Jingmin Yan

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.

Reduced coenzyme Q10 supplementation decelerates senescence in SAMP1 mice

The SAMP1 strain is a mouse model for accelerated senescence and severe senile amyloidosis. We determined whether supplementation with coenzyme Q10 (CoQ10) could decelerate aging in SAMP1 mice and its potential role in aging. Plasma concentrations of CoQ10 and CoQ9 decreased with age in SAMP1 but not in SAMR1 mice. Supplementation with reduced CoQ10 (CoQH2, 250 mg/kg/day) for one week increased plasma CoQ10 concentrations, with an accompanying decrease in plasma CoQ9 concentrations. In two series of experiments, lifelong supplementation with CoQH2 decreased the senescence grading scores from 10 to 14 months, 7 to 15 months, and at 17 months of age. The body weight of female mice increased from 2 to 10 months of age versus controls in the second series of experiments. Lifelong CoQH2 supplementation did not prolong or shorten the lifespan, nor did it alter the murine senile amyloid (AApoAII) deposition rate or cancer incidence. In the second series of experiments, urinary levels of 8-hydroxydeoxyguanosine did not change with age or long-term supplementation with CoQH2. Urinary levels of acrolein (ACR)-lysine adduct increased significantly with age in SAMP1 mice; however, CoQH2 had no effect. Thus, lifelong dietary supplementation with CoQH2 decreased the degree of senescence in middle-aged SAMP1 mice.

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, freezethaw, 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. FASEB J. 20, E211–E221 (2006)

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.

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 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