Tatsumi Korenaga

Induction of AApoAII amyloidosis by various heterogeneous amyloid fibrils

Preformed amyloid fibrils accelerate conformational changes of amyloid precursor proteins and result in rapid extension of amyloid fibrils in vitro. We injected various kinds of amyloid fibrils into mice with amyloidogenic apoAII gene (Apoa2C ). The most severe amyloid depositions were detected in the tissues of mice injected with mouse AApoAII(C) amyloid fibrils. Mild amyloid depositions were also detected in the tissues of mice that were injected with other types of fibrils, including synthetic peptides and recombinant proteins. However, no amyloid depositions were found in mice that were injected with non‐amyloid fibril proteins. These results demonstrated that a common structure of amyloid fibrils could serve as a seed for amyloid fibril formation in vivo.

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.

Tissue Distribution, Biochemical Properties, and Transmission of Mouse Type A AApoAII Amyloid Fibrils

In mouse strains with the amyloidogenic apolipoprotein A-II (ApoA-II) gene (Apoa2c), the type C ApoA-II protein (APOAIIC) associates to form amyloid fibrils AApoAII(C) that lead to development of early onset and systemic amyloidosis with characteristic heavy amyloid deposits in the liver and spleen. We found age-associated heavy deposition of amyloid fibrils [AApoAII(A)] composed of type A ApoA-II protein (APOAIIA) in BDF1 and C57BL/6 mice reared at one of our institutes. AApoAII(A) fibrils were deposited in the intestine, lungs, tongue, and stomach but not in the liver or spleen. AApoAII(A) fibrils were isolated, and morphological, biochemical, and structural characteristics distinct from those seen in AApoAII(C) and mouse AA amyloid fibrils were found. Transmission electron and atomic force microscopy showed that the majority of isolated AApoAII(A) amyloid fibrils featured fine, protofibril-like shapes. AApoAII(A) fibrils have a much weaker affinity for thioflavine T than for AApoAII(C), whereas APOAIIA protein contains less of the beta-pleated sheet structure than does APOAIIC. The injection of AApoAII(A) fibrils induced amyloid deposition in C57BL/6 and DBA2 mice (Apoa2a) as well as in R1.P1-Apoa2c mice (Apoa2c), but AApoAII(A) induced more severe amyloidosis in Apoa2a strains than in the Apoa2c strain. It was found that AApoAII(A) fibrils isolated from mice with mildly amyloidogenic APOAIIA protein have distinct characteristics. Induction of amyloidosis by heterologous amyloid fibrils clearly showed interactions between amyloid protein monomers and fibrils having different primary structures.

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)

Extrahepatic expression of apolipoprotein A-II in mouse tissues: Possible contribution to mouse senile amyloidosis

Apolipoprotein A-II (apoA-II), an apolipoprotein in serum high-density lipoprotein, is a precursor of mouse senile amyloid fibrils. The liver has been considered to be the primary site of synthesis. However, we performed nonradioactive in situ hybridization analysis in tissue sections from young and old amyloidogenic (R1.P1-Apoa2 c ) and amyloid-resistant (SAMR1) mice and revealed that other tissues in addition to the liver synthesize apoA-II. We found a strong hybridization signal in the basal cells of the squamous epithelium and the chief cells of the fundic gland in the stomach, the crypt cells and a small portion of the absorptive epithelial cells in the small intestine, the basal cells of the tongue mucosa, and the basal cells of the epidermis and hair follicles in the skin in both mouse strains. Expression of apoA-II mRNA in those tissues was also examined by RT-PCR analysis. Immunolocalization of apoA-II protein also indicated the cellular localization of apoA-II. ApoA-II transcription was not observed in the heart. Amyloid deposition was observed around the cells expressing apoA-II mRNA in the old R1.P1-Apoa2 c mice. These results demonstrate that the apoA-II mRNA is transcribed and translated in various extrahepatic tissues and suggest a possible contribution of apoA-II synthesized in these tissues to amyloid deposition.

Amyloidosis Modifier Genes in the Less Amyloidogenic A/J Mouse Strain

Apolipoprotein A-II is deposited as an amyloid fibril in aged mice (senile AApoAII amyloidosis). Although mouse strains with the apolipoprotein A-II c allele (Apoa2c) generally develop early-onset and severe senile amyloidosis, the A/J strain shows significantly less amyloid deposition. To identify genes that modify spontaneous amyloidosis development in the A/J mouse, we performed a genome-wide screening using hybrid mice derived from A/J and SAMP1 mice, which have Apoa2c and age-associated severe amyloid deposition. Our genetic analysis revealed that the lower levels of amyloidosis in the A/J strain were polygenically controlled. We found two chromosome locations associated with amyloidosis. One of these regions was in the chromosome 19 telomeric region, where the A/J alleles modify amyloidosis in an additive manner. The second region was in the chromosome 4 telomeric region, where the A/J alleles modify amyloidosis in a dominant manner. Perlecan and group II secretory phospholipase A2, located on the significantly linked region of chromosome 4, were compared in this study. These findings are for understanding the genetic mechanism of amyloidosis-related diseases and their prevention.

Induction of AApoAII and AA amyloidosis by the injections of various amyloid fibrils

Abstract Preformed amyloid fibrils accelerate conformational changes of amyloid precursor proteins and result in a rapid extension of amyloid fibrils in vitro. In this paper, we show the induction of AApoAII amyloidosis in vivo by various human and mouse amyloid fibrils isolated from human and mouse tissues or formed in vitro from synthetic peptides and recombinant proteins. These fibrils were injected intravenously into R1.P1- Apoa2 c mice. At 3 and 6 months after injection, amyloid deposition was examined. Severe amyloid depositions were detected in the tissues of mice injected with AApoAII (C) amyloid fibrils. Mild or slight amyloid depositions were also detected in the tissues of mice, which were injected with other types of fibrils, including synthetic peptides. However, no amyloid depositions were found in the control mice, which were injected with non-amyloid fibril proteins. In addition, AA amyloidosis was accelerated by the various amyloid fibrils in C57BL/6 mice, which were injected simultaneously and subcutaneously with 1.0% AgNO 3 . These results demonstrated that various exogenous substances with a common structure could work as seeds for amyloid fibril formation in vivo. These findings suggested a prion-like mechanism of protein conformation in the pathogenesis of mouse AApoAII and AA amyloidosis.