Kumiko Kogishi

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.

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.

MANAGEMENT AND DESIGN OF THE MAINTENANCE OF SAM MOUSE STRAINS : AN ANIMAL MODEL FOR ACCELERATED SENESCENCE AND AGE-ASSOCIATED DISORDERS

The Senescence-Accelerated Mouse (SAM) was established by inbreeding and pedigree selection based on the life span, degree of senescence, as well as the incidence and degree of several age-associated disorders. At first, SAM strains were developed under conventional conditions, but now some strains are also maintained under specific pathogen-free conditions. There are many methods used to maintain such strains of mice; our methods will be introduced as one example of how to develop and maintain strains of mice used in aging research.

Induction of Protein Conformational Change in Mouse Senile Amyloidosis

Aggregated amyloid fibrils can induce further polymerization of precursor proteins in vitro, thus providing a possible basis for propagation or transmission in the pathogenesis of amyloidoses. Previously, we postulated that the transmission of amyloid fibrils induces conformational changes of endogenous amyloid protein in mouse senile amyloidosis (Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo Z., Hosokawa, M., Mori, M., and Higuchi, K. (2001) Lab. Invest.81, 493–499). To further characterize this transmissibility, we injected amyloid fibrils (AApoAII(C)) of amyloidogenic C type apolipoprotein A-II (APOAIIC) intravenously into 2-month-old SAMR1 mice, which have B type apolipoprotein A-II (APOAIIB), and develop few if any amyloid deposits spontaneously. 10 months after amyloid injection, deposits were detected in the tongue, stomach, intestine, lungs, heart, liver, and kidneys. The intensity of deposition increased thereafter, whereas no amyloid was detected in distilled water-injected SAMR1 mice, even after 20 months. The deposited amyloid was composed of endogenous APOAIIB with a different amyloid fibril conformation. The injection of these amyloid fibrils of APOAIIB (AApoAII(B)) induced earlier and more severe amyloidosis in SAMR1 mice than the injection of AApoAII(C) amyloid fibrils. Thus, AApoAII(C) from amyloidogenic mice could induce a conformational change of less amyloidogenic APOAIIB to a different amyloid fibril structure, which could also induce amyloidosis in the less amyloidogenic strain. These results provide important insights into the pathogenesis of amyloid diseases.

A monoclonal antibody to the 15,000 Dalton protein associated with porcine pulmonary surfactant

From a surface active fraction of porcine lung lavage fluid, separated by discontinuous sucrose density gradient ultracentrifugation, a protein with a nominal molecular weight (MW) of 15,000 daltons was isolated by sequential extraction with several buffers, including one containing deoxycholate. A monoclonal antibody was prepared from a hybrid cell (8B5E) obtained by fusing a myeloma cell, X63.Ag8.653, with spleen cells of BALB/c mice immunized with the protein. With immunoblotting technique, the antibody was found to be specific to the 15,000 dalton protein and did not react with another surfactant-associated protein with a nominal MW of 38,000 daltons. The antibodys IgG subclass was IgG1 and the light chain was kappa. In immunohistochemical studies using biotinylated antibody, peroxidase reaction products were localized selectively at inclusions of alveolar wall cells which were located chiefly at the alveolar corners. These results strongly suggest that this 15,000 dalton protein was localized in inclusions of alveolar wall cells and did not originate from other larger surfactant-associated proteins degraded after secretion into alveolar space.

Genetic typing of the Senescence-Accelerated Mouse (SAM) strains with microsatellite markers

Abstract. The Senescence-Accelerated Mouse (SAM) strains constitute a murine model of accelerated senescence originating from the ancestral AKR/J strains and consist of nine senescence-prone (SAMP) strains and four senescence-resistant (SAMR) strains. The chromosomes (Chrs) of the SAM strains were typed with 581 microsatellite markers amplified by PCR, and the fundamental genetic information of the SAM strains was obtained. One-third of the examined markers displayed polymorphism among the strains, and only two alleles were detected in almost all loci among the SAM and AKR/J strains. However, in 12 loci (5.6% of total 215 polymorphic markers), the third allele was detected among the SAM strains. The genetic typing and developmental history suggested that the SAM strains were related inbred strains developed by the accidental crossing between the AKR/J strain and other unknown strain(s). Comparison of the distribution of the loci in the SAMP and the SAMR series revealed notable differences in the four regions on Chrs 4, 14, 16, and 17. This indicated that some of these chromosomal sites might contain the genes responsible for accelerated senescence in the SAMP series.

Development of congenic strains of mice carrying amyloidogenic apolipoprotein A-II (Apoa2c): Apoa2c reduces the plasma level and the size of high density lipoprotein

A congenic strain of mice with amyloidogenic apolipoprotein A‐II (Apoa2c) on the genetic background of the amyloidosis‐resistant SAM‐R/1 strain was produced by 12 generations of backcrossing. Genome mapping using endogenous murine leukemia proviral markers was done in the congenic strain, termed R1·P1‐Apoa2c. We confirmed that only a small region surrounding the apoA‐II gene on chromosome 1 was transferred from the genome of the donor SAM‐P/1 strain. The level and particle size of plasma high density lipoprotein were decreased in RI · Pl‐Apoa2c mice compared to those in the progenitor SAM‐R/1 mice. The function of apoA‐II can be studied using this strain of mice.

AGE-ASSOCIATED CHANGES IN ELASTIN AND COLLAGEN CONTENT AND THE PROPORTION OF TYPES I AND III COLLAGEN IN THE LUNGS OF MICE

We investigated the effect of aging on the extracellular matrix of the lungs by examining age-associated changes in the elastin and collagen content, and the proportion of types I and III collagen, in the whole lungs of BALB/c and SAMR1 male mice between the ages of 3 and 24 months. The elastin content was determined by the hot alkali method. The hydroxyproline content was measured and assessed to be the collagen content. The relative proportion of types I and III collagen was assessed by cyanogen bromide digestion, followed by separation of the resultant peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The elastin content did not change significantly with age in either strain of mice. The total collagen content of the whole lung was significantly higher at 24 months of age, although there were no significant changes with aging in the hydroxyproline content per dry lung weight nor in the proportion of type III to type I collagen. We conclude that in terms of the extracellular matrix, the lungs of aged mice are not very different in feature from the lungs of younger mice, and this is probably the simple consequence of growth of the lungs of young mice.

Polymorphisms of mouse apolipoprotein A-11: seven alleles found among 41 inbred strains of mice

In mice, apolipoprotein A-II (apoA-II) associates to form amyloid fibrils in an age-associated manner. We determined the complete nucleotide sequences of the apoA-II gene (Apoa2) cDNA in 41 inbred strains of mice including Mus musculus domesticus (laboratory mouse), Mus musculus castaneus, Mus musculus molossinus, Mus musculus musculus and Mus spretus. Among these strains we identified 7 alleles (Apoa2a1, Apoa2a2, Apoa2b, Apoa2c, Apoa2d, Apoa2e and Apoa2f). Polymorphisms of nucleotides at 15 positions were detected and amino acid substitutions were found at 8 positions. Apoa2a1 was found in all mouse subspecies, but Apoa2b and Apoa2c were found only in Mus musculus domesticus. Two strains of Mus spretus have the unique alleles Apoa2eand Apoa2f which resemble Apoa2c. We confirmed that VICS in which we found severe amyloidosis here and other amyloidoneic strains in published reports have Apoa2c allele. We determined the plasma concentrations of total and HDL cholesterol in the strains of Mus musculus domesticus with the Apoa2a1, Apoa2b and Apoa2c alleles. Significantly higher concentrations of plasma cholesterol were observed in mouse strains with the Apoa2b allele. These findings provide fundamental data on mouse Apoa2 alleles. Furthermore, differences in these alleles likely have considerable influence on traits related to amyloidosis and lipid metabolism.

Mouse senile amyloid deposition is suppressed by adenovirus-mediated overexpression of amyloid-resistant apolipoprotein A-II.

Apolipoprotein A-II (apoA-II), the second most abundant apolipoprotein of serum high density lipoprotein, deposits as an amyloid fibril (AApoAII) in old mice. Mouse strains with a high incidence of senile amyloidosis have the type C apoA-II gene (Apoa2(c)), whereas the strains with a low incidence of amyloidosis have the type B apoA-II gene (Apoa2(b)). In this study, to investigate whether the type B apoA-II protein inhibits the extension of amyloid fibrils, we constructed an adenovirus vector bearing the Apoa2(b) cDNA (Adex1CATApoa2(b)), which is expressed under the control of a hepatocyte-specific promoter. The mice were infected with Adex1CATApoa2(b) before induction of amyloidosis by the injection of AApoAII amyloid fibril seeds. Compared with the mice infected with the control virus, amyloid deposition was suppressed significantly in the mice infected with Adex1CATApoa2(b). Fluorometry using thioflavine T also revealed that AApoAII fibril extension was inhibited by the addition of type B apoA-II in vitro. Thus, we propose that Apoa2(b) contributes as an active inhibitor of amyloid fibril extension and overexpression of amyloid-resistant gene variant may be an attractive therapeutic target in amyloidosis.