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（NF-L）N端头部区域PKC和PKA的磷酸化位点。通过使用亲和柱制备针对这些磷酸化位点的位点特异性磷酸化依赖性抗体（abNFL 51和abNFL 55）。这些抗体的体外特异性通过斑点和蛋白质印迹分析来评价。然后，在原代培养的大鼠神经元中研究体内特异性。在这些细胞中发现了少量的被abNFL 55识别的磷酸化NF-L，并且被冈田酸处理的数量增加。然而，毛喉素并没有增加的量，而同时处理毛喉素和冈田酸显着增加it. These研究结果表明，一小部分的NF-L是磷酸化PKA和磷酸化NF-L的量调节冈田酸敏感的磷酸酶，这意味着NF-L在神经元中的动力学是由激酶和磷酸酶调节。另一方面，在培养的神经元中没有发现abNFL 51的信号，表明NF-L在神经元中没有被PKC磷酸化。通过abNFL 51和abNFL 55对阿尔茨海默病和对照脑的15微米厚的自由漂浮切片进行了化学染色研究。在两个脑中，abNFL 51染色神经元胞体结构，abNFL 55染色神经元胞体和神经突。

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.