School of Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, UK.
Invest Ophthalmol Vis Sci. 2011 Jun 21;52(7):4369-80. doi: 10.1167/iovs.10-6406.
To investigate the developmental and ocular expression of Opa3 in a mouse model of 3-methylglutaconicaciduria type III and the effect of mutation on protein localization and mitochondrial morphology.
The B6 C3-Opa3(L122P) mouse carrying a missense mutation in exon 2 (c.365T>C; p.L122P) of Opa3, which displays features of recessive 3-methylglutaconic aciduria type III was studied. The expression of Opa3 was determined with RT-PCR, quantitative PCR, and Western blot, in embryos (embryonic day [E]8 to postnatal day [P]0) and adult tissues, and by ocular immunohistochemistry. Mitochondria were stained using a mitochondrion-selective probe in mouse embryonic fibroblasts from Opa3(-/-) mutants and imaged by electron microscopy of the retinas.
The splice variants Opa3a and Opa3b were expressed in the lenses and the retinas in the Opa3(-/-) mice, with the expression of the Opa3a isoform predominant. Opa3 was expressed throughout embryonic development, with high levels of expression in the developing brain, retina, optic nerve, and lens. Opa3 localized to the mitochondria, and the L122P mutant protein did not mislocalize. Neither protein localized to the peroxisome. Opa3(-/-) mice displayed disrupted mitochondrial morphology in the retina. Wild-type Opa3 protein increased as the lenses aged, despite the reduction in Opa3 mRNA occurring as a part of lens differentiation. However, mutant Opa3 mRNA was upregulated in homozygous mutant lenses, suggesting a compensatory increase in expression, which may further increase Opa3 protein levels.
Mutant Opa3 protein retains its mitochondrial localization and induces disrupted mitochondrial morphology. Opa3 accumulates in the lens. The results may reflect a slow turnover of Opa3 protein in vivo and may be important in normal lens physiology.
研究 3-甲基戊烯二酸尿症 III 型小鼠模型中 Opa3 的发育和眼部表达，以及突变对蛋白定位和线粒体形态的影响。
研究携带 Opa3 外显子 2 （c.365T>C; p.L122P）错义突变的 B6 C3-Opa3（L122P）小鼠，该突变显示出隐性 3-甲基戊烯二酸尿症 III 型的特征。通过 RT-PCR、定量 PCR 和 Western blot，在胚胎（胚胎日 [E]8 至出生后第 [P]0 天）和成年组织中，以及通过眼部免疫组织化学，确定 Opa3 的表达。使用线粒体选择性探针对 Opa3（-/-）突变体的小鼠胚胎成纤维细胞中的线粒体进行染色，并通过视网膜的电子显微镜对其进行成像。
Opa3a 和 Opa3b 剪接变体在 Opa3（-/-）小鼠的晶状体和视网膜中表达，Opa3a 同工型的表达占优势。Opa3 在整个胚胎发育过程中表达，在发育中的大脑、视网膜、视神经和晶状体中表达水平较高。Opa3 定位于线粒体，L122P 突变蛋白没有定位错误。两种蛋白都没有定位于过氧化物酶体。Opa3（-/-）小鼠的视网膜中线粒体形态受损。尽管作为晶状体分化的一部分，Opa3 mRNA 的减少，但随着晶状体老化，野生型 Opa3 蛋白增加。然而，在纯合突变体晶状体中，突变型 Opa3 mRNA 上调，表明表达代偿性增加，这可能进一步增加 Opa3 蛋白水平。
突变型 Opa3 蛋白保留其线粒体定位，并诱导线粒体形态破坏。Opa3 在晶状体中积累。结果可能反映了体内 Opa3 蛋白的缓慢周转，这在正常晶状体生理学中可能很重要。
