Hepatic production of transthyretin L12P leads to intracellular lysosomal aggregates in a new somatic transgenic mouse model

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.     

Clusterin regulates transthyretin amyloidosis

Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.     

Bifunctional crosslinking ligands for transthyretin

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR–ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.     

Natural polyphenols inhibit different steps of the process of transthyretin (TTR) amyloid fibril formation

Several natural polyphenols with potent inhibitory effects on amyloid fibril formation have been reported. Herein, we studied modulation of transthyretin (TTR) fibrillogenesis by selected polyphenols. We demonstrate that both curcumin and nordihydroguaiaretic acid (NDGA) bind to TTR and stabilize the TTR tetramer. However, while NDGA slightly reduced TTR aggregation, curcumin strongly suppressed TTR amyloid fibril formation by generating small "off-pathway" oligomers and EGCG maintained most of the protein in a non-aggregated soluble form. This indicates alternative mechanisms of action supported by the occurrence of different non-toxic intermediates. Moreover, EGCG and curcumin efficiently disaggregated pre-formed TTR amyloid fibrils. Our studies, together with the safe toxicological profile of these phytochemicals may guide a novel pharmacotherapy for TTR-related amyloidosis targeting different steps in fibrillogenesis.     

Uncovering the Mechanism of Aggregation of Human Transthyretin

The tetrameric thyroxine transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify β-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy.     

Binding of epigallocatechin-3-gallate to transthyretin modulates its amyloidogenicity

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.

Structured summary

MINT-7294529: TTR (uniprotkb:P02766) and TTR (uniprotkb:P02766) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs

The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.     

The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal. Direct analysis by fluorescence-detected analytical ultracentrifugation

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys(10)) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.     

Cardiac amyloidosis: the need for early diagnosis

Amyloidosis is a collection of systemic diseases characterised by misfolding of previously soluble precursor proteins that become infiltrative depositions, thereby disrupting normal organ structure and function. In the heart, accumulating amyloid fibrils lead to progressive ventricular wall thickening and stiffness, resulting in diastolic dysfunction gradually progressing to a restrictive cardiomyopathy. The main types of cardiac amyloidosis are amyloid light chain (AL) amyloidosis caused by an underlying plasma cell dyscrasia, amyloid transthyretin (TTR) amyloidosis of wild-type (normal) TTR at older age (ATTRwt) and hereditary or mutant amyloid TTR (ATTRm) in which a genetic mutation leads to an unstable TTR protein. Overall survival is poor once heart failure develops, underlining the need for early referral and diagnosis. Treatment for AL amyloidosis has improved markedly over the last decades, and TTR amyloidosis gene silencers and orally available transthyretin stabilisers are ready to enter the clinical arena after recent positive outcome trials. Novel therapies aiming at fibril degradation with monoclonal antibodies are under investigation. In this review, we focus on ‘red flag’ signs and symptoms, diagnosis and management of cardiac amyloidosis which differs considerably from the general management of heart failure. Only by increasing awareness, prognosis for patients with this devastating disease can be improved.

     

Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions

Conservative mutation of transthyretin's surface residues can predispose an individual to familial amyloidosis by dramatically changing the energetics of misfolding. Senile systemic amyloidosis (SSA), however, cannot be explained in this fashion because wild-type (WT) transthyretin (TTR) misfolds and misassembles into amyloid. Since various modifications of the SH functionality of Cys10 have been reported in humans, we sought to understand the extent to which these modifications alter the stability and amyloidosis of WT TTR as a possible explanation for SSA. Homotetrameric Cys10 TTR variants, including TTR-Cys, TTR-GSH, TTR-CysGly, and S-sulfonated TTR, were chemically synthesized starting with WT TTR. The TTR-Cys, TTR-GSH, and TTR-CysGly isoforms are more amyloidogenic than WT at the higher end of the acidic pH range (pH 4.4-5.0), and they are similarly destabilized relative to WT TTR toward urea denaturation. They exhibit rates of urea-mediated tetramer dissociation (pH 7) and MeOH-facilitated fibril formation similar to those of WT TTR. Under mildly acidic conditions (pH 4.8), the amyloidogenesis rates of the mixed disulfide TTR variants are much faster than the WT rate. S-Sulfonated TTR is less amyloidogenic and forms fibrils more slowly than WT under acidic conditions, yet it exhibits a stability and rates of tetramer dissociation similar to those of WT TTR when subjected to urea denaturation. Conversion of the Cys10 SH group to a mixed disulfide with the amino acid Cys, the CysGly peptide, or glutathione increases amyloidogenicity and the amyloidogenesis rate above pH 4.6, conditions under which TTR probably forms fibrils in humans. Hence, these modifications may play an important role in human amyloidosis.     

Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid

Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date. After incubation for 15 days at 37 °C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation in a biologically relevant environment, opening new avenues of investigation into the molecular mechanisms of LA.     

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.     

The Flavonoid Luteolin,but Not Luteolin-7-O-Glucoside,Prevents a Transthyretin Mediated Toxic Response

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.     

D18G transthyretin is monomeric,aggregation prone,and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis?

Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date. Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR. Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT. It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade. Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation; however, benign systemic deposition is also observed. Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR. The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease. CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate. Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T(4)) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T(4) CSF concentration. Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.     

Transthyretin Pro 36 associated with familial amyloidotic polyneuropathy in an Ashkenazic Jewish kindred

Summary Mutations in the serum protein transthyretin (TTR) cause amyloidosis involving the peripheral nerves, heart, and other organs. In Ashkenazic Jews, the only TTR variant described to date has been TTR Ile 33. We have studied DNA from another Ashkenazic Jewish kindred with familial amyloidotic polyneuropathy. Singlestrand conformation polymorphism analysis, DNA sequencing, and restriction analysis indicated that this kindred has the TTR Pro 36 variant, previously described only in a Greek kindred.     

Frequency and genetic background of the position 122 (Val----Ile) variant transthyretin gene in the black population.

Transthyretin (TTR) (122 Val----Ile), caused by a point mutation which destroys a MaeIII restriction site, is associated with cardiac amyloidosis in black individuals. To estimate the frequency of the MaeIII(-) gene in the black population without overt cardiac disease, DNA from 177 black individuals without amyloidosis was amplified by the PCR around TTR codon 122 and was digested with MaeIII. The MaeIII(-) gene frequency was 4/354 (1.1%; 95% confidence interval 0.32%2.7%), suggesting that the variant is relatively common in blacks. HLA genotype testing did not suggest that the TTR (122 Val----Ile) heterozygotes were of a closely related genetic background.     

Cardiac amyloid in patients with familial amyloid polyneuropathy consists of abundant wild-type transthyretin

Patients with familial amyloid polyneuropathy (FAP) are now cured by liver transplantation, but cardiac amyloidosis would further progress even after liver transplantation in some patients. To clarify the pathological mechanism of the progress of cardiac amyloidosis in FAP, we investigated cardiac tissues obtained from 6 FAP patients with 3 different types of TTR mutations. One of them had undergone liver transplantation and one year later died of cardiac amyloidosis. We determined clinical severity of cardiac involvement of those patients and characterized amyloid fibril proteins depositing in their cardiac muscles by immunohistochemistry, mass spectrometry and isoelectric focusing. All the patients had cardiac dysfunction and increased cardiac weight. Diffuse deposition of TTR-related amyloid was seen in their myocardium on microscopic examination. Amyloid fibrils of the heart were composed of wild-type TTR as well as variant TTR at a ratio of about 1:1 in 5 patients without liver transplantation. In the patient with a transplanted liver, about 80% of the cardiac amyloid consisted of wild-type TTR. Wild-type TTR contributes greatly to the development of amyloid deposition in the heart of FAP patients regardless of the types of TTR mutations.     

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe --> Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.     

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design,synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer’s disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal.     

Iodine Atoms: A New Molecular Feature for the Design of Potent Transthyretin Fibrillogenesis Inhibitors

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis.