TTR Aggregation Research Articles

Plasmalogens Alter the Aggregation Rate of Transthyretin and Lower Toxicity of Transthyretin Fibrils.

Heart tissue can experience a progressive accumulation of transthyretin (TTR), a small four subunit protein that transports holoretinol binding protein and thyroxine. This severe pathology is known as transthyretin amyloid cardiomyopathy. Numerous experimental studies indicated that the aggregation rate and toxicity of TTR fibrils could be altered by the presence of lipids; however, the role of plasmalogens in this process remains unknown. In this study, we investigate the effect of choline plasmalogens (CPs) with different lengths and saturations of fatty acids (FAs) on TTR aggregation. We found that CPs with saturated and unsaturated FAs strongly suppressed TTR aggregation. We also found that CPs with saturated FAs did not change the morphology of TTR fibrils; however, much thicker fibrillar species were formed in the presence of CPs with unsaturated FAs. Finally, we found that CPs with C16:0, C18:0, and C18:1 FAs substantially lowered the cytotoxicity of TTR fibrils that were formed in their presence.

Structures and Dynamics of β-Rich Oligomers of ATTR (105-115) Assembly.

Transthyretin (TTR) is a tetrameric homologous protein that can dissociate into monomers. Misfolding and aggregation of TTR can lead to amyloid transthyretin amyloidosis (ATTR), which can cause many diseases (e.g., senile systemic amyloidosis, familial amyloid cardiomyopathy, and familial amyloid polyneuropathy). Despite growing evidence indicating that small oligomers play a critical role in regulating cytotoxicity, the structures of these oligomeric intermediates and their conformational transformations are still unclear, impeding our understanding of neurodegenerative mechanisms and the development of therapeutics targeting early aggregation species. The TTR monomer protein consists of various fragments prone to self-aggregation, including the residue 105-115 sequence. Therefore, our study investigated the assembly progress of ATTR (105-115) peptides using all-atom molecular dynamics simulations. The findings indicate that the probability of β-sheet content increases with increasing numbers of peptides. Additionally, interactions between hydrophobic residues L110 and L111 are crucial for the formation of a β-rich oligomer formation. These β-rich oligomers may adopt β-barrel conformations, potentially toxic oligomer species. Free-energy analysis reveals that β-barrel conformations serve as intermediates for these β-rich oligomers. Our insights into the structural ensemble dynamics of ATTR (105-115) contribute to understanding the physical mechanisms underlying the β-barrel oligomers of ATTR. These findings may shed light on the pathological role of ATTR in neurodegenerative diseases and offer potential therapeutic targets.

An ultra performance liquid chromatography method for transthyretin variants screening and heart failure assisting diagnosis

BackgroundTransthyretin (TTR) gene mutations are associated with hereditary amyloidosis (ATTR) caused by mutant TTR protein dissociation, misfolding, aggregation, and insoluble fibrils deposition. Herein, we reported a chromatographic approach for quantification and identification of TTR tetramer in human blood serum by ultra performance liquid chromatography (UPLC). MethodsTTR proteins and serum were incubated with a fluorescent TTR tetramer sensor (A2). The A2 sensor specifically reacted with tetrameric TTR and released stoichiometric fluorescence that was detected by fluorescence detector coupled to UPLC. The external standard was used for quantification, the chromatographic peak parameters were used to identification certain mutation types. ResultsUPLC correctly distinguished 18 types of mutant TTR proteins from wild type. The results were consistent with follow-up analysis of two ATTR patients’ blood serum samples. In addition, the tetrameric TTR of 30 heart failure (HF) patients showed strongly correlation (r = −0.63, p < 0.00) with NT-proBNP, a HF clinical biomarker. ConclusionsUPLC method has sufficient accuracy to eliminate the necessity of sequencing for certain types of TTR mutations and allows for facile initial screening of ATTR amyloidosis patients, carriers, and healthy individuals for time-saving and economical purposes. TTR tetramer may serve as a diagnostic biomarker to evaluate the risk of HF diseases.

Cholesterol and Sphingomyelin Uniquely Alter the Rate of Transthyretin Aggregation and Decrease the Toxicity of Amyloid Fibrils.

Transthyretin (TTR) is a small tetrameric protein that aggregates, forming highly toxic oligomers and fibrils. In the blood and cerebrospinal fluid, TTR can interact with various biomolecules, phospho- and sphingolipids, and cholesterol on the red blood cell plasma membrane. However, the role of these molecules in TTR aggregation remains unclear. In this study, we investigated the extent to which phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (Cho), important components of plasma membranes, could alter the rate of TTR aggregation. We found that PC and SM inhibited TTR aggregation whereas Cho strongly accelerated it. The presence of these lipids during the stage of protein aggregation uniquely altered the morphology and secondary structure of the TTR fibrils, which changed the toxicity of these protein aggregates. These results suggest that interactions of TTR with red blood cells, whose membranes are rich with these lipids, can trigger irreversible aggregation of TTR and cause transthyretin amyloidosis.

Abstract 18558: Modeling Cardiac Amyloidosis: Insights Into Pathogenesis and Therapeutic Strategies

Introduction: Cardiac transthyretin (TTR) amyloidosis is an underdiagnosed cause of heart failure and cardiac arrhythmias. The lack of relevant human cardiac tissue models hinders understanding of the underlying mechanisms and development of effective treatments. Hypothesis: We aimed to establish an in vitro model of cardiac amyloidosis using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) and aggregated TTR and investigate cellular changes and potential therapeutic targets. Methods: Wild-type and mutated TTR proteins were aggregated and added to hiPSC-CM cultures directly or embedded in Matrigel used for culture-plate coating. Single-cell and two- and three-dimensional hiPSC-CM models were used for real-time microscopy, calcium and voltage imaging, immunostaining, apoptosis, ROS assays, and proteomics studies with mass spectrometry. Results: Immunohistochemical and histological staining confirmed the deposition of TTR aggregates within the hiPSC-CM tissue models. Cells exposed to TTR aggregates exhibited increased apoptosis and increased production of reactive oxygen species (ROS). TTR-treated cells also displayed irregular calcium transients, characterized by oscillating calcium peaks and double-humped signals. The presence of TTR also induced morphological changes, including cell shrinkage at the single-cell level and the formation of holes in 2D hiPSC-CM cell sheets due to cell migration and apoptosis. These holes led to conduction abnormalities and the development of reentrant arrhythmias, specifically spiral waves. The severity of these abnormalities was markedly increased when mutant TTR aggregates were used. Proteomics studies suggest a potential involvement of the mTOR signaling pathway in the pathogenesis of cardiac amyloidosis. Conclusions: Our in vitro model successfully recapitulated key features of cardiac amyloidosis and revealed significant protein expression changes. These findings provide valuable insights into disease pathogenesis and potential therapeutic targets and highlight the utility of our model for studying cardiac amyloidosis for the development and evaluation of novel therapeutic strategies.

Saturation of fatty acids in phosphatidic acid uniquely alters transthyretin stability changing morphology and toxicity of amyloid fibrils

Transthyretin (TTR) is a small, β-sheet-rich tetrameric protein that transports thyroid hormone thyroxine and retinol. Phospholipids, including phosphatidic acid (PA), can uniquely alter the stability of amyloidogenic proteins. However, the role of PA in TTR aggregation remains unclear. In this study, we investigated the effect of saturation of fatty acids (FAs) in PA on the rate of TTR aggregation. We also reveal the extent to which PAs with different length and saturation of FAs altered the morphology and secondary structure of TTR aggregates. Our results showed that TTR aggregation in the equimolar presence of PAs with different length and saturation of FAs yielded structurally and morphologically different fibrils compared to those formed in the lipid-free environment. We also found that PAs drastically lowered the toxicity of TTR aggregates formed in the presence of this phospholipid. These results shed light on the role of PA in the stability of TTR and transthyretin amyloidosis.

EGCG-Mediated Protection of Transthyretin Amyloidosis by Stabilizing Transthyretin Tetramers and Disrupting Transthyretin Aggregates.

Transthyretin amyloidosis (ATTR) is a progressive and systemic disease caused by the misfolding and amyloid aggregation of transthyretin (TTR). Stabilizing the TTR tetramers and disrupting the formed TTR aggregation are treated as a promising strategy for the treatment of ATTR. Previous studies have reported that epigallocatechin gallate (EGCG) can participate in the whole process of TTR aggregation to prevent ATTR. However, the interaction mechanism of EGCG in this process is still obscure. In this work, we performed molecular dynamics simulations to investigate the interactions between EGCG and TTR tetramers, and between EGCG and TTR aggregates formed by the V30M mutation. The obtained results suggest that EGCG at the binding site of the V30M TTR tetramer can form stable hydrogen bonds with residues in the flexible AB-loop and EF-helix-loop, which reduces the structural mobility of these regions significantly. Additionally, the polyaromatic property of EGCG contributes to the increasement of hydrophobicity at the binding site and thus makes the tetramer difficult to be solvated and dissociated. For V30M-TTR-generated aggregates, EGCG can promote the dissociation of boundary β-strands by destroying key residue interactions of TTR aggregates. Moreover, EGCG is capable of inserting into the side-chain of residues of neighboring β-strands and disrupting the highly structured aggregates. Taken together, this study elucidates the role of EGCG in preventing TTR amyloidosis, which can provide important theoretical support for the future of drug design for ATTR.

P203 PLASMA TRANSTHYRETIN FORMS IN PATIENTS WITH WILD–TYPE TRANSTHYRETIN CARDIAC AMYLOIDOSIS: INFLUENCED OF TAFAMIDIS TREATMENT

Abstract Introduction Transthyretin (TTR) is a plasma protein that transports thyroxin and retinol complexed to retinol–binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid in the myocardium, causing TTR cardiac amyloidosis (ATTR–CA). Aim of this study is to develop a native electrophoretic method to characterize circulating TTR forms in plasma samples of ATTR–CA patients, before and after administration of tafamidis, a TTR stabilizer. Material &amp; Methods Plasma samples from patients affected by wild–type ATTR amyloidosis (ATTRwt, n=10) were collected before (T0) and during tafamidis treatment at 14, 30 and 90 days. Plasma samples of sex– and age–matched healthy controls (n=6) were also collected. All samples were separated on a native 4–20% Tris–Gly polyacrylamide gel. Western blot analysis was performed with anti–TTR or anti–RBP antibodies. Proteins were detected by Clarity ECL substrate. ImageLab software was used for image acquisition and densitometric analysis of the blots. Total TTR (mg/dl) and RBP (mg/dl) were quantified by nephelometry. Results In both groups, the most represented forms were: TTR dimers or trimers (37–50kDa), TTR tetramers complexed with RBP protein in 1:1 (75kDa,) or 1:2 ratio (100kDa), and high molecular weight aggregates (&amp;gt;150kDa). RBP protein was detectable in association with TTR tetramers and some of the TTR aggregates (250kDa, 480kDa). Based on TTR electrophoretic pattern at T0, patients could be divided in 2 groups: ATTR–1, characterised by the presence of TTR tetramers (55kDa), and ATTR–2, characterised by its absence. The ATTR–1 group showed also higher levels of total TTR in comparison with ATTR–2 (mean±SD 25.5±3.8, 16.6±4.2 respectively, p=0.004), while plasma RBP levels were similar (5.1±0.7, 5.1±1.4 respectively). The electrophoretic patterns of ATTR–1 at T0, as well as TTR and RBP levels were comparable to those of controls (TTR 22.6±3.8, RBP 4.4±0.9). Tafamidis treatment was associated with a progressive increase of total TTR levels both in ATTR–1 and –2 (T90 31.4±5.8, 28.7±6.8) but the 2 groups continued to be distinguishable by the electrophoretic pattern. Conclusions The native electrophoresis allowed us to detect diverse circulating TTR forms and to classify patients in 2 groups according to their electrophoretic pattern. TTR quali/quantitative evaluation by electrophoresis could represent a valid tool to assess the individual pharmacological response to tafamidis.

Amyposomes, a nanotechnological chaperone with anti-amyloidogenic activity

Aim The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid β1–40 (Aβ1–40), transthyretin (TTR) variant S52P, human β2microglobulin (β2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). Methods The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. Results The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aβ1–40, β2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aβ1–40, β2 D76N and TTR. SPR assays show that liposomes bind Aβ1–40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of β2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the β-stranded peptide regions responsible for the amyloid formation. Conclusion Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases. Key message Amyloidosis is a group of diseases, each due to a specific protein misfolding. Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders. Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.

Human transthyretin gene expression is markedly increased in repair Schwann cells in an in vitro model of hereditary transthyretin amyloidosis.

Hereditary transthyretin (TTR) amyloidosis (ATTRv) is characterized by TTR amyloid deposition in the peripheral nervous system. It remains unknown why variant TTR preferentially deposits in the peripheral nerves and dorsal root ganglia. We previously detected low levels of TTR expression in Schwann cells and established an immortalized Schwann cell line, TgS1, derived from a mouse model of ATTRv amyloidosis expressing the variant TTR gene. In the present study, the expression of TTR and Schwann cell marker genes was investigated in TgS1 cells by quantitative RT-PCR. TTR gene expression was markedly upregulated in TgS1 cells incubated in non-growth medium-Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The expression levels of c-Jun, Gdnf and Sox2 were increased, while Mpz was downregulated, suggesting that TgS1 cells exhibit a repair Schwann cell-like phenotype in the non-growth medium. Western blot analysis revealed that TTR protein was produced and secreted by the TgS1 cells. Furthermore, downregulation of Hsf1 with siRNA induced TTR aggregates in the TgS1 cells. These findings indicate that TTR expression is markedly increased in repair Schwann cells, likely to promote axonal regeneration. Therefore, aged dysfunctional repair Schwann cells may cause the deposition of variant TTR aggregates in the nerves of patients with ATTRv.

Thyroxine metabolite-derived 3-iodothyronamine (T1AM) and synthetic analogs as efficient suppressors of transthyretin amyloidosis

Aggregation and fibrillization of transthyretin (TTR) is a fatal pathogenic process that can cause cardiomyopathic and polyneuropathic diseases in humans. Although several therapeutic strategies have been designed to prevent and treat related pathological events, there is still an urgent need to develop better strategies to improve potency and wider applicability. Here, we present our study demonstrating that 3-iodothyronamine (T1AM) and selected thyronamine-like compounds can effectively prevent TTR aggregation. T1AM is one of the thyroid hormone (TH) metabolites, and T1AM and its analogs, such as SG2, SG6, and SG12, are notable molecules for their beneficial activities against metabolic disorders and neurodegeneration. Using nuclear magnetic resonance (NMR) spectroscopy and biochemical analysis, we confirmed that T1AM analogs could bind to and suppress acid-induced aggregation of TTR. In addition, we employed computational approaches to further understand the detailed mechanisms of the interaction between T1AM analogs and TTR. This study demonstrates that T1AM analogs, whose beneficial effects against several pathological processes have already been proven, may have additional benefits against TTR aggregation and fibrillization. Moreover, we believe that our work provides invaluable insights to enhance the pleiotropic activity of T1AM and structurally related analogs, relevant for their therapeutic potential, with particular reference to the ability to prevent TTR aggregation.

Pro-inflammatory cytokine secretion induced by amyloid transthyretin in human cardiac fibroblasts

Extracellular aggregates of wild-type human transthyretin are associated with heart diseases such as wild-type transthyretin (TTR)-derived amyloidosis (ATTR-wt). Due to their strategic location, cardiac fibroblasts act as sentinel cells that sense injury and activate the inflammasome. No studies of the effects of TTR amyloid aggregation on the secretion of inflammatory factors by primary human cardiac fibroblasts (hCFs) have been reported yet. The intracellular internalization of TTR aggregates, which correspond to the early stage of ATTR-wt, were determined using immunofluorescence and Western blotting of cell lysates. A further objective of this study was to analyze the secretion of inflammatory factors by hCFs after analysis of TTR amyloid aggregation using X-MAP® Luminex Assay techniques. We show that TTR aggregates are internalized in hCFs and induce the secretion of both Brain Natriuretic Peptide (BNP) and N-terminal pro B-type Natriuretic Peptide(NT-proBNP). Also, pro-inflammatory mediators such as interleukin-6 (IL-6) and IL-8 are secreted without significant changes in the levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). In conclusion, these findings suggest that IL-6 and IL-8 play important roles in the development of ATTR-wt, and indicate that IL-6 in particular could be a potentially important therapeutic target in patients with ATTR-wt.

276 CIRCULATING FORMS OF PLASMA TRANSTHYRETIN IN PATIENTS WITH WILD-TYPE TRANSTHYRETIN AMYLOIDOSIS AND EFFECTS OF TREATMENT WITH TAFAMIDIS

Abstract Introduction Transthyretin (TTR) is a homotetrameric 55-KDa plasma protein that transports thyroxin and retinol complexed to retinol-binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid (ATTR) representing one of the most frequent forms of amyloidosis in elderly. Aim of this study is to develop a native electrophoretic method to characterize circulating TTR in plasma samples of ATTR patients before and during treatment with tafamidis, a TTR stabilizer. Methods Plasma from ATTR patients (n=6), collected before (T0) and during tafamidis treatment, and plasma of healthy controls (n=6) were obtained from Fondazione Toscana G. Monasterio (Pisa, Italy). Plasma samples were separated on a native 4–20% Tris-Gly polyacrylamide gel. Western blot analysis was performed with anti-TTR (DAKO) or anti-RBP (Siemens Healthineer) antibodies. Proteins were detected by Clarity ECL substrate (BioRad). Results Circulating forms of TTR were qualitatively similar between ATTRwt patients at T0 and controls. In both groups, the most represented forms were: TTR dimers or trimers (∼37-50 kDa), TTR tetramers complexed with RBP protein in 1:1 ratio (∼80 kDa,) or 1:2 ratio (∼100 kDa), and high molecular weight (MW) aggregates (&amp;gt;150 kDa). Neither TTR monomers nor the tetramers were visible. RBP protein was detectable in association with TTR tetramers and some of the higher MW fractions (∼150 kDa, &amp;gt;250 kDa). Following tafamidis treatment, all ATTRwt patients displayed a progressive increase of the intensity of the band corresponding to TTR-RBP complexes, in agreement with the drug stabilizing action on TTR tetramers. Interestingly dimers and trimers, detectable at T0, were progressively lost during tafamidis treatment. Conclusions The native electrophoretic allowed us to detect several circulating TTR fractions. Data suggest a similar pattern of circulating TTR both in patients and healthy control: TTR tetramer exists only complexed with RBP and in equilibrium with low and high MW forms. Furthermore, the method allowed to appreciate the stabilizing effect of tafamidis on circulating TTR tetramers complexed with RBP. The study of circulating TTR fractions can expand our knowledge on mechanisms triggering its destabilization even when not mutated.

Liver-directed drugs for transthyretin-mediated amyloidosis.

Transthyretin-mediated amyloidosis (ATTR) is a rare, under-recognized, progressively debilitating, fatal disease caused by the aggregation and extracellular deposition of amyloid transthyretin (TTR) fibrils in multiple organs and tissues throughout the body. TTR is predominantly synthesized by the liver and normally circulates as a homotetramer, while misfolded monomers aggregate to form amyloid fibrils. One strategy to treat ATTR amyloidosis is to reduce the amount of TTR produced by the liver using drugs that directly target the TTR mRNA or gene. This narrative review focuses on how TTR gene silencing tools act to reduce TTR production, describing strategies for improved targeted delivery of these agents to hepatocytes where TTR is preferentially expressed. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), termed RNA silencers, cause selective degradation of TTR mRNA, while a TTR gene editing tool reduces TTR expression by introducing nonsense mutations into the TTR gene. Two strategies to facilitate tissue-specific delivery of these nucleic acid-based drugs employ endogenous receptors expressed by hepatocytes. Lipid nanoparticles (LNPs) that recruit apolipoprotein E support low-density lipoprotein receptor-mediated uptake of unconjugated siRNA and are now used for CRISPR gene editing tools. Additionally, conjugating N-acetylgalactosamine (GalNAc) moieties to ASOs or siRNAs facilitates receptor-mediated uptake by the asialoglycoprotein receptor. In summary, ATTR is a progressive disease with various clinical manifestations due to TTR aggregation, deposition, and amyloid formation. Receptor-targeted ligands (eg, GalNAc) and nanoparticle encapsulation (eg, LNPs) are technologies to deliver ASOs, siRNAs, and gene editing tools to hepatocytes, the primary location of TTR synthesis.

Feasibility Study of Dendrimer-Based TTR-CRISPR pDNA Polyplex for Ocular Amyloidosis in Vitro.

Hereditary amyloidgenic transthyretin (ATTR) amyloidosis is caused by a genetic point-mutated transthyretin such as TTR Val30Met (TTR V30M), since it forms protein aggregates called amyloid resulting in the tissue accumulation and functional disorders. In particular, ATTR produced by retinal pigment epithelial cells often causes ATTR ocular amyloidosis, which elicits deterioration of ocular function and ultimately blindness. Therefore, development of novel therapeutic agents is urgently needed. Genome-editing technology using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas9) system is expected to be a therapeutic approach to treat genetic diseases, such as ATTR amyloidosis caused by a point mutation in TTR gene. Previously, we reported that glucuronylglucosyl-β-cyclodextrin conjugated with a polyamidoamine dendrimer (CDE) had excellent gene transfer ability and that underlying dendrimer inhibited TTR aggregation. Conversely, folate receptors are known to be highly expressed in retina; thus, folate has potential as a retinal target ligand. In this study, we prepared a novel folate-modified CDE (FP-CDE) and investigated its potential as a carrier for the retinal delivery of TTR-CRISPR plasmid DNA (pDNA). The results suggested that FP-CDE/TTR-CRISPR pDNA could be taken up by retinal pigment epithelial cells via folate receptors, exhibited TTR V30M amyloid inhibitory effect, and suppressed TTR production via the genome editing effect (knockout of TTR gene). Thus, FP-CDE may be useful as a novel therapeutic TTR-CRISPR pDNA carrier in the treatment of ATTR ocular amyloidosis.

Tafamidis polyneuropathy amelioration requires modest increases in transthyretin stability even though increases in plasma native TTR and decreases in non-native TTR do not predict response

Background TTR aggregation causes hereditary transthyretin (TTR) polyneuropathy (ATTRv-PN) in individuals with destabilised TTR variants. ATTRv-PN can be treated with ligands that bind TTR and prevent aggregation. One such ligand, tafamidis, is widely approved to treat ATTRv-PN. We explore how TTR stabilisation markers relate to clinical efficacy in 210 ATTRv-PN patients taking tafamidis. Methods TTR concentration in patient plasma was measured before and after tafamidis treatment using assays for native or combined native + non-native TTR. TTR tetramer dissociation kinetics, which are slowed by tafamidis binding, were also measured. Results Native TTR levels increased by 56.8% while combined native + non-native TTR levels increased by 3.1% after 24 months of tafamidis treatment, implying that non-native TTR decreased. Accordingly, the fraction of native TTR increased from 0.54 to 0.71 with tafamidis administration. Changes in native and non-native TTR levels were uncorrelated with clinical response to tafamidis. TTR tetramer dissociation generally slowed to an extent consistent with ∼40% of TTR being tafamidis-bound. Male non-responders had a lower extent of binding. Conclusions Native and non-native TTR concentration changes cannot be used as surrogate measures for therapeutic efficacy. Also, successful tafamidis therapy requires only moderate TTR stabilisation. Male patients may benefit from higher tafamidis doses.

Selective recognition of human small transthyretin aggregates by a novel monoclonal antibody

Biochemical characterisation of transthyretin variant TTR Y78F showed that this variant adopts a tetrameric conformation as normal TTR but exhibits some of the characteristics of an intermediate structure in the fibrillogenesis pathway. It was hypothesised that native Y78F might represent an early event in TTR amyloidogenesis. We immunised TTR knock out mice with recombinant variant TTR Y78F. One stable hybridoma named CE11, of the IgM isotype, was tested for reactivity towards several soluble recombinant TTR variants both amyloidogenic and non-amyloidogenic. CE11 only recognises the highly amyloidogenic TTR variants L55P, S52P, A97S, Y78F or acidified TTR wt preparations. At the same time, this clone was negative for TTR V30M, soluble wild type protein or TTR T119M. The reactivity increased with oligomer formation and decreased as mature fibrils grow. After size exclusion chromatography (SEC) followed by sandwich ELISA and native immunoblotting, the mAb recognised two peaks (i) peak 1 present in acidified and in soluble variant proteins preparations with material above 146 KDa (ii) peak 2 only present in soluble L55P and S52P TTR preparations with material between 66 and 146 KDa. mAb CE11 may be a potential tool to survey therapeutical agents against TTR aggregation.

Evidence From Human Placenta, Endoplasmic Reticulum-Stressed Trophoblasts, and Transgenic Mice Links Transthyretin Proteinopathy to Preeclampsia.

We have demonstrated that protein aggregation plays a pivotal role in the pathophysiology of preeclampsia and identified several aggregated proteins in the circulation of preeclampsia patients, the most prominent of which is the serum protein TTR (transthyretin). However, the mechanisms that underlie protein aggregation remain poorly addressed. We examined TTR aggregates in hypoxia/reoxygenation-exposed primary human trophoblasts (PHTs) and the preeclampsia placenta using complementary approaches, including a novel protein aggregate detection assay. Mechanistic analysis was performed in hypoxia/reoxygenation-exposed PHTs and Ttr transgenic mice overexpressing transgene-encoded wild-type human TTR or Ttr-/- mice. High-resolution ultrasound analysis was used to measure placental blood flow in pregnant mice. TTR aggregation was inducible in PHTs and the TCL-1 trophoblast cell line by endoplasmic reticulum stress inducers or autophagy-lysosomal disruptors. PHTs exposed to hypoxia/reoxygenation showed increased intracellular BiP (binding immunoglobulin protein), phosphorylated IRE1α (inositol-requiring enzyme-1α), PDI (protein disulfide isomerase), and Ero-1, all markers of the unfolded protein response, and the apoptosis mediator caspase-3. Blockade of IRE1α inhibited hypoxia/reoxygenation-induced upregulation of Ero-1 in PHTs. Excessive unfolded protein response activation was observed in the early-onset preeclampsia placenta. Importantly, pregnant human TTR mice displayed aggregated TTR in the junctional zone of the placenta and severe preeclampsia-like features. High-resolution ultrasound analysis revealed low blood flow in uterine and umbilical arteries in human TTR mice compared with control mice. However, Ttr-/- mice did not show any pregnancy-associated abnormalities. These observations in the preeclampsia placenta, cultured trophoblasts, and Ttr transgenic mice indicate that TTR aggregation is an important causal contributor to preeclampsia pathophysiology.

A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway

Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the 19F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the 19F linewidth and R2 relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of 19F probes at additional TTR sites yielded distinct 19F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The 19F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway.

Santalol Isomers Inhibit Transthyretin Amyloidogenesis and Associated Pathologies in Caenorhabditis elegans.

Transthyretin (TTR) is a homotetrameric protein found in human serum and is implicated in fatal inherited amyloidoses. Destabilization of native TTR confirmation resulting from mutation, environmental changes, and aging causes polymerization and amyloid fibril formation. Although several small molecules have been reported to stabilize the native state and inhibit TTR aggregation, prolonged use can cause serious side effects. Therefore, pharmacologically enhancing the degradation of TTR aggregates and kinetically stabilizing the native tetrameric structure with bioactive molecule(s) could be a viable therapeutic strategy to hinder the advancement of TTR amyloidoses. In this context, here we demonstrated α- and β-santalol, natural sesquiterpenes from sandalwood, as a potent TTR aggregation inhibitor and native state stabilizer using combined in vitro, in silico, and in vivo experiments. We found that α- and β-santalol synergize to reduce wild-type (WT) and Val30Met (V30M) mutant TTR aggregates in novel C. elegans strains expressing TTR fragments fused with a green fluorescent protein in body wall muscle cells. α- and β-Santalol extend the lifespan and healthspan of C. elegans strains carrying TTRWT::EGFP and TTRV30M::EGFP transgene by activating the SKN-1/Nrf2, autophagy, and proteasome. Moreover, α- and β-santalol directly interacted with TTR and reduced the flexibility of the thyroxine-binding cavity and homotetramer interface, which in turn increases stability and prevents the dissociation of the TTR tetramer. These data indicate that α- and β-santalol are the strong natural therapeutic intervention against TTR-associated amyloid diseases.