McLean W G, Pekiner C, Cullum N A, Casson I F
Department of Pharmacology and Therapeutics, University of Liverpool, UK.
Mol Neurobiol. 1992 Summer-Fall;6(2-3):225-37. doi: 10.1007/BF02780555.
Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.
已知轴突运输在实验性糖尿病大鼠的周围神经中受损。由于轴突运输依赖于神经元细胞骨架的完整性，我们研究了实验性糖尿病中大鼠脑和神经细胞骨架蛋白的改变方式。通过注射链脲佐菌素（STZ）使大鼠患糖尿病。在长达六周后，取出坐骨神经、脊髓和脑，用于制备神经丝、微管和细胞骨架蛋白的粗制品。通过与3H-硼氢化钠孵育，然后在二维聚丙烯酰胺凝胶上分离并对分离的蛋白质进行亲和层析，评估脑微管蛋白和周围神经微管蛋白的非酶糖基化程度。两周糖尿病大鼠和非糖尿病大鼠的脑微管蛋白非酶糖基化没有差异。微管蛋白组装成微管也不受糖尿病状态的影响。另一方面，糖尿病两周后坐骨神经微管蛋白的非酶糖基化显著增加。我们还从两周和六周糖尿病大鼠脑的细胞骨架蛋白制品中鉴定出脑肌动蛋白的电泳迁移率发生改变。还证实了一种额外的新型多肽，其等电点比肌动蛋白略酸性，可用抗肌动蛋白抗体进行免疫染色。相同的多肽可通过体外将纯化的肌动蛋白与葡萄糖孵育产生，从而将其鉴定为非酶糖基化产物。结合糖尿病患者临床研究的数据对这些结果进行了讨论，在该临床研究中我们鉴定出血小板肌动蛋白糖基化增加。STZ诱导的糖尿病还导致糖尿病六周内体内脊髓神经丝蛋白磷酸化增加。这种过度磷酸化以及神经丝相关蛋白激酶活性降低导致在体外将纯化的神经丝蛋白与32P-ATP孵育时，32P掺入减少。我们的综合数据显示神经元细胞骨架蛋白存在多种翻译后修饰，这可能导致实验性糖尿病中轴突运输改变及随后的神经功能障碍。
