STUDY OF THE PATHOPHYSIOLOGY OF MYOTOIC DYSTROPHY TOWARD THE FUTURE THERAPY FROM THE UNDERSPAND]NG OF MUSCLE WASTING MECHANISM

最近的研究发现了强直性肌营养不良症(DM)的一种新的发病机制，即所谓的“功能基因获得”。带有扩展重复序列的突变型RNA转录本会异常地影响各种mRNA的剪接。氯通道和胰岛素受体的剪接中断被认为是糖尿病患者出现肌强直和胰岛素抵抗的原因之一。该项目旨在通过鉴定其他重要转录本的异常剪接来阐明糖尿病最致残症状-肌肉萎缩的原因。我们已经成功地鉴定了几个异常剪接的转录本，它们是骨骼肌功能所必需的；细胞骨架蛋白，dystrophin和α-dystrobrevin，以及肌浆网蛋白，肌浆/内质网钙-ATPase 1(SERCA1)和兰诺定受体1。我们广泛分析了dystrobrevin的剪接异常，dystrobrevin是一种包含dystrophin相关复合体的细胞骨架蛋白。我们已经证明，在1型糖尿病(DM1)肌肉中，dystrobrevin的选择性剪接是失调的，导致合成营养素结合的变化。这些结果表明，对dystrobrevin剪接的影响可能会影响DM1肌肉细胞中的信号转导。有研究表明，突变的mRNA改变了选择性剪接调节因子的功能和定位，如CUG-BP和MENL1，它们对正常的RNA加工至关重要。我们分析了MBNL1和CUG-BP对SERCA1的剪接调控，证明了MBNL1作用于一个内含子基序。这些结果表明，MBNL1被隔离在突变的mRNA的扩展重复序列中，可能导致SERCA1外显子22的排除，增加了在DM1肌肉中观察到的异常亚型。

Recent research has uncovered a new disease mechanism for myotonic dystrophy (DM), so-called "mRNA gain of function". The mutant RNA transcripts with expanded repeats aberrantly affect the splicing of various mRNA. Disrupted splicing of Cl channel and insulin receptor is believed to contribute to myotonia and insulin resistance observed in DM patients. This project has aimed to elucidate the cause of the most disabling symptom of DM, muscle wasting, by identifying the aberrant splicing of other important transcripts. We have successfully identified several abnormally spliced transcripts, which are essential for skeletal muscle function; cytoskeletal proteins, dystrophin and alpha-dystrobrevin, and sarcoplasmic reticulum proteins, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 1 (SERCA1) and ryanodine receptor type1.We have extensively analyzed the splicing abnormalities of dystrobrevin, which is a cytoskeletal protein consisting dystrophin-associated complex. We have shown that alternative splicing of dystrobrevin is dysregulated in DM type 1 (DM1) muscle, resulting in changes in syntrophin binding. These results raise the possibility that effects on dystrobrevin splicing may influence signaling in DM1 muscle cells.It has been suggested mutant mRNA alters the function and localization of alternative splicing regulators, such as CUG-BP and MENL1, which are critical for normal RNA processing. We have analyzed the splicing regulation of SERCA1 by MBNL1 and CUG-BP and demonstrated that MBNL1 acts on an intoronic motif. These results indicate that sequestration of MBNL1 into the expanded repeats of mutant mRNA could cause the exclusion of SERCA1 exon 22, the increase of the abnormal isoform observed in DM1 muscle.