Beiru Zhang

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.

Amyloidosis in transgenic mice expressing murine amyloidogenic apolipoprotein A-II (Apoa2c).

In mice, apolipoprotein A-II (apoA-II) self-associates to form amyloid fibrils (AApoAII) in an age-associated manner. We postulated that the two most important factors in apoA-II amyloidosis are the Apoa2c allele, which codes for the amyloidogenic protein APOA2C (Gln5, Ala38) and transmission of amyloid fibrils. To characterize further the contribution of the Apoa2c allele to amyloidogenesis and improve detection of amyloidogenic materials, we established transgenic mice that overexpress APOA2C protein under the cytomegalovirus (CMV) immediate early gene (CMV-IE) enhancer/chicken β promoter. Compared to transgene negative (Tg−/−) mice that express apoA-II protein mainly in the liver, mice homozygous (Tg+/+) and heterozygous (Tg+/−) for the transgene express a high level of apoA-II protein in many tissues. They also have higher plasma concentrations of apoA-II, higher ratios of ApoA-II/apolipoprotein A-I (ApoA-I) and higher concentrations of high-density lipoprotein (HDL) cholesterol. Following injection of AApoAII fibrils into Tg+/+ mice, amyloid deposition was observed in the testis, liver, kidney, heart, lungs, spleen, tongue, stomach and intestine but not in the brain. In Tg+/+ mice, but not in Tg−/− mice, amyloid deposition was induced by injection of less than 10−8 μg AApoAII fibrils. Furthermore, deposition in Tg+/+ mice occurred more rapidly and to a greater extent than in Tg−/− mice. These studies indicate that increased levels of APOA2C protein lead to earlier and greater amyloid deposition and enhanced sensitivity to the transmission of amyloid fibrils in transgenic mice. This transgenic mouse model should prove valuable for studies of 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.

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 Apoa2cTg+/− and normal R1.P1-Apoa2c 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.

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.

Mouse model to study human A β2M amyloidosis: Generation of a transgenic mouse with excessive expression of human β2-microglobulin

Patients on long-term hemodialysis can develop dialysis-related amyloidosis (DRA) due to deposition of β2-microglobulin (β2m) into amyloid fibrils (Aβ2M). Despite intensive biochemical studies, the pathogenesis of amyloid deposition in DRA patients remains poorly understood. To elucidate the mechanisms that underlie Aβ2M fibril formation in DRA, we generated transgenic mice that overexpress human β2m protein in a mouse β2m gene knockout background (hB2MTg+/+ mB2m+/+). The hB2MTg+/+mB2m−/− mice express a high level of human β2m protein in many tissues as well as a high plasma β2m concentration (192.8 mg/L). This concentration is >100 times higher than that observed in healthy humans and >4 times higher than that detected in patients on dialysis. We examined spontaneous and amyloid fibril-induced amyloid deposition in these mice. Amyloid deposition of β2m protein was not observed in aged or amyloid fibril injected animals. However, mouse senile apolipoprotein A-II amyloidosis (AApoAII) was detected, particularly in the joints of mice that were injected with AApoAII amyloid fibrils. This study demonstrates that this mouse model could be valuable in studying the components and conditions that promote DRA, and indicates that high plasma concentrations of hβ2m as well as seeding with pre-existing amyloid fibrils may not be sufficient to induce Aβ2M.

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

Significance Apolipoprotein (apo) A-II is the most important protein associated with senile amyloidosis. Because some variants of apoA-II protein have been found among inbred strains of mice, we hypothesized that investigating amyloidogenesis of the variants would improve our understanding of the molecular and biological mechanisms of senile amyloidosis. Here, we demonstrate that mice with type F apoA-II (APOA2F) protein were absolutely resistant to senile amyloidosis. Moreover, a selective C-terminal APOA2F peptide inhibited fibril formation of amyloidogenic apoA-II in vitro and prevented senile amyloidosis in mice. We propose an inhibitory model in which the C-terminal APOA2F peptide prevents further fibril extension by blocking the active ends of seeds. This approach could provide a novel therapeutic option for the treatment of senile amyloidosis. 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.

The Toxicity Mechanisms of Action of Aβ25–35 in Isolated Rat Cardiac Myocytes

β-Amyloid (Aβ) is deposited in neurons and vascular cells of the brain and is characterized as a pathologic feature of Alzheimer’s disease (AD). Recently studies have reported that there is an association between cardiovascular risk factors and AD, however the mechanism of this association is still uncertain. In this study we observed Aβ had an effect on cardiovascular cells. We represent as a major discovery that Aβ25–35 had toxicity on isolated rat cardiac myocytes by impacting the cytoskeleton assembly and causing ER stress, ultimately contributing to the apoptosis of the myocytes. Importantly, the activation of ER stress and subsequent cellular dysfunction and apoptosis by Aβ25–35 was regulated by the MAPK pathway, which could be prevented by inhibition of p38 via pharmacological inhibitors. It was noteworthy that Aβ25–35 played a critical role in cardiac myocytes, suggesting that Alzheimer’s disease (AD) had a relation with the heart and understanding of these associations in future will help search for effective treatment strategies.

Mouse apoA-II amyloid fibrils deposit in skeletal muscle and exhibit amyloidosis-enhancing activity

In mouse senile amyloidosis, apolipoprotein A-II (apoA-II) deposits as amyloid fibrils (AApoAII). Mouse AApoAII amyloidosis has been demonstrated to be a transmissible disease by a prion-like infectious process and can be transmitted from one animal to another, both by the feces and milk excreted by mice with amyloidosis. To further characterize the transmissibility, AApoAII amyloid fibrils were injected into transgenic Apoa2cTgþ/7 and normal R1.P1-Apoa2 mice to induce amyloidosis. Two months later, AApoAII amyloid deposits were found in the skeletal muscles of amyloidaffected mice, primarily in the blood vessels and in the interstitial tissues surrounding muscle fibers (endomysium) both in Apoa2cTgþ/7 and R1.P1Apoa2 mice. Amyloid fibril fractions isolated from the muscles demonstrated the structure of amyloid fibrils in electron microscope and induced amyloidosis in young mice [1]. These findings present a possible pathogenesis of amyloidosis, transmission of amyloid fibrils through muscle, and shed new light on the etiology involved in amyloid disorders. Introduction: Mouse AApoAII amyloidosis has been demonstrated to be a transmissible disease by a prion-like infectious process occurring through a seeding-nucleation mechanism [2,3]. In the present study, we found AApoAII amyloid fibrils in the skeletal muscles of AApoAII amyloidaffected mice. Amyloid fibrils isolated from the muscles were demonstrated to be sufficient for the transmission of amyloidosis. Materials and methods: Mouse: In laboratory mice, the Apoa2 allele of the apolipoprotein A-II (apoA-II) gene markedly accelerates the deposition of AApoAII. R1.P1-Apoa2 is a congenic strain of mice with the amyloidogenic Apoa2 allele in the genetic background of the SAMR1 strain. Apoa2 transgenic mice (Apoa2 Tgþ/7) were established in the genetic background of R1.P1-Apoa2 [4]. Mice were raised under specific pathogen-free condition. All experimental procedures were carried out in accordance with the Regulations for Animal Experimentation of Shinshu University. Induction and secondary transmission of amyloidosis: Two-month-old female Apoa2cTgþ/7 and R1.P1-Apoa2 mice were injected intravenously with 1 mg (Apoa2cTgþ/7) and 100 mg (R1.P1-Apoa2) of AApoAII fibrils to induce amyloidosis. Two and 4 months later, the mice were sacrificed and the triceps brachii muscles in the forelimbs, the femoral quadriceps muscles in the hindlimb, the longissimus thoracis muscles in the back, and the greater pectoral muscles from the breast were dissected. Amyloid fibril fractions were isolated from the muscle of amyloid fibril-injected mice by Pras’ method and injected into 2-month-old female R1.P1-Apoa2 mice, and after 2 months, the mice were sacrificed and the intensity of AApoAII amyloid deposition was determined. The amyloid index (AI) was determined by taking the mean value of the scores of amyloid deposition (graded from 0 to 4) in the seven major organs (liver, spleen, tongue, heart, intestine, stomach, and skin) stained with Congo Red as described previously [2]. Results: AApoAII amyloid deposits in the muscle: We intravenously injected 1 mg of AApoAII fibrils into six 2-month-old female Apoa2cTgþ/7 mice. Two months later, amyloid deposition was detected in all four kinds of muscles of Apoa2cTgþ/7 mice displaying heavy amyloid deposits throughout the body. In 100 mg amyloid fibril-injected normal R1.P1-Apoa2 mice, amyloid deposition was observed in one of three mice 2 months after injection (1/3), and all three had amyloid deposits at 4 months after injection (3/3). Amyloid deposits were found mainly in the blood vessels of muscle tissues, but were also found in connective tissues around muscle fibers (endomysium), both in Apoa2cTgþ/7 and R1.P1-Apoa2 mice (Figure 1A,B). AApoAII amyloid deposition was confirmed with anti-apoA-II staining (Figure 1 C,D). Amyloid fibril fractions were isolated from various muscles, and apoA-II protein was detected by Western blot analysis. ApoA-II was detected in all four kinds of muscles of Apoa2cTgþ/7 mice. In R1.P1-Apoa2 mice, 2 months after injection of amyloid fibrils, apoA-II was detected in greater pectoral muscles in the breast of all three mice. Four months after injection, apoA-II deposition expanded to other muscles and amounts of apoA-II increased. The amount of deposition was different among different muscles: greater pectoral muscles from the breast4 longissimus thoracis muscle in the back4 triceps brachii muscles in the fore-limbs4 femoral quadriceps muscles in the pelvic-limb. Secondary transmission of amyloidosis: To elucidate whether AApoAII amyloid transmissibility existed in skeletal muscle, amyloid fibril fractions were isolated from femoral quadriceps muscles of Apoa2cTgþ/7 mouse with AApoAII deposition and 42

Mouse Models to Study Systemic Amyloidoses: Is Prion-Like Transmission a Common Pathogenic Mechanism?

The amyloidoses are a group of protein-misfolding disorders characterized by the accumulation of amyloid fibrils formed from a variety of proteins. Currently, twentyeight different kinds of human and animal proteins, in intact or fragmented forms, have been found to be associated with pathological disorders such as Alzheimer’s disease, type II diabetes, prion diseases, dialysis-related amyloidosis, and various familial, senile and sporadic amyloidosis (Sipe et al., 2010; Benson et al 2008). Amyloidoses have been divided into two major classes: 1) systemic and 2) localized amyloidoses. In systemic amyloidoses, precursor proteins circulating in the blood associate to form amyloid fibrils that are then deposited throughout the body. For example, immunoglobulin light chains form deposits in patients with myeloma in AL amyloidosis. In reactive AA amyloidosis, serum amyloid A (SAA) protein forms deposits in patients with chronic inflammation, and transthyretin (TTR) forms deposits in patients with familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). Patients on long-term hemodialysis develop dialysis-related amyloidosis (DRA) due to the deposition of amyloid fibrils (As2M) of s2-microglobulin (s2m). In contrast to systemic amyloidosis, precursor proteins produced in local organs deposit in one particular area of the body in various localized amyloidoses. In mice, apolipoprotein A-II (apoA-II) in serum high density lipoproteins (HDL) forms amyloid fibrils (AApoAII) in age-associated systemic amyloidosis (senile AApoAII amyloidosis). AA amyloidosis, known as reactive or secondary amyloidosis associated with inflammation, is generally recognized as the predominant form of systemic amyloidosis that occurs in humans, mice, domestic animals and many species in the animal kingdom. These amyloidoses are characterized by the systemic deposition of extracellular fibrils composed of apoA-II in AApoAII amyloidosis or SAA (serum AA) in AA amyloidosis, primarily in the spleen, liver, heart, kidney, vessels walls, and to a lesser extent in other organs. In most