Hereditary Systemic Amyloidosis Research Articles

Phospholipid-induced secondary structural changes of lysozyme polymorphic amyloid fibrils studied using vacuum-ultraviolet circular dichroism.

The hallmark of amyloidosis, such as Alzheimer's disease and Parkinson's disease, is the deposition of amyloid fibrils in various internal organs. The onset of the disease is related to the strength of cytotoxicity caused by toxic amyloid species. Furthermore, amyloid fibrils show polymorphism, where some types of fibrils are cytotoxic while others are not. It is thus essential to understand the molecular mechanism of cytotoxicity, part of which is caused by the interaction between amyloid polymorphic fibrils and cell membranes. Here, using amyloid polymorphs of hen egg white lysozyme, which is associated with hereditary systemic amyloidosis, showing different levels of cytotoxicity and liposomes of DMPC and DMPG, changes in the secondary structure of the polymorphs and the structural state of phospholipid membranes caused by the interaction were investigated using vacuum-ultraviolet circular dichroism (VUVCD) and Laurdan fluorescence measurements, respectively. Analysis has shown that the more cytotoxic polymorph increases the antiparallel β-sheet content and causes more disorder in the membrane structure while the other less cytotoxic polymorph shows the opposite structural changes and causes less structural disorder in the membrane. These results suggest a close correlation between the structural properties of amyloid fibrils and the degree of structural disorder of phospholipid membranes, both of which are involved in the fundamental process leading to amyloid cytotoxicity.

Development of an in vitro aggregation assay for long synthetic polypeptide, amyloidogenic gelsolin fragment AGelD187N 173-242.

Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173-242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173-242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173-242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.

Fibrinogen Aα-chain amyloidosis outbreaks in Japanese squirrels (Sciurus lis): a potential disease model.

Fibrinogen Aα-chain amyloidosis is a hereditary systemic amyloidosis characterized by glomerular amyloid depositions, which are derived from the fibrinogen Aα-chain variant in humans. Despite its unique pathology, the pathogenic mechanisms of this disease are only partially understood. This is in part because comparative pathological studies on fibrinogen Aα-chain amyloidosis are currently unavailable as there is a lack of reported cases in animals other than humans. In this study, mass spectrometry-based proteomic analyses of Japanese squirrels (Sciurus lis) that died in five Japanese zoos showed that they developed glomerular-associated fibrinogen Aα-chain amyloidosis with an extremely high incidence rate (29/38 cases, 76.3%). The condition was found to be age-dependent in the Japanese squirrels, with 89% of individuals over 4 years of age affected. Mass spectrometry revealed that the C-terminal region of the fibrinogen Aα-chain was involved in amyloidogenesis in Japanese squirrels as well as humans. No gene variations were identified between amyloid-positive and amyloid-negative squirrels, which contrasted with the available data for humans. The results indicate that fibrinogen Aα-chain amyloidosis is a senile amyloidosis in Japanese squirrels. The results have also provided comparative pathological support that the amyloidogenic C-terminal region of the fibrinogen Aα-chain is involved in the characteristic glomerular pathology, regardless of the animal species. This study elucidates the potential causes of death in Japanese squirrels and will contribute to future comparative pathological studies of fibrinogen Aα-chain amyloidosis. © 2023 The Pathological Society of Great Britain and Ireland.

Hydrogen bonds connecting the N-terminal region and the DE loop stabilize the monomeric structure of transthyretin.

Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF β-sheets during the unfolding of the TTR monomer by stabilizing the interaction between β-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.

Pathogenic D76N Variant of β2-Microglobulin: Synergy of Diverse Effects in Both the Native and Amyloid States.

Simple SummaryElevated β2-microglobulin (β2m) serum levels cause serious complications in patients on long-term kidney dialysis by depositing in the form of amyloid fibrils in the osteoarticular system. Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m mutant exhibiting normal serum levels and a distinct, visceral deposition pattern. D76N β2m showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Despite the extensive research, the molecular bases of the aberrant aggregation of β2m in vivo remains elusive. Here, using a variety of biophysical techniques, we investigated the role of the pathogenic D76N mutation in the amyloid formation of β2m by point mutations affecting the stabilizing ion-pairs of β2m. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular matrix proteins. Understanding the underlying molecular mechanisms might help to find target points for effective treatments against diseases associated with the deleterious aggregation of proteins.β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.

Severe ocular involvement in hereditary gelsolin amyloidosis.

Hereditary gelsolin amyloidosis is a rare subtype of hereditary systemic amyloidosis. An old male presented with the characteristic triad of symptoms, including bilateral facial palsy, cutis laxa, and corneal lattice amyloidosis. The diagnosis was confirmed by the detection of the mutation in gelsolin gene located on chromosome 9. Although the presenting symptoms were highly suggestive of the disease, reports of severe ocular involvement are scarce in the literature.

The Clinical Impact of Proteomics in Amyloid Typing

The Clinical Impact of Proteomics in Amyloid Typing

SYSTEMIC AMYLOIDOSIS IN YOUNG MICE INDUCED BY HUMAN SALIVA ADMINISTRATION

The urgency of the study lies in the fact that for the first time it was proved in the experiment that intraperitoneal administration of human saliva to young mice causes the development of systemic amyloidosis, comparable to hereditary systemic amyloidosis in humans, induced by mutagenic changes in saliva lysozyme. The aim of the research: to test human saliva as an amyloidogen in a model experiment on young mice in comparison with the albumin model of amyloidosis that we developed earlier. White male mice weighing 20.0–25.0 g were divided into six groups: Intact mice (5); 30 days 1 time every other day were intraperitoneally administered to groups: 1 (5) soy cream substitute TU 9199-004-58706213-10 15 10 g/100 ml water 0.1 ml/10 g; 2 (3) saliva of a healthy person (SHP) 0.5 ml; 3 (3) SHP and Ciprofloxacin (C) 0.05 mg/10 g of weight; 4 (3) saliva of a person with chronic tonsillitis (remission, SCT); 5 (3) SCT+C. Kidney weight changed from 176.0±21 mg in intact mice (IM) to 197±43,0, 195,0±18,0, 195,0±18,0, 183,0±44,0, 153,0±25,0 mg, respectively. The number of cells per visual field of the tubular epithelium decreased by 1.3, 1.2, 1.6, 1.4, 1.4 times from the number of cells per visual field in IM 380.0±84.0 (p = 0.00), glomerular cells – by 1.4, 1.1, 1.3, 1.2, 1.2 times from the number of glomerular cells in IM 34.0±11.0 (p = 0.02), in groups 1-5, respectively. The glomerular area decreased by 2.4, 2.7, 2.3, 2.4, 3.0 times from the average glomerular area in IM (3163.7±832.7 mm2, p = 0.0000). The relative area of amyloid lesion when stained with Congo red: 18.4±8.2% (group 1), 26,7±10,6, 35,0±11,9, 45,7±13,2, 63,6±14,0% (groups 1-5). The spleen mass and length increased from 133.0±16.0 mg (IM) by 1.7, 1.7, 1.8, 2.1, 1.7 times and from 17.0±2.0 mm (IM) by 1.2, 1.2, 1.3, 1.4, 1.2 times, the number of cells per visual field decreased from 1318±205 (IM) by 1.7, 2.0, 3.3, 2.1, 2.0 times, groups 1-5, respectively (p < 0.01). The relative area of amyloid lesion in Congo red staining: 11,4±9,4%, 28,2±16,0, 27,9±20,8, 20,9±12,2, 16,5±8,3% (groups 1-5). The liver mass changed from 1.4±0.15 g (IM) to 1,57±0,26, 1,45±0,18, 1,53±0,1, 1,71±0,3, 1,36±0,4 g, the number of cells per visual field decreased from 67.0±15.0 (IM) by 1.9, 1.4, 1.8, 2.5, 1.9 times, the area of hepatocyte nuclei – from 54.0±12.3 µm (IM) by 3.1, 3.6, 4.2, 3.1, 3.3 times (groups 1-5, respectively, p < 0.01). The relative area of amyloid lesion in Congo red staining: 6,8±4,6%, 15,3±11,0, 24,5±12,8, 9,8±8,5, 12,6±2,3% (groups 1-5). Conclusions: 1) human saliva when administered intraperitoneally causes severe systemic amyloidosis in young mice with damage to the liver, kidneys and the spleen, which manifests more by cytotoxic effect than the volume of amyloid deposition; 2) human saliva contains a more active amyloidogen than albumin in combination with fillers of the soy cream substitute formulation; 2) human saliva of a person with chronic tonsillitis in remission is more amyloidogenic than the saliva of a healthy person; 3) Ciprofloxacin, administered in a therapeutic dose during the amyloid model formation, moderately increases the severity of amyloid organ damage; 4) human saliva can be used to simulate amyloidosis in an experiment on young mice; 5) the liver may contain enzymatic systems that perform the function of amyloidoclasia.

Plasma growth differentiation factor 15: a novel tool to detect early changes of hereditary transthyretin amyloidosis.

AimsHereditary transthyretin (ATTRv) amyloidosis is the most frequent and representative form of autosomal dominant hereditary systemic amyloidosis. Disease‐modifying treatments of the disease are more effective during the early stages, and we require biomarkers to detect early pathological changes for prompt diagnosis. This study aimed to investigate whether plasma growth differentiation factor 15 (GDF‐15) levels could aid detection of early pathological changes in ATTRv amyloidosis.Methods and resultsWe retrospectively studied 32 patients with ATTRv amyloidosis, eight asymptomatic TTR mutation carriers, and eight healthy volunteers. We evaluated plasma GDF‐15 levels in these subjects as related to levels of brain natriuretic peptide and high‐sensitivity troponin T, echocardiographic features, 99mTc‐pyrophosphate (PYP) scans, and cardiac magnetic resonance imaging findings. Plasma GDF‐15 levels significantly increased even in asymptomatic TTR mutation carriers compared with healthy volunteers (P < 0.01). Plasma GDF‐15 levels were significantly correlated with plasma brain natriuretic peptide values (P < 0.01), serum high‐sensitivity troponin T values (P < 0.05), and interventricular septal thickness at end‐diastole (P < 0.01) in patients with ATTRv amyloidosis. Plasma GDF‐15 levels in patients with PYP‐positive ATTRv amyloidosis were significantly higher than those in patients with PYP‐negative ATTRv amyloidosis (P < 0.01). Plasma GDF‐15 levels in patients with late gadolinium enhancement‐positive ATTRv amyloidosis were significantly higher than those in patients with late gadolinium enhancement‐negative ATTRv amyloidosis (P < 0.01). Groups of patients with different TTR genotypes manifested different plasma GDF‐15 levels.ConclusionsGrowth differentiation factor 15 may reflect early pathological changes of ATTRv amyloidosis.

The Early Phase of β2m Aggregation: An Integrative Computational Study Framed on the D76N Mutant and the ΔN6 Variant.

Human β2-microglobulin (b2m) protein is classically associated with dialysis-related amyloidosis (DRA). Recently, the single point mutant D76N was identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. To get insight into the early stage of the β2m aggregation mechanism, we used molecular simulations to perform an in depth comparative analysis of the dimerization phase of the D76N mutant and the ΔN6 variant, a cleaved form lacking the first six N-terminal residues, which is a major component of ex vivo amyloid plaques from DRA patients. We also provide first glimpses into the tetramerization phase of D76N at physiological pH. Results from extensive protein–protein docking simulations predict an essential role of the C- and N-terminal regions (both variants), as well as of the BC-loop (ΔN6 variant), DE-loop (both variants) and EF-loop (D76N mutant) in dimerization. The terminal regions are more relevant under acidic conditions while the BC-, DE- and EF-loops gain importance at physiological pH. Our results recapitulate experimental evidence according to which Tyr10 (A-strand), Phe30 and His31 (BC-loop), Trp60 and Phe62 (DE-loop) and Arg97 (C-terminus) act as dimerization hot-spots, and further predict the occurrence of novel residues with the ability to nucleate dimerization, namely Lys-75 (EF-loop) and Trp-95 (C-terminus). We propose that D76N tetramerization is mainly driven by the self-association of dimers via the N-terminus and DE-loop, and identify Arg3 (N-terminus), Tyr10, Phe56 (D-strand) and Trp60 as potential tetramerization hot-spots.

Binding of Monovalent and Bivalent Ligands by Transthyretin Causes Different Short- and Long-Distance Conformational Changes.

The wild type protein, transthyretin (TTR), and over 120 genetic TTR variants are amyloidogenic and cause, respectively, sporadic and hereditary systemic TTR amyloidosis. The homotetrameric TTR contains two identical thyroxine binding pockets, occupation of which by specific ligands can inhibit TTR amyloidogenesis in vitro. Ligand binding stabilizes the tetramer, inhibiting its proteolytic cleavage and its dissociation. Here, we show with solution-state NMR that ligand binding induces long-distance conformational changes in the TTR that have not previously been detected by X-ray crystallography, consistently with the inhibition of the cleavage of the DE loop. The NMR findings, coupled with surface plasmon resonance measurements, have identified dynamic exchange processes underlying the negative cooperativity of binding of “monovalent” ligand tafamidis. In contrast, mds84, our prototypic “bivalent” ligand, which is a more potent stabilizer of TTR in vitro that occupies both thyroxine pockets and the intramolecular channel between them, has greater structural effects.

Hereditary renal amyloidosis with a variant lysozyme p.Trp82Arg in a Chinese family: case report and literature review

BackgroundLysozyme amyloidosis is a rare hereditary systemic amyloidosis with amyloid deposits in various tissues leading to progressive organ failure. It has been mainly reported in developed countries since 1993. Here we report a lysozyme amyloidosis family with variant lysozyme p.Trp82Arg in a Chinese family.Case presentationThe main clinical manifestation of this case was dominant kidney involvement presenting with proteinuria and decreased renal function. Biopsy of the kidney showed massive amyloid deposits in the glomerular mesangium and subendothelium. Immunohistochemistry and mass spectrometry of renal tissue confirmed the lysozyme nature of the amyloid. DNA sequencing of the peripheral blood leukocytes revealed a single base-pair transition from T to C (TGG/ CGG) of codon 82, leading to the replacement of tryptophan by arginine in the mature protein (p.Trp82Arg). The affected patients in this family also presented with dominant kidney involvement, one of them has been confirmed by IHC and mass spectrometry on his renal biopsy and gene testing as well. As there is no radical therapy for lysozyme amyloidosis, patients were given symptomatic treatment such as antihypertensive drugs and antibiotics. To our knowledge, this is the first report of lysozyme amyloidosis in a Chinese family.ConclusionsHereditary amyloidosis with a variant lysozyme of p.Trp82Arg presented with dominant kidney involvement was firstly reported in a Chinese family.

Hereditary systemic transthyretin amyloidosis: a clinical case and an opinion on the problem

This article presents a clinical case of a patient with hereditary systemic hereditary transthyretin amyloidosis. Difficulties of diagnostics and differential diagnostics of this disease are shown in a patient with arterial hypertension, a clinic of angina, coronary artery disease, cardiac rhythm and conduction disturbances, cardiomegaly, heart failure and renal dysfunction. It required a comprehensive approach using the methods of routine clinical and laboratory examination, as well as nuclear cardiology, morphological studies and molecular DNA diagnostics.

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.

A transgenic mouse model reproduces human hereditary systemic amyloidosis

Amyloidoses are rare life-threatening diseases caused by protein misfolding of normally soluble proteins. The fatal outcome is predominantly due to renal failure and/or cardiac dysfunction. Because amyloid fibrils formed by all amyloidogenic proteins share structural similarity, amyloidoses may be studied in transgenic models expressing any amyloidogenic protein. Here we generated transgenic mice expressing an amyloidogenic variant of human apolipoprotein AII, a major protein of high density lipoprotein. According to amyloid nomenclature this variant was termed STOP78SERApoAII. STOP78SER-APOA2 expression at the physiological level spontaneously induced systemic amyloidosis in all mice with full-length mature STOP78SER-ApoAII identified as the amyloidogenic protein. Amyloid deposits stained with Congo red were extracellular, and consisted of fibrils of approximately 10 nm diameter. Renal glomerular amyloidosis was a major feature with onset of renal insufficiency occurring in mice older than six months of age. The liver, heart and spleen were also greatly affected. Expression of STOP78SER-APOA2 in the liver and intestine in mice of the K line but not in other amyloid-laden organs showed they present systemic amyloidosis. The amyloid burden was a function of STOP78SER-APOA2 expression and age of the mice with amyloid deposition starting in two-month-old high-expressing mice that died from six months onwards. Because STOP78SER-ApoAII conserved adequate lipid binding capacity as shown by high STOP78SER-ApoAII amounts in high density lipoprotein of young mice, its decrease in circulation with age suggests preferential deposition into preformed fibrils. Thus, our mouse model faithfully reproduces early-onset hereditary systemic amyloidosis and is ideally suited to devise and test novel therapies.

Antiamyloid activity of functionalized cerium oxide nanoparticle on lysozyme fibrillation: Spectroscopic and microscopic investigation

Accumulation of amyloid fibrils of proteins in different organs is a pathological indication for various neurogenerative disorders. Recently nanotechnology is at the centre of interest to tackle with those disorders. In this study we have chosen hen egg white lysozyme as our model protein which is responsible for hereditary systemic Amyloidosis disease. Here we have reported effect of cerium oxide nanoparticle (CeONP), folic acid functionalized CeONP (FA-CeONP) and polyacrylic acid functionalized CeONP (PAA-CeONP) (all of them having size ∼18 nm) on fibrillation of hen egg white lysozyme (Lyz). The amyloid growth is monitored by Thioflavin T (ThT), Congo Red (CR), 8-anilinonapthalene-1-sulfonic acid (ANS) assay, time resolved fluorescence anisotropy, intrinsic fluorescence, circular dichroism, atomic force microscopy and fluorescence microscopy study. Results demonstrate that all nanoparticle (NP) shows antiamyloid activity by broadening lag phase and suppressing growth phase of Lyz amyloid growth, but PAA-CeONP shows maximum inhibitory effect. These bare and functionalized CeONP shows dose dependent antiamyloid activity. We have also demonstrated that such inhibition of amyloid formation is associated with reduction of β-sheet content of protein. The inhibition efficiency is quantified by half maximal inhibition concentration IC50 values. A mechanistic pathway is explained on the basis of electrostatic interaction between NP and Lyz by ζ- potential measurement. This interaction is also analyzed theoretically by docking analysis. Thus our study provides a basis for use of CeONP as a promising candidate in therapeutic application in amyloid related disorders.

On-Line Immunoaffinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Serum Transthyretin.

The analysis of low abundant proteins in biological fluids by capillary electrophoresis (CE) is particularly problematic due to the typically poor concentration limits of detection of microscale separation techniques. Another important issue is sample matrix complexity that requires an appropriate cleanup. Here, we describe an on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS) method for the immunoextraction, preconcentration, separation, detection, and characterization of serum transthyretin (TTR). TTR is a protein biomarker related to diverse types of amyloidosis, such as familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis.

Development of transgenic Caenorhabditis elegans expressing human transthyretin as a model for drug screening

Familial amyloid polyneuropathy is a hereditary systemic amyloidosis caused by a mutation in the transthyretin (TTR) gene. Amyloid deposits in tissues of patients contain not only full-length TTR but also C-terminal TTR fragments. However, in vivo models to evaluate the pathogenicity of TTR fragments have not yet been developed. Here, we generated transgenic Caenorhabditis elegans strains expressing several types of TTR fragments or full-length TTR fused to enhanced green fluorescent protein in the body wall muscle cells and analyzed the phenotypes of the worms. The transgenic strain expressing residues 81–127 of TTR, which included the β-strands F and H, formed aggregates and caused defective worm motility and a significantly shortened lifespan compared with other strains. These findings suggest that the C-terminal fragments of TTR may contribute to cytotoxicity of TTR amyloidosis in vivo. By using this C. elegans model system, we found that (−)-epigallocatechin-3-gallate, a major polyphenol in green tea, significantly inhibited the formation of aggregates, the defective motility, and the shortened lifespan caused by residues 81–127 of TTR. These results suggest that our newly developed C. elegans model system will be useful for in vivo pathological analyses of TTR amyloidosis as well as drug screening.

Recent Advances in Oligonucleotide-Based Therapy for Transthyretin Amyloidosis: Clinical Impact and Future Prospects.

Transthyretin (TTR) amyloidosis, also known as transthyretin-related familial amyloidotic polyneuropathy (ATTR-FAP), is a fatal hereditary systemic amyloidosis caused by mutant forms of TTR. Although conventional treatments for ATTR-FAP, such as liver transplantation (LT) and TTR tetramer stabilizer, reportedly halt the progression of clinical manifestation, these therapies have several limitations. Oligonucleotide-based therapy, e.g. small interfering RNA (siRNA)- and antisense oligonucleotides (ASOs)-based therapy, hold enormous potential for the treatment of intractable diseases such as ATTR-FAP, by specifically regulating the gene responsible for the disease. Clinical evidence strongly suggests that LT inhibits mutant TTR production, thus improving the manifestation of ATTR-FAP. Therefore, an oligonucleotide-based therapy for ATTR-FAP, which reduces the production of TTR by the liver, has recently been developed in preclinical and clinical studies. This review focuses on recent advances in oligonucleotide-based therapy and future prospects of next-generation oligonucleotide-based drugs for therapeutic use against ATTR-FAP.

Membrane interactions of fibrillar lysozyme: Effect of lipid bilayer composition

Protein polymerization into amyloid fibrils underlies a number of human disorders, including hereditary systemic amyloidosis arising from fibrillization of the mutated lysozyme. Cell membranes represent one of the main targets for the toxic action of amyloid aggregates. In the present study the impact of monomeric and fibrillar lysozyme on the structural state and physicochemical properties of the model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin and cholesterol was investigated using the fluorescence spectroscopy technique. Probing the protein-lipid interactions with the fluorescent probes Laurdan and pyrene showed that both forms of lysozyme give rise to the dehydration of lipid bilayer and decrease of its free volume, but the magnitude of these effects was much more pronounced for the fibrillar protein. The membrane-modifying propensity of lysozyme was found to be strongly modulated by the composition of lipid bilayer, with the membrane responses being enhanced by cardiolipin and diminished by cholesterol.