Gene therapy approach to FAP: in vivo influence of T119M in TTR deposition in a transgenic V30M mouse model.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of amyloid fibrils composed by mutated transthyretin (TTR) mainly in the peripheral nervous system. At present, liver transplantation is still the standard treatment to halt the progression of clinical symptoms in FAP, but new therapeutic strategies are emerging, including the use of TTR stabilizers. Here we propose to establish a new gene therapy approach using adeno-associated virus (AAV) vectors to deliver the trans-suppressor TTR T119M variant to the liver of transgenic TTR V30M mice at different ages. This TTR variant is known for its ability to stabilize the tetrameric protein. Analysis of the gastrointestinal tract of AAV-treated animals revealed a significant reduction in deposition of TTR non-fibrillar aggregates in as much as 34% in stomach and 30% in colon, as well as decreased levels of biomarkers associated with TTR deposition, namely the endoplasmic reticulum stress marker BiP and the extracellular matrix protein MMP-9. Moreover, we showed with different studies that our approach leads to an increase in tetrameric and more stable forms of TTR, in favor of destabilized monomers. Altogether our data suggest the possibility to use this gene therapy approach in a prophylactic manner to prevent FAP pathology.

17beta-estradiol induces transthyretin expression in murine choroid plexus via an oestrogen receptor dependent pathway.

Oestrogen protects against AD by multiple mechanisms, including the enhancement of Abeta clearance. Transthyretin (TTR) is a homotetrameric protein mainly synthesized by the liver and choroid plexus (CP) of the brain that sequesters the amyloid beta (Abeta) peptide. In this study we examined the effects of 17beta-estradiol (E2) on TTR protein and mRNA levels, in primary cultures of rat CP epithelial cells (CPEC) by Western blot and Real Time PCR, respectively. Moreover, the localization of oestrogen receptors alpha (ERalpha) and beta (ERbeta) in response to E2 treatment was analysed by confocal microscopy in these cells. The expression of TTR, ERalpha and ERbeta was also compared in the CP of castrated female mice treated with E2 to vehicle-treated animals by Real Time PCR. TTR concentration in the CSF of all these animals was measured by radioimmunoassay. E2 treatment induced TTR transcription and increased TTR protein content in CPEC. Pre-treatment with ICI 182,780 (ICI) abrogated E2-induced TTR expression suggesting that, TTR is up-regulated via an ER-dependent pathway. Confocal microscopy demonstrated extranuclear ERalpha and ERbeta localization in untreated CPEC. Upon E2 treatment, translocation of ERalpha to the nucleus occurred, while ERbeta remained in the cytosol. These data was concurrent with the up-regulation of TTR expression detected in the CP of castrated female mice subjected to E2 treatment. Our results highlight the importance of E2 on the regulation of TTR, which may participate in the oestrogen-induced decrease in Abeta levels and deposition described in the literature.

Transthyretin binding to A-Beta peptide--impact on A-Beta fibrillogenesis and toxicity.

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.

Extracellular matrix markers for disease progression and follow-up of therapies in familial amyloid polyneuropathy V30M TTR-related.

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.

5Alpha-dihydrotestosterone up-regulates transthyretin levels in mice and rat choroid plexus via an androgen receptor independent pathway.

Transthyretin (TTR) is a 55 kDa plasma homotetrameric protein mainly synthesized in the liver and choroid plexuses (CPs) of the brain that, functions as a carrier for thyroxin and retinol binding protein. It sequesters amyloid beta (Abeta) peptide, and TTR levels in the cerebrospinal fluid (CSF) appear to be inversely correlated with Alzheimer's disease (AD) onset and progression. Androgen deprivation increases plasma Abeta levels, which indicate that androgens may reduce the levels of soluble Abeta, the peptide widely implicated in the initiation of AD pathogenesis; however, the underlying mechanisms are still poorly understood. In this study we examined the effects of 5alpha-dihydrotestosterone (DHT) on TTR protein and mRNA levels, in primary cultures of rat CPs epithelial cells (CPEC) by Western blot, and real time PCR, respectively. Moreover, TTR concentrations were measured in the CSF of castrated wild-type, and transgenic mice expressing human TTR subjected to DHT treatment, by radioimmunoassay and ELISA, respectively. TTR mRNA expression was also compared in the CPs, of the animals from each experimental group by real time PCR. DHT treatment increased TTR protein levels in CPEC, and induced TTR transcription in these cells. The combination of flutamide with DHT in the treatment of CPEC did not abrogate DHT-induced TTR levels, suggesting that TTR is up-regulated via an androgen receptor independent pathway. In the CPs of both mice strains, DHT also increased TTR mRNA levels, but no significant differences in TTR protein levels were detected in the CSF of these animals. These findings open a wide range of possibilities for future studies on Abeta deposition and cognitive function, in response to androgen induction of TTR in animal models of AD.

Biochemical marker in familial amyloidotic polyneuropathy, Portuguese type. Family studies on the transthyretin (prealbumin)-methionine-30 variant.

A transthyretin variant with a methionine for valine substitution at position 30 [TTR(Met30)] is found in Portuguese patients with familial amyloidotic polyneuropathy (FAP). Effective, rapid, small- and semimicro-scale (immunoblotting) procedures were developed to determine whether or not TTR(Met30) is present in the plasma of an individual subject. The immunoblotting procedure employs only 0.10 ml of serum and can serve as a reliable procedure for the screening of large numbers of persons for the presence of TTR(Met30). In family studies of seven FAP kindreds, TTR(Met30) was found in 21 out of 41 asymptomatic FAP offspring, and its presence was not related to either age or sex. Thus, the mutant TTR segregated in accordance with the known autosomal dominant mode of inheritance of FAP. Total plasma TTR levels were not reduced in asymptomatic FAP offspring who were carriers of TTR(Met30), and no difference was observed between carriers and noncarriers of the mutant TTR. The ratios of the variant to normal TTR in plasma were estimated in asymptomatic FAP offspring and were similar to those found in FAP patients. In contrast, TTR(Met30) was relatively enriched in cerebrospinal fluid samples from two FAP patients. The significance of this finding is not known, but might relate to the preferential deposition of amyloid in the nervous system in FAP. A limited study was conducted involving simultaneous analysis of both stored (collected in 1975) and fresh serum from 20 FAP offspring, all of whom had been asymptomatic in 1975. In every subject, the results obtained with the stored and the fresh serum samples were in agreement. Six of these subjects developed clinical FAP since 1975; TTR(Met30) was present in each of these subjects. These several studies strongly suggest that the presence of TTR(Met30) in plasma constitutes a predictive biochemical marker of FAP in the preclinical phase of the disease.

Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin).

Amyloid fibril protein in patients with familial amyloidotic polyneuropathy is known to be chemically related to transthyretin (TTR), the plasma protein that is usually referred to as prealbumin. A genetically abnormal TTR may be involved in this disease. Studies were conducted on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with familial amyloidosis, and on TTR isolated from sera of patients with this disease. AFp, purified by affinity chromatography on retinol-binding protein linked to Sepharose, resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and retinol-binding protein. The structural studies included: (a) comparative peptide mappings by reverse-phase high performance liquid chromatography (HPLC) after trypsin digestion; (b) cyanogen bromide cleavage studies; and (c) amino acid microsequence analysis of selected tryptic and CNBr peptides. On the basis of the known amino acid sequence of TTR, comparative tryptic peptide maps showed the presence of a single aberrant tryptic peptide (peptide 4, residues 22-34) in AFp as compared with TTR. This aberrant peptide contained a methionine residue, not present in normal tryptic peptide 4. CNBr cleavage of AFp produced two extra peptide fragments, which were demonstrated, respectively, by HPLC analysis and by sodium dodecyl sulfate-gel electrophoresis. Sequence analyses indicated the presence of a methionine-for-valine substitution at position 30 in AFp as compared with TTR. Thus, the purified amyloid fibril protein comprised a TTR variant with a methionine-forvaline substitution at position 30. A single nucleotide change in a possible codon for valine 30 could explain the substitution. The variant TTR was also present in the TTR isolated from the pooled sera of amyloidoses patients, together with larger (four- to six-fold) amounts of the normal TTR. Thus, in these patients, the variant TTR was circulating in plasma, along with larger amounts of normal TTR. We suggest that the variant TTR represents the specific biochemical cause of the disease, and that this abnormal form of TTR selectively deposits in tissues as the amyloid characteristic of the disease.

Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius.

The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.

Doxycycline disrupts transthyretin amyloid: evidence from studies in a FAP transgenic mice model.

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.

Transthyretin provides trophic support via megalin by promoting neurite outgrowth and neuroprotection in cerebral ischemia.

Transthyretin (TTR) is a protein whose function has been associated to binding and distribution of thyroid hormones in the body and brain. However, little is known regarding the downstream signaling pathways triggered by wild-type TTR in the CNS either in neuroprotection of cerebral ischemia or in physiological conditions. In this study, we investigated how TTR affects hippocampal neurons in physiologic/pathologic conditions. Recombinant TTR significantly boosted neurite outgrowth in mice hippocampal neurons, both in number and length, independently of its ligands. This TTR neuritogenic activity is mediated by the megalin receptor and is lost in megalin-deficient neurons. We also found that TTR activates the mitogen-activated protein kinase (MAPK) pathways (ERK1/2) and Akt through Src, leading to the phosphorylation of transcription factor CREB. In addition, TTR promoted a transient rise in intracellular calcium through NMDA receptors, in a Src/megalin-dependent manner. Moreover, under excitotoxic conditions, TTR stimulation rescued cell death and neurite loss in TTR KO hippocampal neurons, which are more sensitive to excitotoxic degeneration than WT neurons, in a megalin-dependent manner. CREB was also activated by TTR under excitotoxic conditions, contributing to changes in the balance between Bcl2 protein family members, toward anti-apoptotic proteins (Bcl2/BclXL versus Bax). Finally, we clarify that TTR KO mice subjected to pMCAO have larger infarcts than WT mice, because of TTR and megalin neuronal downregulation. Our results indicate that TTR might be regarded as a neurotrophic factor, because it stimulates neurite outgrowth under physiological conditions, and promotes neuroprotection in ischemic conditions.

Familial amyloid polyneuropathy: receptor for advanced glycation end products-dependent triggering of neuronal inflammatory and apoptotic pathways.

Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder associated with extracellular deposition of mutant transthyretin (TTR) amyloid fibrils, particularly in the peripheral nervous system. We have hypothesized that binding of TTR fibrils to the receptor for advanced glycation end products (RAGE) on critical cellular targets is associated with a destructive stress response underlying peripheral nerve dysfunction. Analysis of nerve biopsy samples from patients with FAP (n = 16) at different stages of disease (0-3), compared with age-matched controls (n = 4), by semiquantitative immunohistology and in situ hybridization showed increased levels of RAGE, beginning at the earliest stages of the disease (FAP 0; p < 0.02) and especially localized in axons. Upregulation of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-1beta) (approximately threefold; p < 0.02) and the inducible form of nitric oxide synthase (iNOS) ( approximately 2.5-fold; p < 0.04) was also observed in a distribution overlapping RAGE expression. Tyrosine nitration and increased activated caspase-3 in axons from FAP patients (p < 0.03) were apparent. Although these data suggest the presence of ongoing neuronal stress, there was no upregulation of neurotrophins (nerve growth factor and neurotrophin-3) in FAP nerves. Studies on cultured neuronal-like, Schwann, and endothelial cells incubated with TTR fibrils displayed RAGE-dependent expression of cytokines and iNOS at early times (6 and 12 hr, respectively), followed by later (24 hr) activation of caspase-3 and DNA fragmentation. We propose that the interaction of TTR fibrils with RAGE may contribute to cellular stress and toxicity in FAP. Furthermore, there is an apparent lack of responsiveness of Schwann cells in FAP nerve to provide neurotrophic factors.

'In vitro' amyloid fibril formation from transthyretin: the influence of ions and the amyloidogenicity of TTR variants.

The mechanisms of amyloid formation in Familial Amyloidotic Polyneuropathy (FAP) are unknown, as well as the factors determining the development of this pathology. To get some insights into this process, we have first tested a fluorimetric assay with thioflavine T, as a quantitative method for transthyretin (TTR) amyloid estimation, using amyloid isolated from post-mortem tissues of a FAP patient. Then production of amyloid fibrils from soluble TTR was achieved by acidification and optimized for protein concentration and pH. The effect of different ions such as metal and sulphate ions in the process of amyloid formation from wild type TTR was compared using a kinetic assay. Under the conditions tested sulphate diminishes the amount of amyloid formed from wild type TTR and in addition appears to promote aggregation of preexisting amyloid fibrils. The relative amyloidogenicity of three TTR variants, TTR Met30, TTR Pro55 and TTR Met119 respectively, was evaluated using a pH dependent assay. It was shown that the Pro55 variant is highly susceptible to amyloid formation as compared to the wild type protein; on the contrary, the Met119 variant is more resistant than the other TTR proteins towards precipitation into amyloid. These results are in agreement with the pathological conditions associated with these mutations. This type of assay has a wide application for testing the influence of other factors, such as therapeutical agents, on amyloid formation.

Characterization of a basic transthyretin variant--TTR Arg 102--in the German population.

A basic transthyretin (TTR) variant, apparently non-pathogenic, has been reported in a German family. Protein analysis of this TTR variant revealed the substitution of arginine for proline at position 102 of the TTR polypeptide chain. This result was confirmed by DNA analysis of PCR amplified DNA.

Small transthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses.

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.

Transthyretin stability as a key factor in amyloidogenesis: X-ray analysis at atomic resolution.

Transthyretin (TTR) amyloidosis is a conformational disturbance, which, like other amyloidoses, represents a life threat. Here, we report a TTR variant, TTR Thr119Met, that has been shown to have a protective role in the development of clinical symptoms in carriers of TTR Val30Met, one of the most frequent variants among TTR amyloidosis patients. In order to understand this effect, we have determined the structures of the TTR Val30Met/Thr119Met double mutant isolated from the serum of one patient and of both the native and thyroxine complex of TTR Thr119Met. Major conclusions are: (i) new H-bonds within each monomer and monomer-monomer inter-subunit contacts, e.g. Ser117-Ser117 and Met119-Tyr114, increase protein stability, possibly leading to the protective effect of the TTR Val30Met/Thr119Met variant when compared to the single variant TTR Val30Met. (ii) The mutated residue (Met119) extends across the thyroxine binding channel inducing conformational changes that lead to closer contacts between different dimers within the tetramer. The data, at atomic resolution, were essential to detect, for the first time, the subtle changes in the inter-subunit contacts of TTR, and explain the non-amyloidogenic potential of the TTR Thr119Met variant, improving considerably current research on the TTR amyloid fibril formation pathway.

Search for intermediate structures in transthyretin fibrillogenesis: soluble tetrameric Tyr78Phe TTR expresses a specific epitope present only in amyloid fibrils.

Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of TTR into an insoluble beta-sheet. The TTR tetramer is thought to dissociate into monomeric intermediates and subsequently polymerise into the pathogenic amyloid form. The biochemical mechanism behind this transformation is unknown. We characterised intermediate TTR structures in the in vitro amyloidogenesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the TTR tetrameric fold, based on the known crystallographic structure of a Leu55Pro transthyretin variant. We generated a soluble tetrameric form of TTR that is recognised by a monoclonal antibody, previously reported to react only with highly amyloidogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M(-1). We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway.

Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils.

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.

Transthyretin is not necessary for thyroid hormone metabolism in conditions of increased hormone demand.

Thyroid hormones circulate in blood mainly bound to plasma proteins. Transthyretin is the major thyroxine plasma carrier in mice. Studies in transthyretin-null mice revealed that the absence of transthyretin results in euthyroid hypothyroxinemia and normal thyroid hormone tissue distribution, with the exception of the choroid plexus in the brain. Therefore, transthyretin does not influence normal thyroid hormone homeostasis under standard laboratory conditions. To investigate if transthyretin has a buffer/storage role we challenged transthyretin-null and wild-type mice with conditions of increased hormone demand: (i) exposure to cold, which elicits thermogenesis, a process that requires thyroid hormones; and (ii) thyroidectomy, which abolishes thyroid hormone synthesis and secretion and induces severe hypothyroidism. Transthyretin-null mice responded as the wild-type both to changes induced by stressful events, namely in body weight, food intake and thyroid hormone tissue content, and in the mRNA levels of genes whose expression is altered in such conditions. These results clearly exclude a role for transthyretin in thyroid hormone homeostasis even under conditions of increased hormone demand.

Antibody recognition of amyloidogenic transthyretin variants in serum of patients with familial amyloidotic polyneuropathy.

Familial amyloidotic polyneuropathy (FAP) is a late-onset inherited disease characterized by the deposition of amyloid fibrils. FAP is associated with mutations on the transthyretin (TTR) gene. A monoclonal antibody, MAb 39-44, reacting with high molecular weight aggregates of TTR but not with tetrameric TTR has recently been generated and characterized. This antibody recognizes a cryptic epitope that is expressed in isolated recombinant amyloidogenic mutants and in ex vivo amyloid. In the present work we show that this amyloid-specific antibody specifically recognizes in a direct enzyme-linked immunoassay (ELISA) plasma TTR from carriers of various mutations associated with FAP, both in asymptomatic individuals and in patients. In contrast, it does not react with plasma TTR from healthy individuals or that from carriers of nonpathogenic mutations. Using the ELISA developed in this study we identified three different TTR mutations in Portuguese patients with neuropathy of unknown cause, later shown to have amyloid tissue deposition. This antibody recognizes conformations that express cryptic epitopes shared by amyloidogenic TTR variants associated with FAP, not present among nonpathogenic TTR molecules. This antibody will contribute to further identify and characterize intermediates of the amyloidogenic cascade. In addition, it will also be useful for screening amyloidogenic TTR mutations in patients with neuropathy of unknown cause, prior to precise molecular diagnosis using protein and/or DNA analysis.

Transthyretin mutations in hyperthyroxinemia and amyloid diseases.

Over 80 different disease-causing mutations in transthyretin (TTR) have been reported. The vast majority are inherited in an autosomal dominant manner and are related to amyloid deposition, affecting predominantly peripheral nerve and/or the heart. A small portion of TTR mutations are apparently non-amyloidogenic. Among these are mutations responsible for hyperthyroxinemia, presenting high affinity for thyroxine (a TTR ligand). Compound heterozygotic individuals for TTR mutants have been described; noteworthy is the clinically protective effect exerted by a non-pathogenic over a pathogenic mutation. Current TTR mutations and their significance are briefly reviewed here.