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。用亲和层析法制备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)。通过斑点分析和免疫印迹分析，评价这些抗体的体外特异性。此后，在原代培养的大鼠神经元中研究了其体内特异性。其中有少量由abNFL55识别的磷酸化核因子-L，且冈田酸处理使其数量增加。然而，Forskolin并没有增加其数量，而同时处理Forsklin和冈田酸则显著增加了它的数量。这些结果表明，一小部分的NF-L被PKA磷酸化，而磷酸化的NF-L的数量受冈田酸敏感的磷酸酶调节，提示神经元中的NF-L的动力学受激酶和磷酸酶的调节。另一方面，在培养的神经元中，abNFL51没有发现任何信号，表明神经元中的核因子-L不被蛋白激酶C磷酸化。AbNFL51阳性神经纤维结构，abNFL55阳性神经元核膜和神经突起。

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.