Keiichi Higuchi

Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T☆

We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.

Senescence-Accelerated Mouse (SAM): A Novel Murine Model of Accelerated Senescence

n animal model for accelerated senescence named the Senescence-Accelerated Mouse (SAM) was developed in our laboratory (Department of Senescence Biology, formerly Department of Pathology, Chest Disease Research Institute, Kyoto University) beginning in 1970. In this review, the circumstances related to development of SAM, characteristics of aging, pathologic phenotypes, and genetic background of this model are described.

Pathobiology of the senescence-accelerated mouse (SAM)

Routine postmortem examinations and the pathobiological features revealed by systematically designed studies have shown several pathologic phenotypes that are often characteristic enough to differentiate among the various SAM strains: senile amyloidosis in SAMP1, -P2, -P7, -P9, -P10, and -P11; secondary amyloidosis in SAMP2 and -P6; contracted kidney in SAMP1, -P2, -P10, and -P11; immunoblastic lymphoma in SAMR1 and -R4; histiocytic sarcoma in SAMR1 and -R4; ovarian cysts in SAMR1; impaired immune response in SAMP1, -P2, and -P8; hyperinflation of the lungs in SAMP1; hearing impairment in SAMP1; degenerative temporomandibular joint disease in SAMP3; senile osteoporosis in SAMP6; deficits in learning and memory in SAMP8 and -P10; emotional disorders in SAMP8 and -P10; cataracts in SAMP9; and brain atrophy in SAMP10. These are all age-associated pathologies, the incidence and severity of which increase with advancing age. The SAM model in which these pathobiological features have been carefully monitored will be a valuable tool in the clarification of the pathogenic mechanisms of age-associated pathologies and in the research for effective methods to modulate or ameliorate these pathologies.

Identification of peak bone mass QTL in a spontaneously osteoporotic mouse strain

Abstract. The whole genome scan for quantitative trait loci (QTLs) specifying peak bone mass was performed with the F2 intercrosses of SAMP6, an established murine model of senile osteoporosis, exhibiting a significantly lower peak bone mass, and SAMP2, exhibiting a higher peak bone mass. Cortical thickness index (CTI), a parameter of bone mass of femurs, was measured in 488 F2 progeny at 4 months of age, when the animals attained peak bone mass by microphotodensitometry. Genetic markers were typed at 90 loci spanning all chromosomes except the Y. By interval mapping of 246 male F2 mice, two loci were identified with significant linkage to peak bone mass, one on Chromosome (Chr) 11 and another on Chr 13, with a maximum lod score of 10.8 (22.2% of the total variance) and 5.8 (10.0%), respectively. Another locus on the X Chr was suggestive of a QTL associated oppositely with a low peak bone mass to the SAMP2 allele. This association was consistent with the distribution of peak bone mass in the F1 and F2. These findings should be useful to elucidate the genetics of osteoporosis.

Spontaneous age-associated amyloidosis in senescence-accelerated mouse (sam).

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R). Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%). Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm. The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.

Transmission of mouse senile amyloidosis

In mouse senile amyloidosis, apolipoprotein A-II polymerizes into amyloid fibrils (AApoAII) and deposits systemically. Peripheral injection of AApoAII fibrils into young mice induces systemic amyloidosis (Higuchi et al, 1998). We isolated AApoAII amyloid fibrils from the livers of old R1.P1-Apoa2c mice and injected them with feeding needles into the stomachs of young R1.P1-Apoa2c mice for 5 consecutive days. After 2 months, all mice had AApoAII deposits in the lamina propria of the small intestine. Amyloid deposition extended to the tongue, stomach, heart, and liver at 3 and 4 months after feeding. AApoAII suspended in drinking water also induced amyloidosis. Amyloid deposition was induced in young mice reared in the same cage for 3 months with old mice who had severe amyloidosis. Detection of AApoAII in feces of old mice and induction of amyloidosis by the injection of an amyloid fraction of feces suggested the propagation of amyloidosis by eating feces. Here, we substantiate the transmissibility of AApoAII amyloidosis and present a possible pathogenesis of amyloidosis, ie, oral transmission of amyloid fibril conformation, where we assert that exogenous amyloid fibrils act as templates and change the conformation of endogenous amyloid protein to polymerize into amyloid fibrils.

Exercise Effects on Methylation of ASC Gene

Chronic moderate exercise has been reported to reduce pro-inflammatory cytokines. To analyze the molecular mechanisms by which training exerts these effects, the epigenetic influences of age and exercise on the ASC gene, which is responsible for IL-1beta and IL-18 secretion, were investigated by ASC gene methylation. Further, the relationship between carcinogenesis and exercise, and methylation of the P15 tumor suppressive gene was also analyzed. High-intensity interval walking exercise, consisting of 3 min low-intensity walking at 40% of peak aerobic capacity followed by a 3 min high-intensity walking period above 70% of peak aerobic capacity, was continued for 6 months. Peripheral blood DNA extracts from young control (n=34), older control (n=153), and older exercise (n=230) groups were then analyzed by pyrosequencing for DNA methylation. Methylation of ASC decreased significantly with age (young control vs. older control, p<0.01), which is indicative of an age-dependent increase in ASC expression. Compared to the older control group, the degree of ASC methylation was higher in the older exercise group (older control vs. older exercise: p<0.01), and presumably lower ASC expression. Neither exercise nor age affected the methylation of the P15. In summary, chronic moderate exercise appears to attenuate the age-dependent decrease in ASC methylation, implying suppression of excess pro-inflammatory cytokines through reduction of ASC expression.

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.

Secreted frizzled-related protein 4 is a negative regulator of peak BMD in SAMP6 mice.

We segregated a QTL for peak BMD on Chr 13 by generating congenic sublines of the senescence‐accelerated mouse SAMP6. Sfrp 4 within this locus was responsible for lower BMD of SAMP6.

A novel murine model of aging, Senescence-Accelerated Mouse (SAM)

Senescence-Accelerated Mouse (SAM) has been under development by our research team at Kyoto University since 1970, based on the AKR/J strain donated by the Jackson Laboratory in 1968. The SAM mouse has an accelerated senescence and age-associated pathologies such as senile amyloidosis, senile osteoporosis, degenerative joint disease, cataract, deficits in learning and memory, brain atrophy, hyperinflation of lungs, hearing impairment and so on. SAM research is advancing world-wide and attempts are being made to clarify fundamental mechanisms involved in primary aging processes, pathogenesis of age-associated pathologies and effective methods to modulate or ameliorate the advance of senescence and disease processes involved in age-associated pathologies.