C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice.

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.

Extracellular deposition of mouse senile AApoAII amyloid fibrils induced different unfolded protein responses in the liver, kidney, and heart.

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1 µg of isolated AApoAII fibrils into R1.P1-Apoa2(c) mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney.

Supplementation with a Probiotic Formula Having ß-Glucuronidase Activity Modulates Serum Estrogen Levels in Healthy Peri- and Postmenopausal Women.

Declines in estrogen levels occur in women transitioning to menopause. Estrogen hormones play important roles in multiple systems of the body, and estrogen loss is associated with a variety of symptoms that can decrease quality of life. The gut microbiota is involved in regulating endogenous estrogen levels. A portion of estrogen glucuronides can be reactivated in the gut by the microbial enzyme ß-glucuronidase, and the resulting free estrogens can return to the bloodstream. Here, we carried out in vitro screening of ß-glucuronidase activities for 84 strains belonging to 16 different species of lactic acid bacteria and bifidobacteria and found that one and three strains of Levilactobacillus brevis and Lacticasebacillus rhamnosus, respectively, can deconjugate estrogens. Among these strains, L. brevis KABP052 had the highest ß-glucuronidase activity. Moreover, in an exploratory, randomized, double-blind, placebo-controlled trial, we demonstrated that serum estrogen levels in healthy peri- and postmenopausal women given a probiotic formula containing KABP052 were maintained over time, whereas levels significantly decreased in the group given a placebo. Significantly higher levels of estradiol (31.62 ± 7.97 pg/mL vs. 25.12 ± 8.17 pg/mL) and estrone (21.38 ± 8.57 pg/mL vs. 13.18 ± 8.77 pg/mL) were observed in the probiotic versus placebo group after 12 weeks of intervention. This clinical study demonstrated for the first time the estrogen modulation capacity of a probiotic formula containing a bacterial strain having ß-glucuronidase activity in women during the menopausal transition and formed the basis for future investigations using probiotics in the menopausal population.

Mouse senile amyloid fibrils deposited in skeletal muscle exhibit amyloidosis-enhancing activity.

Amyloidosis describes a group of protein folding diseases in which amyloid proteins are abnormally deposited in organs and/or tissues as fine fibrils. Mouse senile amyloidosis is a disorder in which apolipoprotein A-II (apoA-II) deposits as amyloid fibrils (AApoAII) and can be transmitted from one animal to another both by the feces and milk excreted by mice with amyloidosis. Thus, mouse AApoAII amyloidosis has been demonstrated to be a "transmissible disease". In this study, to further characterize the transmissibility of amyloidosis, AApoAII amyloid fibrils were injected into transgenic Apoa2(c)Tg(+/-) and normal R1.P1-Apoa2(c) mice to induce AApoAII systemic amyloidosis. Two months later, AApoAII amyloid deposits were found in the skeletal muscles of amyloid-affected mice, primarily in the blood vessels and in the interstitial tissues surrounding muscle fibers. When amyloid fibrils extracted from the skeletal muscles were subjected to Western blot analysis, apoA-II was detected. Amyloid fibril fractions isolated from the muscles not only demonstrated the structure of amyloid fibrils but could also induce amyloidosis in young mice depending on its fibril conformation. These findings present a possible pathogenesis of amyloidosis: transmission of amyloid fibril conformation through muscle, and shed new light on the etiology involved in amyloid disorders.

A probiotic blend improves defecation, mental health, and productivity in healthy Japanese volunteers under stressful situations.

We investigated whether a blend of probiotics (KABP-021, KABP-022, and KABP-023) improved diarrhea-related problems in healthy Japanese adults who routinely lived under stressful conditions. Twenty-six females and 34 males were divided randomly into the probiotic and placebo groups in this double-blind, placebo-controlled, parallel-group comparison study. All participants ingested 1 capsule of probiotics or placebo per day for 4 weeks. Intervention with probiotics significantly reduced diarrhea-related problems assessed by the Izumo scale compared with placebo treatment (P < 0.001). In the Short Form-8 questionnaire, probiotic intervention improved mental component scores (P = 0.002), role emotional scores (P = 0.002), and mental health scores (P < 0.001). Treatment with probiotics also reduced the effects of diarrhea on daily activities (P < 0.001) and overall working habits (P = 0.010), including missing work (absenteeism) and impaired productivity (presenteeism), as assessed by the Work Productivity and Activity Impairment Questionnaire: General Health. Furthermore, there was a correlation between improved scores for diarrhea on the Izumo scale and increased abundance of Faecalibacterium, a butyric acid-producing bacterium, in the gut in the probiotic group (P = 0.047), whereas no such a correlation or trend was found in the placebo group. Our strategy of supplementation for 4 weeks with a specific blend of probiotics reduced diarrhea-related symptoms and may improve the mental health and daily activities of healthy individuals under stress.

Exercise suppresses mouse systemic AApoAII amyloidosis through enhancement of the p38 MAPK signaling pathway.

Exercise interventions are beneficial for reducing the risk of age-related diseases, including amyloidosis, but the underlying molecular links remain unclear. Here, we investigated the protective role of interval exercise training in a mouse model of age-related systemic apolipoprotein A-II amyloidosis (AApoAII) and identified potential mechanisms. Mice subjected to 16 weeks of exercise showed improved whole-body physiologic functions and exhibited substantial inhibition of amyloidosis, particularly in the liver and spleen. Exercise activated the hepatic p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway and the downstream transcription factor tumor suppressor p53. This activation resulted in elevated expression and phosphorylation of heat shock protein beta-1 (HSPB1), a chaperone that defends against protein aggregation. In amyloidosis-induced mice, the hepatic p38 MAPK-related adaptive responses were additively enhanced by exercise. We observed that with exercise, greater amounts of phosphorylated HSPB1 accumulated at amyloid deposition areas, which we suspect inhibits amyloid fibril formation. Collectively, our findings demonstrate the exercise-activated specific chaperone prevention of amyloidosis, and suggest that exercise may amplify intracellular stress-related protective adaptation pathways against age-associated disorders, such as amyloidosis.

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis.

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1⁻/⁻) mice were used. Apoa1⁻/⁻ mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1⁻/⁻ mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1⁻/⁻ mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.

Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease.

AA amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction, but little is known about the underlying mechanisms. Given the transmissible characteristics of AA amyloidosis, transmission between captive cheetahs may be a possible mechanism involved in the high incidence of AA amyloidosis. In this study of animals with AA amyloidosis, we found that cheetah feces contained AA amyloid fibrils that were different from those of the liver with regard to molecular weight and shape and had greater transmissibility. The infectious activity of fecal AA amyloid fibrils was reduced or abolished by the protein denaturants 6 M guanidine.HCl and formic acid or by AA immunodepletion. Thus, we propose that feces are a vehicle of transmission that may accelerate AA amyloidosis in captive cheetah populations. These results provide a pathogenesis for AA amyloidosis and suggest possible measures for rescuing cheetahs from extinction.

Glavonoid-rich oil supplementation reduces stearoyl-coenzyme A desaturase 1 expression and improves systemic metabolism in diabetic, obese KK-Ay mice.

AIMS: Glavonoid-rich oil (GRO) derived from ethanol extraction of licorice (Glycyrrhiza glabra Linne) root has been reported to have beneficial effects on health. In this study, we aimed to determine the effect of long-term administration of GRO on metabolic disorders and to elucidate the molecular mechanism. MAIN METHODS: Female obese, type 2 diabetic KK-Ay mice were fed diets supplemented with 0.3% or 0.8% GRO (w/w) for 4-12 weeks. Mice were euthanized and autopsied at 20 weeks old. The effects of GRO on lipid and glucose metabolism were evaluated by measuring physiological and biochemical markers using mRNA sequencing, quantitative reverse-transcription PCR, and western blot analyses. KEY FINDINGS: Compared to mice fed the control diet, GRO-supplemented mice had reduced body and white adipose tissue weights, serum levels of triglycerides and cholesterol, and improved glucose tolerance, while food intake was not affected. We found remarkable reductions in the gene expression levels of stearoyl-coenzyme A desaturase 1 (Scd1) and pyruvate dehydrogenase kinase isoenzyme 4 (Pdk4) in the liver, in addition to decreased expression of fatty acid synthase (Fasn) in inguinal white adipose tissue (iWAT). These results suggest that GRO supplementation improves lipid profiles via reduced de novo lipogenesis in the liver and white adipose tissue. Glucose metabolism may also be improved by increased glycolysis in the liver. SIGNIFICANCE: Our analysis of long-term supplementation of GRO in obese and diabetic mice should provide novel insight into preventing insulin resistance and metabolic syndromes.

Glavonoid, a possible supplement for prevention of ATTR amyloidosis.

Transthyretin (TTR) is an amyloidogenic protein associated with hereditary and nonhereditary transthyretin amyloidoses (ATTR). Dissociation of the tetramer of TTR to the monomer induces TTR misfolding, which leads to amyloid fibril formation and triggers the onset of ATTR amyloidosis. Stabilizers of tetrameric TTR have been accepted as an effective ATTR amyloidosis treatment while effect is limited and they are too expensive. The aim of our study was to find more effective and cheep natural compound to suppress TTR amyloid formation. Glabridin, a prenylated isoflavan isolated from Glycyrrhiza glabra L., stabilized the TTR tetramer in vitro. The effects of licorice-derived flavonoid oil-Glavonoid, a natural substance that includes glabridin and several polyphenols-on stabilizing the TTR tetramer must still be elucidated. To examine plasma TTR stabilization by Glavonoid in vitro, we investigated the feasibility of utilizing glabridin plus Glavonoid to prevent TTR amyloid fibril formation. Glavonoid mixed with human plasma samples at 24 h incubation in vitro increased the tetramer level (P < 0.05) and reduced the monomer level (P < 0.01) and the monomer/tetramer ratio (P < 0.05) of TTR compared to those without Glavonoid by immunoblot analysis, such effect could not observe in the presence of glabridin. Oral Glavonoid (300 mg for 12 weeks) in 7 healthy volunteers effectively increased the plasma glabridin concentration. Glavonoid increased the TTR tetramer level and reduced the monomer/tetramer ratio of TTR (P < 0.05) in plasma at 12 weeks in healthy volunteers compared to those of age matched control subjects without the supplement. Thus, oral Glavonoid may effectively prevent TTR amyloid fibril formation via TTR tetramer stabilization. Glavonoid may become a promising supplement before onset of ATTR amyloidosis.

Amyloid fibrils formed by selective N-, C-terminal sequences of mouse apolipoprotein A-II.

In mice, amyloidogenic type C apolipoprotein A-II (apoA-II) forms amyloid fibrils in age-associated amyloidosis. To understand the mechanism of amyloid fibril formation by apoA-II, we examined the polymerization of synthetic partial peptides of apoA-II in vitro. None of the partial apoA-II peptides polymerized into amyloid fibrils when tested as a single species mixture. We found a unique mechanism in which N- and C-terminal peptides associated into amyloid fibrils in a 1:1 ratio at pH 2.5. The 11-residue amino acid sequence (6-16), which is a common sequence of type B apoA-II and type C apoA-II proteins in amyloidosis-resistant mice and amyloidosis-susceptible mice, respectively, was critical for polymerization into amyloid fibrils. The 18-residue-long amino acid sequence (48-65) is also necessary for nucleation, but not for the extension phase. These findings suggest that there may be different mechanisms underlying the nucleation and extension phases of apoA-II amyloid fibril formation. We also found that amino acid substitutions between type B apoA-II (Pro5, Val38) and type C apoA-II (Gln5, Ala38) did not affect either phase. The strategy of using synthetic partial peptides of amyloidogenic proteins in vitro is a useful system for understanding amyloid fibril formation and for the development of novel therapies.

The reduced form of coenzyme Q10 mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice.

Studies in humans and mice indicate a role for coenzyme Q(10) (CoQ(10)) in gene expression. To analyze this function in relation to metabolism, SAMP1 mice were supplemented with the reduced (ubiquinol) or oxidized (ubiquinone) form of CoQ(10) (500 mg/kg BW/d) for 14 months. Microarray analyses in liver tissues of SAMP1 mice identified 946 genes as differentially expressed between ubiquinol-treated and control animals (≥1.5-fold, P < 0.05). Text mining analyses revealed for a part of the ubiquinol-regulated genes, a functional connection in PPARα and LXR/RXR signalling pathways. Because these pathways are involved in cholesterol homeostasis, relevant metabolites were determined by gas chromatography/mass spectrometry (GC/MS). We found a significant increase of desmosterol (2.0-fold, P < 0.001) in the liver of ubiquinol-supplemented SAMP1 mice when related to control animals. In agreement, cholesterol concentrations were also distinctly increased (1.3-fold, P = 0.057). The Q(10)H(2)-induced PPARα and LXR/RXR gene expression signatures and effects on cholesterol metabolism were not apparent for the oxidized form of CoQ(10). In conclusion, the reduced form of CoQ(10) mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice.

Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat.

The Shumiya cataract rat (SCR) is a hereditary cataractous strain. It is thought that the continuous occurrence of poorly differentiated epithelial cells at the bow area of the lens forms the pathophysiological basis for cataract formation in SCRs. In this study, we attempted to identify the genes associated with cataract formation in SCRs by positional cloning. Genetic linkage analysis revealed the presence of a major cataract locus on chromosome 20 as well as a locus on chromosome 15 that partially suppressed cataract onset. Hypomorphic mutations were identified in genes for lanosterol synthase (Lss) on chromosome 20 and farnesyl diphosphate farnesyl transferase 1 (Fdft1) on chromosome 15, both of which function in the cholesterol biosynthesis pathway. A null mutation for Lss was also identified. Cataract onset was associated with the specific combination of Lss and Fdft1 mutant alleles that decreased cholesterol levels in cataractous lenses to about 57% of normal. Thus, cholesterol insufficiency may underlie the deficient proliferation of lens epithelial cells in SCRs, which results in the loss of homeostatic epithelial cell control of the underlying fiber cells and eventually leads to cataractogenesis. These findings may have some relevance to other types of cataracts, inborn defects of cholesterol synthesis, and the effects of cholesterol-lowering medication.

Suppression of Mouse AApoAII Amyloidosis Progression by Daily Supplementation with Oxidative Stress Inhibitors.

Amyloidosis is a group of diseases characterized by protein misfolding and aggregation to form amyloid fibrils and subsequent deposition within various tissues. Previous studies have indicated that amyloidosis is often associated with oxidative stress. However, it is not clear whether oxidative stress is involved in the progression of amyloidosis. We administered the oxidative stress inhibitors tempol and apocynin via drinking water to the R1.P1-Apoa2c mouse strain induced to develop mouse apolipoprotein A-II (AApoAII) amyloidosis and found that treatment with oxidative stress inhibitors led to reduction in AApoAII amyloidosis progression compared to an untreated group after 12 weeks, especially in the skin, stomach, and liver. There was no effect on ApoA-II plasma levels or expression of Apoa2 mRNA. Detection of the lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) revealed that the antioxidative effects of the treatments were most obvious in the skin, stomach, and liver, which contained higher levels of basal oxidative stress. Moreover, the unfolded protein response was reduced in the liver and was associated with a decrease in oxidative stress and amyloid deposition. These results suggest that antioxidants can suppress the progression of AApoAII amyloid deposition in the improved microenvironment of tissues and that the effect may be related to the levels of oxidative stress in local tissues. This finding provides insights for antioxidative stress treatment strategies for amyloidosis.

Characterization of the cheetah serum amyloid A1 gene: critical role and functional polymorphism of a cis-acting element.

Amyloid A (AA) amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction. For practical conservation of this species, therefore, it is critical to elucidate the etiology of AA amyloidosis, especially to understand the mechanisms of transcriptional regulation of serum amyloid A (SAA), a precursor protein of the AA protein. In this study, the structure and nucleotide sequence of the cheetah SAA1 gene including the 5'-flanking promoter/enhancer region was determined. Putative nuclear factor kappa-B (NF-kappaB) and CCAAT/enhancer binding protein beta (C/EBPbeta) cis-acting elements, which play key roles in SAA1 transcriptional induction in response to inflammation, were identified in the 5'-flanking region of the cheetah SAA1 gene. Fortuitously, a single nucleotide polymorphism was identified in the captive cheetah cohort in the putative NF-kappaB cis-acting element and had a remarkable effect on SAA1 transcriptional induction. These results provide a foundation not only for clarifying the etiology of AA amyloidosis in the cheetah but also for contriving a strategy for conservation of this species.

Coenzyme Q10 Prevents Senescence and Dysfunction Caused by Oxidative Stress in Vascular Endothelial Cells.

Oxidative damage in endothelial cells is proposed to play an important role in endothelial dysfunction and atherogenesis. We previously reported that the reduced form of coenzyme Q10 (CoQ10H2) effectively inhibits oxidative stress and decelerates senescence in senescence-accelerated mice. Here, we treated human umbilical vein endothelial cells (HUVECs) with H2O2 and investigated the protective effect of CoQ10H2 against senescence, oxidative damage, and reduction in cellular functions. We found that CoQ10H2 markedly reduced the number of senescence-associated ß-galactosidase-positive cells and suppressed the expression of senescence-associated secretory phenotype-associated genes in H2O2-treated HUVECs. Furthermore, CoQ10H2 suppressed the generation of intracellular reactive oxygen species (ROS) but promoted NO production that was accompanied by increased eNOS expression. CoQ10H2 prevented apoptosis and reductions in mitochondrial function and reduced migration and tube formation activity of H2O2-treated cells. The present study indicated that CoQ10H2 protects endothelial cells against senescence by promoting mitochondrial function and thus could delay vascular aging.

Amyloidosis-inducing activity of blood cells in mouse AApoAII amyloidosis.

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II (APOA2) deposits as amyloid fibrils (AApoAII) in many organs. We previously reported that AApoAII amyloidosis can be transmitted by feces, milk, saliva and muscle originating from mice with amyloid deposition. In this study, the ability of blood components to transmit amyloidosis was evaluated in our model system. Blood samples were collected from SAMR1.SAMP1-Apoa2c amyloid-laden or amyloidosis-negative mice. The samples were fractionated into plasma, white blood cell (WBC) and red blood cell (RBC) fractions. Portions of each were further separated into soluble and insoluble fractions. These fractions were then injected into recipient mice to determine amyloidosis-induction activities (AIA). The WBC and RBC fractions from amyloid-laden mice but not from amyloidosis-negative mice induced AApoAII amyloid deposition in the recipients. The AIA of WBC fraction could be attributed to AApoAII amyloid fibrils because amyloid fibril-like materials and APOA2 antiserum-reactive proteins were observed in the insoluble fraction of the blood cells. Unexpectedly, the plasma of AApoAII amyloidosis-negative as well as amyloid-laden mice showed AIA, suggesting the presence of substances in mouse plasma other than AApoAII fibrils that could induce amyloid deposition. These results indicated that AApoAII amyloidosis could be transmitted across tissues and between individuals through blood cells.

Comprehensive proteomic profiles of mouse AApoAII amyloid fibrils provide insights into the involvement of lipoproteins in the pathology of amyloidosis.

Amyloidosis is a disorder characterized by extracellular fibrillar deposits of misfolded proteins. The amyloid deposits commonly contain several non-fibrillar proteins as amyloid-associated proteins, but their roles in amyloidosis pathology are still unknown. In mouse senile amyloidosis, apolipoprotein A-II (ApoA-II) forms extracellular amyloid fibril (AApoAII) deposits with other proteins (AApoAII-associated proteins) in many organs. We previously reported that R1.P1-Apoa2c mice provide a reproducible model of AApoAII amyloidosis. In order to investigate the sequential alterations of AApoAII-associated protein, we performed a proteomic analysis of amyloid fibrils extracted from mouse liver tissues that contained different levels of AApoAII deposition. We identified 6 AApoAII-associated proteins that constituted 20 of the top-ranked proteins in mice with severe AApoAII deposition. Although the amount of AApoAII-associated proteins increased with the progression of amyloidosis, the relative abundance of AApoAII-associated proteins changed little throughout the progression of amyloidosis. On the other hand, plasma levels of these proteins showed dramatic changes during the progression of amyloidosis. In addition, we confirmed that AApoAII-associated proteins were significantly associated with lipid metabolism based on functional enrichment analysis, and lipids were co-deposited with AApoAII fibrils from early stages of development of amyloidosis. Thus, these results demonstrate that lipoproteins are involved in AApoAII amyloidosis pathology. SIGNIFICANCE: This study presented proteomic profiles of AApoAII amyloidosis during disease progression and it revealed co-deposition of lipids with AApoAII deposits based on functional analyses. The relative abundance of AApoAII-associated proteins in the amyloid fibril fractions did not change over the course of development of AApoAII amyloidosis pathology. However, their concentrations in plasma changed dramatically with progression of the disease. Interestingly, several AApoAII-associated proteins have been found as constituents of lipid-rich lesions of other degenerative diseases, such as atherosclerosis and age-related macular degeneration. The common protein components among these diseases with lipid-rich deposits could be accounted for by a lipoprotein retention model.

Apolipoprotein A-II induces acute-phase response associated AA amyloidosis in mice through conformational changes of plasma lipoprotein structure.

During acute-phase response (APR), there is a dramatic increase in serum amyloid A (SAA) in plasma high density lipoproteins (HDL). Elevated SAA leads to reactive AA amyloidosis in animals and humans. Herein, we employed apolipoprotein A-II (ApoA-II) deficient (Apoa2 -/- ) and transgenic (Apoa2Tg) mice to investigate the potential roles of ApoA-II in lipoprotein particle formation and progression of AA amyloidosis during APR. AA amyloid deposition was suppressed in Apoa2 -/- mice compared with wild type (WT) mice. During APR, Apoa2 -/- mice exhibited significant suppression of serum SAA levels and hepatic Saa1 and Saa2 mRNA levels. Pathological investigation showed Apoa2 -/- mice had less tissue damage and less inflammatory cell infiltration during APR. Total lipoproteins were markedly decreased in Apoa2 -/- mice, while the ratio of HDL to low density lipoprotein (LDL) was also decreased. Both WT and Apoa2 -/- mice showed increases in LDL and very large HDL during APR. SAA was distributed more widely in lipoprotein particles ranging from chylomicrons to very small HDL in Apoa2 -/- mice. Our observations uncovered the critical roles of ApoA-II in inflammation, serum lipoprotein stability and AA amyloidosis morbidity, and prompt consideration of therapies for AA and other amyloidoses, whose precursor proteins are associated with circulating HDL particles.

Transmissibility of mouse AApoAII amyloid fibrils: inactivation by physical and chemical methods.

AApoAII amyloid fibrils have exhibited prion-like transmissibility in mouse senile amyloidosis. We have demonstrated that AApoAII is extremely active and can induce amyloidosis following doses less than 1 pg. We tested physical and chemical methods to disrupt AApoAII fibrils in vitro as determined by thioflavin T binding and electron microscopy (EM) as well as inactivating the transmissibility of AApoAII fibrils in vivo. Complete disruption of AApoAII fibrils was achieved by treatment with formic acid, 6 M guanidine hydrochloride, and autoclaving in an alkaline solution. Injection of these disrupted AApoAII fibrils did not induce amyloidosis in mice. Disaggregation with 6 M urea, autoclaving, and alkaline solution was incomplete, and injection of these AApoAII fibrils induced mild amyloidosis. Treatment with formalin, delipidation, freeze-thaw, and RNase did not have any major effect. A distinct correlation was obtained between the amounts of amyloid fibrils and the transmissibility of amyloid fibrils, thereby indicating the essential role of fibril conformation for transmission of amyloidosis. We also studied the inactivation of AApoAII fibrils by several organic compounds in vitro and in vivo. AApoAII amyloidosis provides a valuable system for studying factors that may prevent transmission of amyloid disease as well as potential novel therapies.