The Amyloid P-Component (Protein AP): an Integral Part of the Amyloid Substance?

The P-component of amyloid (protein AP) appears to be present in all types of amyloid substance regardless of the clinical category of amyloidosis or the chemical class of the amyloid fibril. The role of protein AP in the formation of amyloid substance has not been established. In a patient with primary amyloidosis, significant amounts of protein AP were found closely associated with the amyloid fibril proteins and was released from the latter only after dissociation and reduction of the amyloid fibril preparation. EDTA seemed to be very effective in releasing protein AP, and it is thought that the close association between the amyloid fibrils and protein AP is calcium-dependent. The very close association between the amyloid fibrils and protein AP suggests that the latter is an integral part of the amyloid substance.     

Intralysosomal formation of amyloid fibrils.

Unusual inclusions, which occurred in the reticuloendothelial cells intimately associated with fresh amyloid deposits, were analyzed by electron microscopy. The inclusions were located in the areas rich in the primary lysosome type of dense bodies and the cytoplasmic invaginations containing well-oriented amyloid fibrils. They were single-membrane-bounded, measured 0.3 to 0.8 mu in width and 0.5 to several microns in length, and showed considerable variation in the electron density of their contents. The latter consisted of two different ultrastructural elements: fibrillar profiles and a homogeneous or finely granular electron-dense substance. The fibrillar profiles were virtually identical in ulstrastructure to the amyloid fibrils and were well-oriented parallel to the long axis of the inclusion. The homogeneous or finely granular electron-dense substance appeared to be comparable to that composing the dense body matrix. The inclusions were usually acid phosphatase positive, but did not take up intravenously injected Thorotrast particles. These data led us to conclude that these inclusions were transitional forms from the usual dense bodies to the deep cytoplasmic invaginations containing well-oriented amyloid fibrils (which are accepted by most investigators as the sites of amyloid formation) and thus constitute direct evidence for the involvement of lysosomes in amyloid fibril formation.     

Contribution of human smooth muscle cells to amyloid angiopathy in AL (light-chain) amyloidosis

Objective: Amyloid light-chain (AL) amyloidosis is a disease process that often compromises the peripheral vascular system and leads to systemic end-organ dysfunction. Although amyloid formation in vessel walls is a multifaceted process, the assembly of the native light chains (LCs) into amyloid fibrils is central to its pathogenesis. Recent evidence suggests that endocytosis and endolysosomal processing of immunoglobin LCs by host cells is essential to the formation of amyloid fibrils that are deposited in at least some tissues. The aim of this study was to elucidate the role of vascular smooth muscle in amyloid angiopathy. Methods: Human coronary artery smooth muscle cells (SMCs) were grown on coverslips, four chamber glass slides, and growth factor-reduced Matrigel matrix in the presence of 10 µg/ml of ALs (λ and κ isotypes), nonamyloidogenic LCs, and culture medium (negative control) for 48 and 72 hours. Thereafter, a detailed light microscopic, immunohistochemical, and ultrastructural evaluation was conducted to verify amyloid deposition and characterize the role of SMCs in the formation of amyloid deposits in the various experimental conditions. Results: Amyloid deposits were detected extracellulary as early as 48 hours after exposure of vascular smooth muscle cells (VSMCs) to AL-LCs (amyloidogenic light chains) as confirmed by affinity to Congo red dye, thioflavin T fluorescence, and transmission electron microscopy. No amyloid was present in the cultures of SMCs treated with medium alone or nonamyloidogenic LCs. SMCs associated with amyloid deposits exhibited CD68, lysosome-associated membrane protein 1-1, and intracellular lambda light chain expression and only focal smooth muscle actin and muscle-specific actin positivity. Electron microscopy revealed these cells to have an expanded mature lysosomal compartment closely associated with deposits of newly formed amyloid fibrils. Conclusions: The interaction of amyloidogenic LCs with VSMCs is necessary for the formation of amyloid fibrils that are deposited in peripheral vessels. VSMCs participate in the formation of amyloid by the intracellular processing of AL-LCs, which is possible due to their transformation from a smooth muscle to a macrophage phenotype. The formation of amyloid fibrils occurs in the mature lysosomal compartment of transformed cells. The amyloid that is formed is then extruded into the extracellular matrix.     

Kinetic analysis of amyloid fibril polymerization in vitro

We investigated the polymerization kinetics of murine senile amyloid fibrils (fASSAM) in vitro. When sonicated murine senile amyloid fibrils was incubated with its constituent monomer protein, the extension of amyloid fibrils was observed in an electron microscopic analysis. Quantitative fluorometric analysis with thioflavine T (Naiki H, Higuchi K, Hosokawa M, Takeda T: Anal Biochem 177:244, 1989) revealed that (a) extension of amyloid fibrils occurred by a pseudo-first-order exponential increase in the fluorescence of thioflavine T; (b) the rate of extension was maximal around pH 7.5, and was inhibited with the increase in KCl or NaCl concentration in the reaction mixture; (c) the rate of polymerization was proportional to the product of the murine senile amyloid fibrils number concentration and the constituent monomer protein concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization with the equilibrium association constant K of 5 x 10(7) M-1. These results show that amyloid fibril formation can apparently be explained by a first-order kinetic model: that is, extension of amyloid fibrils proceeds by consecutive association of precursor proteins onto the ends of existing fibrils.     

PATHOLOGICAL STUDY ON AMYLOIDOSIS

Five biopsy materials of human amyloidosis and experimental amyloidosis in mice induced by-, casein injection were studied by an electron microscope, and the following results were obtained. 1. Amyloid consisted of amyloid fibrils with an approximate width of 100 Å. These fibrils were found in both biopsy materials of human cases and experimental amyloidosis in mice. Amyloid was produced by the reticulo-endothelial cells which were functionally-disturbed. 3. The process of amyloid production in the reticuro-endothelial cells was essentially similar to that of collagen synthesis in the fibroblast. The protein synthesized in the granular reticulum was transported to the Golgi complex, and polymerization occurred in the lysosome, and finally complete amyloid fibrils were released from the cell.     

Binding of serum amyloid P-component (SAP) by amyloid fibrils.

Serum amyloid P-component (protein SAP) was found to bind in vitro to isolated amyloid fibrils of both primary and secondary types. The binding was strictly calcium-dependent, optimal uptake requiring at least 0.5 mM calcium ion. Using normal human serum as the source of protein SAP different fibril preparations became saturated with between 5--20 micrograms of SAP per mg dry weight of fibril. Isolated pure protein SAP bound in greater amounts. In control experiments SAP did not bind significantly to collagen fibrils, sheep erythrocytes, plastic shavings, or the following immobilized proteins: human kappa or lambda Bence-Jones proteins; human; rabbit or mouse IgG; human serum albumin. C-reactive protein, which resembles protein SAP structurally but has calcium-dependent specificity for different ligands, bound significantly to only one of five different amyloid fibril preparations.     

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis.

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.     

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.     

Cortical angiopathy in Alzheimer's disease: The formation of dystrophic perivascular neurites is related to the exudation of amyloid fibrils from the pathological vessels

Summary We studied the organization of dystrophic neurites around pathological vessels in Alzheimer cortex. Two techniques were used simultaneously on serial sections: thioflavine staining of amyloid substance and immunohistochemistry with immune sera against Paired Helical Filaments (anti-PHF) and native Tau proteins (anti-Tau). We observed different distributions of dystrophic neurites (immunolabelled with anti-PHF or anti-Tau) around thioflavine-stained angiopathic arterioles. The wall of the vessels with large diameter (>100 µm) presented a congophilic angiopathy without neuropil reaction. In vessels with lesser diameter (<100 µm), dystrophic neurites constituted a discontinuous sleeve around vessels, always in close contact with amyloid substance outside the wall (dyshoric angiopathy). We observed structures similar to senile plaques around capillaries (diameter: 10–15 µm). The sleeve of dystrophic neurites with aggregated Tau proteins were always observed in the close vicinity of the amyloid substance which exuded from the pathological blood vessels. Thus, the exudation of these amyloid fibrils seems to induce the formation of dystrophic neurites (neuritic reaction).     

Transformation from SAA2-fibrils to AA-fibrils in amyloid fibrillogenesis: In vivo observations in murine spleen using anti-SAA and anti-AA antibodies

Early amyloid fibrillogenesis from serum amyloid A protein (SAA) has been observed in the murine spleen after an injection of casein-Freund's complete adjuvant in the presence of amyloid enhancing factor, using anti-SAA C-terminal (anti-SAA) and anti-amyloid A (AA) antibodies. In Western immunoblotting of sera, both SAA1 and SAA2 reached a maximum after 24 h and began to decrease after 48 h. In spleen extracts, SAA2, but not SAA1 or AA, was found from 48 h, when amyloid was first deposited in the marginal zone. Electron microscopic immunohistochemistry of this stage showed reaction products from SAA in the marginal zone as fine granules along the cell membrane of mononuclear cells and focal intercellular aggregates, which contained fine fibrils originating from the cell membrane. Amyloid nodules, surrounded by mononuclear cells, developed from this stage. In the nodules, fibrils were positive for anti-SAA only in the vicinity of the cell membrane, while anti-AA stained fibrils throughout. Our hypothesis for fibrillogenesis is thus as follows: Serum SAA2 is specifically deposited on mononuclear cells in the marginal zone and polymerized extracellularly into fibrils, retaining its antigenicity (SAA2 amyloid fibrils); these fibrils are then processed to AA amyloid fibrils in situ by cleavage of the C-terminal portion of SAA2.     

Beta amyloid is focally deposited within the outer basement membrane in the amyloid angiopathy of Alzheimer''s disease. An immunoelectron microscopic study.

The fine structure of cerebral amyloid angiopathy, especially in small and presumably early deposits, was examined by immunolabeling of the beta/A4 protein in semithin and ultrathin sections from brains with Alzheimer's disease. The following findings emerged: 1) in large leptomeningeal arteries, small, focal amyloid deposits appear to consist of clusters of delicate (approximately 8 nm diameter) amyloid fibrils, not previously described, in the outermost part of the basement membrane (BM) at the media-adventitia junction; 2) in small leptomeningeal arteries and perforating cortical arterioles, small foci of delicate amyloid fibrils were observed within the BM. They appeared mostly in the outer portion of the BM, around intact smooth muscle cells, rather than in the subendothelial region. In larger and presumably more advanced deposits, coarse amyloid fibrils (approximately 10 nm) occupied the abluminal BM, and adjacent smooth muscle cells showed degeneration; and 3) in capillaries, small amounts of delicate (approximately 8 nm) amyloid fibrils, not previously described, were seen within the BM in the smallest discernible deposits. The BM at these sites was abnormally folded and layered. In larger deposits, amyloid fibrils appeared to extravasate from the outer BM of the capillary into the neuropil and were surrounded by astrocytic foot processes and/or microglia. Our results suggest that vascular amyloid fibrils may first be formed within the abluminal vascular BM, that is, outside of cells. The BM may trap degradative intermediates of the amyloid precursor protein that contain the beta/A4 region, and local proteases may then cleave them further to yield amyloidogenic fragments.     

Properties and possible significance of substance P and insulin fibrils

The neuropeptide substance P forms polymeric fibrils similar to those previously reported for the hormone, insulin. Structural and chemical aspects of these two fibrillary forms have been compared and their possible existence in vivo considered. Numerous substance P fibrils are readily formed in vitro in mM solutions under conditions which are physiological with respect to salt concentration, pH and temperature, whereas more severe conditions (heat and acid) are apparently required for the rapid formation of numerous insulin fibrils. Morphologically, both fibrils appear to be relatively long and unbranched and the neuropeptide fibrils are similar in size to such naturally occurring structures as neurofilaments. Disaggregation of the neuropeptide fibril follows dilution (1000-fold) whilst more stringent (alkaline) treatment is apparently necessary for insulin fibril dissociation. These observations are discussed in relation to the role of an insulin-like peptide in the formation of certain types of amyloid and the possibility that fibrillary or similar polymeric forms of substance P may exist in normal tissue.     

Cross-seeding and cross-competition in mouse apolipoprotein A-II amyloid fibrils and protein A amyloid fibrils

Murine senile [apolipoprotein A-II amyloid (AApoAII)] and reactive [protein A amyloid (AA)] amyloidosis are reported to be transmissible diseases via a seeding mechanism similar to that observed in the prion-associated disorders, although de novo amyloidogenesis and the progression of AApoAII or AA amyloidosis remain unclear. We examined the effect of co-injection of AApoAII and AA fibrils and multiple inflammatory stimuli in R1.P1-Apoa2(c) mice with the amyloidogenic Apoa2(c) allele. Both AApoAII and AA amyloidosis could be induced in this system, but the two types of amyloid fibrils preferentially promote the formation of the same type of fibrils while inhibiting the formation of the other. Furthermore, we demonstrate that AA or AApoAII amyloidosis could be cross-seeded by predeposited AApoAII or AA fibrils and that the predeposited amyloid fibrils were degraded when the fibril formation was reduced or stopped. In addition, a large proportion of the two amyloid fibrils colocalized during the formation of new fibrils in the spleen and liver. Thus, we propose that AApoAII and AA can both cross-seed and cross-compete with regard to amyloid formation, depending on the stage of amyloidogenesis. These results will aid in the clarification of the mechanisms of pathogenesis and progression of amyloid disorders.     

Ubiquitin profile in inflammatory leukocytes and binding of ubiquitin to murine AA amyloid: Immunocytochemical and immunogold electron microscopic studies

Lysosomes in activated murine monocytoid cells have been implicated in AA amyloid formation. The pathophysiology of this process is not well understood. Previous studies into the nature of the relationship between ubiquitin (UB), possessing intrinsic amyloid enhancing factor (AEF) activity; serum amyloid A (SAA), the precursor protein of AA amyloid; and activated monocytoid cells have indicated a temporal and spatial relationship between these proteins and tissue AA amyloid deposits. To extend these findings, we have examined murine peritoneal leukocytes and splenic tissues during the early amyloid deposition phase by immunocytochemical and immunogold electron microscopic methods using monospecific anti-ubiquitin and anti-mouse AA amyloid antibodies. We show here enrichment of endosome–lysosome-like (EL) vesicles in the activated monocytoid cells with UB and SAA, and the presence of UB-bound AA amyloid fibrils in the EL vesicles, perikarya, and interstitial spaces. The importance of these findings is emphasized by the fact that activated monocytoid cells, containing UB in the EL vesicles, sequester and eventually localize SAA in their EL vesicles, and that UB binds to the EL-contained AA amyloid fibrils. These findings may also have functional consequences for studies on the role of EL and UB in amyloidogenesis.     

Insular Amyloid in a Case of Type HI Glycogenosis with a Special Reference to the Origin of Amyloid Fibrils

Amyloid depositions of pancreatic islets were investigated with electron microscopy in a case of type III glycogenosis.

Beta cells adjoining small amyloid depositions were shown to have cytoplasmic invaginations where closely packed amyloid fibrils were disclosed regularly orientated amyloid bundles. In the cytoplasm of the beta cells, some membrane-bounded vesicles contained amyloid fibrils and a few beta granules directly transformed into the fibrils within the vesicles.

These findings indicate that, at least in this case, the beta cells play a crucial role in the formation of insular amyloid.     

An Analysis of the Close Relationship of Lysosomes to Early Deposits of Amyloid: Ultrastructural Evidence in Experimental Mouse Amyloidosis

On the basis of recent morpholgic and biochemical studies which suggested the possible involvement of reticuloendothelial (RE) cells and proteolytic enzymes in amyloidogenesis, the present study was undertaken to examine the ultrastructural interrelationship between lysosomes and amyloid fibrils at the sites of very early amyloid deposition. In the spleen, liver and kidney of the experimental mouse model, foci of amyloid deposits were closely associated with the RE cells. The lysosomal enzyme activity, as marked by cytochemical demonstration of acid glycerophosphatase activity, was localized in the primary type lysosomes (as defined by their electron microscopic appearance), in the Golgi complexes, in the small cytoplasmic vesicles and occasionally widespread in the cytoplasm. They showed an intimate relationship to the amyloid fibrils. The findings were interpreted as favoring the hypothesis that the hydrolases play a role in amyloid fibril formation. The enzyme activity was also demonstrated in the secondary type lysosomes which occasionally contained amyloid fibrils that appeared to be phagocytized.     

Relationship between amyloid deposition and intracellular structural changes in familial amyloidotic polyneuropathy

Transthyretin-related familial amyloidotic polyneuropathy is a systemic amyloidosis caused by mutations in the transthyretin gene. Extracellular deposition of amyloid is the common pathologic hallmark of amyloidoses including Alzheimer disease, AL amyloidosis, AA amyloidosis, and familial amyloidotic polyneuropathy. However, the exact relationship between amyloid deposition and cell death has not yet been clarified. To elucidate this relationship, we studied the effect of transthyretin amyloid fibrils and prefibrillar aggregates on cells by using autopsy tissues obtained from 8 patients with familial amyloidotic polyneuropathy, as well as cultured cell lines. Ultrastructural studies of amyloid-laden cardiomyocytes showed that intracellular structural changes correlated with the degree of amyloid deposition and may reflect metabolic disturbances caused by physical limitations imposed by the amyloid deposits. Amyloid-laden vascular endothelial cells, mesangial cells, smooth muscle cells, Schwann cells, and cardiomyocytes, however, had well-preserved cell nuclei and showed no apoptotic changes, even when cells were completely surrounded by prefibrillar transthyretin aggregates and amyloid fibrils. Synthesized prefibrillar transthyretin aggregates, transthyretin fibrils, and amyloid fibrils obtained from patients with familial amyloidotic polyneuropathy evidenced no cytotoxicity in cell culture experiments. Our data thus indicate that neither transthyretin amyloid fibrils nor prefibrillar transthyretin aggregates directly induced apoptosis. However, cellular metabolic disturbances caused by cells' being physically confined by amyloid deposits may induce cell degeneration.     

Seminal vesicle amyloid: the first example of exocrine cell origin of an amyloid fibril precursor.

Amyloid fibrils have been extracted from seminal vesicles, and a dominant 14 kD amyloid fibril protein has been identified. An antiserum to this protein reacted both with amyloid and with epithelial cells in some normal seminal vesicles. These reactions were blocked with seminal vesicle secretion and seminal vesicle amyloid fibril protein, but not by degraded amyloid fibrils or fibril protein from other types of amyloid. It is concluded that seminal vesicle amyloid is derived from secretory proteins of the seminal vesicles. As such, it is the first amyloid described which appears to be the product of an exocrine secretory cell.     

Induction of murine AA amyloidosis by various homogeneous amyloid fibrils and amyloid-like synthetic peptides

We investigated amyloid-enhancing factor (AEF) activity of amyloid fibrils extracted from amyloid-laden livers of mice, cow, cheetah, cat and swan. All amyloid fibrils were confirmed to be amyloid protein A (AA) by an immunohistochemical analysis. We found that these fibrils accelerated the deposition of amyloid in an experimental mouse model of AA amyloidosis. Furthermore, the degree of deposition was dependent on the concentration of fibrils. When we compared the minimal concentration of amyloid fibrils needed to induce deposition, we found that these fibrils showed different efficiencies. Murine amyloid fibril induced amyloid deposition more efficiently than cow, cat, cheetah or swan amyloid fibrils. These data suggest that amyloid deposition is preferentially induced by amyloid fibrils with the same primary sequence as the endogenous amyloid protein. We then analysed the AEF activity of synthetic peptides, synthesized corresponding to amino acids 1-15 of mouse SAA (mSAA), 2-15 of cow SAA (bSAA), 1-15 of cat SAA (cSAA), which was the same as cheetah, and the common amino acids 33-45 of these four SAA (aSAA). We found that mSAA, bSAA and cSAA formed amyloid-like fibrils in morphology and showed similar AEF properties to those of native amyloid fibrils. Although aSAA also formed highly ordered amyloid-like fibrils, it showed weaker AEF activity than the other synthetic fibrils. Our results indicate that amyloidosis is transmissible between species under certain conditions; however, the efficiency of amyloid deposition is species-specific and appears to be related to the primary amino acid sequence, especially the N-terminal segment of the amyloid protein.     

Chain reaction of amyloid fibril formation with induction of basement membrane in familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is characterized by extracellular deposition of amyloid fibrils caused by a point mutation in the transthyretin (TTR) gene. Despite data from a number of in vitro studies of TTR amyloidogenesis, many questions, including where and how these fibrils form in vivo and what is the impact of amyloid deposition on tissues, remain unanswered. Here, we analysed the relationship between amyloid fibril formation and micro‐environmental changes by using autopsy cardiac tissues from 11 patients with FAP and a smooth muscle cell line. Ultrastructural studies of cardiomyocytes and vascular smooth muscle cells showed that amyloid fibrils formed first at the basement membrane and that amorphous non‐fibrillar TTR deposits and premature fibrils predominated during the early stage of amyloid deposition. Immunohistochemical analyses revealed that expression of major components of the basement membrane, such as collagen IV, laminin, and fibronectin, increased in parallel with the accumulation of TTR amyloid fibrils. In vitro studies with a vascular smooth muscle cell line revealed that synthetic TTR aggregates increased expression of these basement membrane components. Serum levels of collagen IV in FAP patients were significantly higher than those in healthy controls. Our data thus indicate that TTR amyloid fibrils formed first at the basement membrane and that expression of basement membrane components that was induced by amyloid deposition contributed to further amyloid deposition. This chain reaction may have important implications for FAP pathogenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.