Effect Of Pin1 Inhibition On Tau Phosphorylation And Its Underlying Mechanisms

One of the most important neuropathological hallmarks of Alzheimer's disease is the neurofibrillary tangles(NFTs), which contains paired helical filaments (PHFs) composed of the microtubule-associated protein Tau. The microtubule associated protein Tau, is hyperphosphorylated in PHFs and this hyperphosphorylation abolishes its microtubule binding ability.The human parvulin Pin1 is a member of the peptidyl–prolyl cis–trans isomerase proteins, whose gene is highly conserved. Pin1 recognises a specific motif of a phosphorylated serine or threonine residue preceding a proline (Ser/Thr-Pro) and specifically catalyzes the cis/trans isomerization of phosphorylated Ser/Thr-Pro peptide bonds in various phosphoproteins. The conformational conversion mediated by Pin1 modulates the assembly, folding, stability/activity, structure and transport of it's target proteins at different subcellular locations. For example, Pin1 can recognize the Phospho-Thr212-Pro213 and Phospho-Thr231–Pro sites of Tau protein, thereby inducing conformational changes in Tau and facilitate it's dephosphorylation by PP2A. However, in Alzheimer brains, especially in hippocampus, Pin1 is unactivated because of oxidative damage, also its level is declined compare with normal brains.The Pin1 inhibitor juglone(5-hydroxy-1,4-naphthoquinone), which is also named as Hutaokun in Chinese, can be extracted from walnut Hulls. Since the first knowledge that Juglone can irreversibly inhibit and decrease parvulins includes human Pin1, then it is often used to study the function of Pin1 in animal and cell experiments.In our experiment, we use juglone as Pin1 inhibitor in vivo and vitro to modify the declined Pin1 condition in AD brains. Then observe its influence and protein molecule change. Results: 1. Fed rats with juglone through water for 5 weeks to develop the chronic animal model. Then we use stepdown inhibitory avoidance task to study it's behavior change and found that these animals show impaired study and memory retention in a dosage dependent way compare with controls. Also, along with Pin1 decrease, the unphosphorylated Tau protein declined, and PT231 enhanced greatly.2. We intrahippocampal injection different concentration juglone to rats to develop the acute animal model. Also found hyperphosphorylated Tau in their brains.3. In vitro, after treated with juglone of different dosages, Pin1 decreased, while Tau was hyperphosphorylated in N2a/wt cells.As reported the Thr231 site plays a critical regulatory role in Tau founction, for its phosphorylation greatly diminishes the ability of Tau to bind and stabilize micro-tubules in the cell. Also it is well known that the Thr231 site could be phosphorylated by GSK-3βas many papers have already reported. So we further use GSK-3βassociated antibodies to examine it's change and assure its role.Results: 1. Both chronic and acute model animals show significant increasement of GSK-3βactivity when high dosage juglone(100 uM) was given.2. After treated with juglone in different concentrations, GSK-Y216 increased while GSK-S9 declined in a dosage dependent way in N2a/wt cells.Considering the fact that activate GSK-3βcan inhibit LTP(long-term potentiation), and impairs the spatial memory of animals, we got the following conclusions:1. Use juglone as Pin1 inhibitor in vivo can induce GSK-3βover activation.2. The declined Pin1 probably induced Tau hyperphosphorylation through over-activate GSK-3β.3. Through over-activate GSK-3β, the declined Pin1 probably induced learning and memory impairment of rats in stepdown inhibitory avoidance task.