Stieber Anna, Gonatas J O, Moore J S, Bantly Andrew, Yim H-S, Yim M B, Gonatas N K
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Medical Center, 609 Stellar-Chance Labs., 422 Curie Boulevard, Philadelphia, PA 19104-6100, USA.
J Neurol Sci. 2004 Apr 15;219(1-2):45-53. doi: 10.1016/j.jns.2003.12.004.
The Golgi apparatus of motor neurons (GA) is fragmented in sporadic amyotrophic lateral sclerosis (ALS), in familial ALS with SOD1 mutations, and in mice that express SOD1G93A of familial ALS, in which it was detected months before paralysis. In paralyzed transgenic mice expressing SOD1G93A or SOD1G85R, mutant proteins aggregated not only in the cytoplasm of motor neurons, but also in astrocytes and oligodendrocytes. Furthermore, aggregation of the G85R protein damaged astrocytes and was associated with rapidly progressing disease. In order to gain insight into the functional state of the fragmented GA, we examined the effects of S0D1 mutants G93A and G85R in Chinese Hamster Ovary Cells (CHO). In contrast to cells expressing the wt and G93A, the G85R expressers had no SOD1 activity. However, cells expressing both mutants, and to a lesser degree the wt, showed decreased survival, fragmentation of the GA, and dysfunction of the secretory pathway, which was assessed by measuring the amount of cell surface co-expressed CD4, a glycoprotein processed through the GA. The G93A and wt proteins were partially recovered in detergent insoluble fractions; while the recovery of G85R was minimal. Both mutants showed equal reductions of cell survival and function of the secretory pathway, in comparison to the wt and cells expressing mutant alsin, a protein found in rare cases of fALS. These results are consistent with the conclusion that the two SOD1 mutants, by an unknown mechanism, promote the dispersion of the GA and the dysfunction of the secretory pathway. This and other in vitro models of mutant SOD1 toxicity may prove useful in the elucidation of pathogenetic mechanisms.
在散发性肌萎缩侧索硬化症（ALS）、携带超氧化物歧化酶1（SOD1）突变的家族性ALS以及表达家族性ALS的SOD1G93A的小鼠中，运动神经元的高尔基体（GA）会发生碎片化，在瘫痪前数月就能检测到这种情况。在表达SOD1G93A或SOD1G85R的瘫痪转基因小鼠中，突变蛋白不仅在运动神经元的细胞质中聚集，还在星形胶质细胞和少突胶质细胞中聚集。此外，G85R蛋白的聚集会损害星形胶质细胞，并与疾病快速进展相关。为了深入了解碎片化GA的功能状态，我们检测了SOD1突变体G93A和G85R在中国仓鼠卵巢细胞（CHO）中的作用。与表达野生型和G93A的细胞不同，表达G85R的细胞没有SOD1活性。然而，表达这两种突变体的细胞，以及程度较轻的表达野生型的细胞，显示出存活率降低、GA碎片化以及分泌途径功能障碍，这是通过测量细胞表面共表达的CD4（一种通过GA加工的糖蛋白）的量来评估的。G93A和野生型蛋白部分存在于去污剂不溶性组分中；而G85R的回收率极低。与野生型和表达突变体alsin（在罕见的家族性ALS病例中发现的一种蛋白质）的细胞相比，这两种突变体在细胞存活率和分泌途径功能方面的降低程度相同。这些结果与以下结论一致：这两种SOD1突变体通过未知机制促进GA的分散和分泌途径的功能障碍。这种以及其他突变体SOD1毒性的体外模型可能有助于阐明发病机制。
