FEBS Letters 586 (2012) 2891–2896

journal homepage: www.FEBSLetters .org

Review

Clearance of extracellular misfolded proteins in systemic amyloidosis: Experiencewith transthyretin

Maria Rosário Almeida ⇑, Maria João Saraiva ⇑IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, PortugalICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal

a r t i c l e i n f o

Article history:Received 2 July 2012Revised 11 July 2012Accepted 11 July 2012Available online 20 July 2012

Edited by Miguel De la Rosa, Felix Wielandand Wilhelm Just

Keywords:TransthyretinAmyloidClusterinDoxycyclineEGCG (epigallocatechin gallate)

0014-5793/$36.00 � 2012 Federation of European Biohttp://dx.doi.org/10.1016/j.febslet.2012.07.029

⇑ Corresponding authors. Address: Molecular NeuAlegre 823, 4150, Porto, Portugal. Fax: +351 22 60991

E-mail addresses: ralmeida@ibmc.up.pt (M.R. Alm(M.J. Saraiva).

a b s t r a c t

Increasing evidence indicates that accumulation of misfolded proteins in the form of oligomers,protofibrils or amyloid fibrils, and their consequences in triggering intracellular signaling cascadeswith toxic consequences represent unifying events in many of slowly progressive neurodegenerativedisorders. Studies with small compounds or molecules, known to recognize and disrupt amyloido-genic structures, have proven efficient in promoting clearance of protein aggregates in experimentalmodels of systemic and localized forms of amyloidoses. Doxycycline and EGCG were efficient inremoving aggregates in pre-clinical studies in a transgenic mouse model for transthyretin (TTR) sys-temic amyloidosis and represent an opportunity to address mechanisms and key players in depositremoval. Extracellular chaperones, such as clusterin and metalloproteinases play an important rolein this process.� 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Extracellular protein misfolding and aggregation occurring insystemic amyloidosis triggers inflammation, oxidative stress, ma-trix remodeling, the unfolded-protein-response and ER pathwaysthat resemble in many aspects, including common molecular play-ers and scenarios, to those described in local amyloidoses affectingfor example the central nervous system (CNS), such as AlzheimerDisease. Thus, similarities and dissimilarities in toxicity found be-tween the CNS and the periphery are very useful to pinpoint andguide us to the treatment of aging-associated neurodegenerativedisorders. Understanding the two-way crosstalk between theextracellular milieu and the cell is a major trend in diseases relatedto protein aggregation. In particular, mechanisms involved in theclearance of protein aggregates, both extra and intracellular, arepivotal and need detailed analyses for the development of thera-peutic strategies. Studies with small compounds or molecules,such as antibodies [1,2] known to recognize and disrupt amyloido-genic structures, have proven efficient in removing and promotingclearance of protein aggregates in studies with experimental mod-els of misfolding disorders. However, the mechanisms and key

chemical Societies. Published by E

robiology, IBMC, R. Campo57.eida), mjsaraiv@ibmc.up.pt

players in these processes are largely unknown. Extracellularmolecular chaperones are capable to repair or target damaged pro-teins to degradation by binding to extracellular misfolded proteinsand by promoting their disposal either by endocytosis for intracel-lular degradation or degradation by the extracellular matrix.

Herein, we give examples of studies designed to approach the is-sue of clearance of extracellular deposition of transthyretin (TTR), akey protein associated with familial amyloidotic polyneuropathy(FAP). FAP is a fatal neurodegenerative disorder characterised bythe extracellular deposition of aggregates and fibrils of mutantforms of TTR, particularly in nerves and ganglia of the peripheralnervous system (PNS). The most common TTR mutation is a substi-tution of Valine for Methionine at position 30 (V30M TTR) that pre-disposes TTR to form aggregates and fibrils. FAP initially presentswith symptoms that are associated with sensory and autonomicnervous system (ANS) dysfunction. These include loss of pain andtemperature sensation in the distal limbs, impotence, gastrointesti-nal disturbances, bladder dysfunction and postural hypotension. AsFAP progresses, sensory deficiencies extend to more proximal re-gions of limbs and cardiac insufficiency and mal-absorption fromthe gut become common. In addition, motor involvement resultsin a progressive loss of reflexes and muscle wasting in later stagesof FAP [3]. Over 600 kindreds have been identified in Portugalwhich constitutes the largest world focus of the disease.

TTR is a transport protein for thyroid hormones (namely, thy-roxine – T4) and vitamin A (through a complex with retinol binding

lsevier B.V. All rights reserved.

2892 M.R. Almeida, M.J. Saraiva / FEBS Letters 586 (2012) 2891–2896

protein – RBP) and is predominantly synthesized in the liver. Be-cause most circulating mutant TTR is synthesized in the liver, livertransplant has been used to treat FAP since 1990. However, univer-sal and less invasive therapeutics are mandatory. Proposed phar-maco-therapeutic strategies for FAP are similar to the approachestaken for other systemic amyloidoses, such as lowering the levelsor stabilizing the native structure of the plasma protein precursor,inhibiting aggregation, disrupting TTR amyloid by selective mole-cules, and counteracting cellular toxicity.

Based on the knowledge gathered from the various in vitro andin vivo approaches to elucidate TTR aggregation and toxicity, dif-ferent multicenter clinical trials are underway including RNAisilencing methodology to lower liver TTR expression – ALN-TTR01 – (www.Alnylam.com); in a transgenic mice model for thehuman TTR V30M (described below), this compound showed dura-ble suppression, as much as 90%, for both TTR mRNA and proteinlevels in plasma and reduction of deposition in extra-hepatic tis-sues [4]. Phase I/II human clinical studies are underway to evaluatethe safety and tolerability of this drug in V30M FAP patients.Stabilization of the native structure of plasma TTR is achieved byTafamidis (now proprietary of Pfizer) as explained below; resultsfrom an 18-month phase II/III randomized double-blind trialshowed improvement of body mass index and slight stabilizationin nerve function [5]. As referred to previously, once the clinicalsymptoms are triggered in FAP, disease evolution takes severalyears; thus, the initial studies have been extended to assess theusefulness of this drug in FAP treatment and also to investigatethe mechanisms underlying the results found in non-responders.

It is clear that more than a single approach to treat FAP effec-tively is mandatory; in this regard, the experience achieved sofar with clearance based approaches is very promising and nextdescribed.

2. Modulation of the heat-shock response: extracellularchaperones

The heat-shock response was investigated in FAP. Up-regulationof Hsp27 and Hsp70 expression related to the presence of extracel-lular TTR aggregates was documented in human FAP biopsies ascompared to normal controls. TTR aggregates did not co-localizewith Hsps suggesting that extracellular TTR tissue deposits are ableto induce an intracellular stress response. Moreover, the heat-shock transcription factor-1 (HSF-1) was up-regulated and local-ized to the nucleus [6].

It was hypothesized that HSF-1 could be involved in FAP path-ogenesis as a cell-defense mechanism against the presence ofextracellular TTR deposits and that disruption of the heat-shock re-sponse would aggravate TTR deposition. A mouse model express-ing the human TTR-V30M in an HSF-1-null background wascharacterized. The lack of HSF-1 expression lead to extensive andearlier non-fibrillar TTR deposition, particularly in the gastrointes-tinal tract, evolving into fibrillar material in distinct organs, includ-ing the peripheral and autonomic nervous systems; in contrast,V30M animals in the wild-type background do not display deposi-tion in the PNS and ANS. As in the human disease, liver, brain, andspinal cord did not present deposition in this animal model. Fur-thermore, inflammatory stress and a reduction in unmyelinatednerve fibers were observed, as in human patients, indicating thatHSF-1-regulated genes are involved in FAP, modulating TTR tissuedeposition [7].

Clusterin, also known as apolipoprotein J, is an ubiquitoushighly conserved secreted protein. Clusterin inhibits protein aggre-gation in an ATP independent manner after binding to misfoldedproteins forming soluble, high molecular weight complexes [8,9].It was shown that clusterin, much like small heat shock proteins,

does not have the capability to refold non-native enzymes by itself;however clusterin is able to keep non-native proteins in a stabi-lized state until Hsp70 can refold them. Clusterin is present in mostphysiologic fluids and described to act as a molecular chaperone inthe extracellular milieu [10]. Several functions have been recog-nized for clusterin other than being a molecular chaperone; thisprotein has a role in: (i) control of cell–cell and cell–matrix inter-actions; (ii) apoptosis regulation; (iii) lipid transport and comple-ment regulation. Clusterin gene expression is induced by avariety of factors including stress, cellular growth, differentiationand ageing. HSF-1 is also responsible for clusterin expressioninduction [11]. In vitro data reported that clusterin is able to inhi-bit fibril formation of several different proteins, including b-amy-loid and a-synuclein [12,13]. Clusterin binds b-amyloid andforms a complex that is internalized for degradation by megalin(low-density lipoprotein receptor related protein 2 – LRP2); uptakeof these complexes lowers amyloid toxicity [14]. Facilitated clear-ance of extracellular misfolded proteins by clusterin was also doc-umented in vivo [15]. Thus, the available data support the notionthat besides a role as a molecular chaperone, stabilizing proteinsand inhibiting their aggregation, clusterin also has an importantrole in the clearance of extracellular aggregates through megalinmediated endocytosis. In the case of FAP, we have shown clusterinoverexpression in tissues with TTR deposition of V30M transgenicmice with a full HSF-1 response; by double immunohistochemis-try, clusterin co-localizes with both fibrillar and non-fibrillar TTRdeposits in human nerve and is identified in TTR fibrils extractedfrom human kidney. In vitro studies revealed that incubation ofSH-SY5Y neuroblastoma cells with TTR oligomers leads to intracel-lular clusterin over-expression. Moreover, clusterin secretion wasalso significantly increased in the medium of cells incubated withTTR oligomers and association of clusterin with TTR aggregates ob-served. Furthermore, clusterin modulates TTR aggregation, as clus-terin deprivation in cellular models stimulates extracellular TTRaggregation [16].

Further details of clusterin chaperone activity, scavenger prop-erties and mechanisms of other extracellular chaperones acting inFAP are warranted.

3. Pharmacological agents

3.1. Clearance by disaggregation of TTR amyloid fibrils: doxycycline

Some years ago it was reported that 40-iodo-40-doxy doxorubi-cin inhibits amyloid formation and promotes the reabsorption ofamyloid deposits [17]. Subsequently, tetracycline antibiotics werescreened for these properties based on their structural homologieswith the aglycone moiety of the anthracyclines. The first demon-stration was by in vitro studies with the human prion protein(PrP) whereby tetracyclines were shown to bind to human PrP,to hinder assembly into amyloid fibrils, and to prevent neuronaldeath and astrocyte proliferation induced by PrP peptides‘‘in vitro’’ [18]. It was found that doxycycline was particularly po-tent in disrupting TTR-like amyloid and that the generated assem-blies did not display cytotoxic properties in caspase-3 activationassays [19]. In vivo studies corroborated the in vitro findings; whendoxycycline was administrated to 23–28 month old V30M TTRtransgenic animals in the drinking water over a period of 3 months,immunohistochemistry revealed that Congo red positive materialwas only observed in the control, non-treated group. Moreover,immunohistochemistry for several markers associated with TTRamyloid deposition such as matrix metalloprotease 9 (MMP-9)[20] and serum amyloid P component (SAP) was performed. Signif-icantly lower levels of MMP-9 were found in the treated animalswhen compared with the control group and mouse SAP was absent

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in treated animals, being only observed in non-treated animalswith congophilic deposits. These results indicated that doxycyclineis capable of disrupting Congo red positive TTR amyloid depositsand decreases standard markers associated with fibrillar deposi-tion, being suggested as a potential drug in the treatment of amy-loidosis [21]. Doxycycline can also disassemble preformed b2microglobulin amyloid fibrils and at the same inhibits their forma-tion [22].

More recently, in vitro studies demonstrated that doxycyclinedirectly disrupted the formation of recombinant light chains (LC)amyloid fibrils. Furthermore, treatment of ex vivo LC amyloid fi-brils with doxycycline reduced the number of intact fibrils andled to the formation of large disordered aggregates; in vivo studiesin a CMV-k6 transgenic model for AL amyloidosis showed clear-ance of amyloid deposits in the stomach after doxycycline treat-ment [23].

All together these evidences led to the approval by the Euro-pean Medicines Agency of the application of doxycycline as an or-phan drug in the treatment of FAP (EU/3/12/955) and b2microglobulin systemic amyloidosis (EU/3/12/961). Detailed anal-yses on the mechanism of action of doxycycline on amyloid fibrilsawait elucidation.

3.2. Clearance by modulation of the TTR aggregation pathway: EGCG

It is widely accepted that the mechanism of TTR amyloid forma-tion involves destabilization of the TTR tetramer, due to environ-mental or genetic conditions, namely mutations, leading todissociation in monomers with an altered conformation that aremore prone to aggregation and fibril formation [24]. Consideringthis mechanism, one of the most explored approaches in TTR amy-loidosis was the search for compounds that specifically bind toTTR, in particular at T4 binding sites, stabilizing the tetramer andblocking the cascade of aggregation and fibril formation. Thus awide variety of small compounds presenting structural similaritieswith thyroxine, the natural TTR ligand, have been proposed andtested as TTR amyloid inhibitors [25,26]. Those compounds arevery specific in their binding at the TTR–T4 binding sites establish-ing interactions with different TTR monomers and promoting TTRstabilization; such is the case of Tafamidis, above referred.

Recently, several compounds have been referred to act as non-specific aggregation inhibitors of different proteins involved inmisfolding diseases as is the case of Ab and a-synuclein [27]. Thenon-specific inhibitors hinder formation of oligomeric and/or pre-fibrillar species, probably recognizing common structural or con-formational properties and through a still unknown mechanismof action may also disrupt pre-existing amyloid fibrils [28]. Thisconcept of general modulators of the aggregation pathway is gain-ing increasing relevance. Among those modulators, particularinterest has been raised by natural polyphenols such as epigalloca-techin gallate (EGCG), from green tea, and curcumin [29]. Based onthis concept of general modulators of amyloid formation, EGCGand curcumin have also been tested in TTR-related amyloidosis.Studies in vitro and in a cell culture system using different amyloi-dogenic TTR variants demonstrated that EGCG and curcumin bindto TTR, increase its tetrameric conformational stability, modulatethe intermediary species formed and inhibit TTR aggregation[30,31] (Fig. 1a). However, contrary to other polyphenols, EGCGdoes not bind at the T4 binding channel on TTR [30]. Crystal struc-ture of the TTR-EGCG complex demonstrated that there are 3 dif-ferent binding sites for EGCG on TTR [32]. One of these sites islocated in a region of the TTR tetramer that allows contact of EGCGwith both TTR dimers, indicating that the interaction of EGCG withTTR at this binding site contributes to the TTR tetramer stabiliza-tion. This site seems to be the preferential binding site for EGCG.The other two binding sites are located at the surface of different

monomers in the TTR molecule and might be involved in the olig-omerization of TTR tetramers, leading to the formation of non-toxic, off-pathway aggregates [30,32].

EGCG was also demonstrated to act as amyloid fibril disruptorin vitro as assayed by transmission electron microscopy (TEM)and dynamic light scattering (DLS) (Fig. 1b) [33]. These resultswere further confirmed by in vivo studies using two FAP micemodels, the transgenic mice expressing the human TTR V30Mand the transgenic TTR V30M in a HSF-1 null background, at differ-ent stages of TTR deposition. The results obtained revealed thatEGCG inhibited TTR deposition when treated in a precocious stage,when TTR starts to deposit (at 3–4 months of age) in the gastroin-testinal tract and in the PNS. Inhibition of TTR deposition, evalu-ated by immunohistochemistry and immunoblotting of micetissues after EGCG treatment for 6 weeks, was accompanied by adecrease of the biomarkers associated with the disease such asER-stress markers BiP and P-eIF2a, Fas and 3-nitrotyrosine(Fig. 1c) [34]. Furthermore, as pointed out by several authors, EGCGinhibitory effect on the amyloid formation cascade is not limited toearly intermediates of fibrillogenesis, since EGCG is also able toefficiently remodel mature fibrils made from a variety of amyloido-genic proteins into smaller, non-toxic unstructured protein aggre-gates [30,34]. Therefore, when similar in vivo studies wereperformed with aged TTR V30M mice that simultaneously presentnon-fibrillar and fibrillar TTR forms deposited in tissues, EGCG wasalso found to disaggregate amyloid deposits. This was particularlyevident by Congo red birefringence analysis of stomach sections(Fig. 1d). These results were further supported by substantial de-crease of remodeling extracellular matrix markers, MMP-9 andSAP, in EGCG treated mice, which was concomitant with amyloidclearance and indicative of inflammation reduction and matrixrecovery.

Preliminary results from similar studies in vivo with curcuminindicate that it also acts as an effective TTR aggregation modulator[33].

Very recently, based on the above referred in vitro and animalstudies with EGCG and also on studies on patients with AL amyloi-dosis [35,36] a preliminary study was conducted on a cohort of TTRamyloidosis patients, including systemic senile amyloidosis (SSA),where normal TTR deposits in the heart of old people, and variantTTR amyloidosis [37]. After one year of treatment with green tea orgreen tea extract, 86% of the treated patients presented a decreaseof cardiac wall thickness and mass as revealed mainly by cardiacmagnetic resonance imaging (CMRI) indicating that green tea,mainly EGCG, halted TTR deposition and increased TTR amyloiddeposits clearance. Though a randomized placebo-controlled studyis necessary, as proposed by the authors of the study [37], the re-sults point to a positive therapeutic effect of EGCG on TTR amyloi-dosis, in particular in cardiac forms. Moreover, it must be stressedthat EGCG possesses radical scavenging properties and it is rele-vant as an antioxidant agent [38]. Thus we should consider thatthese characteristics might potentiate its neuroprotective actionas an anti-amyloid compound.

Besides these natural modulators, synthetic compounds havebeen designed to interact with general particular small sequencesof amino acids in aggregated proteins and not with specific pro-teins [28]. Those synthetic compounds designated by ‘‘moleculartweezers’’ interfere with different kinds of interactions in aggre-gated proteins namely electrostatic, hydrogen or van der Wallsinteractions, and modulate the aggregation pathway, remodel theintermediary species formed and also disrupt pre-existent amyloidfibrils. Some molecular tweezers have already been tested in vivoin mice models for a-synuclein and Ab aggregation [39] and consti-tute also great candidates for TTR amyloidosis.

Detailed knowledge of the mechanism of action of all thesemodulators of aggregation will contribute to disclose unknown

Fig. 1. EGCG inhibits TTR aggregation and disrupts TTR fibrils. (a) DLS (dynamic light scattering) analysis of TTR (TTR Y78F, amyloidogenic variant) in the presence of EGCG(red line) and in its absence (control) (black line). EGCG inhibits TTR aggregation as evident by the very small percentage of TTR aggregates and by the increase of thepercentage of soluble TTR, as compared to control. (b) DLS analysis of pre-formed aggregates and fibrils of the amyloidogenic TTR variant TTR L55P (t = 0) and after incubationfor 4 days with EGCG. Aggregates and fibrils were disrupted and converted to a polydisperse size particle population (bottom graph). (c) Immunohistochemistry analysis oftissues from mice treated with EGCG and controls. Top panels: transgenic mice TTR V30M treated with EGCG present a decrease of approximately 50% of non-fibrillar TTRdeposition in the gastro-intestinal tract as compared to non-treated mice (control). Bottom panels, transgenic TTR V30M mice in HSF-1 deficient background treated withEGCG present a decrease of TTR deposition of approximately 66% in dorsal root ganglia (DRG). (d) Analysis of tissues from old (17 months of age) TTR V30M transgenic micetreated with EGCG and controls. Top panels: immunohistochemistry analysis shows an evident clearance of the TTR deposited in tissues (reduction of approximately 42% inTTR deposition) as compared to non-treated mice. Bottom panels: Congo red staining of stomach. Congo red positive deposits were decreased in about 40% of mice treatedwith EGCG.

Fig. 2. Combination therapies in the treatment of TTR amyloidosis. Lowering thelevels or stabilizing the native structure of TTR, inhibiting aggregation, disruptingTTR amyloid, and counteracting cellular toxicity are potential therapies for TTRamyloidosis. Combination therapies by administration of selected compounds thatact synergistically, such as doxycycline and TUDCA should be emphasized, as wellas compounds with different modes of action such as EGCG.

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steps of the processes involved in deposition and clearance of toxicintermediates in amyloid formation and to design more potent andefficient therapeutic agents for TTR amyloidosis.

4. Combination therapies

Another drug, which was tested in TTR transgenic animals andwas effective to counteract TTR aggregation, was tauroursodeoxy-cholic acid (TUDCA). TUDCA is a unique natural compound thatacts as a potent anti-apoptotic and anti-oxidant agent, reducingcytotoxicity in several neurodegenerative diseases [40]. Since oxi-dative stress, apoptosis, and inflammation are associated with TTRdeposition in FAP [41], the possible therapeutic application of TUD-CA in this disease was investigated. It was shown by semi-quanti-tative immunohistochemistry and western blotting thatadministration of TUDCA to a transgenic mouse model of FAP de-creased apoptotic and oxidative biomarkers namely the ER chaper-one BiP, the Fas death receptor, and oxidation products such as 3-nitrotyrosine. Most importantly, TUDCA treatment significantly re-duced TTR toxic aggregates as much as 75% [42].

We recently showed that combined cyclic drug administrationof doxycycline and TUDCA, resulted in significant reduction inTTR deposition and associated tissue markers in the TTR V30Mtransgenic mouse model [43]. The observed synergistic effect ofdoxycycline/TUDCA within the range of human-tolerable quanti-ties prompted their application in FAP, particularly in the early

stages of disease. For this purpose, a phase-II clinical study forTTR amyloidosis is underway in Italy (www.clinicaltrials.gov –transthyretin amyloidosis) with preliminary very promising re-sults. Thus, the neuropathy impairment score in 20 enrolled pa-tients in the lower limbs remained stable during the time of thetrial (1 year) and no clinical progression of cardiac involvement

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was observed [44]. These studies are currently extended to differ-ent TTR amyloidosis populations.

As summarized in Fig. 2, one of the main messages of this par-ticular study is that combination therapies, such as the combina-tion of doxycycline and TUDCA should be applied in thetreatment of TTR systemic amyloidosis. In this regard, pre-clinicalstudies in animal models are pivotal not only in the assessment ofpotential synergistic effects between individual drugs presentingdiverse modes of action as well as single drugs with differentmodes of action, such as EGCG.

Acknowledgements

Work of the authors is supported by Grants from the Portu-guese Fundação para a Ciência e Tecnologia (FCT), European Union(Framework Programme 6), Gulbenkian Foundation and the LionsClub.

References

[1] Bodin, K., Ellmerich, S., Kahan, M.C., Tennent, G.A., Loesch, A., Gilbertson, J.A.,Hutchinson, W.L., Mangione, P.P., Gallimore, J.R., Millar, D.J., Minogue, S.,Dhillon, A.P., Taylor, G.W., Bradwell, A.R., Petrie, A., Gillmore, J.D., Bellotti, V.,Botto, M., Hawkins, P.N. and Pepys, M.B. (2010) Antibodies to human serumamyloid P component eliminate visceral amyloid deposits. Nature 468 (7320),93–97.

[2] Terazaki, H., Ando, Y., Fernandes, R., Yamamura, K., Maeda, S. and Saraiva, M.J.(2006) Immunization in familial amyloidotic polyneuropathy: Counteractingdeposition by immunization with a Y78F TTR mutant. Lab. Invest. 86, 23–31.

[3] Sousa, M.M. and Saraiva, M.J. (2003) Neurodegeneration in familialamyloidotic polyneuropathy: from pathology to molecular signaling. Prog.Neurobiol. 71, 385–400.

[4] Alvarez, R., Borland, T., Chen, Q., Nilstein, S., Nguyen, T., Hinkle, G.,Kuchimanchi, S., Costigan, J., Ristoiu, V., Wang, G., Cole, G., Dorkin, R., Akinc,A., Nechev, L., Kosovrasti, V., Tchangov, L., Tracy, M., Jeffs, L., MaqLachlan, I.,Lutwyche, P., Martins, D., Costelha, S., Saraiva, M.J. and Sah, D.W.Y. (2010)ALN-TTR, an RNAi therapeutic for the treatment of transthyretin-mediatedamyloidosis. Amyloid. J. Protein Fold. Disord. 17 (Supplement), 51.

[5] Coelho, T., Maia, L.F., Silva, A.M., Cruz, M.W., Plante-Bordeneuve, V., Lozeron, P.,Suhr, O.B., Campistol, J.M., Conceição, I.M., Schmidt, H.H-J., Trigo, P., Kelly, J.W.,Labaudinière, Chan, J., Packman, J., Wilson, A., Grogan, D.R. (2011). Tafamidisfor transthyretin familial amyloid polyneuropathy. A randomized, controlledtrial, Neurology, in press.

[6] Santos, S.D., Magalhães, J. and Saraiva, M.J. (2008) Activation of the heat shockresponse in familial amyloidotic polyneuropathy. J. Neuropathol. Exp. Neurol.67, 449–455.

[7] Santos, S.D., Fernandes, R. and Saraiva, M.J. (2010) The heat shock responsemodulates transthyretin deposition in the peripheral and autonomic nervoussystems. Neurobiol. Aging 31 (2), 280–289.

[8] Poon, S., Easterbrook-Smith, S.B., Rybchyn, M.S., et al. (2000) Clusterin is anATP-independent chaperone with very broad substrate specificity thatstabilizes stressed proteins in a folding-competent state. Biochemistry 39(51), 15953–15960.

[9] Humphreys, D.T., Carver, J.A., Easterbrook-Smith, S.B., et al. (1999) Clusterinhas chaperone-like activity similar to that of small heat shock proteins. J. Biol.Chem. 274 (11), 6875–6881.

[10] Wilson, M.R. and Easterbrook-Smith, S.B. (2000) Clusterin is a secretedmammalian chaperone. Trends Biochem. Sci. 25 (3), 95–98.

[11] Michel, D., Chatelain, G., North, S., et al. (1997) Stress-induced transcription ofthe clusterin/apoJ gene. Biochem. J. 328 (Pt 1), 45–50.

[12] Yerbury, J.J., Poon, S., Meehan, S., et al. (2007) The extracellular chaperoneclusterin influences amyloid formation and toxicity by interacting withprefibrillar structures. FASEB J. 21 (10), 2312–2322.

[13] Matsubara, E., Soto, C., Governale, S., et al. (1996) Apolipoprotein J andAlzheimer’s amyloid beta solubility. Biochem. J. 316 (Pt 2), 671–679.

[14] Yerbury, J.J. and Wilson, M.R. (2010) Extracellular chaperones modulate theeffects of Alzheimer’s patient cerebrospinal fluid on Abeta(1–42) toxicity anduptake. Cell Stress Chaperones 15 (1), 115–121.

[15] Wyatt, A.R., Yerbury, J.J., Berghofer, P., Greguric, I., Katsifis, A., Dobson, C.M.and Wilson, M.R. (2011) Clusterin facilitates in vivo clearance of extracellularmisfolded proteins. Cell. Mol. Life Sci. 68 (23), 3919–3931.

[16] Magalhães, J. and Saraiva, M.J. (2011) Clusterin overexpression and putativerole in transthyretin deposition in familial amyloidotic polyneuropathy. J.Neurop. Exp. Neurol. 70, 1097–1106.

[17] Merlini, G., Ascari, E., Amboldi, N., Bellotti, V., Arbustini, E., Perfetti, V., Ferrari,M., Zorzoli, I., Marinone, M.G., Garini, P., et al. (1995) Interaction of theanthracycline 4’-iodo-4’-deoxydoxorubicin with amyloid fibrils: inhibition ofamyloidogenesis. Proc. Natl. Acad. Sci. U S A 92 (7), 2959–2963.

[18] Tagliavini, F., Forloni, G., Colombo, L., Rossi, G., Girola, L., Canciani, B.,Angeretti, N., Giampaolo, L., Peressini, E., Awan, T., De Gioia, L., Ragg, E.,

Bugiani, M. and Salmona, M. (2000) Tetracycline affects abnormal propertiesof synthetic PrP peptides and PrP(Sc) in vitro. J. Mol. Biol. 300 (5), 1309–1322.

[19] Cardoso, I., Merlini, G. and Saraiva, M.J. (2003) 40-Iodo-40-Deoxydoxorubicinand tetracyclines disrupt transthyretin amyloid fibrils in vitro producing non-cytotoxic species. Screening for TTR fibril disrupters. FASEB J. 17, 803–809.

[20] Cardoso, I., Brito, M. and Saraiva, M.J. (2008) Extracellular matrix markers fordisease progression and follow-up of therapies in familial amyloidpolyneuropathy V30M TTR-related. Dis. Markers 25, 37–47.

[21] Cardoso, I. and Saraiva, M.J. (2006) Doxycycline disrupts transthyretinamyloid: evidence from studies in a FAP transgenic mice model. FASEB J. 20,234–239.

[22] Giorgetti, S., Raimondi, S., Pagano, K., Relini, A., Bucciantini, M., Corazza, A.,Fogolari, F., Codutti, L., Salmona, M., Mangione, P., Colombo, L., De Luigi, A.,Porcari, R., Gliozzi, A., Stefani, M., Esposito, G., Bellotti, V. and Stoppini, M.(2011) Effect of tetracyclines on the dynamics of formation anddestructuration of beta2-microglobulin amyloid fibrils. J. Biol. Chem. 286(3), 2121–2131.

[23] Ward, J.E., Ren, R., Toraldo, G., Soohoo, P., Guan, J., O’Hara, C., Jasuja, R.,Trinkaus-Randall, V., Liao, R., Connors, L.H. and Seldin, D.C. (2011) Doxycyclinereduces fibril formation in a transgenic mouse model of AL amyloidosis. Blood118, 6610–6617.

[24] Quintas, A., Vaz, D.C., Cardoso, I., Saraiva, M. and Brito, R.M. (2001) Tetramerdissociation and monomer partial unfolding precedes protofibril formation inamyloidogenic transthyretin variants. J. Biol. Chem. 276, 27207–27213.

[25] Almeida, M.R., Gales, L., Damas, A.M., Cardoso, I. and Saraiva, M.J. (2005) Smalltransthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses.Curr. Drug Targets CNS Neurol. Disord. 5, 587–596.

[26] Johnson, S.M., Wiseman, R.L., Sekijima, Y., Green, N.S., Adamski-Werner, S.L.and Kelly, J.W. (2005) Native state kinetic stabilization as a strategy toameliorate protein misfolding diseases: a focus on the transthyretinamyloidoses. Acc. Chem. Res. 38 (12), 911–921.

[27] Ehrnhoefer, D.E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., et al. (2008)EGCG redirects amyloidogenic polypeptides into unstructured, off-pathwayoligomers. Nat. Struct. Mol. Biol. 15 (6), 558–566.

[28] Liu, T. and Bitan, G. (2012) Modulating self-assembly of amyloidogenicproteins as a therapeutic approach for neurodegenerative diseases: strategiesand mechanisms. ChemMedChem 5,7 (3), 359–374.

[29] Bieschke, J., Russ, J., Friedrich, R.P., Ehrnhoefer, D.E., Wobst, H., Neugebauer, K.and Wanker, E.E. (2010) EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity. Proc. Natl. Acad. Sci. U S A 107 (17),7710–7715.

[30] Ferreira, N., Cardoso, I., Domingues, M.R., Vitorino, R., Bastos, M., Bai, G.,Saraiva, M.J. and Almeida, M.R. (2009) Binding of epigallocatechin-3-gallate totransthyretin modulates its amyloidogenicity. FEBS Lett. 583, 3569–3576.

[31] Pullakhandam, R., Srinivas, P.N., Nair, M.K. and Reddy, G.B. (2009) Binding andstabilization of transthyretin by curcumin. Arch. Biochem. Biophys. 485 (2),115–119.

[32] Miyata, M., Sato, T., Kugimiya, M., Sho, M., Nakamura, T., Ikemizu, S., Chirifu,M., Mizuguchi, M., Nabeshima, Y., Suwa, Y., Morioka, H., Arimori, T., Suico,M.A., Shuto, T., Sako, Y., Momohara, M., Koga, T., Morino-Koga, S., Yamagata, Y.and Kai, H. (2010) The crystal structure of the green tea polyphenol (-)-epigallocatechin gallate-transthyretin complex reveals a novel binding sitedistinct from the thyroxine binding site. Biochemistry 49 (29), 6104–6114.

[33] Ferreira, N., Saraiva, M.J. and Almeida, M.R. (2011) Natural polyphenols inhibitdifferent steps of the process of transthyretin (TTR) amyloid fibril formation.FEBS Lett. 585 (15), 2424–2430.

[34] Ferreira, N., Saraiva, M.J. and Almeida, M.R. (2012) Epigallocatechin-3-gallateas a potential therapeutic drug for TTR-related amyloidosis: in vivo evidencefrom FAP mice models. PLoS One 7 (1), e29933.

[35] Hunstein, W. (2007) Epigallocathechin-3-gallate in AL amyloidosis: a newtherapeutic option? Blood 110 (6), 2216.

[36] Mereles, D., Buss, S.J., Hardt, S.E., Hunstein, W. and Katus, H.A. (2010) Effects ofthe main green tea polyphenol epigallocatechin-3-gallate on cardiacinvolvement in patients with AL amyloidosis. Clin. Res. Cardiol. 99 (8), 483–490.

[37] Kristen, A.V., Lehrke, S., Buss, S., Mereles, D., Steen, H., Ehlermann, P., Hardt, S.,Giannitsis, E., Schreiner, R., Haberkorn, U., Schnabel, P.A., Linke, R.P., Röcken,C., Wanker, E., Dengler, T.J., Altland, K. and Katus, H.A. (2012) Green tea haltsprogression of cardiac transthyretin amyloidosis: an observational report.Clin. Res. Cardiol. [Epub ahead of print].

[38] Mandel, S. and Youdim, M.B. (2004) Catechin polyphenols: neurodegenerationand neuroprotection in neurodegenerative diseases. Free Radic. Biol. Med. 37(3), 304–317.

[39] Sinha, S., Lopes, D.H., Du, Z., Pang, E.S., Shanmugam, A., Lomakin, A., Talbiersky,P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P.Y., Ehrmann, M., Benedek,G.B., Loo, J.A., Klärner, F.G., Schrader, T. and Wang, C. (2011) Lysine-specificmolecular tweezers are broad-spectrum inhibitors of assembly and toxicity ofamyloid proteins. J. Am. Chem. Soc. 26,133 (42), 16958–16969.

[40] Keene, C.D., Rodrigues, C.M., Eich, T., Chhabra, M.S., Steer, C.J. and Low, W.C.(2002) Tauroursodeoxycholic acid, a bile acid, is neuroprotective in atransgenic animal model of Huntington’s disease. Proc. Natl. Acad. Sci. U S A99 (16), 10671–10676.

[41] Sousa, M.M., Yan, S.D., Fernandes, R., Guimarães, A., Stern, D. and Saraiva,M.J.M. (2001) Familial amyloid polyneuropathy: RAGE-dependent triggeringof neuronal inflammatory and apoptotic pathways. J. Neurosci. 21, 7576–7586.

2896 M.R. Almeida, M.J. Saraiva / FEBS Letters 586 (2012) 2891–2896

[42] Macedo, B., Batista, A.R., Ferreira, N., Almeida, M.R. and Saraiva, M.J. (2008)Anti-apoptotic treatment reduces transthyretin deposition in a transgenicmouse model of Familial Amyloidotic Polyneuropathy. Biochem. Biophys. Acta1782 (9), 517–522.

[43] Cardoso, I., Martins, D., Ribeiro, T., Merlini, G. and Saraiva, M.J. (2010) Synergyof combined Doxycycline/TUDCA treatment in lowering Transthyretin

deposition and associated biomarkers: studies in FAP mouse models. J.Transl. Med. 8, 74–78.

[44] Obici, L., Cortese, A., Lozza, A., Lucchetti, J., Gobbi, M., Palladini, G., Perlini, S.,Saraiva, M.J. and Merlini, G. (2012) Doxycycline plus tauroursodeoxycholicacid for transthyretin amyloidosis: a phase II study. Amyloid 19 (Suppl. 1), 34–36.