Study of disfunctions of axons and abnormal phosphorylation of neurofilament in Alzheimer barins

Ser-51和Ser-55分别是68 kDa神经丝亚单位蛋白n端头区PKC和PKA的磷酸化位点。利用亲和柱法制备了针对这些磷酸化位点的位点特异性磷酸化依赖抗体（abNFL51和abNFL55）。通过斑点和免疫印迹分析鉴定了这些抗体的体外特异性。随后，在大鼠原代培养的神经元中进行了体内特异性研究。在这些细胞中发现少量被abNFL55识别的磷酸化NF-L，经冈田酸处理后，磷酸化NF-L的数量增加。然而，福斯克林并没有增加其数量，而福斯克林和冈田酸同时处理则显著增加了其数量。这些发现表明，一小部分NF-L被PKA磷酸化，磷酸化的NF-L的数量受冈田酸敏感磷酸酶的调节，这意味着神经元中NF-L的动态受激酶和磷酸酶的调节。另一方面，在培养的神经元中未发现abNFL51的信号，表明神经元中NF-L不被PKC磷酸化。用abNFL51和abNFL55免疫组织化学方法研究了阿尔茨海默氏症和对照组大脑15微米厚的自由漂浮切片。abNFL51染色两脑的神经原纤维结构，abNFL55染色神经元核周和神经突。

Ser-51 and Ser-55 are the phosphorylation site of PKC and PKA, respectively, in he N-terminal head region of 68 kDa neurofilament subunit protein (NF-L). Site-specific phosphorylation-dependent antibodies (abNFL51 and abNFL55) against those phosphorylation sites were prepared by using affinity-column. The in vitro specificity of those antibodies were appreciated by dot and western blot analyses. Thereafter, The in vivo specificity was studied in rat primary cultured neurons. A little amount of phosphorylated NF-L recognized by abNFL55 was found in them, and the amount was increased by okadaic acid treatment. However, forskolin did not increase the amount, while simultaneous treatment of forskolin and okadaic acid dramatically increased it. These findings suggest that a small part of NF-L is phosphorylated by PKA and that the amount of the phosphorylated NF-L is regulated by okadaic acid-sensitive phosphatase, implying that NF-L dynamics in neurons is regulated by kinases and phosphatases. On the other hand, no signal was found by abNFL51 in cultured neurons, indicating that NF-L is not phosphorylated by PKC in neurons.15 micrometer thick free-floating sections of Alzheimer and control brains were immunohistochemically studied by abNFL51 and abNFL55. abNFL51 stained neurofibrillary structures, and abNFL55 stained neuronal perikarya and neurites in both brains.