Effects Of O-Glycosylation On The Phosphory-lation And Function Of The Neurofilaments In

Aims Neurofilaments protein, assembled from three subunits of NF-L, NF-M and NF-H, is the major cytoskeletal protein of nerve cells and play a significant role in stabilizing morphology, structure of cells and maintaining axonal transport. Hyperphosphorylation of neurofilaments, one of major components of NFT and the early pathological change of AD, affects the function of axonal transport of nerve cells. Glycosylation is another important modification of neurofilaments protein. Similar to phosphorylation, they modify the same serine/threonine residues of protein. The study demonstrated that O-Glycosylation of neurofilaments decreased and phosphorylation increased in AD brains. We furthermore detected the effects of O-Glycosylation of neurofilaments protein on its phosphorylatjon and the function of axonal transportion in vitro. Methods Different drugs were used to intervene the level of O-Glycosylation of the SK-N-SH cells through different mechanisms; MTT assay was used to decide the proper concentration of drugs and observe the changes of the cell viability. Western blot and immunofluorescence were used to observe the changes of O-Glycosylation and phosphorylation of neurofilaments protein. PI/Hoechst staining and DAPI staining were used to observe the way of the cell death. Meanwhile, transfection of GFP-NFM to the SK-N-SH cells and fluorescence photobleaching recovery were used to observe the effects of O-Glycosylation on the function of axonal transport of neurofilaments protein. Results Compared to the control, alloxan treatment decreased the cell viability, and increased the phosphorylation of neurofilaments protein, accumulated in the cell bodies, but it decreased the level of O-Glycosylation in cells. The cells pretreated with20μM NAG-Ae for12hours, the viability was not affected, but it the level of O-Glycosylation increased, and the hyperphosphorylation and accumulation of neurofilaments protein induced by alloxan was decreased. The cell death induced by alloxan has two ways, apoptosis and necrosis, while, the cell apoptosis and necrosis induced by alloxan could be protected by the pretreatment with20μM NAG-Ae for12hours.The velocity of recovery after fluorescence photobleaching was slowed in the cells with the treatment of6mM alloxan, while, the velocity of recovery after fluorescence photobleaching was accelerated in the cells with pretreatment of20μm NAG-Ae. Similarly, in cells treated with DON, the level of O-Glycosylation decreased and the phophorylation of neurofilaments protein increased, and the velocity of recovery after fluorescence photobleaching was slowed. Conclusions The decrease of O-Glycosylation could induce AD like hyperphosphorylation and the decrease of cell viability, and affect the function of axonal transport, but increase the O-Glycosylation could improve the phosphorylation of cells, and protect the AD like neurodegeneration and the function of neurofilaments proteins.