Characterization of splenic glycosaminoglycans accumulated in vivo in experimentally induced amyloid-susceptible and amyloid-resistant mice.

The pathogenesis of the amyloid deposition diseases is poorly understood. The CE/J mouse, which is naturally protected from amyloid A (AA) protein amyloidosis, has provided a tool to study mechanisms that may be implicated in amyloid deposition diseases by means of comparison of findings with those in an AA-susceptible mouse strain. We have compared proteoglycan/glycosaminoglycan accumulation in vivo in amyloid-protected CE/J mouse strain and in AA-susceptible CBA/J mouse strain in homeostasis and when injected with amyloid-inducing agents. Results indicate that there is an overall increase in [(35)S]proteoglycan/glycosaminoglycan accumulation in the spleens of both strains of mice, but with a specific increase in heparan sulfate in only CBA/J mouse spleens that are rich in amyloid. Further, we report the absence of heparan sulfate in the splenic perifollicular areas of amyloid-free CE/J mouse, whereas in the amyloid-laden CBA/J mouse there is co-localization of heparan sulfate with the AA deposits. We have also examined the glycosaminoglycan disaccharide products in both these strains of mice for their sulfation positions and found no differences in the disaccharide composition of chondroitin/dermatan sulfate and heparan sulfate isolated from the control CBA/J and control CE/J mice. There were no differences in chondroitin/dermatan sulfate in both strains after experimental induction. However, analysis of the heparan sulfate disaccharides by means of capillary high-performance liquid chromatography linked to microelectrospray ionization time-of-flight mass spectrometry indicated that the disaccharide composition of the splenic heparan sulfate obtained from the treated CBA/J mice that had developed amyloid was markedly different from that obtained from the control CBA/J mice and the treated amyloid-resistant CE/J mice. These findings suggest that unique heparan sulfates play a fundamental role in the pathogenesis of amyloid.

Diet, amyloid enhancing factor (AEF) and amyloidogenesis: an hypothesis.

At least two forms of amyloidosis, amyloid A (AA) and prion protein (PrP), can be transmitted by dietary ingestion of an agent(s) present in crude mammalian tissues. Although the incubation time for PrP or scrapie-induced diseases to develop in experimental animals extends over months or years, AA or secondary amyloidosis in mice is inducible within a week. In response to inflammatory stimuli we hypothesize that dietary factor(s) modulate the rate at which beta-pleated sheet fibrils accumulate in most forms of amyloidosis. The critical importance of precursor protein polymorphism, cell surface proteoglycans (PG), lipids and apolipoprotein metabolism has also been addressed in this hypothesis.

Serum amyloid A, an acute-phase protein, modulates proteoglycan synthesis in cultured murine peritoneal macrophages.

Cultured peritoneal macrophages obtained from azocasein-injected mice were found to produce several fold more cell-associated and medium proteoglycans than peritoneal macrophages from untreated mice. Since serum amyloid A (an acute-phase protein) is also upregulated following injections of azocasein, we questioned whether its production was the immediate agent stimulating proteoglycan formation. Cultured peritoneal macrophages from untreated mice were then incubated with varying concentrations of SAA, resulting in a similar dose-dependent several fold increase in proteoglycan production. Of particular note was a disproportionate increase in cell-associated heparan sulfate proteoglycans in both experimental groups and of dermatan sulfate and chondroitin sulfate proteoglycans when cells were incubated in the presence of SAA in the culture medium. These results indicate a potentially important function of SAA in directing specific modifications in inflammatory conditions where increase in macrophage proteoglycans may play direct roles.

The acute phase response in apolipoprotein A-1 knockout mice: apolipoprotein serum amyloid A and lipid distribution in plasma high density lipoproteins.

In plasma, the bulk of apoSAA, a positive acute phase reactant protein, is transported in high density lipoproteins (HDL), especially HDLH (apoA1-rich HDL). In this study we tested whether apoA1 deficiency would adversely affect apoSAA concentration and lipid distribution in mouse plasma lipoproteins. Acute phase response (APR) was induced in C57BL/6J (apoA1+/+) and apoA1-knockout mice (apoA1-/-) by a subcutaneous injection of silver nitrate. The APR increased cholesterol concentrations in LDL of apoA1-/- mice and apoA1+/+ mice in a like manner. In contrast to apoA1+/+ mice, concentrations of cholesterol, phospholipids and proteins in both HDLL (1.063

A unique amyloidogenic apolipoprotein serum amyloid A (apoSAA) isoform expressed by the amyloid resistant CE/J mouse strain exhibits higher affinity for macrophages than apoSAA1 and apoSAA2 expressed by amyloid susceptible CBA/J mice.

CBA/J and other inbred strains of mice that express the amyloidogenic apolipoprotein serum amyloid A (apoSAA) apoSAA2, together with apoSAA1, are susceptible to amyloid A (AA) amyloidosis, whereas CE/J mice that express a single unique isoform, apoSAACEJ, are resistant. Studies indicate that CBA/JxCE/J hybrid mice that express apoSAA2 in the presence of apoSAACEJ are protected from amyloidogenesis. To define a mechanism by which expression of apoSAACEJ may protect from AA formation in the presence of apoSAA2, binding of recombinant apoSAA (r-apoSAA) isoforms, validated by N-terminal sequencing, to a murine macrophage cell line was investigated. Maximal specific binding occurred after incubation of radiolabeled apoSAA with IC-21 macrophages (1x105 cells/ml) for 30 min at 4 degreesC. The binding of 125I-r-apoSAA1, 125I-r-apoSAA2 and 125I-r-apoSAACEJ was specific and saturable, with an affinity (Kd) of about 2.8, 3.2 and 1.3 nM, respectively, and approximately 2-4x106 sites per cell. Competitive binding experiments indicate apoSAACEJ binds with higher affinity to macrophages than does either apoSAA1 or apoSAA2. We suggest that greater cellular affinity of apoSAACEJ compared to apoSAA2 may contribute to protection from AA amyloid in certain CBA/JxCE/J hybrid mice by interfering with interaction of apoSAA2 by macrophages and hence either membrane associated or intracellular degradation.

Catabolism of lipid-free recombinant apolipoprotein serum amyloid A by mouse macrophages in vitro results in removal of the amyloid fibril-forming amino terminus.

Serum amyloid A fibrils are formed when the normally rapid catabolism of the acute-phase reactant apolipoprotein serum amyloid A (apoSAA) is incomplete; thus amyloidosis may be viewed as a condition of dysregulated proteolysis. There is evidence that apoSAA is dissociated from plasma high-density lipoprotein (HDL) prior to fibril formation. The objective of this study was to investigate degradation of lipid-free apoSAA by tissue macrophages derived from amyloid-susceptible CBA/J mice in vitro. Peritoneal macrophages derived from untreated (normal) mice converted apoSAA (12 kDa) to a single 4 kDa C-terminal peptide while splenic macrophages converted apoSAA to 10, 7 and 4 kDa C-terminal peptides and a 4 kDa peptide that lacked the C- and N-terminal regions. Similar patterns of proteolysis occurred when peritoneal and splenic macrophages from amyloidotic CBA/J mice were used. Conditioned medium prepared from peritoneal, but not splenic macrophages, degraded apoSAA. Specific sites of cleavage indicated activity of cathepsin G- and elastase-like neutral proteases. The data indicate that lipid-free apoSAA can be degraded by secreted or cell-associated neutral proteases that are generated by macrophages to yield peptides that lack fibrillogenic potential.

Amyloid enhancing factor and dietary transmission in accelerated amyloid A amyloidosis.

The etiology and pathogenesis of amyloid A (AA) amyloidosis that may occur as an occasional complication of chronic inflammatory and infectious diseases are poorly understood. The preamyloid phase of experimentally induced AA amyloidosis can be greatly shortened in recipient animals by intravenous or intraperitoneal transfer of amyloid enhancing factor (AEF) when there is a concomitant inflammatory episode. AEF is an operational term applied to poorly characterized tissue extracts and increased AEF activity that precedes amyloid deposition. We now report that AA is rapidly formed in mice following oral administration of an AEF preparation that does not contain AA peptides. This finding indicates that a transmissible agent present in diet may be a contributory factor in amyloid fibril formation.

In vitro amyloid fibril formation by synthetic peptides corresponding to the amino terminus of apoSAA isoforms from amyloid-susceptible and amyloid-resistant mice.

Specific proteins of the apolipoprotein serum amyloid (apoSAA) family that are synthesized in large quantities during the acute, early phase of inflammation can serve as the proteinaceous precursors for amyloid fibrils. To model fibrillogenesis in such inflammatory diseases, we have used electron microscopy and X-ray diffraction to examine the structures formed by synthetic peptides corresponding in sequence to the 11 amino-terminal amino acids of murine apoSAA1, apoSAAcej, and apoSAA2 and to the 15 amino-terminal amino acids of apoSAA2. This region is reported to be the major fibrillogenic determinant of apoSAA isoforms. Both in 1 mM Tris buffer and in 35% acetonitrile, 0.1% trifluoracetic acid (ACN/TFA), all of the peptides formed macromolecular assemblies consisting of twisted, approximately 40- to 60-A-thick ribbons, which varied in width from around 40-70 A (for 11-mer apoSAA2 in Tris) up to 900 A (for the other peptides). X-ray diffraction patterns recorded from lyophilized peptides, vapor-hydrated samples, and solubilized/dried samples showed hydrogen bonding and intersheet reflections typical of a beta-pleated sheet conformation. The coherent lengths measured from the breadths of the X-ray reflections indicated that with hydration the growth of the assemblies in the intersheet stacking direction was comparable to that in the hydrogen-bonding direction, and analysis of oriented samples showed that the beta-strands were oriented perpendicular to both the long axis and the face of the assemblies. These X-ray results are consistent with the ribbon- or plate-like morphology of the individual aggregates and emphasize the polymorphic nature of amyloidogenic peptides. Our findings demonstrate that X-ray diffraction measurements on vapor-hydrated or solubilized/dried versus lyophilized, amyloidogenic peptides are a good indicator of their fibrillogenic potential. For example, from the highest to the lowest potential, the peptides examined here were ranked as: Abeta1-28 > Abeta1-40 > apoSAA1 approximately apoSAAcej > apoSAA2 > Abeta17-42. Experiments in which the three different 11-mer apoSAA isoforms were solubilized in ACN/TFA and then combined as binary mixtures showed that the ribbon morphology was not affected but that the extent of hydrogen bonding in the assemblies was substantially reduced. Our observations on the in vitro assembly of apoSAA analogs emphasize that amyloid fibril formation and morphology depend on primary sequence, length of polypeptide chain, the presence of additional fibrillogenic polypeptides, and solvent conditions.

Apolipoprotein E and apolipoprotein A-1 knock-out mice readily develop amyloid A protein amyloidosis.

Studies have identified apolipoprotein E (apoE) ubiquitously in biochemically distinct amyloid deposits including amyloid A protein (AA) in secondary amyloidosis and amyloid beta protein (A beta) amyloid in Alzheimer's disease (AD). Apolipoprotein A-1 (apoA-1) has been identified in cortical plaques derived from the tissues of patients with AD. To determine if apoE is essential for and apoA-1 may be a factor in AA-amyloidogenesis we investigated induction of secondary amyloidosis in mutant C57BL/6J mice that lack either apoE or apoA-1. Induction of secondary amyloidosis in nonmutant C57BL/6J mice that are AA amyloid-susceptible were the AA positive control. Discreet deposits of AA amyloid were detected in the perifollicular regions of spleens derived from mutant and nonmutant strains. The findings clearly demonstrate that generation of AA fibrils can occur independently of apoE and ApoA-1 expression.

Polymorphism of acute-phase serum amyloid A isoforms and amyloid resistance in wild-type Mus musculus czech.

Until CE/J mice and their offspring were characterized as amyloid-resistant, all mice were thought to be amyloid-susceptible to multiple injections of azocasein or a single injection of silver nitrate following administration of amyloid enhancing factor. We now report, for the first time, that wild-type Mus musculus czech and F1 hybrids bred by crossing M. musculus czech with amyloid-susceptible CBA/J mice are also amyloid resistant. Based on the derived amino acid sequences of two serum amyloid A (SAA) cDNA clones, we describe two unusual SAA gene isoforms in M. musculus czech, one of which differs from four previously characterized acute-phase apoSAA isoforms at several amino acid residues. Our findings support the hypothesis that protection against amyloid fibril formation in wild-type M. musculus czech mice and their offspring is linked to apoSAA gene mutations (molecular motif).

Degradation of serum amyloid A in amyloid-susceptible and amyloid-resistant mouse strains.

Degradation of serum amyloid A (apoSAA) by resident peritoneal cells (RPCS) and conditioned medium (CDM), prepared with RPCS, from amyloid-susceptible CBA/J mice, amyloid-resistant CE/J mice and their amyloid-resistant CBA/J x CE/J F1 progeny was investigated in vitro. Serum amyloid A was derived from murine acute phase (AP) plasma and associated with high density lipoprotein (HDL). Degradation of apoSAA by RPCS and CDM from CBA/J mice was complete while degradation by RPCS and CDM from CE/J mice did not occur. Degradation of apoSAA by RPCS and CDM from CBA/J x CE/J F1 hybrid mice was indistinguishable from that by RPCS and CDM from the CBA/J parent. Intermediate fragments were not detected with either RPCS or CDM from CBA/J mice or CBA/J x CE/J F1 hybrid mice. Degradation of apoSAA was inhibited by phenylmethanylsulfonyl fluoride (PMSF) indicating that the enzyme, secreted into the fluid phase, was a serine esterase. Unlike apoSAA, HDL-associated apoA-1 remained intact. It was thus concluded that while selective degradation of HDL-associated apoSAA (apoSAA-HDL) by RPCS from the CBA/J and CE/J mice was significantly different, the genetic study did not support the hypothesis that there was direct linkage between impaired degradation of apoSAA-HDL in the CE/J mouse strain and protection against amyloid fibril formation. As amyloid resistance in CBA/J x CE/J F1 hybrid mice is not attributable to failure to express the amyloidogenic isoform apoSAA2, the study supports the original hypothesis that amyloid resistance may be linked to expression of apoSAAcej.

Linkage of protection against amyloid fibril formation in the mouse to a single, autosomal dominant gene.

Inbred strains of mice provide a model for studies of the pathogenesis of amyloid A (AA) amyloidosis. All susceptible strains of mice described to date codominantly express two serum amyloid A (apoSAA) isoforms, apoSAA1 and apoSAA2, of which only apoSAA2 serves as a precursor for amyloid fibrils. In previous studies, we have shown that the CE/J strain, which produces a single, novel apoSAA isoform, apoSAACE/J, is amyloid resistant. In the present study amyloid-resistant CE/J females were mated with amyloid-susceptible CBA/J males to produce F1 hybrid offspring which were then backcrossed to the parental CBA/J mouse strain. Amyloid susceptibility was determined in 30 backcrossed mice 72 h after injection of murine amyloid enhancing factor and silver nitrate. ApoSAA isoforms in plasma were separated by isoelectric focusing gel electrophoresis and visualized after immunoblotting with anti-AA antiserum. Amyloid A fibrils in spleen homogenates were denatured by formic acid and AA protein was quantified by ELISA using anti-mouse apoSAA antibodies. Values < 5 apoSAA equivalent units were considered negative. 13 mice expressed an apoSAA1 and apoSAA2 doublet characteristic of CBA/J mice, whereas 17 mice, expressed the apoSAACE/J isoform codominantly with apoSAA1 and apoSAA2. The correlation of amyloid resistance to expression of the apoSAACE/J isoform was absolute (17/17 were negative; mean score 2.6 +/- 0.17 [standard error of the mean] apoSAA equivalent units) and the correlation between amyloid susceptibility and the expression of apoSAA2/apoSAA1 was also striking (12/13 were amyloid positive; mean score 47.9 +/- 9.0 [standard error of the mean] apoSAA equivalent units (P < 0.001). This is not significantly different from the 50% segregation of apoSAA phenotypes expected for linkage to a single gene. These results indicate that a single gene governs apoSAACE/J expression and thus confers protection against amyloid deposition even in the presence of apoSAA1 and apoSAA2 isoforms and show for the first time that resistance to AA amyloidosis is a dominant trait governed by a single gene.