Analysis of the roles of POMGnT1 in development and maintenance of certral nervous system and skeletal muscle

蛋白质O-连接甘露糖β1，2-N-乙酰氨基葡萄糖转移酶1是一种将N-乙酰氨基葡萄糖转移到糖蛋白O-甘露糖的酶，是营养不良蛋白-糖蛋白复合体中的一个组分--α-营养不良糖链的O-甘露糖基糖基化(O-甘露醇糖基化)所必需的。POMGNT1基因突变是导致肌肉-眼-脑(MEB)疾病的原因之一，该疾病的特征是严重的先天性肌营养不良以及眼睛和中枢神经系统(CNS)的异常。为了更好地了解MEB病的发病机制，我们利用靶向技术对POMGNT1基因进行了突变：1)骨骼肌突变的肌肉显示α-DG和α-DG的异常糖基化，突变的肌肉在结合实验中未能与层粘连蛋白结合。POMGNT1^&lt；-/-&gt；肌肉表现出轻微的病变，血清肌酸激酶水平非常低，并正常形成肌肉基底板。重要的是，POMGNT1^&lt；-/-&卫星细胞增殖缓慢，但有效地分化了…更多的细胞在体外进入多核肌管。将逆转录病毒载体介导的POMGNT1基因转入POMGNT1成肌细胞，可完全恢复α-DG的糖基化，但细胞的增殖未见改善。我们的结果提示α-DG不直接参与成肌细胞的增殖和分化，但其糖基化对于维持卫星细胞在出生后阶段的增殖能力是重要的。2)中枢神经系统POMGNT1^&lt；-/-&gt；小鼠大脑皮层出现神经元异常迁移、胶质细胞界限破坏和反应性胶质增生。在突变的新皮质中，reelin的表达普遍上调。我们测试了POMGNT1在神经元中表达的恢复是否导致了沿着放射状胶质细胞的正常迁移。为此，我们将POMGNT1表达载体电穿孔到E12和E15胚胎的脑室带内。令人惊讶的是，POMGNT1表达恢复后，POMGNT1缺陷神经元的迁移模式并没有改变。结果提示，POMGNT1-/-&GT；神经元的异常迁移不是由于神经元中POMGn1活性的缺乏，而可能是由于放射状胶质细胞(Bergmann Glia)的异常所致，该细胞具有从脑室带(VZ)延伸到软膜表面的长径向突起，为神经元迁移提供了轨道。较少

Protein O-linked mannose β1, 2-N-acetylglucosaminyltransferase 1(POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins, and is indispensable for (O-mannosyl glycosylation of alpha-dystroglycan(α-DG), a component of the dystrophin-glycoprotein complex. Mutation of the POMGnT1 gene is a cause of muscle-eye-brain(MEB) disease, which is characterized by severe congenital muscular dystrophy and abnormalities of the eyes and central nervous system(CNS). To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique.1) Skeletal muscle The mutant muscle showed aberrant glycosylation of α-DG and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^<-/-> muscle showed minimal pathological changes with very low serum creatinine kinase levels, and had normally formed muscle basal lamina. Importantly, POMGnT1^<-/-> satellite cells proliferated slowly, but efficiently differen … More tiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^<-/-> myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that α-DG is not directly involved in the proliferation and differentiation of myoblasts but that its glycosylation is important for maintenance of the proliferative capacity of satellite cells in the postnatal stage.2) CNS The cerebral cortex of POMGnT1^<-/-> mice showed abnormal migration of neurons, disrupted glia limitans and reactive gliosis. The expression of reelin was widely up-regulated in the mutant neocortex. We tested whether the restoration of POMGnT1 expression in neuron results in normal migration along the radial glia. To this end, we electroporated a POMGnT1-expressing plasmid into the ventricular zone of E12 and E15 embryos in utero.Surprisingly, the migration pattern of POMGnT1-deficient neuron did not change after restoration of POMGnT1 expression. The results suggest that the abnormal migration of POMGnT1^<-/-> neurons is not due to deficiency ogf POMGn1 activity in neuron but likely due to abnormality of radial glial cells(Bergmann glia), which have long radial processes extending from the ventricular zone(VZ) to the pial surface and serve a railroad of neuronal migration. Less