Induction of protein conformational change in mouse senile amyloidosis

Aggregated amyloid fibrils can induce further polymerization of precursor proteins in vitro, thus providing a possible basis for propagation or transmission in the pathogenesis of amyloidoses. Previously, we postulated that the transmission of amyloid fibrils induces conformational changes of endogenous amyloid protein in mouse senile amyloidosis (Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo Z., Hosokawa, M., Mori, M., and Higuchi, K. (2001) Lab. Invest. 81, 493-499). To further characterize this transmissibility, we injected amyloid fibrils (AApoAII(C)) of amyloidogenic C type apolipoprotein A-II (APOAIIC) intravenously into 2-month-old SAMR1 mice, which have B type apolipoprotein A-II (APOAIIB), and develop few if any amyloid deposits spontaneously. 10 months after amyloid injection, deposits were detected in the tongue, stomach, intestine, lungs, heart, liver, and kidneys. The intensity of deposition increased thereafter, whereas no amyloid was detected in distilled water-injected SAMR1 mice, even after 20 months. The deposited amyloid was composed of endogenous APOAIIB with a different amyloid fibril conformation. The injection of these amyloid fibrils of APOAIIB (AApoAII(B)) induced earlier and more severe amyloidosis in SAMR1 mice than the injection of AApoAII(C) amyloid fibrils. Thus, AApoAII(C) from amyloidogenic mice could induce a conformational change of less amyloidogenic APOAIIB to a different amyloid fibril structure, which could also induce amyloidosis in the less amyloidogenic strain. These results provide important insights into the pathogenesis of amyloid diseases.     

Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1

We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.     

Deposition of amyloid in the liver of hamsters: an enzyme-histochemical and electron-microscopical study.

In induced amyloidosis the amyloid was first deposited in the portal areas, then in the centre of the lobule and disseminated through it in the spaces of Disse. No intracellular amyloid was found in the predeposit phase. In the hepatic lobule both mononuclear phagocytes and hepatocytes showed a topographic relationship to the first deposits of amyloid. Some macrophages showed invaginations or vacuoles containing amyloid fibrils. Between the microvilli of hepatocytes, parallel amyloid fibrils occurred. Between the amyloid fibrils were 30-50 nm membrane-bounded spherical particles which may have been lipoprotein aggregates. There was no large-scale phagocytosis of amyloid during the induction period or after survival without casein treatment up to 18 weeks. Lysosomal enzyme activity was seen in the deposits of extracellular amyloid.     

Pathogenesis, diagnosis and treatment of systemic amyloidosis

Amyloidosis is a disorder of protein folding in which normally soluble proteins are deposited as abnormal, insoluble fibrils that disrupt tissue structure and cause disease. Although about 20 different unrelated proteins can form amyloid fibrils in vivo, all such fibrils share a common cross-beta core structure. Some natural wild-type proteins are inherently amyloidogenic, form fibrils and cause amyloidosis in old age or if present for long periods at abnormally high concentration. Other amyloidogenic proteins are acquired or inherited variants, containing amino-acid substitutions that render them unstable so that they populate partly unfolded states under physiological conditions, and these intermediates then aggregate in the stable amyloid fold. In addition to the fibrils, amyloid deposits always contain the non-fibrillar pentraxin plasma protein, serum amyloid P component (SAP), because it undergoes specific calcium-dependent binding to amyloid fibrils. SAP contributes to amyloidogenesis, probably by stabilizing amyloid fibrils and retarding their clearance. Radiolabelled SAP is an extremely useful, safe, specific, non-invasive, quantitative tracer for scintigraphic imaging of systemic amyloid deposits. Its use has demonstrated that elimination of the supply of amyloid fibril precursor proteins leads to regression of amyloid deposits with clinical benefit. Current treatment of amyloidosis comprises careful maintenance of impaired organ function, replacement of end-stage organ failure by dialysis or transplantation, and vigorous efforts to control underlying conditions responsible for production of fibril precursors. New approaches under development include drugs for stabilization of the native fold of precursor proteins, inhibition of fibrillogenesis, reversion of the amyloid to the native fold, and dissociation of SAP to accelerate amyloid fibril clearance in vivo.     

Enhancement of AA-amyloid formation in mice by transthyretin amyloid fragments and polyethylene glycol

The mechanism behind amyloid formation is unknown in all types of amyloidosis. Several substances can enhance amyloid formation in animal experiments. To induce secondary systemic amyloid (AA-type amyloid) formation, we injected silver nitrate into mice together with either amyloid fibrils obtained from patients with familial polyneuropathy (FAP) type I or polyethylene glycol (PEG). Mice injected with silver nitrate only served as controls. Amyloid deposits were detectable at day 3 in animals injected with amyloid fibrils and in those injected with PEG, whereas in control mice, deposits were not noted before day 12. Our results indicate that amyloid fibrils from FAP patients and even a non-sulfate containing polysaccharide (PEG) have the potential to act as amyloid-enhancing factors.     

Colocalization of apolipoprotein AI in various kinds of systemic amyloidosis.

Apolipoprotein AI (apoAI), a major component of high-density lipoproteins, is one of the major amyloid fibril proteins and a minor constituent of the senile plaques observed in Alzheimer's disease. We examined colocalization of apoAI in various kinds of systemic amyloidosis in this study. Forty-three of 48 formalin-fixed paraffin-embedded heart specimens with various forms of systemic amyloidosis reacted immunohistochemically with anti-human apoAI antibody. ApoAI was also detected in water-extracted amyloid material by immunoblotting. In addition, we observed colocalization of apoAI and murine amyloid A (AA) amyloidosis in human apoAI transgenic mice. This is the first report of colocalization of apoAI with amyloid deposits in various forms of human systemic amyloidosis and murine AA amyloidosis in human apoAI transgenic mice. ApoAI may not always be a major component of amyloid fibrils, even when it is present in systemic amyloid deposits.     

Islet amyloid development in a mouse strain lacking endogenous islet amyloid polypeptide (IAPP) but expressing human IAPP

BACKGROUND: Several mouse strains expressing human islet amyloid polypeptide (IAPP) have been created to study development of islet amyloid and its impact on islet cell function. The tendency to form islet amyloid has varied strongly among these strains by factors that have not been elucidated. Because some beta cell granule components are known to inhibit IAPP fibril formation in vitro, we wanted to determine whether a mouse strain expressing human IAPP but lacking the nonamyloidogenic mouse IAPP is more prone to develop islet amyloidosis. MATERIALS AND METHODS: Such a strain was created by cross-breeding a transgenic mouse strain and an IAPP null mouse strain. RESULTS: When fed a fat-enriched diet, male mice expressing only human IAPP developed islet amyloid earlier and to a higher extent than did mice expressing both human and mouse IAPP. Supporting these results, we found that mouse IAPP dose-dependently inhibits formation of fibrils from human IAPP. CONCLUSIONS: Female mice did not develop amyloid deposits, although small extracellular amorphous IAPP deposits were found in some islets. When cultivated in vitro, amyloid deposits occurred within 10 days in islets from either male or female mice expressing only human IAPP. The study shows that formation of islet amyloid may be dependent on the environment, including the presence or absence of fibril inhibitors or promoters.     

Senile cardiac amyloid: demonstration of a unique fibril protein in tissue sections.

Antisera were raised against degrading amyloid fibrils isolated from the heart of a patient with senile cardiac amyloidosis (SCA), and from a medullary carcinoma of the thyroid (MCT). The antisera were absorbed and used in indirect immunofluorescence to identify an amyloid fibril protein (ASCA) in heart tissue from patients with senile cardiac amyloidosis and to identify the amyloid fibril protein (AMCT) found in association with medullary carcinomas of the thyroid. Absorbed anti-ASCA antiserum did not react with normal tissue such as heart, liver, spleen, and striated muscle, or with amyloid tissue known to contain amyloid fibril proteins AA, AlambdaI, AlambdaIV, AlambdaV, AMCT or with pancreatic tissue containing islet amyloid deposits. The reactions with senile amyloid he,rt tissue could be blocked completely by degraded amyloid fibrils extracted from senile amyloid heart tissue or by amyloid fibril protein ASCA isolated from such fibrils. The anti-AMCT antiserum showed a similar specific reaction restricted to amyloid associated with MCT. In addition, antisera specific for amyloid fibril proteins AA, AlambdaI, AlambdaIV, and AlambdaV failed to react with senile cardiac amyloid, pancreatic islet amyloid, or medullary thyroid amyloid.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

Amyloid protein in familial amyloidosis (Finnish type) is homologous to gelsolin, an actin-binding protein

Familial amyloidosis, Finnish type, is clinically characterized by cranial neuropathy and lattice corneal dystrophy. It is an autosomal dominant form of systemic amyloidosis with small deposits of congophilic material occurring in most tissues, particularly in association with blood vessel walls and basement membranes. Amyloid fibrils were extracted from the kidney of patient VUO, and rabbit antiserum raised against the 12 kDa purified amyloid subunit displayed strong immunohistochemical reactivity with the amyloid deposits. The amino terminal sequence of this 12 kDa amyloid protein (ATEVPVSWESFNNGD) showed homology with gelsolin (or actin depolymerizing factor), a 93 kDa plasma protein. The amyloid peptide is a degradation product, starting at position 173, of the gelsolin molecule.     

Flow-induced alignment of amyloid protofilaments revealed by linear dichroism

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi* transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.     

Fibrillar islet amyloid polypeptide (amylin) is internalised by macrophages but resists proteolytic degradation

Pancreatic islet amyloid, formed from islet amyloid polypeptide, is found in 96% of Type II (non-insulin-dependent) diabetic patients. Islet amyloidosis is progressive and apparently irreversible. Fibrils immunoreactive for islet amyloid polypeptide are found in macrophages associated with amyloid, suggesting that deposits can be phagocytosed. To determine the mechanism for the recognition and internalisation of fibrils, mouse peritoneal macrophages were cultured with fibrillar synthetic human islet amyloid polypeptide. Fibrils did not exert a cytotoxic effect over 72 h of culture. The uptake and degradation of fibrils was analysed by quantitative light-and electron-microscopic immunocytochemistry and immunoreactivity was detectable in 86±3% cells within 6 h of culture. Neither polyinosinic acid (200 µg/ml) nor nocodazole (10 µg/ml) inhibited fibril uptake, suggesting that internalisation is not blocked by poly-ions and is independent of microtubule assembly. Inhibition of pseudopodia formation by cytochalasin B blocked fibriI uptake. Fibril aggregates became condensed in lysosomes to form protofilaments and were resistant to intracellular proteolysis. Fibrils can be phagocytosed by macrophages in vitro but amyloid-associated factors may block the recognition of fibrils in vivo preventing the removal of islet amyloid in diabetes.     

Amyloid Fibrils Trigger the Release of Neutrophil Extracellular Traps (NETs), Causing Fibril Fragmentation by NET-associated Elastase

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.     

Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling

The human heart can be frequently affected by an organ-limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long-standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo-red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10-nm-thick filaments were strictly extra-cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.     

Apolipoprotein A-I induced amyloidosis

Amyloidosis is characterized by extracellular deposits of protein fibrils with a high content of β-sheets in secondary structure. The protein forms together with proteoglycans amyloid fibrils causing organ damage and serious morbidity. Intact apolipoprotein A-I (apoA-I) is an important protein in lipid metabolism regulating the synthesis and catabolism of high density lipoproteins (HDL). Usually, apoA-I is not associated with amyloidosis. However, four naturally occuring mutant forms of apoA-I are known so far resulting in amyloidosis. The most important feature of all variants is the very similar formation of N-terminal fragments which were found in the amyloid deposits (residues 1–83 to 1–94). The new insights in the understanding of the association of apoA-I with HDL, its metabolism, and its hypothesized structural findings may explain a common mechanism for the genesis of apoA-I induced amyloidosis. Here we summarized the specific features of all known amyloidogenic variants of apoA-I and speculate about its metabolic pathway, which may have general implications for the metabolism of apoA-I.     

Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation

Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.     

MALDI-Mass Spectrometry Imaging Identifies Vitronectin as a Common Constituent of Amyloid Deposits

Amyloids are pathological intra- and extracellular fibrillar aggregates of polypeptides with a cross-β-sheet structure and characteristic tinctorial properties. The amyloid deposits commonly enclose several non-fibrillar components of the extracellular matrix. Their potential to regulate the formation and aggregation process of amyloid fibrils is still poorly understood. For a better understanding of the role of the extracellular matrix in amyloidosis, it is essential to gain deeper insights into the composition of amyloid deposits. Here, we utilized matrix-assisted laser desorption and ionization mass spectrometry imaging to identify extracellular matrix compounds in amyloid deposits. Using this technique, we identified and determined the spatial distribution of vitronectin within AApoAI-, ALλ-, ATTR- and AIns amyloid deposits and, using immunohistochemistry, validated the spatial overlap of vitronectin with amyloids in 175 cases with diverse types of amyloid in several different tissues.     

Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology.

Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet beta-cells and converted to amyloid deposits in type 2 diabetes. Conversion from soluble monomer, IAPP 1-37, to beta-sheet fibrils involves changes in the molecular conformation, cellular biochemistry and diabetes-related factors. In addition to the recognised amyloidogenic region, human IAPP (hIAPP) 20-29, the peptides human or rat IAPP 30-37 and 8-20, assume beta-conformation and form fibrils. These three amyloidogenic regions of hIAPP can be modelled as a folding intermediate with an intramolecular beta-sheet. A hypothesis is proposed for co-secretion of proIAPP with proinsulin in diabetes and formation of a 'nidus' adjacent to islet capillaries for subsequent accumulation of secreted IAPP to form the deposit. Although intracellular fibrils have been identified in experimental systems, extracellular deposition predominates in animal models and man. Extensive fibril accumulations replace islet cells. The molecular species of IAPP that is cytotoxic remains controversial. However, since fibrils form invaginations in cell membranes, small non-toxic IAPP fibrillar or amorphous accumulations could affect beta-cell stimulus-secretion coupling. The level of production of hIAPP is important but not a primary factor in islet amyloidosis; there is little evidence for inappropriate IAPP hypersecretion in type 2 diabetes and amyloid formation is generated in transgenic mice overexpressing the gene for human IAPP only against a background of obesity. Animal models of islet amyloidosis suggest that diabetes is induced by the deposits whereas in man, fibril formation appears to result from diabetes-associated islet dysfunction. Islet secretory failure results from progressive amyloidosis which provides a target for new therapeutic interventions.     

Amyloidosis: new strategies for treatment

Amyloidosis is a disorder of protein folding in which normally soluble proteins are deposited extracellularly as insoluble fibrils, impairing tissue structure and function. Over 20 unrelated proteins form amyloid fibrils in vivo, with fibrils sharing a lamellar cross-β sheet structure, composed of non-covalently associated protein or peptide subunits. Amyloidosis may be acquired or hereditary and local or systemic, and is defined according to the precursor protein. Of note, local amyloid deposition occurs in Alzheimer’s disease (AD) and maturity onset diabetes but their precise role in the pathogenesis of these diseases remains uncertain. Glycosaminoglycans (GAG) and the pentraxin protein, serum amyloid P (SAP) component, are universal non-fibrillar constituents of amyloid deposits that contribute to fibrillogenesis. We review potential therapies for amyloidosis, which include measures to reduce the production of amyloidogenic precursor proteins, interference with fibrillogenesis, and enhancement of amyloid clearance, either by active or passive immunisation or by destabilising deposits through removal of serum amyloid P component.     

The protofilament substructure of amyloid fibrils

Tissue deposition of normally soluble proteins, or their fragments, as insoluble amyloid fibrils causes the usually fatal, acquired and hereditary systemic amyloidoses and is associated with the pathology of Alzheimer's disease, type 2 diabetes and the transmissible spongiform encephalopathies. Although each type of amyloidosis is characterised by a specific amyloid fibril protein, the deposits share pathognomonic histochemical properties and the structural morphology of all amyloid fibrils is very similar. We have previously demonstrated that transthyretin amyloid fibrils contain four constituent protofilaments packed in a square array. Here, we have used cross-correlation techniques to average electron microscopy images of multiple cross-sections in order to reconstruct the sub-structure of ex vivo amyloid fibrils composed of amyloid A protein, monoclonal immunoglobulin lambda light chain, Leu60Arg variant apolipoprotein AI, and Asp67His variant lysozyme, as well as synthetic fibrils derived from a ten-residue peptide corresponding to the A-strand of transthyretin. All the fibrils had an electron-lucent core but the packing arrangement comprised five or six protofilaments rather than four. The structural similarity that defines amyloid fibres thus exists principally at the level of beta-sheet folding of the polypeptides within the protofilament, while the different types vary in the supramolecular assembly of their protofilaments.