both inhibit-ing and disrupting fibril formation. Co-incubation of the twomolecules resulted in the abolishment of A-Beta toxicity. Our re-sults confirmed TTR as an A-Beta ligand and indicated the inhi-bition/disruption of A-Beta fibrils as a possible mechanismunderlying the protective role of TTR in AD.   2008 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved. Keywords:  Alzheimer  s disease; A-Beta peptide; Transthyretin;Inhibitor; Disrupter; Neuroprotector 1. Introduction Alzheimer  s disease (AD) presently affects 20–30 millionindividuals worldwide and accounts for most cases of dementiathat are diagnosed after the age of 60. Histopathologically, thisdisease is characterized by two lesions: senile or neuritic pla-ques and neurofibrillary tangles (NFTs) [1]. Neuritic plaques are mainly constituted by extracellular deposits of the 40-and 42-amino acid beta-amyloid (A-Beta) peptides. The pep-tide in these plaques is in the form of insoluble amyloid fibrilsmixed with a poorly defined array of non-fibrillar forms of thepeptide [2]. A-Beta, a 37–43 amino acid peptide with 4 kDa, isproduced by proteolytic cleavage of a large transmembraneprecursor, the amyloid precursor protein (APP) [3]. Severalmolecules were identified and suggested as A-Beta carriers[4,5]. Among them, TTR has recently received a large attention[6 – 9]. TTR is a homotetrameric 55 kDa protein produced mainly in the liver and in the choroid plexus of the brain[10] that is responsible for thyroid hormone and retinol trans-port [11]. Over 100 TTR mutations have been identified andassociated with TTR related amyloid deposition in familialamyloidotic polyneuropathy, affecting the peripheral nervoussystem.Schwarzman and co-workers used human cerebrospinalfluid (CSF) incubated with synthetic A-Beta (1–40) to identifythe interacting proteins and to evaluate the peptide aggrega-tion levels [4]. The authors concluded that TTR was the majorA-Beta binding protein in the CSF; a decrease in the aggrega-tion state of the peptide as well as in its toxicity in the presenceof TTR was also observed [4,12]. The sequestration hypothesis was put forward: normally produced A-Beta is sequestered bycertain extracellular proteins, thereby preventing amyloid for-mation and A-Beta cytotoxicity; formation of amyloid and theconsequent toxicity occurs when sequestration fails [12].  In vivo experiments were also performed in order to investigate theprotective effect of TTR. In  Caenorhabditis elegans  expressinghuman A-Beta (1–42), TTR rescued the neurodegenerationtriggered by the toxic peptide [13]. Studies in transgenic mice overexpressing mutant APP revealed slow disease progressionand lack of neurodegeneration attributed to TTR expression[7]. In the absence of TTR, A-Beta deposition was accelerated[9].The discussion on TTR/A-Beta interaction and consequentinhibition of aggregation and toxicity reduction raised thehypothesis that mutations in the TTR gene or conformationalchanges in the protein induced by aging, could affect thesequestration properties. However, no mutations in the TTRgene have been found in AD patients [14].To summarize, TTR may prove to be a useful therapeuticagent capable of preventing or retarding cerebral amyloid pla-que formation in AD. Nevertheless, the precise mechanism bywhich TTR acts against the amyloidogenicity and toxicity of the A-Beta peptide is unknown and thus further investigationis necessary. 2. Materials and methods  2.1. Production of A-Beta species A-Beta peptide (1–42) was purchased from BioSource, dissolved inHFIP and kept at room temperature for 1–2 h. HFIP was then re-moved under a stream of nitrogen until a clear film remained in theEppendorf tube. The residue was then dissolved in DMSO at 2 mMconcentration. Soluble A-Beta was prepared by instantly diluting thepeptide in the appropriate buffer and used immediately for competitionbinding assays; to prepare oligomers, A-Beta peptide was diluted to Abbreviations:  AD, Alzheimer  s disease; WT TTR, wild-type trans-thyretin; V30M TTR, transthyretin with a valine substitute by amethionine at position 30; L55P TTR, transthyretin with a leucinesubstitute by a praline at position 55; Y78F TTR, transthyretin with atyrosine substituted by a phenylalanine at position 78; T119M TTR,transthyretin with a threonine substituted by a methionine at position119 * Corresponding author. Fax: +351 22 6074905. E-mail address:  icardoso@ibmc.up.pt (I. Cardoso).0014-5793/$34.00    2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.febslet.2008.02.034FEBS Letters 582 (2008) 936–942  100  l M in F12 cell media for 48 h at 4   C, whereas for fibril formationthe peptide was incubated at 37   C for 8 days; samples were then ana-lyzed by transmission electron microscopy (TEM).  2.2. TTR production and purification Recombinant TTRs were produced in a bacterial expression systemusing  Escherichia coli   BL21 [15] and purified as previously described[16]. Briefly, after growing the bacteria, the protein was isolated andpurified by preparative gel electrophoresis after ion-exchange chroma-tography. Protein concentration was determined using the Lowrymethod [17].  2.3. Competition binding assays Recombinant wild-type transthyretin (WT TTR) was iodinated withNa 125 I (NEN) using the Iodogen (Sigma) method following the sup-plier  s instructions. The reaction mixture was subsequently desaltedby Sephadex G50 gel filtration. 96 well plates (Maxisorp, Nunc) werecoated with soluble, oligomeric or fibrillar A-Beta peptide prepared asdescribed (5  l g/well) in coating buffer (0.1 M bicarbonate/carbonatebuffer, pH 9.6) and incubated overnight at 4   C. Unoccupied sites wereblocked by incubation with 5% non-fat dried milk in PBS for 2 h at37   C. For competition studies, a constant amount of   125 I-labelledWT TTR was added to each well alone or in the presence of the indi-cated molar excess of unlabelled competitors. Specific binding was de-fined as that observed with  125 I-labelled protein alone minus  125 I-labelled protein in the presence of 100-fold molar excess unlabelledprotein. Kd assays were performed as described elsewhere [18]. Bindingdata were fit to a one-site model and analyzed by the method of Klotzand Hunston using non-linear regression analysis with the Prism pro-gram (GraphPad Software Inc.). Results are shown as percent of max-imal binding ± S.D. and are representative of a minimum of twoindependent experiments.T 4  binding competition assays were based on a gel filtration proce-dure as previously described [19]. Briefly, 50  l l of a diluted sample(30 nM TTR) was incubated with 50  l l of either cold T 4  solutions orA-Beta peptide (1–42) solutions of variable concentrations rangingfrom 0 to 1000 nM and with a constant amount of labeled  125 I-T 4 (  50,000 cpm). This solution was counted in a gamma spectrometerand incubated at 4   C overnight. Protein bound  125 I-T 4  and free  125 I-T 4  were separated by gel filtration through a 1 ml BioGel P6DG(Bio-Rad) column. The bound fraction was eluted while free T 4  was re-tained on the BioGel matrix. The eluate containing the bound T 4  wascollected and counted. Bound T 4  was expressed as percentage of totalT 4  added. Each assay was performed in triplicate. Analysis of the bind-ing data was performed with the GraphPad Prism program (version2.0, San Diego, CA).  2.4. Influence of TTR in A-Beta aggregation and fibril disruption A-Beta peptide 100  l M, prepared as described, was incubated withdifferent TTR variants (10  l M), and incubated at 37   C for differentperiods of time. Alternatively, A-Beta 100  l M was incubated aloneat 37   C for 8 days and then 10  l M TTR, either WT or L55P, wasadded and further incubated at 37   C for the desired time. Sampleswere analyzed by TEM.  2.5. Transmission electron microscopy For visualization by TEM, sample aliquots were absorbed to glow-discharged, carbon-coated collodion film supported on 200-mesh cop-per grids, and negatively stained with 1% uranyl acetate. The gridswere exhaustively visualized with a Zeiss microscope (modelEM10C), operated at 60 kV.  2.6. Cell culture and caspase-3 assay SH-SY5Y cells (human neuroblastoma cell line) were propagated in25-cm 2 flasks and maintained at 37   C in a 95% humidified atmosphereand 5% CO 2 . Cells were grown in Dulbecco  s minimal essential med-ium supplemented with 10% fetal bovine serum (Gibco BRL). Activa-tion of caspase-3 was measured using the CaspACE fluorimetric 96-well plate assay system (Sigma), following the manufacturer  s instruc-tions. Briefly, 10  l M A-Beta peptide pre-incubated for 48 h in F12media (Gibco BRL) with or without 2  l M TTR at 4   C with shaking,were added to 80% confluent cells in Dulbecco  s minimal essentialmedium with 1% fetal bovine serum, and further incubated for 48 h,at 37   C. Subsequently, each well was trypsinized and the cell pelletwas lysed in 100  l l of hypotonic lysis buffer (Sigma). Forty microlitresof each cell lysate was used in duplicates for determination of caspase-3activation. The remaining cell lysate was used to measure total cellularprotein concentration with the Bio-Rad protein assay kit (Bio-Rad),using BSA as standard. Values shown are the mean of duplicatesand the experiment was performed three times. Comparison betweengroups was made using the Student  s  t -test. A  P   value of less than0.05 was considered statistically significant. 3. Results 3.1. Assessment of WT TTR binding to different species of A-Beta It has been previously reported that TTR acts as an A-Betacarrier [4,6]. To further characterize this interaction we per- formed competition binding assays using soluble A-Beta pep-tide (1–42) and recombinant  125 I-TTR. Using this techniquewe were able not only to confirm the interaction between thetwo molecules but also to determine a Kd of 28 ± 5 nM(Fig. 1A). We next produced A-Beta amyloidogenic species,oligomers and fibrils, upon incubation of the peptide at 4   Cor 37   C, respectively, for different periods of time. Oligomersappeared as short and thin protofilaments together with roundparticles, 4–5 nm wide (Fig. 1 C, upper panel), while A-Betaincubated at 37   C for 8 days formed long fibrils, 10–13 nmof diameter (Fig. 1C, lower panel). These species were then as-sessed for binding to TTR and results showed that they alsobind to TTR (Fig. 1B), with affinities similar to the solublecounterpart. 3.2. Interaction of different TTR variants with A-Beta We next assessed the ability of different TTR mutations,amyloidogenic (transthyretin with a valine substitute by amethionine at position 30 (V30M TTR), transthyretin with aleucine substitute by a proline at position 55 (L55P TTR)and transthyretin with a tyrosine substituted by a phenylala-nine at position 78 (Y78F TTR)) and non-amyloidogenic(transthyretin with a threonine substituted by a methionineat position 119 (T119M TTR)) to bind the A-Beta peptide,using the same approach as above. The competition bindingassays allow us to conclude that the diverse variants bind dif-ferently to the peptide in the following manner: T119MTTR > WT > V30M  P  Y78F > L55P, as displayed in Fig. 2.These results suggest an inverse relation between the amyloido-genic potential of TTR and affinity to the peptide. Table 1 dis-plays the relative affinities found for the different TTR variantswhen binding to soluble A-Beta.We also investigated if binding of A-Beta peptide to TTRinvolved the T 4  binding channel by performing competitionassays with  125 I-T 4 . Our results showed no competitionbetween T 4  and A-Beta peptide towards binding to TTR (datanot shown). 3.3. Effect of TTR in A-Beta fibrillization To ascertain if TTR binding to A-Beta peptide has anyimpact on the fibrillization of the peptide, we performedultrastructural analysis of co-incubated WT TTR and solubleA-Beta preparations; visualization of the samples by TEM re-vealed a decrease in the number and length of the fibrils whencompared to preparations of A-Beta incubated alone (Fig. 3,middle and left panels, respectively), indicating that TTR R. Costa et al. / FEBS Letters 582 (2008) 936–942  937  was inhibiting A-Beta fibrillogenesis; under the conditions of the experiment WT TTR does not aggregate [20]. Extendedincubations at 37   C for at least 7 days revealed that TTRdid not lose the inhibitory ability, resulting in a more pro-nounced effect as only small oligomers were observable underthe microscope. Control samples of WT TTR alone were alsoprepared and analysed; no fibrils or aggregates were seen (datanot shown) but only round particles representing the nativeprotein.We next studied the effect of the TTR variants, previouslyshown to bind the peptide, on A-Beta fibrillization; L55Pand T119M were chosen due to their lowest and highest bind-ing capacities, respectively. The peptide was incubated with orwithout the TTR variant, at 37   C, and the samples were ana-lysed at different stages (day 1, day 5 and day 7), similarly tothe study performed with WT TTR. In Fig. 3, right panels,the effects of L55P TTR on A-Beta fibrils are noticeable,revealing that this variant retained the ability to inhibit A-Betafibril formation; comparable data was gathered using T119MTTR (not shown) and thus, no significant differences werefound between TTR variants and the WT counterpart in theircapacity to inhibit A-Beta fibril formation. The discrepancyobserved between binding and effect on fibrillization for thedifferent TTR variants used, may reflect that different mecha-nisms are implicated in the two processes; it can also indicatea limitation of the technique used since TEM is not a quanti-tative approach and therefore may fail to detect the differences. Fig. 1. Characterization of TTR and A-Beta interaction. (A) Binding of   125 I-WT TTR to soluble A-Beta peptide; a Kd of 28 ± 5 nM was calculatedas described in Section 2. (B) Binding of   125 I-WT TTR to different amyloidogenic species of A-Beta peptide: soluble (A-Beta soluble), oligomersformed at 4   C for 48 h (oligomers 48 h) and fibrils formed at 37   C for 8 days (fibrils 8d) demonstrating similar affinities. (C) Morphologiccharacterization assessed by TEM of A-Beta oligomeric (upper panel) and fibrillar (lower panel) species. Scale bar = 200 nm.Fig. 2. Competition binding assays with TTR variants. Displacementcurves of   125 I-WT TTR from soluble A-Beta by different TTR variants(WT TTR, V30M, Y78F, L55P and T119M).Table 1Binding affinity of different TTR variants towards A-Beta, relative tothe WT counterpartTTR variant Relative affinityWT 1T119M 3.7V30M 0.6Y78F 0.5L55P 0.07938  R. Costa et al. / FEBS Letters 582 (2008) 936–942  Additionally, we also analyzed preparations of A-Beta pre-formed fibrils where TTR, either WT or L55P, was addedand further incubated at 37   C. Surprisingly and contrarilyto a previous report [12], our data indicated that TTR was also Fig. 3. Influence of TTR in A-Beta fibrillization. A-Beta peptide was incubated with or without TTR during different periods of time and analyzedby TEM at days 1, 5 and 7. The data revealed a decrease in the number and the length of fibrils when the peptide was co-incubated with WT TTR(middle panels) when compared with A-Beta incubated alone (left panels). No significant differences were detected among the TTR variants tested, asshown here for L55P TTR (right panels), thus showing inhibition of A-Beta fibrillization. Scale bar = 100 nm.Fig. 4. Disruption of A-Beta fibrils by TTR. A-Beta fibrils were grown for 8 days at 37   C as described in Section 2; then TTR was added and furtherincubated at 37   C and analyzed at different time points by TEM. A-Beta alone generated long fibrils 10–13 nm wide (A), whereas addition of WTTTR resulted in shorter fibrils after 2 days of co-incubation (B); continued incubation at 37   C resulted in even shorter fibrils (C, 4 days) and in smallaggregates and oligomers (D, 14 days). Co-incubation of L55P TTR with A-Beta peptide for 4 days at 37   C indicated that this variant retained thedisrupter activity (E). Scale bar = 100 nm. R. Costa et al. / FEBS Letters 582 (2008) 936–942  939  able to disrupt A-Beta fibrils as shorter fibrils were observed 2days after the addition of WT TTR, as depicted in Fig. 4B,when compared with control samples containing A-Beta incu-bated alone (Fig. 4A); extended incubations for 4 and 14 daysresulted in even shorter fibrils (Fig. 4C) and small aggregatesand oligomers (Fig. 4D), respectively. TTR alone was alsoincubated for the same time periods and observed under theelectron microscope showing no fibrils or aggregates; againonly round particles similar to the native protein were ob-served (data not shown). No significant differences were foundbetween TTR WT and L55P, the most amyloidogenic TTRvariant used in their ability do disaggregate A-Beta peptide fi-brils (Fig. 4E). Taken together, our results indicate TTR notonly as an A-Beta fibril inhibitor but also as a fibril disrupter. 3.4. TTR abolishes A-Beta toxicity in cell culture A-Beta peptide (1–42) is toxic to cells and leads to apoptosisand cellular death; previous works performed  in vitro  and in vivo  demonstrated that the oligomeric form of the peptidehas the highest toxicity [21]. Here we tested the hypothesis thatTTR could protect against this neurotoxicity. We measuredcaspase-3 activity occurring in SH-SY5Y cells incubated withTTR pre-incubated with A-Beta 1–42, TTR alone or A-Betaalone (Fig. 5). In the latter, significant caspase-3 activationwas observed, in opposition to cells incubated with TTR aloneor A-Beta pre-incubated with TTR which presented levels of caspase-3 activation similar to the non-treated control cells.Therefore, TTR abolishes A-Beta oligomers toxicity. 4. Discussion A-Beta peptide deposition in the brain is the biochemicalhallmark in AD and although APP processing and A-Betageneration have been intensely investigated, the reasons whythe peptide deposits extracellularly in the brain are not com-pletely understood. TTR has been suggested as an A-Beta car-rier [12,22] and attempts to relate TTR/A-Beta levels in CSF and AD have been made [23,24]. Previous  in vitro  studiesestablished differences between TTR variants in their bindingability towards the A-Beta peptide [25].In the present work, the WT TTR/A-Beta peptide interac-tion was further investigated and the respective Kd determinedas 28 ± 5 nM. We also observed that TTR interacted withother forms of A-Beta, namely oligomers and fibrils with sim-ilar affinities (Fig. 1B). TTR interaction with A-Beta peptide in vivo  in CSF refers to the soluble form of the peptide withconsequent rescue of A-Beta aggregation. Thus, binding of TTR to aggregated forms of A-Beta peptide (oligomers and fi-brils) with similar strengths was unexpected, since in these spe-cies, especially in the fibrils, the availability of the epitopesshould be lower than in the soluble counterpart. Other authorshave observed binding of TTR to aggregated forms of the pep-tide [26]. In their work, binding of TTR to A-Beta (1–40) wasinvestigated and results indicated that TTR suppressed growthof A-Beta aggregates but did not inhibit the initial assembly.These authors also hypothesize that TTR binds preferentiallyto growing A-Beta rather than to soluble non-aggregatedpeptide and estimated an association constant of    2300 ±100 M  1 . Although A-Beta aggregates were visible uponTTR incubation, inhibition of the initial A-Beta assembly can-not be ruled out due to the great tendency for aggregation of the synthetic peptide. As mentioned above we observed similarbinding affinities for all the A-Beta species tested, and whereasLiu and co-workers did not specifically tested binding to ma-ture fibrils, we used A-Beta (1–42) which can account for thedifferences observed, including a stronger interaction (Kd28 ± 5 nM).The influence of TTR mutations, such as T119M, Y78F,V30M and L55P, in the binding to A-Beta was also investi-gated. TTR variants bound differently to A-Beta in thefollowing order: T119M > WT > V30M  P  Y78F > L55P,suggesting that the ability to rescue the peptide is different be-tween them, with possible implications in A-Beta deposition inthe brain. Our observation suggests an inverse relationbetween the amyloidogenic potential of TTR and affinity toA-Beta peptide, i.e. a direct relation with protein stability.X-ray diffraction studies of L55P TTR suggested this variantas an amyloidogenic intermediate in the process of TTR fibrilformation. The mutation leads to the disruption of the Dstrand due to the disturbance of the hydrogen bonds betweenstrands D and A, thus residues 54–56 are part of a long surfaceloop that connects strands C and E [27]. This variant, the lessstable and most amyloidogenic among the tested ones, showedthe lowest affinity to the peptide, whereas the anti-amyloido-genic one, T119M, showed the highest affinity, indicating that Fig. 5. Caspase-3 activation in cell culture. A-Beta with or without TTR or TTR alone incubated at 4   C for 48 h were then added to SH-5YSYcultured cells and further incubated for another 48 h at 37   C. A-Beta was used at a final concentration of 10  l M and TTR at 2  l M. Significantcaspase-3 activation was observed in the presence of A-Beta (A-Beta oligomers), when compared to non-treated (NT) and TTR-treated (WT TTR)cells; toxicity of A-Beta was prevented by pre-incubating the peptide with TTR (A-Beta + WT TTR).  * P   < 0.05.940  R. Costa et al. / FEBS Letters 582 (2008) 936–942
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