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

蛋白O-linked甘露糖β 1,2 - n-乙酰氨基葡萄糖氨基转移酶1（POMGnT1）是一种将n-乙酰氨基葡萄糖转移到糖蛋白的o -甘露糖的酶，是α-肌营养不良-糖蛋白复合物的组成部分α-DG的o -甘露糖基化不可缺少的酶。POMGnT1基因突变是导致肌肉-眼-脑（MEB）疾病的原因之一，其特征是严重的先天性肌肉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疾病的发病机制，我们利用靶向技术突变了小鼠的POMGnT1基因。1)骨骼肌突变肌α-DG出现异常糖基化，α-DG在结合实验中无法结合层粘连蛋白。POMGnT1^<-/->肌病理变化很小，血清肌酐激酶水平很低，肌基板形成正常。重要的是，POMGnT1^<-/->卫星细胞增殖缓慢，但有效地分化成多核肌管。将逆转录病毒载体介导的POMGnT1基因转移到POMGnT1^<-/->肌母细胞中，α-DG的糖基化完全恢复，但细胞的增殖并未得到改善。我们的研究结果表明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出生后卫星细胞的增殖能力很重要。2)中枢神经系统POMGnT1^<-/->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细胞增生。在突变体的新皮层中，reelin的表达广泛上调。我们测试了POMGnT1在神经元中表达的恢复是否会导致沿径向胶质细胞的正常迁移。为此，我们在子宫内将表达pomgnt1的质粒电穿孔到E12和E15胚胎的心室区。令人惊讶的是，POMGnT1缺陷神经元的迁移模式在恢复POMGnT1表达后没有改变。结果表明，POMGnT1^<-/->神经元的异常迁移不是由于神经元中POMGn1活性的缺乏，而可能是由于径向胶质细胞（Bergmann胶质细胞）的异常，这些细胞具有从心室区（VZ）延伸到脑顶表面的长径向突起，并为神经元迁移提供了一条铁路。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