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Identification of expression cells and elucidation of a function for a novel gene encoding a PrP-like protein newly separated and identified

编码新分离和鉴定的 PrP 样蛋白的新基因的表达细胞的鉴定和功能的阐明

基本信息

批准号：
12670207
负责人：
SHIGEMATSU Kazuto
金额：
$ 2.24万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

We identified aberrant mRNA species in the brain of PrP knockout mice (Prnp-/- Nagasaki), which mRNAs were the novel sequence encoding PrP-like protein (Dopple/PrPLP), a putative membrane glycoprotein with 23 % identity to PrpC in the primary amino acid structure. The expression tissues and cells for this gene were identified, and the following knowledge was acquired.(1) In adult wild-type mice, Dopple/PrPLP mRNA was physiologically expressed at a high level by testis and heart, bat was barely detectable in brain. However, transient expression of Dopple/PrPLP mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry Identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the Dopple/PrPLP mRNA localized in capillaries of spleen and lamina propria mucosa of gut. These findings suggested a role of Dopple/PrPLP in angiogenesis, in particular blood-brain barrier maturation.(2) In a testis of neonatal mice, the expression of mRNA was found in the leaf cells specializing in the lymph vessel in the future. On the other hand, from the 3rd week after birth, the spermatogonia at the stage IV-VI day of the spermatogenic cycle became to strongly express Dopple/PrPLP mRNA. Hence, Dopple/PrPLP may play an important role in the spermatogenesis and blood-testis barrier.(3) Northern blotting demonstrated unregulated expression of the genes for GFAP and LM in the brains of PrP knockout mice. A transgene for normal mouse PrP^C- successfully rescued PrP knockout mice from the glial activation. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. These findings strongly suggest that ectopic Dopple/PrPLP in the absence of Prp^C is actively involved in the glial-cell activation in the brain.

我们在PrP基因敲除小鼠（Prnp-/- Nagasaki）的大脑中发现了异常mRNA，这些mRNA是编码PrP样蛋白（Dopple/PrPLP）的新序列，PrP样蛋白是一种假定的膜糖蛋白，其初级氨基酸结构与PrpC的同源性为23%。鉴定了该基因的表达组织和细胞，获得了以下知识。(1)在成年野生型小鼠中，Dopple/PrPLP mRNA在睾丸和心脏中有高水平的生理表达，而在大脑中几乎检测不到。然而，在新生野生型小鼠的大脑中，通过Northern blotting检测到Dopple/PrPLP mRNA的瞬时表达，在出生后1周左右达到最大表达。原位杂交与免疫组织化学配对鉴定了表达转录本的脑内皮细胞。此外，在新生野生型小鼠中，Dopple/PrPLP mRNA定位于脾脏毛细血管和肠道固有层粘膜。这些发现提示Dopple/PrPLP在血管生成，特别是血脑屏障成熟中的作用。(2)在新生小鼠的睾丸中，mRNA在未来专门用于淋巴管的叶细胞中表达。另一方面，从出生后第3周开始，生精周期第IV-VI天的精原细胞开始强烈表达Dopple/PrPLP mRNA。因此，Dopple/PrPLP可能在精子发生和血睾丸屏障中发挥重要作用。(3) Northern blotting显示，PrP敲除小鼠大脑中GFAP和LM基因的表达不受调控。正常小鼠PrP^C-的转基因成功地将PrP敲除小鼠从神经胶质活化中拯救出来。此外，在浦肯野细胞变性发生之前，胶质细胞的活化是显著的。这些发现有力地表明，在缺乏Prp^C的情况下，异位Dopple/PrPLP积极参与脑内胶质细胞的激活。