Amyloidotic Mice Research Articles

AA amyloid fibrils from diseased tissue are structurally different from in vitro formed SAA fibrils

Systemic AA amyloidosis is a world-wide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein. Using cryo electron microscopy we here show that amyloid fibrils which were purified from AA amyloidotic mice are structurally different from fibrils formed from recombinant SAA protein in vitro. Ex vivo amyloid fibrils consist of fibril proteins that contain more residues within their ordered parts and possess a higher β-sheet content than in vitro fibril proteins. They are also more resistant to proteolysis than their in vitro formed counterparts. These data suggest that pathogenic amyloid fibrils may originate from proteolytic selection, allowing specific fibril morphologies to proliferate and to cause damage to the surrounding tissue.

Amyloid persistence in decellularized liver: biochemical and histopathological characterization

Systemic amyloidoses are a group of debilitating and often fatal diseases in which fibrillar protein aggregates are deposited in the extracellular spaces of a range of tissues. The molecular basis of amyloid formation and tissue localization is still unclear. Although it is likely that the extracellular matrix (ECM) plays an important role in amyloid deposition, this interaction is largely unexplored, mostly because current analytical approaches may alter the delicate and complicated three-dimensional architecture of both ECM and amyloid. We describe here a decellularization procedure for the amyloidotic mouse liver which allows high-resolution visualization of the interactions between amyloid and the constitutive fibers of the extracellular matrix. The primary structure of the fibrillar proteins remains intact and the amyloid fibrils retain their amyloid enhancing factor activity.

Tissue remodeling after RNAi-mediated knockdown of TTR in a Familial Amyloidotic Polyneuropathy mouse model

Background Transthyretin (TTR) deposition in the peripheral nervous system (PNS) is the hallmark of Familial Amyloidotic Polyneuropathy (FAP). Mice expressing human TTR with the V30M mutation in a heterozygous heat shock factor 1 (Hsf-1) background show extensive TTR deposits in PNS and gastrointestinal tract, as well as extracellular matrix (ECM) remodeling, similar to those seen in human FAP patients. Currently, liver transplantation is the only available treatment to halt the progression of clinical symptoms in FAP. Due to the limitations of this procedure, it is of utmost importance to develop alternative therapeutic strategies. In this regard, an RNAi therapeutic targeting TTR for the treatment of FAP is currently in Phase 3 clinical development. An ongoing phase 2 clinical trial in FAP patients demonstrated promising results as a mean plasma TTR reduction of 80%, sustained for over nine months, led to a decrease in neuropathy progression compared to historical data.

Generation and Characterization of Anti-AA Amyloid-Specific Monoclonal Antibodies

AA amyloidosis results from the pathologic deposition in the kidneys and other organs of fibrils composed of N-terminal fragments of serum amyloid A protein (SAA). Given that there are only limited means to visualize these deposits, we have developed a series of mAbs, 2A4, 7D8, and 8G9, that bind specifically with nanomolar affinity to a carboxy-terminal epitope generated following proteolysis of SAA that yields the predominant component of AA amyloid deposits. Notably, these antibodies do not recognize native SAA, they retain their immunoreactivity when radiolabeled with I-125 and, after injection into AA amyloidotic mice, localize, as evidenced by autoradiography and micro-single photon emission computed tomography imaging, to histologically confirmed areas of amyloid deposition; namely, spleen, liver, and pancreas. The results of our in vitro and in vivo studies demonstrate the AA fibril-selectivity of mAbs 2A4, 7D8, and 8G9 and warrant further investigation into their role as novel diagnostic agents for patients with AA amyloidosis.

Amyloid-enhancing factor mediates amyloid formation on fibroblasts via a nidus/template mechanism.

To determine the mechanism by which amyloid-enhancing factor (AEF) promotes amyloid deposition, and to test whether AEF seeds deposition of serum amyloid A (SAA) and facilitates conversion to beta-sheet structure. Fibroblasts were cultured with mouse recombinant SAA1.1 and AEF, SAA1.1, or AEF. AEF was prepared as a glycerol extract of spleen from amyloidotic mice. Amyloid was identified by staining with Congo red and examining for green birefringence under polarized light. SAA was localized immunohistochemically. Texas Red-labeled SAA was visualized in living cultures by fluorescence confocal microscopy. AEF was characterized by Western blot analysis using anti-SAA antiserum and N-terminal sequence analysis. Subunits comprising amyloid in fibroblast cultures were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyloid was produced in fibroblast cultures by an AEF-dependent mechanism. AEF, added to culture medium as insoluble protein precipitates, adhered to fibroblast monolayers. SAA bound preferentially to the adherent precipitates. Coincident with SAA binding, precipitates developed an affinity for Congo red. Over time, as more SAA was added, networks of Congo red-positive material producing bright green birefringence also developed outward from AEF precipitates. Amyloid built upon AEF in this manner was composed of full-length SAA. No amyloid was produced in cultures treated with either SAA or AEF alone. SAA and SAA peptides processed in the C-terminal region were the most prominent proteins in the glycerol-extracted AEF preparation. AEF binds to fibroblast monolayers and acts as a sink for SAA. SAA that collects on AEF assembles into an amyloid structure. Thus, it is concluded that AEF serves as both a nidus and a template for amyloid formation.

Pregnancy and amyloidogenesis: I. Offspring of amyloidotic mice are not predisposed to develop amyloidosis

Amyloid enhancing factor (AEF) is a substance formed during amyloidogenesis that accelerates amyloid deposition in tissues. The administration of AEF followed by AgNO3 (inflammatory stimulus) to mice results in amyloidosis within 6 days. The purpose of the study was to determine whether the offspring of amyloidotic mice are exposed to maternal AEF during pregnancy and therefore become predisposed to the development of amyloidosis on inflammatory stimulus. To that end female mice were made amyloidotic by the administration of AEF and AgNO3, made pre-amyloidotic (a condition associated with self-generation of AEF) with a short course of casein, or treated with exogenous AEF without AgNO3; then mating and conception took place. The possible priming of offspring with maternal AEF was studied by the administration of AgNO3 alone (without AEF) to the offspring and the determination of the presence of amyloid deposits in their spleens. Despite the long-term effect of AEF and its high activity, amyloidosis did not develop in any of the studied offspring, implying that the newborn mice were not primed by maternal AEF. These findings suggest that amyloidotic mothers do not predispose their offspring to the risk of developing amyloidosis, probably because maternal AEF does not cross the placenta.

Immunolocalization of serum amyloid A and AA amyloid in lysosomes in murine monocytoid cells: confocal and immunogold electron microscopic studies.

Murine AA amyloid (AA) protein represents the amino-terminal two-third portion of SAA2, one of the isoforms of serum amyloid A. Whether plasma membrane-bound or lysosomal enzymes in activated murine monocytoid cells degrade SAA2 to generate amyloidogenic AA-like peptides is not clearly understood, although AA has been localized in the lysosomes. Here we show, using confocal and immunogold microscopy (IEM), that both SAA and AA localize in lysosomes of activated monocytoid cells from amyloidotic mice. Rabbit anti-mouse AA IgG (RAA) and two monoclonal antibodies against murine lysosome-associated membrane proteins (LAMP-1 and LAMP-2) were used to immunolocalize SAA/AA and lysosomes, respectively. Confocal analysis co-localized both anti-RAA and anti-LAMP-1/LAMP-2 reactivities in the perikaryal organelles which by IEM proved to be electron-dense lysosomes. LAMP-1/LAMP-2-specific gold particles were also localized on lysosomal and perikaryal AA. The results suggest sequestration of SAA into the lysosomes. Since monocytoid cells are not known to phagocytose native amyloid fibrils, our results implicate lysosomes in AA formation.

Intraperitoneal amyloid formation by amyloid enhancing factor--rich macrophages in ascitic fluid.

Although resident peritoneal cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing the precursor protein of secondary amyloidosis, serum amyloid A (SAA) to amyloid fibrils, the peritoneum is a rare site for amyloid deposition. This is considered to be due to a deficiency of SAA in the peritoneum. To increase the supply of SAA to the peritoneum, ascitic fluid containing about the same protein constituents as in the serum was induced in mice. Amyloidotic peritoneal cells were packed in a microchamber which was shielded with filter membranes, and cultured in ascitic fluid supplemented with additional inflammatory factors. On the 7th day, Congo red-positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or -SAA immunostaining, amyloid deposits and the cell surfaces of macrophages were positive. Immunologic depletion of T- and B-lymphocytes from the amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers. These results suggest that either ascitic fluid containing sufficient amounts of SAA, or peritoneal macrophages with a high amyloid enhancing factor (AEF) activity are indispensable for AA amyloid fibrillogenesis in the peritoneum.

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Although peritoneal resident cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing serum amyloid A (SAA) to amyloid fibrils, the original residence peritoneum is a rare place of amyloid deposition. This is considered to occur due to deficiency of blood supply in the peritoneum. To increase blood supply in the peritoneum, ascitic fluid which contained about the same proteins as those in the serum was induced in mice.Amyloidotic peritoneal cells were packed in a microchamber and cultured in ascites with additional inflammatory stimuli which increased the SAA concentration of ascites to about 1% of the highest serum level.On the 7 th day, Congo red positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or-SAA immunostaining, amyloid deposits as well as cell surface of adjucent macrophages were positively stained. Immunologic depletion of T and B lymphocytes from amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers.These results suggest that both ascitic fluid containing sufficient amount of SAA and peritoneal macrophages with high AEF activity are indispensable agents for AA amyloid fibrillogenesis in the peritoneum.

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis.

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.

Binding of ubiquitin to experimentally induced murine AA amyloid

Amyloid enhancing factor (AEF) activity has recently been demonstrated in ubiquitin purified from amyloidotic murine tissues and Alzheimer brain extract. Since AEF is known to bind to amyloid fibrils and 'fibril-AEF' on passive transfer induces accelerated amyloidogenesis in the recipient animals, it was of interest to investigate whether ubiquitin binds to amyloid. Immunohistological studies were carried out on liver sections from amyloidotic mice. Biotin-strepavidin-peroxidase methods using monospecific rabbit anti-mouse AA amyloid IgG (RAAG) and rabbit anti-bovine ubiquitin IgG (RABU) antibodies were employed to immunostain the amyloid and ubiquitin deposits, respectively. RABU-treated liver sections were counterstained with thioflavine S. RAAG reacted strongly with the amyloid, indicating that it is AA type, and RABU-positive immunodeposits were found bound to the thioflavine-S-positive AA deposits. Treatment of the liver sections with 0.1 M sodium acetate containing 0.5 M NaCl, pH 4, for 2-3 h at 37 degrees C nearly completely desorbed the AA amyloid-bound ubiquitin. Since ubiquitin demonstrates AEF activity in vivo and binds non-covalently to AA amyloid, we suggest that ubiquitin may indeed be 'fibril-AEF' and may play a crucial role in the pathogenesis of amyloidosis. To our knowledge, this is the first time that ubiquitin bound to extracellularly deposited amyloid has been demonstrated.

Murine tissue macrophages synthesize and secrete amyloid proteins different to amyloid A (AA)

We recently demonstrated that serum amyloid A (SAA) gene expression can be induced in extrahepatic sites with exception of the brain. Furthermore we demonstrated that tissue macrophages express SAA-gene constitutively and that SAA-gene expression can be increased by endotoxin. Until now the protein corresponding to the SAA-specific mRNA contained in macrophages has not been identified. We compared proteins precipitated from endogenously labelled samples by three antisera against amyloid A (AA) raised in different laboratories. Radiolabelled samples were derived from murine hepatocyte cultures, from cell-free translation of acute phase liver RNA or hepatocytes RNA and from peritoneal macrophages as well as Kupffer cells. All three antisera recognize a protein of 12.5 kDa Mr produced by hepatocytes (SAA), and a major protein of 14.3 kDa Mr contained in the cell-free translation products; this is the precursor of the mature SAA as demonstrated by cleavage experiments with canine pancreas microsomal enzymes. The antisera also recognize two proteins--a major one of 14.5 kDa Mr and a second of 12.5 kDa Mr contained in the supernatants and cell lysates of liver and peritoneal macrophages. New antisera raised against the two proteins do not recognize any protein, either of hepatocyte or of cell-free translation samples; they specifically precipitate two proteins from macrophage samples with the same molecular mass as that of the proteins precipitated by the anti SAA antisera. Murine acute phase sera do not react with the new antisera. However, amyloid deposits of amyloidotic mice specifically react with the new antisera. We describe two new components of murine amyloid produced by tissue macrophages.

Imaging of experimental amyloidosis with 131I-labeled serum amyloid P component.

131I-labeled human serum amyloid P component, which was injected into mice with experimentally induced systemic AA amyloidosis and into controls, became specifically localized and was retained in amyloidotic organs. In comparison, it was rapidly and completely eliminated from unaffected tissues and from control animals. Distinctive images of this amyloid-specific deposition of labeled serum amyloid P component were derived from whole body scanning, in vivo, of amyloidotic mice. These findings suggest that such imaging may have applications for the diagnosis and quantitation of amyloid deposits in humans.

Demonstration of amyloid in murine and human secondary amyloidosis by the immunoperoxidase technique.

A specific rabbit anti-murine amyloid A protein (anti-AA) has been used in the immunoperoxidase (IP) technique for the detection of amyloid in formalin-fixed, paraffin-embedded, tissue obtained from amyloidotic mice and a post-mortem kidney specimen from a patient with amyloidosis secondary to osteomyelitis. Amyloidosis was induced in five out of 20 white mice by weekly intraperitoneal injections of complete Freund's adjuvant. Amyloid deposits were clearly demonstrated both by the Congo Red and by the IP techniques. The antibody against murine AA was found to be cross-reactive with human AA. These preliminary results suggest that the IP technique with the use of specific antibodies against the different amyloid proteins might be extremely useful for the histological dianosis and classification of amyloidosis.

The effect of levamisole in experimental murine amyloidosis.

In casein-induced murine amyloidosis various lines of investigation have implicated immunodeficiency as playing a role in amyloid formation. In this study, the immunopotentiating agent levamisole failed to prevent amyloidogenesis or to accelerate resolution of preformed amyloid deposits in the mouse model. The serum precursor of amyloid, serum amyloid protein A (SAA), was increased by levamisole in both normal and amyloidotic mice.

Delayed hypersensitivity during casein-induced murine amyloidosis

Cellular immunity has been studied in mice at various times during the induction of amyloidosis following multiple injections of casein. The assay system used was one which measured delayed hypersensitivity (DH) in vivo, by injecting antigen into the left ear lobe of sensitized mice, followed by the intravenous administration of 125I-deoxyuridine ( 125I-UdR). The ears were then cut off and the L R 125I-UdR ratio provided a measure of DH. It was found that DH to casein appeared in pre-amyloidotic mice, remained through the stages of mild and moderate amyloidosis and disappeared in severely amyloidotic mice. DH to fowl IgG disappeared after three injections of casein and remained absent at all times thereafter, likely due to antigenic competition. In contrast, DH to DNFB persisted at all times, even in the face of severe amyloidosis. These results have been interpreted to indicate that, using this assay, DH is normal during casein-induced murine amyloidosis.

Casein-induced murine amyloidosis: amyloid and immunoglobulin production and proliferative capacity of splenocytes.

We have examined some aspects of lymphocyte and macrophage function in experimental murine amyloidosis. Casein-induced murine amyloidosis is a good model for studying secondary human amyloidosis while myeloma-associated murine amyloidosis is a poor model for human myeloma-associated amyloidosis. The amyloid of casein-induced and myeloma-associated murine amyloidosis cross-reacted immunologically. Neither form of amyloidosis was associated with L chain fragments or excess L chain production. Cellular immunologic reactivity of casein-induced amyloidotic mice, as assessed by lymphocyte transformation with mitogens, was abnormal using spleen lymphocytes but completely normal when thymus and peripheral blood lymphocytes were examined. The depressed activity could be attributed to splenic amyloid deposits. Intracellular amyloid was detected in the spleens of casein-injected mice prior to extracellular amyloid deposits. Amyloid containing cells could also be cultured from the spleen in a much higher proportion than that found in vivo. These cells may represent a subpopulation committed to amyloid synthesis.

Lymphocyte transformation in casein-induced murine amyloidosis

Spleen lymphocytes from casein-induced amyloidotic mice demonstrate diminished transformation in vitro to PHA-P, concanavalin A, and pokeweed mitogen but a normal response to lipopolysaccharide. Thymic and peripheral blood lymphocytes respond normally to these mitogens. The amyloidogen casein acted as a mitogen in both normal and casein injected mice. The diminished PHA responsiveness of spleen lymphocytes in vitro could have resulted from an inhibitory effect of amyloid fibrils on lymphocyte proliferation and did not indicate a generalized diminished cellular immunologic responsiveness of amyloidotic mice.

Murine Amyloidosis: Immunologic Characterization of Amyloid Fibril Protein

Abstract Immunization of rabbits with amyloid-laden murine tissue denatured with 6 M guanidine-HC1 produced antibodies to denatured purified murine amyloid fibril protein. Attempted immunization with native amyloid-laden murine tissues, however, failed to produce antibodies to any component unique to amyloid tissues, indicating that native murine amyloid was not immunogenic. Radioimmunoassay and immunofluorescence studies, nonetheless, demonstrated that native amyloid possessed antigenic determinants in common with denatured amyloid. A high degree of immunologic cross-reactivity was detected between extracts of amyloidotic tissues from different strains of mice regardless of the method of amyloid induction. Sera from amyloidotic mice were shown to contain a component capable of inhibiting the reaction between specific antibodies and purified amyloid protein.

Homograft reaction in amyloidotic mice.

Homograft reaction in amyloidotic mice.