The Effects Of Chronic Restraint Stress On Rat Brain Tau Protein Phosphorylation And Distribution

Chronic stress has been reported to contribute to a series of dysfunctions of central nerves system, which leads to the acceleration of aging process, impairment of learning and memory, Alzheimer disease, depression and so on. Cytoskeletal abnormality has been observed in a variety of neurodegenerative diseases, among many of these diseases, tau protein related pathological changes are often observed, such as the neurofibrillary tangles in Alzheimer disease, suggesting the correlation between chronic stress and tau protein abnormality. Hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and causes microtubule disassembly. The exact molecular basis of toxicity caused by abnormal tau remains to be an area of intense research. Whether the loss of biological function by the hyperphosphorylation or the aggregated NFTs contributes to neurodegeneration is a key issue, which may shed some light on the prevention and treatment of neurodegenerative diseases.We employed the chronic restraint stress rat model to detect the mechanism of stress-induced tau hyperphosphorylation and the abnormality of MAP2. To determine tau solubility, sequential fractionation procedures were performed. The extractions of RAB buffer, RIPA buffer and 70% FA (Formic acid) represent soluble hyperphosphorylated tau, insoluble PHF-tau and NFTs-tau respectively. Western blot was used to investigate the chronic restraint stress induced hyperphosphorylation of tau and the loss of MAP2, immunohistochemical staining and immunofluorescence staining were performed to detect the distribution of hyperphosphorylated tau and MAP2. We got the following results:1. Chronic restraint stress induced hyperphosphorylation of tau proteinWe applied immunoblot method with two phosphorylation dependent site-specific anti-tau antibodies to confirm stress-induced phosphorylation of tau after 1 hour each day,14 consecutive days' chronic restraint stress. Results showed that after stress, soluble phosphorylated tau increased strikingly, but insoluble phosphorylated tau changed slightly. No detectable phosphorylated tau but ATP synthase a subunit was observed in the FA fractionSoluble hyperphosphorylated tau changed slightly after 6 hours each day,21 consecutive days' chronic restraint stress. The insoluble phosphorylated tau in neocortex and hippocampus was increased signifcantly. In addition, the phosphorylated tau bands were detected in the FA fraction when the loading sample was increased.The detction of APP (amyloid precursor protein) in brain with immunoblotting showed that high molecular weight APP was decreased and low molecular APP increased after stress. The change was enhanced when the stress simulation was increased.The results indicated that after 1 hour each day,14 consecutive days' chronic restraint stress, soluble phosphorylated tau increased strikingly, while no insoluble PHF-tau and NFTs-tau formed at this time point. In contrast, after 6 hours each day, 21 consecutive days'chronic restraint stress, the phosphorylated tau in brain was mainly present in insoluble fraction.2. Chronic restraint stress changes the distribution patterns of phosphorylated tau and MAP2The results of immunohistochemistry showed that after 1 hour each day,14 consecutive days' chronic restraint stress, robust increase of phosphorylated tau were seen in cortex, hippocampus, thalamus and piriform cortex, but weak positive staining was seen in the amygdala. The change of phosphorylated tau distribution pattern from axonal location to somatodendritic compartment, was also appeared. After stress stimulation, MAP2 in dendritic compartment was greatly decreased, and beady patches were seen in the cortex and CA3 of hippocampus. Immunofluorescence double labeling showed that the neurons with the increase of hyperphosphorylated tau appeared the decrease of MAP2, indicating the relationship between these changes after chronic restraint stress. In addition, Thioflavin S staining showed the deposition ofβ-sheet protein structure in the molecule stratum and the mossy fiber in the hippocampus of the animals after 1 hour each day,14 consecutive days of chronic restraint stress.3. GSK-3 is one of the main kinases involved in tau phosphorylation. After chronic restraint stress, immunoblotting and immunohistochemistry ananlyzed GSK-3βphosphorylated at Ser 9 which represents the inhibitory form of GSK-3β, and showed an increase of phosphorylated GSK-3βdistribution in cortex and hippocampus. However, the results showed that GSK-3αphosphorylated at Ser21 which represents the inhibitory form of GSK-3αwas decresed after stress stimulation. We also noted that the decreased distribution of the inhibitory form of GSK-3αin hippocampus was not completely meet the distribution of phosphorylated tau, implying that other kinases besides GSK-3 involved in the mechanism by which chronic restraint stress phosphorylate tau.Summary:1.1 hour each day,14 consecutive days' chronic restraint stress induced the increase of soluble hyperphosphorylated tau but no PHF-tau and NFTs, dendritic MAP2 decreased dramatically at the same time. The stronger stimulation of restraint stress caused the increase of insoluble phosphorylated tau.2. Increased hyperphosphorylated tau mainly distributed to the somatodendritic compartment of the affected neuron after chronic restraint stress, meanwhile the MAP2 in dendrites was decreased, among which some dendrites appeared neuritc degeneration. The decrease of dendritic MAP2 is related to the hyperphosphorylation of tau.3. After chronic restraint stress, inhibitory form of GSK-3βwas increased meanwhile the inhibitory form of GSK-3αwas decreased, implying that GSK-3αbut not GSK-3βmay take part in tau phosphorylation.Conclusion:In the present study, we provided evidence that chronic restraint stress induces the increase of hyperphosphorylated tau and the loss of MAP2 in the same brain regions, suggesting a relationship between these two changes. The activation of GSK-3αwas involved in the mechanism of tau phosphorylation after stress. The strength of repeated restraint stress determined phospholated tau in soluble or insoluble state.