TTR Exon-Humanized Mice as a Model for Genotype-Dependent Transthyretin Deposition: Supportive Evidence for RBP4 as a Natural Stabilizer
Abstract
Zhenghua Li, Hideki Kanazashi, Yuya Murakami, Rie Fujikawa, Naoko Matsushita, Toru Takeo, Aki Sugano, Mika Ohta, Yutaka Takaoka and Kenichi Yamamura
Transthyretin amyloidosis is caused by destabilization, dissociation, and tissue deposition of transthyretin (TTR). Although several transgenic and knock-in mouse models have been generated, many models are limited by non- physiological copy number, low expression, or the formation of hybrid tetramers between mouse and human TTR. We previously generated TTR exon-humanized mice in which mouse Ttr exons were replaced with corresponding human TTR exons while preserving the mouse genomic structure. In the present study, we introduced the Val30Met mutation into the TTR exon-humanized allele and generated three genotypes: TtrhTTRV30e/hTTRV30e, TtrhTTRM30e/hTTRM30e, and TtrhTTRV30e/hTTRM30e mice. We examined non-fibrillar human TTR deposition by immunohistochemistry in ileum, kidney, and sciatic nerve at 26 and 52 weeks of age, using a semi-quantitative scoring system. Genotype-dependent TTR deposition was observed in multiple tissues. TTR deposition was prominent in V30/V30 and V30/M30 mice, whereas M30/M30 mice consistently showed lower deposition scores. Notably, TTR-positive deposits were detected in the sciatic nerve at 52 weeks of age. Direct fast scarlet staining did not detect amyloid fibrils, indicating that the observed deposits represented non-fibrillar or pre-amyloid TTR deposition. Serum human TTR levels showed modest genotype-dependent differences, whereas serum mouse RBP4 levels differed markedly among genotypes. Supportive computer simulation analysis suggested that the predicted binding stability between mouse RBP4 and TTR was highest for mouse TTR, intermediate for human TTR Met30, and lowest for human TTR Val30. These findings indicate that TTR exon-humanized mice provide a useful model for evaluating genotype-dependent TTR deposition and support the possibility that RBP4 functions as a natural stabilizer of circulating TTR.