Ghandour M Said, Feutz Anne-Catherine, Jalabi Walid, Taleb Omar, Bessert Denise, Cypher Maria, Carlock Leon, Skoff Robert P
UMR 7004 ULP/CNRS, Strasbourg, France.
Glia. 2002 Dec;40(3):300-11. doi: 10.1002/glia.10122.
The synthesis, transport, and insertion of jimpy proteolipid protein and DM20 were studied in normal (158N) and jimpy (158JP) immortalized oligodendrocyte lines. Four different expression vectors encoding fusion proteins composed of native PLP and DM20 or jimpy PLP or DM20 were linked to enhanced green fluorescent protein (EGFP). All four transfected fusion proteins had similar distributions in the cell bodies and processes of the two cell types. Both normal and jimpy PLP-EGFP and DM20-EGFP were detected in both cell lines as far as 200 microM from the cell body, indicating synthesis and transport of mutated PLP and DM20 toward the plasma membrane. Immunocytochemistry of fixed normal and jimpy cells with the O10 antibody, which recognizes a conformationally sensitive PLP/DM20 epitope, confirmed that normal and jimpy PLP and DM20 were transported to the plasma membrane. Live staining of normal and jimpy cells transiently transfected with the native PLP showed positive staining, indicating PLP was correctly inserted into the membrane of both normal and jimpy oligodendrocytes. However, live staining of normal and jimpy cells transiently transfected with jimpy PLP showed no positive staining, indicating the mutated protein is abnormally inserted into the plasma membrane. Electrophysiological recordings of the resting membrane potential measured in the whole cell mode of the patch-clamp technique showed the absence of a developmentally regulated negative shift in the membrane potential in jimpy cells compared to normal native or immortalized oligodendrocytes. Treatment of 158N cells and native oligodendrocytes with dibutyryl cAMP (dbcAMP) caused morphological and biochemical differentiation, but failed to do so in 158JP cells, suggesting an abnormal signaling pathway in jimpy. The defect in cAMP signaling in jimpy oligodendrocytes was associated with the suppression of increase in mRNA level of the inducible cAMP early repressor (ICER). When the jimpy oligodendrocyte line was transfected with normal PLP or DM20 and exposed to dbcAMP, the cells failed to differentiate. This finding suggests that improper insertion of jimpy protein into the plasma membrane alters the membrane in such a way that certain signaling pathways are permanently altered. The abnormal insertion of jimpy PLP/DM20 into the plasma membrane may be the basis for the lack of cell signaling and abnormal resting potential in jimpy oligodendrocytes.
在正常（158N）和jimpy（158JP）永生化少突胶质细胞系中研究了jimpy蛋白脂质蛋白和DM20的合成、运输及插入过程。四种不同的表达载体编码由天然PLP和DM20或jimpy PLP或DM20组成的融合蛋白，并与增强型绿色荧光蛋白（EGFP）相连。在两种细胞类型的细胞体和突起中，所有四种转染的融合蛋白都有相似的分布。正常和jimpy PLP - EGFP以及DM20 - EGFP在两种细胞系中均在距离细胞体200微米处被检测到，这表明突变的PLP和DM20能够合成并运输至质膜。用识别构象敏感的PLP/DM20表位的O10抗体对固定的正常和jimpy细胞进行免疫细胞化学检测，证实正常和jimpy PLP以及DM20都被运输到了质膜。对瞬时转染天然PLP的正常和jimpy细胞进行活细胞染色显示为阳性，表明PLP正确插入到了正常和jimpy少突胶质细胞的膜中。然而，对瞬时转染jimpy PLP的正常和jimpy细胞进行活细胞染色未显示阳性，表明突变蛋白异常插入到了质膜中。采用膜片钳技术的全细胞模式测量静息膜电位的电生理记录显示，与正常天然或永生化少突胶质细胞相比，jimpy细胞的膜电位不存在发育调控的负向偏移。用二丁酰环磷腺苷（dbcAMP）处理158N细胞和天然少突胶质细胞会导致形态和生化分化，但在158JP细胞中却未能如此，这表明jimpy细胞存在异常的信号通路。jimpy少突胶质细胞中cAMP信号通路的缺陷与诱导型cAMP早期阻遏物（ICER）mRNA水平升高的抑制有关。当jimpy少突胶质细胞系转染正常PLP或DM20并暴露于dbcAMP时，细胞未能分化。这一发现表明，jimpy蛋白异常插入质膜会以某种方式改变膜结构，使得某些信号通路被永久性改变。jimpy PLP/DM20异常插入质膜可能是jimpy少突胶质细胞缺乏细胞信号传导和静息电位异常的原因。
