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Transthyretin (TTR) is a plasma- and cerebrospinal fluid-circulating protein. Besides the primordially attributed systemic role as a transporter molecule of thyroxine (T 4 ) and retinol (through the binding to retinol-binding protein [RBP]), TTR has been recognized as a protein with important functions in several aspects of the nervous system physiology. TTR has been shown to play an important role in behavior, cognition, amidated neuropeptide processing and nerve regeneration. Furthermore, it has been proposed that TTR is neuroprotective in Alzheimer's disease and cerebral ischemia. Mutations in TTR are a well-known cause of familial amyloidotic polyneuropathy, an autosomal dominant neurodegenerative disorder characterized by systemic deposition of TTR amyloid fibrils, particularly in the peripheral nervous system. The purpose of this review is to highlight the roles of TTR in the nervous system, beyond its systemic role as a transporter molecule of T 4 and RBP-retinol.

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

Therapies for Alzheimer disease that reduce the production of pathogenic amyloid β (Aβ) peptides have been associated with a range of unwanted effects. For this reason, alternative strategies that promote the clearance of the peptide by preventing its aggregation and deposition in the brain have been favored. In this context we have studied doxycycline, a member of the tetracycline family of antibiotics that has shown neuroprotective effects in a number of models of neurodegenerative disease. We investigated the neuroprotective potential of doxycycline in a Drosophila model of Aβ toxicity and sought to correlate any effects with the aggregation state of the peptide. We found that administration of doxycycline to Aβ42-expressing flies did not improve their lifespan but was able to slow the progression of their locomotor deficits. We also measured the rough eye phenotype of transgenic flies expressing the E22G variant of Aβ42 and showed that doxycycline administration partially rescued the toxicity of Aβ in the developing eye. We correlated these in vivo effects with in vitro observations using transmission electron microscopy, dynamic light scattering, and thioflavin T binding. We found that doxycycline prevents Aβ fibrillization and favors the generation of smaller, non-amyloid structures that were non-toxic as determined by the lack of caspase 3 activation in a neuroblastoma cell line. Our confirmation that doxycycline can prevent amyloid β toxicity both in vitro and in vivo supports its therapeutic potential in AD.

Background: Allergic disorders, such as seasonal rhinitis and asthma, are increasing causes of morbidity worldwide and often result from exposure to airborne pollen. Pollen allergy has a remarkable clinical impact all over Europe. In fact, epidemiological longitudinal studies confirm that pollen species usually considered with low allergenic potential became more recently responsible for intense allergic reactions. In this study, we aimed to characterize major pollen proteolytic activity and evaluate its contribution to the immunologic and inflammatory response to airborne allergens., Methods: Proteolytic activity in four pollen diffusates with distinct allergenicity, Olea europaea, Dactylis glomerata, Cupressus sempervirens and Pinus sylvestris, was evaluated through several enzymatic assays. The action of pollen proteases on the paracellular integrity of Calu-3, grown at the air-liquid interphase, was evaluated through a transepithelial permeability assay. Immunoblot and immunofluorescence experiments were performed to analyse the disruption of intercellular complexes. Degradation of bioactive peptides by pollen crude extracts was assessed by mass spectrometry., Results: All pollen diffusates were shown to have high molecular weight proteases with serine and/or aminopeptidase activity. These proteases increased Calu-3 transepithelial permeability through disruption of transmembrane adhesion proteins: occludin, claudin-1 and E-cadherin. Moreover, they were able to degrade airway bioactive peptides and were not blocked by endogenous protease inhibitors., Conclusion: Pollen grains with distinct allergenic abilities release proteases that might be involved in the sensitization to a range of airborne allergens by facilitating allergen delivery across the epithelium and also contribute directly to the inflammation characteristic of allergic diseases., (© 2011 John Wiley & Sons A/S.)

The importance of establishing the ecological quality of estuarine systems has been widely acknowledged and led to the development of several fish community-based multimetric indices. Nevertheless, a question rose about the accuracy of these tools when natural disturbance is acting upon the organization of the systems' communities. Four multimetric indices were used to examine their ability to differentiate the ecological status of five small estuarine systems (southern Portugal), and also to test if they reflected the level of anthropogenic pressures. Fish assemblages from Mira, Odeceixe and Aljezur (in the Southwest coast), Gilão and Bensafrim (in the South coast) estuaries were sampled seasonally for one year, and anthropogenic sources of pressure were identified and quantified. We found that although the applied indices provided information on ecological condition differentiation among systems, they are unable to explain different classes of ecological status in systems with equivalent pressure levels., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder affecting tens of millions of people worldwide, with women being at greater risk of developing the disease. A growing body of evidence suggests transthyretin (TTR) as an important modulator of AD pathogenesis. Aiming at providing further insight into the potential neuroprotective role of TTR and gender differences in AD, we crossed transgenic AβPPswe/PS1A246E mice with TTR-null mice and investigated both male and female AβPPswe/PS1A246E/TTR+/+, AβPPswe/PS1A246E/TTR+/-, and AβPPswe/PS1A246E/TTR-/- animals for brain amyloid-β (Aβ) levels and deposition. The levels of circulating TTR between non-transgenic and AD mice were evaluated. Decreased levels of circulating TTR in AD mice as compared to non-transgenic littermates were observed in early stages of AD-like neuropathology, but not at later stages where an opposite relationship was found. Elevated brain levels of Aβ42 were observed in AβPPswe/PS1A246E/TTR+/- female mice as compared to AβPPswe/PS1A246E/TTR+/+ female littermates; no significant differences were found among males of different TTR genotypes. We subsequently quantified the brain levels of testosterone and 17β-estradiol in these animals and verified that AβPPswe/PS1A246E/TTR+/- female mice present reduced brain levels of both hormones as compared to AβPPswe/PS1A246E/TTR+/+ females; no significant differences were detected among males of different TTR genotypes. Our results provide evidence for a gender-associated modulation of brain Aβ levels and brain sex steroid hormones by TTR, and suggest that reduced levels of brain testosterone and 17β-estradiol in female mice with TTR genetic reduction might underlie their increased AD-like neuropathology.

The amyloid-β peptide (Aβ) plays a central role in the mechanism of Alzheimer's disease, being the main constituent of the plaque deposits found in AD brains. Aβ amyloid formation and deposition are due to a conformational switching to a β-enriched secondary structure. Our strategy to inhibit Aβ aggregation involves the re-conversion of Aβ conformation by adsorption to nanoparticles. NPs were synthesized by sulfonation and sulfation of polystyrene, leading to microgels and latexes. Both polymeric nanostructures affect the conformation of Aβ inducing an unordered state. Oligomerization was delayed and cytotoxicity reduced. The proper balance between hydrophilic moieties and hydrophobic chains seems to be an essential feature of effective NPs.

The amyloid-beta peptide (Abeta) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. Soluble oligomers that precede fibril formation have been proposed as the main neurotoxic species that contributes to neurodegeneration and dementia. We hypothesize that oligomerization and cytotoxicity can be repressed by nanoparticles (NPs) that induce conformational changes in Abeta42. We show here that fluorinated and hydrogenated NPs with different abilities to change Abeta42 conformation influence oligomerization as assessed by atomic force microscopy, immunoblot and SDS-PAGE. Fluorinated NPs, which promote an increase in alpha-helical content, exert an antioligomeric effect, whereas hydrogenated analogues do not and lead to aggregation. Cytotoxicity assays confirmed our hypothesis by indicating that the conformational conversion of Abeta42 into an alpha-helical-enriched secondary structure also has antiapoptotic activity, thereby increasing the viability of cells treated with oligomeric species.

Transthyretin (TTR), an amyloid-beta (Abeta) scavenger protein, and metallothioneins 2 and 3 (MT2 and MT3), low molecular weight metal-binding proteins, have recognized impacts in Abeta metabolism. Because TTR binds MT2, an ubiquitous isoform of the MTs, we investigated whether it also interacts with MT3, an isoform of the MTs predominantly expressed in the brain, and studied the role of MT2 and MT3 in human TTR-Abeta binding. The TTR-MT3 interaction was characterized by yeast two-hybrid assays, saturation-binding assays, co-immunolocalization and co-immunoprecipitation. The effect of MT2 and MT3 on TTR-Abeta binding was assessed by competition-binding assays. The results obtained clearly demonstrate that TTR interacts with MT3 with a K(d) of 373.7 +/- 60.2 nm. Competition-binding assays demonstrated that MT2 diminishes TTR-Abeta binding, whereas MT3 has the opposite effect. In addition to identifying a novel ligand for TTR that improves human TTR-Abeta binding, the present study highlights the need to clarify whether the effects of MT2 and MT3 in human TTR-Abeta binding observed in vitro have a relevant impact on Abeta deposition in animal models of Alzheimer's disease.

More than 100 transthyretin (TTR) variants are associated with hereditary amyloidosis. Approaches for TTR amyloidosis that interfere with any step of the cascade of events leading to fibril formation have therapeutic potential. In this study we tested (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, as an inhibitor of TTR amyloid formation. We demonstrate that EGCG binds to TTR "in vitro" and "ex vivo" and that EGCG inhibits TTR aggregation "in vitro" and in a cell culture system. These findings together with the low toxicity of the compound raise the possibility of using EGCG in a therapeutic approach for familial amyloidotic polyneuropathy, the most frequent form of hereditary TTR amyloidosis.

The sequence LPFFD (iAbeta(5)) prevents amyloid-beta peptide (Abeta) fibrillogenesis and neurotoxicity, hallmarks of Alzheimer's disease (AD), as previously demonstrated. In this study iAbeta(5) was covalently linked to poly(ethylene glycol) (PEG) and the activity of conjugates was assessed and compared to the activity of the peptide alone by in vitro studies. The conjugates were characterized by MALDI-TOF. Competition binding assays established that conjugates retained the ability to bind Abeta with similar strength as iAbeta(5). Transmission electron microscopy analysis showed that iAbeta(5) conjugates inhibited amyloid fibril formation, which is in agreement with binding properties observed for the conjugates towards Abeta. The conjugates were also able to prevent amyloid-induced cell death, as evaluated by activation of caspase 3. These results demonstrated that the biological activity of iAbeta(5) is not affected by the pegylation process.

We report the amyloid-like properties of Escherichia coli transthyretin-like protein (TLP). TLP is 32% homologous to human transthyretin (hTTR), and is also tetrameric. In contrast to hTTR, TLP does not bind thyroxine. TLP orthologues are found in several prokaryotes, lower eukaryotes and vertebrates. TLP carries a signal peptide that targets the protein to the periplasmic space. We found that TLP and hTTR tetramers dissociate into monomers under similar conditions, although TLP monomers have different association properties. Like hTTR, TLP forms aggregates, small fibrillar structures of 8 nm width, and annular structures of 8 nm diameter which present amyloid-like properties and are toxic to cells.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

It has been suggested that transthyretin (TTR) is involved in preventing A-Beta fibrillization in Alzheimer's disease (AD). Here, we characterized the TTR/A-Beta interaction by competition binding assays. TTR binds to different A-Beta peptide species: soluble (Kd, 28 nM), oligomers and fibrils; diverse TTR variants bind differentially to A-Beta. Transmission electron microscopy (TEM) analysis demonstrated that TTR is capable of interfering with A-Beta fibrillization by both inhibiting and disrupting fibril formation. Co-incubation of the two molecules resulted in the abolishment of A-Beta toxicity. Our results confirmed TTR as an A-Beta ligand and indicated the inhibition/disruption of A-Beta fibrils as a possible mechanism underlying the protective role of TTR in AD.

Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. Several extracellular matrix proteins have been found elevated in tissues from FAP patients, namely metalloproteinase-9 (MMP-9), neutrophil gelatinase associated lipocalin (NGAL) and biglycan. In this work we assessed the levels of MMP-9, tissue inhibitor of metalloproteinase 1 (TIMP-1), NGAL, biglycan and chondroitin sulphate (CSPG) in an FAP V30M TTR-related transgenic mouse model at different stages of TTR deposition and after two different treatment approaches to remove fibrillar deposits. Immunohistochemistry or RT-PCR analysis showed that biglycan was already increased in animals presenting TTR deposited in a non-fibrillar state, whereas MMP-9, TIMP-1, NGAL and CSPG were elevated only in mice with TTR amyloid deposits. Mice treated with doxycycline, a TTR fibril disrupter, presented lower levels of MMP-9, TIMP-1 and NGAL, suggestive of matrix recovery. Mice immunized with TTR Y78F to remove TTR deposition showed significantly lower levels of all the five tested markers, suggesting removal of fibrillar and non-fibrillar deposits. Cellular studies using oligomeric TTR showed induction of MMP-9 when compared to soluble TTR, large aggregates or fibrils. Furthermore, this induction was neutralized by an anti-receptor for advanced glycation end products (RAGE) antibody, indicating RAGE engagement in this process. Further studies in a larger number of tissue samples will indicate the application of these ECM markers in parallel with Congo Red staining in tissue characterization of pre-clinical and clinical stages in FAP and other amyloidoses.

Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins.

The ubiquitin-proteasome system (UPS) has been associated with neurodegenerative disorders of intracellular protein aggregation. We have studied the UPS in familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder caused by extracellular deposition of mutant transthyretin (TTR). The studies were conducted in TTR-synthesizing and non-synthesizing tissues from affected individuals, in transgenic mouse models for FAP, and in neuronal or Schwannoma cell lines cultured with TTR aggregates. In human FAP tissues presenting extracellular TTR aggregates, ubiquitin-protein conjugates were up-regulated, the proteasome levels were decreased and parkin and alpha-synuclein expression were both decreased. A similar response was detected in mouse models for TTR V30M or L55P. On the other hand, the liver, which normally synthesizes variant TTR V30M, did not show this response. Furthermore, transgenic mice immunized to decrease TTR deposition showed a significant reduction in ubiquitin levels and an increase in parkin and alpha-synuclein levels in comparison to control mice. Studies performed in cell lines with aggregates in the medium resulted in increased ubiquitin and decreased parkin levels. The overall results are indicative of TTR deposition as an external stimulus to an intracellular UPS response in FAP.

Transthyretin (TTR), a carrier protein for thyroxine and retinol in plasma and cerebrospinal fluid (CSF), has been shown to bind the amyloid beta peptide. Accordingly, TTR has been suggested to protect against amyloid beta deposition, a key pathological feature in Alzheimer's disease (AD). Supporting this view are the reduced TTR levels found in CSF of patients with AD, as well as reports of altered TTR expression in the cortex and hippocampus of AD rodent models. Importantly, early characterization of TTR distribution revealed the choroid plexus as the site of TTR synthesis within the brain. To resolve this controversy we used precise laser microdissection technology to assay for TTR mRNA expression. Our results clearly demonstrate that TTR is not produced in the brain parenchyma of wild-type mice nor in two different transgenic mouse models of AD, suggesting that contamination by choroid plexus contributed to the recent results indicating TTR production in various brain regions. The relevance of TTR to AD should now take into consideration TTR production by the choroid plexus and its ability, in the CSF, to sequester the amyloid beta peptide.

Familial amyloidotic polyneuropathy is a neurodegenerative disorder characterized by systemic extracellular deposition of transthyretin (TTR) amyloid fibrils. The latter have been proposed to trigger neurodegeneration through engagement of the receptor for advanced glycation end products (RAGE). Here we show that TTR interaction with RAGE is conserved across mouse and human species and is not dependent on RAGE glycosylation. Moreover, strand D of TTR structure seems important for the TTR-RAGE interaction as well as a motif in RAGE (residues 102-118) located within the V-domain; this motif suppressed TTR aggregate-induced cytotoxicity in cell culture.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by the extracellular deposition of transthyretin (TTR), especially in the PNS. Given the invasiveness of nerve biopsy, salivary glands (SG) from FAP patients were used previously in microarray analysis; mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) was down-regulated in FAP. Results were validated by RT-PCR and immunohistochemistry both in SG and in nerve biopsies of different stages of disease progression. MKP-3 was also down-regulated in FAP SG biopsies. Given the relationship between MKPs and MAPKs, the latter were investigated. Only extracellular signal-regulated kinases 1/2 (ERK1/2) displayed increased activation in FAP SG and nerves. ERK1/2 kinase (MEK1/2) activation was also up-regulated in FAP nerves. In addition, an FAP transgenic mouse model revealed increased ERK1/2 activation in peripheral nerve affected with TTR deposition when compared to control animals. Cultured rat Schwannoma cell line treatment with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by the receptor for advanced glycation end-products (RAGE). Moreover, caspase-3 activation triggered by TTR aggregates was abrogated by U0126, a MEK1/2 inhibitor, indicating that ERK1/2 activation is essential for TTR aggregates-induced cytotoxicity. Taken together, these data suggest that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration.

Familial amyloidotic polyneuropathy is an autosomal dominant disorder mainly characterized by the extracellular deposition of transthyretin, with special involvement of the peripheral nerve. Several animal models have been generated, including transgenic mice carrying the most prevalent TTR mutation (TTR Val30Met). TTR-Val30Met mice without endogenous TTR (TTR-Val30Met X TTR-KO) were previously analyzed in our laboratory and approximately 60% of the animals over 1 year of age were found to have deposition as amyloid, i.e., with Congo red (CR) -positive material, constituting a good tool to investigate the effect of drugs on TTR deposition and fibrillogenesis. We recently showed that the drug doxycycline acts in vitro as a TTR fibril disrupter. In the present work we assessed the activity of this drug in vivo in the TTR-Met30Val X TTR-KO mice. Doxycycline was administrated in the drinking water to 23- to 28-month-old mice over a period of 3 months. Immunohistochemistry analyses revealed no differences in nonfibrillar TTR deposition between treated (n=11) and untreated mice (n=11). However, CR-positive material was observed only in the control group (untreated) whereas none of the animals treated with doxycycline was CR-positive. Immunohistochemistry for several markers associated with amyloid, such as matrix metalloproteinase-9 (MMP-9) and serum amyloid P component (SAP), was performed. MMP-9 was altered with significantly lower levels in treated animals compared with the control group. Mouse SAP was absent in treated animals, being observed only in untreated animals presenting TTR congophilic deposits. These results indicate that doxycycline is capable of disrupting TTR CR-positive amyloid deposits and decreases standard markers associated with fibrillar deposition, being a potential drug in the treatment of amyloidosis.

In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. More than eighty TTR variants are known, most of them being pathogenic. The mechanism of TTR fibril formation is still not completely elucidated. However it is widely accepted that the amino acid substitutions in the TTR variants contribute to a destabilizing effect on the TTR tetramer molecule, which in particular conditions dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Till now the only efficient therapy available is liver transplant when performed in an early phase of the onset of the disease symptoms. Since this is a very invasive therapy alternatives are desirable. In that sense, several compounds have been proposed to impair amyloid formation or disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4'-iodo-4'-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.

In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

The molecular pathology underlying transthyretin (TTR)-related amyloidosis is largely unknown. It is possible that a common factor in the amyloidogenesis process exists among the different forms; this common factor can involve changes produced by mutations in the three-dimensional structure of TTR, rendering it prone to deposition as amyloid. This amyloidogenic potential, together with other yet unidentified factors, contribute to amyloid deposition. The factors that trigger fibril formation and/or neurodegeneration in TTR-related amyloidosis present central questions for which there are still no available clues. We recently showed in vitro that TTR fibrils trigger NF-kappaB activation, and subsequent studies identified some inflammatory and apoptotic pathways opening perspectives to understand the neurodegeneration process in familial amyloidotic polyneuropathy (FAP). It is current opinion that the modified TTR represents an amyloidogenic intermediate, which integrates the fibril structure; analyses of FAP fibrils have proved that TTR in the fibrils maintains a beta-conformation and have suggested that the TTR monomer is the building block in fibrils. This concept has been questioned recently by investigators, and only future studies on native and synthetic TTR fibrils using high-resolution structural techniques will further elucidate fibril structure and the aggregation pathway. Modulators responsible for phenotypic diversity can be addressed by mice transgenic for different human TTR mutations. Different lines are now available; incomplete penetrance and environmental influence on the deposition of mutant TTR has been observed. Therefore, these animals constitute important tools to address modulators of phenotypic expression and pathophysiological consequences of amyloid deposition at cellular/molecular levels. They are pivotal for testing potential drugs for TTR amyloidosis as well.

Transthyretin Leu55Pro is one of the most aggressive mutations in familial amyloidotic polyneuropathy, an autosomal dominant disorder characterized by extracellular deposition of fibrillar amyloid protein. This variant has the ability to form fibrils in vitro under physiological conditions (PBS, pH 7.4). We studied by transmission electron microscopy the effect of the drug 4'-iodo-4'-deoxydoxorubicin (I-DOX) on the in vitro assembly of TTR Leu55Pro fibrils by following fibril growth over a 15 day period. Our results showed that I-DOX at a concentration of 10-5 M/100 microg fibrils does not inhibit fibril formation in up to 10 days since fibrils identical to the ones present in the untreated sample were observed. However, after 15 days of treatment, only round particles, resembling soluble native TTR, were observed. We also tested the ability of tetracyclines and nitrophenols to interfere with amyloid fibril formation for 17 days; the group of compounds tested showed fibril disruption activity to different extents: doxycycline and 2,4-dinitrophenol resulted in complete disaggregation of fibrils. The species generated upon I-DOX and tetracyclines treatments were nontoxic, as revealed by the lack of significant caspase-3 activation on a Schwannoma cell line, making them potential therapeutic drugs in TTR-related and other amyloidosis.

Transthyretin (TTR) amyloid fibrils are the main component of the amyloid deposits occurring in Familial Amyloidotic Polyneuropathy patients. This is 1 of 20 human proteins leading to protein aggregation disorders such as Alzheimer's and Creutzfeldt-Jakob diseases. The structural details concerning the association of the protein molecules are essential for a better understanding of the disease and consequently the design of new strategies for diagnosis and therapeutics. Disulfide bonds are frequently considered essential for the stability of protein aggregates and since in the TTR monomers there is one cysteine residue, it is important to determine unambiguously the redox state of sulfur present in the fibrils. In this work we used x-ray spectroscopy to further characterize TTR amyloid fibrils. The sulfur K-edge absorption spectra for the wild type and some amyloidogenic TTR variants in the soluble and fibrillar forms were analyzed. Whereas in the soluble proteins the thiol group from cysteine (R-SH) and the thioether group from methionine (R-S-CH(3)) are the most abundant forms, in the TTR fibrils there is a significant oxidation of sulfur to the sulfonate form in the cysteine residue and a partial oxidation of sulfur to sulfoxide in the methionine residues. Further interpretation of the data reveals that there are no disulfide bridges in the fibrillar samples and suggest conformational changes in the TTR molecule, namely in strand A and/or in its vicinity, upon fibril formation.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. No systematic immunohistochemical data exists relating TTR deposition with FAP progression. We assessed nerves from FAP patients in different stages of disease progression (FAP 0 to FAP 3) for TTR deposition by immunohistochemistry, and for the presence of amyloid fibrils by Congo Red staining. The nature of the deposited material was further studied by electron microscopy. We observed that early in FAP (FAP 0), TTR is already deposited in an aggregated nonfibrillar form, negative by Congo Red staining. This suggested that in vivo, preamyloidogenic forms of TTR exist in the nerve, in a stage before fibril formation. Cytotoxicity of nonfibrillar TTR was assessed in nerves of different FAP stages by immunohistochemistry for macrophage colony-stimulating factor. FAP 0 patients already presented increased axonal expression of macrophage colony-stimulating factor that was maintained in all other stages, in sites related to TTR deposition. Toxicity of synthetic TTR fibrils formed in vitro at physiological pH was studied on a Schwannoma cell line by caspase-3 activation assays and showed that early aggregates but not mature fibrils are toxic to cells. Taken together, these results show that nonfibrillar cytotoxic deposits occur in early stages of FAP.

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.

Transthyretin (TTR) is a tetrameric protein synthesized mainly by the liver and the choroid plexus, from where it is secreted into the plasma and the cerebrospinal fluid, respectively. Some forms of polyneuropathy, vitreopathy, and cardiomyopathy are caused by the deposition of normal and/or mutant TTR molecules in the form of amyloid fibrils. Familial amyloidotic polyneuropathy is the most common form of TTR amyloidosis related to the V30M variant. It is still unclear the process by which soluble proteins deposit as amyloid. The treatment of amyloid-related disorders might attempt the stabilization of the soluble protein precursor to retard or inhibit its deposition as amyloid; or aim at the resorption of the deposited amyloid. The anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX) has been shown to reduce the amyloid load in immunoglobulin light-chain amyloidosis. We investigated 1) whether I-DOX has affinity for TTR amyloid in tissues, 2) determined the I-DOX binding constants to TTR synthetic fibrils, and 3) determined the nature of the effect of I-DOX on TTR fibrils. We report that 1) I-DOX co-localizes with amyloid deposits in tissue sections of patients with familial amyloidotic polyneuropathy; 2) I-DOX strongly interacts with TTR amyloid fibrils and presents two binding sites with k(d) of 1.5 x 10(-11) mol/L and 5.6 x 10(-10) mol/L, respectively; and 3) I-DOX disrupts the fibrillar structure of TTR amyloid into amorphous material, as assessed by electron microscopy but does not solubilize the fibrils as confirmed by filter assays. These data support the hypothesis that I-DOX and less toxic derivatives can prove efficient in the treatment of TTR-related amyloidosis.

Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, beta-amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot-blot ligand-binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of beta-sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand beta-sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.

Cinchona officinalis 'Ledgeriana', former called Cinchona ledgeriana, hairy roots were initiated containing constitutive-expression constructs of cDNAs encoding the enzymes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) from Catharanthus roseus, two key enzymes in terpenoid indole and quinoline alkaloid biosynthesis. The successful integration of these genes and the reporter gene gus-int was demonstrated using Southern blotting and the polymerase chain reaction. The products of TDC and STR, tryptamine and strictosidine, were found in high amounts, 1200 and 1950 μg g -1 dry weight, respectively. Quinine and quinidine levels were found to rise up to 500 and 1000 μg g -1 dry weight, respectively. The results show that genetic engineering with multiple genes is well possible in hairy roots of C. officinalis. However, 1 year after analyzing the hairy roots for the first time, they had completely lost their capacity to accumulate alkaloids.