Effects Of O-Glycosylation On The Phosphorylation And Assembly Of Neurofilaments

Objective:Alzheimer’s disease (AD) is a most common neurodegenerative disease and it is characterized by the progressive impairment of cognition and memory as clinical symptom. Neurofilaments (NFs), one of cytoskeleton protein, are neuron-specific intermediate filament, composed of three different molecular weight subunits. Abnormal phosphorylation of neurofilaments accumulated in axon, is one of primary components of neurofibrillary tangles (NFT) and early pathological changes of AD, but its pathogenesis is unclear. Except for phosphorylation, NFs protein is also modificated by O-GlcNAcylation produced by intracellular glucose metabolism. Our latest findings showed that the interregulation between O-Glycosylation and phosphorylation of NFs at cellular level. Abnormal phosphorylation neurofilaments accumulated in cells of the AD brain with reduced O-glycosylation.Based on previous results, our research focus on the effect of neurofilaments O-glycosylation dysfuction on its phosphorylation and assembly to explore pathogenesis of neurofilament hyperphosphorylation.Methods:In vivo and in vitro experiments were used to observe the effects of O-Glycosylation on phosphorylation and assembly of NFs.4-6month-old male SD rats were divided randomly into five groups. The rats were respectively intracerebroventricular (i.c.v.) injected into the brains with alloxan (OGT inhibitor,2mg/kg), NAG-AE (NAG-thiazoline, OGA inhibitor,5mg/kg) group, Don (HBP pathway inhibitor,2mg/kg), OA (PP2A inhibitor,50ng/kg) and0.9%NaCl solution.3rats were set as the blank control.24hours later, the rats were sacrificed and the brain tissue used for western blot to detect NFs protein glycosylation and phosphorylation level in brain tissue. Part of brain homogenate with different treatment and control was also used for study neurofilament assembly in vitro by transmission electron microscopy and western blot method. Furthermore, we tried to remove O-Glycosylation of proteins of brain homogenate with the beta-acetyl glucosamine enzyme to observe neurofilament assemble, further exploring whether NFs glycosylation itself or through a secondary phosphorylation to affect NFs assembly. Results:Our pre-experiments showed that high efficiency extraction and high purity of neurofilament. Electron microscopy observed assembly neurofilament consisted of fiber trunk about10nm in diameter and trunk periodically emitted side arm. The size and structure consisted with neurofilament extracted with other methods. In vivo experiment, compared with the control sample, O-glycosylation levels of neurofilament protein decreased and phosphorylation increased with lower and shoter neurofilament assembly in the sample of Alloxan, DON, OA groups. However, O-glycosylation levels were significantly increased and phosphorylation were declined in the sampe of NAG-Ae-treated group, in which NF showed elongated filaments fibers and higher proportion of assembly. These results suggested that changes of neurofilament O-glycosylation play a certain role on neurofilament assembly. Furthermore, in vitro experiments, the NFs with removed glycosylation showed that NFs still assembled into a long rough wire by electron microscopy suggesting that deglycosylation purely has little effect on NFs assembly.Conclusion:Alteration of O-glycosylation in vivo has effect on phosphorylation and assembly of NFs. Decreased O-glycosylation of NFs may affect its structure and function through its phosphorylation and neurofilament assembly, led to AD-like neuron degeneration. These results provided a new idea for AD pathogenesis and drug therapy.