CAJ | 학술논문

Alzheimer disease (AD) neurofibrillary degeneration is characterized by a disruption of the cytoskeleton. The alteration of microtubule system and the microtubule-associated protein has been extensively investigated in this pathology. In the present study, we decided to explore the role of neurofilament (NF) proteins in AD neurofibrillary degeneration. We first investigated the content and the phosphorylation level of NF proteins in AD brain by using a panel of anti-NF antibodies. It was found by quantitative Western blot that the NF subunits were exclusively detected in an insoluble fraction from AD brain grey matter. The level of phosphorylated (p)-NF-H and (p)-NF-M was increased 1.5 and 1.3 times (P＜0.05) respectively at phosphorylation specific antibody SMI31 epitope in AD as compared to neurological controls of Huntington disease (HD). A 1.6 fold elevation (P＞0.05) of p-NF-H to another phosphate reactive antibody SMI34 was also seen in AD. The level of non-phosphorylated (np)-NF-H/M recognized by SMI33 was similar before alkaline phosphatase (ALP) treatment, but the total level of NF-H/M was 1.5 and 1.6 times (P＜0.01) higher in AD than HD after dephosphorylation. Furthermore, a 1.8 fold increase of NF-M to SMI32 was observed in AD only after ALP treatment, suggesting that the NF-H/M are increased in the phosphorylated form. The amount of NF-L determined by NR-4 was 1.6 fold (P＜0.01) higher in AD than HD. To our knowledge, this is the first biochemical data shown definite abnormality of NF subunits in AD brain. To understand the possible mechanism for the abnormal hyperphosphorylation and elevation of NF in AD brain, we treated human SY5Y neuroblastoma cell with protein phosphatase(PP)-2A and PP-1 inhibitor okadaic acid(OA). Then, we determined the relationship between an AD-like PP-A and PP-1 activity deficiency and NF phosphorylation as well as intracellular translocation in modeled cell system. It was demonstrated that p-NF-H/M detected by SMI31 and SMI34 were increased, and the elevated p-NF-H/M tended to be condensed in the proximal end of the cell processes after treated with 15 nmol/L OA. Further accumulation of p-NF-H/M to the cell plasma and parikarya was seen after increasing the concentration of OA to 30 nmol/L. On the other hand, the majority of np-NF-H/M bound to SMI32 and SMI33 were seen in the cell body although it was also detected in cell processes before OA treatment. The immunoreaction of np-NF-H/M was significantly decreased in the cell body and it became to be condensed in the proximal end of the cell processes after treatment of the cell by 15 nmol/L of OA. Further decreasing of the staining was observed when the concentration of OA was raised to 30 nmol/L. The data demonstrated that an Alzheimer-like inhibition of PP-2A and PP-1 induced hyperphosphorylation and accumulation of NF proteins as seen in AD brain, indicating that abnormality of NF might be involved in AD neurofibrillary degeneration. As SY5Y contains negligible amount of tau protein which was reported to cross-react with p-NF subunits, it might be served as a proper cell model for NF study.