FEBS Letters 586 (2012) 2891–2896
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Clearance of extracellular misfolded proteins in systemic amyloidosis: Experience with transthyretin Maria Rosário Almeida ⇑, Maria João Saraiva ⇑ IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal
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Article history: Received 2 July 2012 Revised 11 July 2012 Accepted 11 July 2012 Available online 20 July 2012 Edited by Miguel De la Rosa, Felix Wieland and Wilhelm Just Keywords: Transthyretin Amyloid Clusterin Doxycycline EGCG (epigallocatechin gallate)
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 cascades with toxic consequences represent unifying events in many of slowly progressive neurodegenerative disorders. Studies with small compounds or molecules, known to recognize and disrupt amyloidogenic structures, have proven efﬁcient in promoting clearance of protein aggregates in experimental models of systemic and localized forms of amyloidoses. Doxycycline and EGCG were efﬁcient in removing aggregates in pre-clinical studies in a transgenic mouse model for transthyretin (TTR) systemic amyloidosis and represent an opportunity to address mechanisms and key players in deposit removal. Extracellular chaperones, such as clusterin and metalloproteinases play an important role in this process. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Extracellular protein misfolding and aggregation occurring in systemic amyloidosis triggers inﬂammation, oxidative stress, matrix remodeling, the unfolded-protein-response and ER pathways that resemble in many aspects, including common molecular players and scenarios, to those described in local amyloidoses affecting for example the central nervous system (CNS), such as Alzheimer Disease. Thus, similarities and dissimilarities in toxicity found between the CNS and the periphery are very useful to pinpoint and guide us to the treatment of aging-associated neurodegenerative disorders. Understanding the two-way crosstalk between the extracellular milieu and the cell is a major trend in diseases related to protein aggregation. In particular, mechanisms involved in the clearance of protein aggregates, both extra and intracellular, are pivotal and need detailed analyses for the development of therapeutic strategies. Studies with small compounds or molecules, such as antibodies [1,2] known to recognize and disrupt amyloidogenic structures, have proven efﬁcient in removing and promoting clearance of protein aggregates in studies with experimental models of misfolding disorders. However, the mechanisms and key ⇑ Corresponding authors. Address: Molecular Neurobiology, IBMC, R. Campo Alegre 823, 4150, Porto, Portugal. Fax: +351 22 6099157. E-mail addresses: [email protected]
(M.R. Almeida), [email protected]
players in these processes are largely unknown. Extracellular molecular chaperones are capable to repair or target damaged proteins to degradation by binding to extracellular misfolded proteins and by promoting their disposal either by endocytosis for intracellular degradation or degradation by the extracellular matrix. Herein, we give examples of studies designed to approach the issue of clearance of extracellular deposition of transthyretin (TTR), a key protein associated with familial amyloidotic polyneuropathy (FAP). FAP is a fatal neurodegenerative disorder characterised by the extracellular deposition of aggregates and ﬁbrils of mutant forms of TTR, particularly in nerves and ganglia of the peripheral nervous system (PNS). The most common TTR mutation is a substitution of Valine for Methionine at position 30 (V30M TTR) that predisposes TTR to form aggregates and ﬁbrils. FAP initially presents with symptoms that are associated with sensory and autonomic nervous system (ANS) dysfunction. These include loss of pain and temperature sensation in the distal limbs, impotence, gastrointestinal disturbances, bladder dysfunction and postural hypotension. As FAP progresses, sensory deﬁciencies extend to more proximal regions of limbs and cardiac insufﬁciency and mal-absorption from the gut become common. In addition, motor involvement results in a progressive loss of reﬂexes and muscle wasting in later stages of FAP . Over 600 kindreds have been identiﬁed in Portugal which constitutes the largest world focus of the disease. TTR is a transport protein for thyroid hormones (namely, thyroxine – T4) and vitamin A (through a complex with retinol binding
0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.029
M.R. Almeida, M.J. Saraiva / FEBS Letters 586 (2012) 2891–2896
protein – RBP) and is predominantly synthesized in the liver. Because most circulating mutant TTR is synthesized in the liver, liver transplant has been used to treat FAP since 1990. However, universal and less invasive therapeutics are mandatory. Proposed pharmaco-therapeutic strategies for FAP are similar to the approaches taken for other systemic amyloidoses, such as lowering the levels or stabilizing the native structure of the plasma protein precursor, inhibiting aggregation, disrupting TTR amyloid by selective molecules, and counteracting cellular toxicity. Based on the knowledge gathered from the various in vitro and in vivo approaches to elucidate TTR aggregation and toxicity, different multicenter clinical trials are underway including RNAi silencing methodology to lower liver TTR expression – ALNTTR01 – (www.Alnylam.com); in a transgenic mice model for the human TTR V30M (described below), this compound showed durable suppression, as much as 90%, for both TTR mRNA and protein levels in plasma and reduction of deposition in extra-hepatic tissues . Phase I/II human clinical studies are underway to evaluate the safety and tolerability of this drug in V30M FAP patients. Stabilization of the native structure of plasma TTR is achieved by Tafamidis (now proprietary of Pﬁzer) as explained below; results from an 18-month phase II/III randomized double-blind trial showed improvement of body mass index and slight stabilization in nerve function . As referred to previously, once the clinical symptoms are triggered in FAP, disease evolution takes several years; thus, the initial studies have been extended to assess the usefulness of this drug in FAP treatment and also to investigate the mechanisms underlying the results found in non-responders. It is clear that more than a single approach to treat FAP effectively is mandatory; in this regard, the experience achieved so far with clearance based approaches is very promising and next described. 2. Modulation of the heat-shock response: extracellular chaperones The heat-shock response was investigated in FAP. Up-regulation of Hsp27 and Hsp70 expression related to the presence of extracellular TTR aggregates was documented in human FAP biopsies as compared to normal controls. TTR aggregates did not co-localize with Hsps suggesting that extracellular TTR tissue deposits are able to induce an intracellular stress response. Moreover, the heatshock transcription factor-1 (HSF-1) was up-regulated and localized to the nucleus . It was hypothesized that HSF-1 could be involved in FAP pathogenesis as a cell-defense mechanism against the presence of extracellular TTR deposits and that disruption of the heat-shock response would aggravate TTR deposition. A mouse model expressing the human TTR-V30M in an HSF-1-null background was characterized. The lack of HSF-1 expression lead to extensive and earlier non-ﬁbrillar TTR deposition, particularly in the gastrointestinal tract, evolving into ﬁbrillar material in distinct organs, including the peripheral and autonomic nervous systems; in contrast, V30M animals in the wild-type background do not display deposition in the PNS and ANS. As in the human disease, liver, brain, and spinal cord did not present deposition in this animal model. Furthermore, inﬂammatory stress and a reduction in unmyelinated nerve ﬁbers were observed, as in human patients, indicating that HSF-1-regulated genes are involved in FAP, modulating TTR tissue deposition . Clusterin, also known as apolipoprotein J, is an ubiquitous highly conserved secreted protein. Clusterin inhibits protein aggregation in an ATP independent manner after binding to misfolded proteins 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 stabilized state until Hsp70 can refold them. Clusterin is present in most physiologic ﬂuids and described to act as a molecular chaperone in the extracellular milieu . Several functions have been recognized for clusterin other than being a molecular chaperone; this protein has a role in: (i) control of cell–cell and cell–matrix interactions; (ii) apoptosis regulation; (iii) lipid transport and complement regulation. Clusterin gene expression is induced by a variety of factors including stress, cellular growth, differentiation and ageing. HSF-1 is also responsible for clusterin expression induction . In vitro data reported that clusterin is able to inhibit ﬁbril formation of several different proteins, including b-amyloid and a-synuclein [12,13]. Clusterin binds b-amyloid and forms a complex that is internalized for degradation by megalin (low-density lipoprotein receptor related protein 2 – LRP2); uptake of these complexes lowers amyloid toxicity . Facilitated clearance of extracellular misfolded proteins by clusterin was also documented in vivo . Thus, the available data support the notion that besides a role as a molecular chaperone, stabilizing proteins and inhibiting their aggregation, clusterin also has an important role in the clearance of extracellular aggregates through megalin mediated endocytosis. In the case of FAP, we have shown clusterin overexpression in tissues with TTR deposition of V30M transgenic mice with a full HSF-1 response; by double immunohistochemistry, clusterin co-localizes with both ﬁbrillar and non-ﬁbrillar TTR deposits in human nerve and is identiﬁed in TTR ﬁbrils extracted from human kidney. In vitro studies revealed that incubation of SH-SY5Y neuroblastoma cells with TTR oligomers leads to intracellular clusterin over-expression. Moreover, clusterin secretion was also signiﬁcantly increased in the medium of cells incubated with TTR oligomers and association of clusterin with TTR aggregates observed. Furthermore, clusterin modulates TTR aggregation, as clusterin deprivation in cellular models stimulates extracellular TTR aggregation . Further details of clusterin chaperone activity, scavenger properties and mechanisms of other extracellular chaperones acting in FAP 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 doxorubicin inhibits amyloid formation and promotes the reabsorption of amyloid deposits . Subsequently, tetracycline antibiotics were screened for these properties based on their structural homologies with the aglycone moiety of the anthracyclines. The ﬁrst demonstration 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 neuronal death and astrocyte proliferation induced by PrP peptides ‘‘in vitro’’ . It was found that doxycycline was particularly potent in disrupting TTR-like amyloid and that the generated assemblies did not display cytotoxic properties in caspase-3 activation assays . In vivo studies corroborated the in vitro ﬁndings; when doxycycline was administrated to 23–28 month old V30M TTR transgenic animals in the drinking water over a period of 3 months, immunohistochemistry revealed that Congo red positive material was only observed in the control, non-treated group. Moreover, immunohistochemistry for several markers associated with TTR amyloid deposition such as matrix metalloprotease 9 (MMP-9)  and serum amyloid P component (SAP) was performed. Significantly lower levels of MMP-9 were found in the treated animals when compared with the control group and mouse SAP was absent
M.R. Almeida, M.J. Saraiva / FEBS Letters 586 (2012) 2891–2896
in treated animals, being only observed in non-treated animals with congophilic deposits. These results indicated that doxycycline is capable of disrupting Congo red positive TTR amyloid deposits and decreases standard markers associated with ﬁbrillar deposition, being suggested as a potential drug in the treatment of amyloidosis . Doxycycline can also disassemble preformed b2 microglobulin amyloid ﬁbrils and at the same inhibits their formation . More recently, in vitro studies demonstrated that doxycycline directly disrupted the formation of recombinant light chains (LC) amyloid ﬁbrils. Furthermore, treatment of ex vivo LC amyloid ﬁbrils with doxycycline reduced the number of intact ﬁbrils and led to the formation of large disordered aggregates; in vivo studies in a CMV-k6 transgenic model for AL amyloidosis showed clearance of amyloid deposits in the stomach after doxycycline treatment . All together these evidences led to the approval by the European Medicines Agency of the application of doxycycline as an orphan drug in the treatment of FAP (EU/3/12/955) and b2 microglobulin systemic amyloidosis (EU/3/12/961). Detailed analyses on the mechanism of action of doxycycline on amyloid ﬁbrils await elucidation. 3.2. Clearance by modulation of the TTR aggregation pathway: EGCG It is widely accepted that the mechanism of TTR amyloid formation involves destabilization of the TTR tetramer, due to environmental or genetic conditions, namely mutations, leading to dissociation in monomers with an altered conformation that are more prone to aggregation and ﬁbril formation . Considering this mechanism, one of the most explored approaches in TTR amyloidosis was the search for compounds that speciﬁcally bind to TTR, in particular at T4 binding sites, stabilizing the tetramer and blocking the cascade of aggregation and ﬁbril formation. Thus a wide variety of small compounds presenting structural similarities with thyroxine, the natural TTR ligand, have been proposed and tested as TTR amyloid inhibitors [25,26]. Those compounds are very speciﬁc in their binding at the TTR–T4 binding sites establishing interactions with different TTR monomers and promoting TTR stabilization; such is the case of Tafamidis, above referred. Recently, several compounds have been referred to act as nonspeciﬁc aggregation inhibitors of different proteins involved in misfolding diseases as is the case of Ab and a-synuclein . The non-speciﬁc inhibitors hinder formation of oligomeric and/or preﬁbrillar species, probably recognizing common structural or conformational properties and through a still unknown mechanism of action may also disrupt pre-existing amyloid ﬁbrils . This concept of general modulators of the aggregation pathway is gaining increasing relevance. Among those modulators, particular interest has been raised by natural polyphenols such as epigallocatechin gallate (EGCG), from green tea, and curcumin . Based on this concept of general modulators of amyloid formation, EGCG and curcumin have also been tested in TTR-related amyloidosis. Studies in vitro and in a cell culture system using different amyloidogenic TTR variants demonstrated that EGCG and curcumin bind to TTR, increase its tetrameric conformational stability, modulate the intermediary species formed and inhibit TTR aggregation [30,31] (Fig. 1a). However, contrary to other polyphenols, EGCG does not bind at the T4 binding channel on TTR . Crystal structure of the TTR-EGCG complex demonstrated that there are 3 different binding sites for EGCG on TTR . One of these sites is located in a region of the TTR tetramer that allows contact of EGCG with both TTR dimers, indicating that the interaction of EGCG with TTR at this binding site contributes to the TTR tetramer stabilization. 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 oligomerization of TTR tetramers, leading to the formation of nontoxic, off-pathway aggregates [30,32]. EGCG was also demonstrated to act as amyloid ﬁbril disruptor in vitro as assayed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) (Fig. 1b) . These results were further conﬁrmed by in vivo studies using two FAP mice models, the transgenic mice expressing the human TTR V30M and the transgenic TTR V30M in a HSF-1 null background, at different stages of TTR deposition. The results obtained revealed that EGCG inhibited TTR deposition when treated in a precocious stage, when TTR starts to deposit (at 3–4 months of age) in the gastrointestinal tract and in the PNS. Inhibition of TTR deposition, evaluated by immunohistochemistry and immunoblotting of mice tissues after EGCG treatment for 6 weeks, was accompanied by a decrease of the biomarkers associated with the disease such as ER-stress markers BiP and P-eIF2a, Fas and 3-nitrotyrosine (Fig. 1c) . Furthermore, as pointed out by several authors, EGCG inhibitory effect on the amyloid formation cascade is not limited to early intermediates of ﬁbrillogenesis, since EGCG is also able to efﬁciently remodel mature ﬁbrils made from a variety of amyloidogenic proteins into smaller, non-toxic unstructured protein aggregates [30,34]. Therefore, when similar in vivo studies were performed with aged TTR V30M mice that simultaneously present non-ﬁbrillar and ﬁbrillar TTR forms deposited in tissues, EGCG was also found to disaggregate amyloid deposits. This was particularly evident by Congo red birefringence analysis of stomach sections (Fig. 1d). These results were further supported by substantial decrease of remodeling extracellular matrix markers, MMP-9 and SAP, in EGCG treated mice, which was concomitant with amyloid clearance and indicative of inﬂammation reduction and matrix recovery. Preliminary results from similar studies in vivo with curcumin indicate that it also acts as an effective TTR aggregation modulator . Very recently, based on the above referred in vitro and animal studies with EGCG and also on studies on patients with AL amyloidosis [35,36] a preliminary study was conducted on a cohort of TTR amyloidosis patients, including systemic senile amyloidosis (SSA), where normal TTR deposits in the heart of old people, and variant TTR amyloidosis . After one year of treatment with green tea or green tea extract, 86% of the treated patients presented a decrease of cardiac wall thickness and mass as revealed mainly by cardiac magnetic resonance imaging (CMRI) indicating that green tea, mainly EGCG, halted TTR deposition and increased TTR amyloid deposits clearance. Though a randomized placebo-controlled study is necessary, as proposed by the authors of the study , the results point to a positive therapeutic effect of EGCG on TTR amyloidosis, in particular in cardiac forms. Moreover, it must be stressed that EGCG possesses radical scavenging properties and it is relevant as an antioxidant agent . Thus we should consider that these characteristics might potentiate its neuroprotective action as an anti-amyloid compound. Besides these natural modulators, synthetic compounds have been designed to interact with general particular small sequences of amino acids in aggregated proteins and not with speciﬁc proteins . Those synthetic compounds designated by ‘‘molecular tweezers’’ interfere with different kinds of interactions in aggregated proteins namely electrostatic, hydrogen or van der Walls interactions, and modulate the aggregation pathway, remodel the intermediary species formed and also disrupt pre-existent amyloid ﬁbrils. Some molecular tweezers have already been tested in vivo in mice models for a-synuclein and Ab aggregation  and constitute also great candidates for TTR amyloidosis. Detailed knowledge of the mechanism of action of all these modulators of aggregation will contribute to disclose unknown
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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 the percentage 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 incubation for 4 days with EGCG. Aggregates and ﬁbrils were disrupted and converted to a polydisperse size particle population (bottom graph). (c) Immunohistochemistry analysis of tissues 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 TTR deposition in the gastro-intestinal tract as compared to non-treated mice (control). Bottom panels, transgenic TTR V30M mice in HSF-1 deﬁcient background treated with EGCG 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 mice treated with EGCG and controls. Top panels: immunohistochemistry analysis shows an evident clearance of the TTR deposited in tissues (reduction of approximately 42% in TTR 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 treated with EGCG.
steps of the processes involved in deposition and clearance of toxic intermediates in amyloid formation and to design more potent and efﬁcient therapeutic agents for TTR amyloidosis. 4. Combination therapies Another drug, which was tested in TTR transgenic animals and was effective to counteract TTR aggregation, was tauroursodeoxycholic acid (TUDCA). TUDCA is a unique natural compound that acts as a potent anti-apoptotic and anti-oxidant agent, reducing cytotoxicity in several neurodegenerative diseases . Since oxidative stress, apoptosis, and inﬂammation are associated with TTR deposition in FAP , the possible therapeutic application of TUDCA in this disease was investigated. It was shown by semi-quantitative immunohistochemistry and western blotting that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers namely the ER chaperone BiP, the Fas death receptor, and oxidation products such as 3nitrotyrosine. Most importantly, TUDCA treatment signiﬁcantly reduced TTR toxic aggregates as much as 75% . We recently showed that combined cyclic drug administration of doxycycline and TUDCA, resulted in signiﬁcant reduction in TTR deposition and associated tissue markers in the TTR V30M transgenic mouse model . The observed synergistic effect of doxycycline/TUDCA within the range of human-tolerable quantities prompted their application in FAP, particularly in the early
stages of disease. For this purpose, a phase-II clinical study for TTR amyloidosis is underway in Italy (www.clinicaltrials.gov – transthyretin amyloidosis) with preliminary very promising results. Thus, the neuropathy impairment score in 20 enrolled patients in the lower limbs remained stable during the time of the trial (1 year) and no clinical progression of cardiac involvement
Fig. 2. Combination therapies in the treatment of TTR amyloidosis. Lowering the levels or stabilizing the native structure of TTR, inhibiting aggregation, disrupting TTR amyloid, and counteracting cellular toxicity are potential therapies for TTR amyloidosis. Combination therapies by administration of selected compounds that act synergistically, such as doxycycline and TUDCA should be emphasized, as well as compounds with different modes of action such as EGCG.
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was observed . These studies are currently extended to different TTR amyloidosis populations. As summarized in Fig. 2, one of the main messages of this particular study is that combination therapies, such as the combination of doxycycline and TUDCA should be applied in the treatment of TTR systemic amyloidosis. In this regard, pre-clinical studies in animal models are pivotal not only in the assessment of potential synergistic effects between individual drugs presenting diverse modes of action as well as single drugs with different modes of action, such as EGCG.
Acknowledgements Work of the authors is supported by Grants from the Portuguese Fundação para a Ciência e Tecnologia (FCT), European Union (Framework Programme 6), Gulbenkian Foundation and the Lions Club.
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