The Study Of Alzheimer-like Pathological Changes And Its Underlying Mechanisms By Homocysteine In Rats

Alzheimer's disease is characterized by intracellular neurofibrillary tangles and extracellular senile plaques, and clinical manifestation is progressive memory impair. Neurofibrillary tangles are composed of aberrantly hyperphosphorylated tau protein, andβ-amyloid (Aβ), a peptide derived from cleavage ofβ-amyloid precursor protein (APP), is the major component of senile plaques. The pathogenesis of AD is still elusive, there are two dominant theories to underscore the pathogenesis of Alzheimer's disease: tau hypothesis and Aβhypothesis. However, neither of the two theories alone can fully explain the neuropathology in AD brain. Although many pathologic processes have been investigated in this disease, but the concrete influence factors and mechanisms remain unclear.Hcy is central to the methyl cycle and plays an important role in cellular methylation for protein,DNA and lipid, and elevated plasma Hcy levels precedes the onset of AD and is an independent risk factor for the disease. Epidemiology and clinical data have demonstrated that there present a positive correlation between elevated plasma homocysteine (Hcy) and the occurrence of AD, and thus hyperhomocysteinemia(HHcy) has been proposed to be an independent risk factor of AD. In this study, we injected Hcy into rats by vena caudalis, or supplement of folate and vit-B12 to investigate whether HHcy could induce AD-like alteration in rats and the potential mechanisms.PartⅠThe study of the hyperphosphorylation of tau and the underlying mechanisms induced by homocysteine in ratsIn this study, in order to reproduce the HHcy model, we selected the health young male SD rats, injected the rats with Hcy by vena caudalis, to explore whether there would be AD-like hyperphosphorylation of tau and the potential mechanisms. And the concrete methods and results are shown as follows:1.Effect of high plasma Hcy on tau phosphorylation in rat hippocampusTo investigate the effect of Hcy on tau phosphorylation, we injected the rats with Hcy (400μg/kg/day) by vena caudalis for 3, 7, 14 and 21 days, respectively. It was shown that tau was hyperphosphorylated at ser 396 and ser199/ser202 site, and the immunoreactivity of hyperphosphorylation increased time-dependently. Then, we injected the rats for 14 days with two different dosages of Hcy (400μg/kg/day or 1600μg/kg/day), and the rats were treated simultaneously with or without supplement of folate and vit-B12. We observed that a significantly increased tau phosphorylation at many sites by western-blotting. Increased phosphorylation of tau was demonstrated by immunohistochemistry and silver staining, it was attenuated by folate/vit-B12. No further elevation of tau phosphorylation except at PT205 and PS214 was observed when the concentration of Hcy was raised from 400μg/kg/day to 1600μg/kg/day. With these results, we selected 400μg/kg/day of Hcy and injected for 14 days in our further studies of the mechanism.2. The level of plasma Hcy was detected by HPLCTo confirm the role of Hcy , we measured the plasma level of Hcy by HPLC. A significant elevation of Hcy was shown after the injection, and the supplement of folate/vit-B12 distinctly attenuated the elevation of plasma Hcy. These data strongly suggest that elevated plasma Hcy can induce tau hyperphosphorylation at multiple AD-sites in the hippocampus of the rat brains.3. Effect of Hcy on the activity and the activity-related modifications of PP2A and GSK-3We measured the activity of PP2A by 32p-ATP and found that the activity of PP2A decreased to ~60% of the control level in Hcy-injected rats . With basically unchanged total protein level of PP2AC, the level of the Leu309-methylated PP2AC (activated form) decreased significantly and the level of the Leu309-demethylated PP2AC (inactivated form) increased after the injection of Hcy. We measured the phosphorylation of PP2AC, and observed that the level of the Tyr307-phosphorylated PP2AC increased and this change was positively correlated with the alteration of the Leu309-demethylated PP2AC. A simultaneous supplement of folate/vit-B12 restored the activity of PP2A to ~85% of the control level with a simultaneous restoration of the methylation and phosphorylation of PP2AC. These data together suggest that Hcy inhibits PP2A through interrupting the methylation of PP2AC and phosphorylation of PP2AC may also contribute to the inactivation of the phosphatase.We also measured the activity of GSK-3 with 32p-ATP , GSK3 is a crucial kinase in AD-like tau hyperphosphorylation, but no obvious change was observed in the activity of GSK-3, as well as the levels of the total and the Ser9-phosphorylated GSK-3 by western-blotting. These results ruled out the involvement of GSK-3 in Hcy-induced tau hyperphosphorylation in the rats.The methylation of PP2AC is regulated by a specific methylesterase (PME) and S-adenosylmethionine-dependent methyltransferase (PPMT), therefore, we detected the expression of these two enzymes. It was shown that the level of PME increased significantly and the level of PPMT was not changed in Hcy-injected rats.This suggested the role of PME in Hcy-induced demethylation of PP2AC. Simultaneous supplement of folate/vit-B12 partially restored the Hcy-induced elevation of PME.4. Effect of Hcy-induced demethylation on the biological function PP2AC To explore the influence of Hcy-induced demethylation on the biological function of PP2AC, we immunoprecipitated with an anti-total PP2AC antibody. With a significantly elevated demethylation/phosphorylation of PP2AC and an unchanged total PP2AC, we observed a remarkable decrease of PP2AB in the precipitates of the Hcy-injected rats, suggesting a decreased binding capacity of PP2AC with PP2AB.Then, we analyzed the phosphorylation status of PP2AC with immunoprecipitation by demethylated-PP2AC antibody. It was shown that the demethylated PP2AC was phosphorylated, and a highly positive correlation between demethylation and phosphorylation was observed. Simultaneous supplement of folate/vit-B12 partly attenuated the Hcy-induced alterations. These results indicate that demethylation may promote phosphorylation of PP2AC and thus inhibits its activity 5. The in situ relationship between modified PP2AC and hyperphosphorylated tau in the hippocampal neurons of the Hcy-injected rats and the AD patientsTo explore the in situ relevance of the methylation of PP2AC and the phosphorylation of tau, we did co-labeling studies using the antibody of methylated PP2AC, demethylated PP2AC and Ser-396-phosphorylated tau. Compared with control subjects, the immunoreactivity of Ser-396-phosphorylated tau was significantly increased in hippocampal CA1 neurons of the Hcy-injected rats; concomitantly, the immunoreactivity of the demethylated PP2AC was increased but that of methylated PP2AC was decreased dramatically, and only the demethylated PP2AC but not the methylated PP2AC was co-localized with the hyperphosphorylated tau. The co-localization was also remarkable in AD brains. These data together strongly suggest that demethylation of PP2AC contributes to tau hyperphosphorylation in rats induced by Hcy and in the AD brains.ConclusionTaken together, we have found in the present study that elevation of plasma Hcy can induce tau hyperphosphorylation through inactivating PP-2A. The underlying mechanism for the inactivation of PP-2A involves demethylation of PP2AC, which favors phosphorylation of PP2AC and disrupts the binding of PP2Ac to PP2AB. The elevation of PME may also contribute to the Hcy-induced demethylation of PP2AC. Simultaneous supplement of folate and vit-B12 restores partially the plasma Hcy level and thus attenuates the abnormal modification/inactivation of PP2AC and tau hyperphosphorylation.PartⅡThe mechanisms of impairment of spatial memory and increase and accumulation of Aβby Hcy in ratsIn this study, in order to reproduce the HHcy model, we injected the rats with Hcy by vena caudalis, to explore memory impairment of rats and Aβaccumulation and the potential mechanisms. The concrete methods and results are shown as follows: 1. The effect of APP catabolism by HHcy in rats hippocampalTo investigate the effect of Hcy on APP catabolism, we injected the rats with Hcy (400μg/kg/day) by vena caudalis for 3, 7, 14 and 21 days, respectively. It was demonstrated that injection of Hcy could induce APP catabolic time dependently at both 4G8 and 4E10 epitopes. Then, we injected the rats for 14 days with two different dosages of Hcy (400μg/kg/day or 1600μg/kg/day), and the rats were treated simultaneously with or without supplement of folate and vit-B12. We observed that a significantly increased APP dissection atβ-sites by western-blotting. It was attenuated by folate/vit-B12. Without further elevation of Aβproduction and accumulation was observed when the concentration of Hcy was raised from 400μg/kg/day to 1600μg/kg/day. With these results, we selected 400μg/kg/day of Hcy and injected for 14 days in our further studies of the mechanism.APP dissection was further demonstrated by immunohistochemistry with the antibody of Aβand Thioflavin-S staining, the results was similar to above,and folate/vit-B12 attenuated the alteration.These results manifested that one of toxicity of Hcy for AD is through on Aβaccumulation.2. The impairment of spatial memory of rats by HcyTo investigate the impairment of Hcy on spatial memory,we detected the rats spatial memory by Morris Water Maze.The ability of spatial memory was impaired significantly by Hcy,especially in lower dosage of Hcy.Folate and vit-B12 could prevent the spatial memory from impairment.3. The level of Aβwas detected by ELISA in rats cortex and hippocampalELISA detects the actually level of Aβ40 in rats cortex and hippocampal. After 14 days Hcy injection, the level of Aβ40 in cortex and hippocampal increased significantly when compared to the control, especially in contex and lower dosage Hcy. While folate and vitB12 were supplement, the level of Aβ40 decreased.4. The alterations of mRNA of APP and its metabolic secretase by RT-PCRWith RT-PCR, we found that besides the PS1 mRNA, other mRNA were not changed by Hcy. Folate and vit-B12 could decrease the PS1 mRNA.This results demonstrated that the increasing of Aβwasn't induced by the expression of APP, but there were other mechanisms involving in it.5. Detection of proteins expression and the modificationSince there was no change on the gene level of APP and its secretase, we detected the expression of BACE andγ-secretase by western-blotting, and no change was observed on the level. Now that the peptide of APP dissection byβ-secretase was strong positive reaction,with the 4G8 and 6E10 detection, and the level of Aβincreased significantly, nextly we explored the phosphorylation modification of APP with the p-Thr668-APP antibody. Comparation to the control, phosphorylation of APP increased significantly, and folate/vit-B12 decreased partially the alteration, The same alterations were visible in rats brain slice by immunohistochemistry staining. The results demonstrated that APP was abnormally phosphorylated by Hcy..6. The influence of APP catabolism by its phosphorylationIn order to further explore the influence of phosphorylation modified APP on itself catabolism, we immunoprecipitated with an anti-p-Thr668-APP antibody, with a significantly elevated phosphorylation of APP, and the dissected phosphorylated peptide of APP could be visible. We observed a remarkable increase of BACE1 and PS2 in the precipitates of the Hcy-injected rats. Simultaneous supplement of folate/vit-B12 partly attenuated the Hcy-induced alterations. The results explained that phosphorylation modificated APP increased the ability and affinity for APP to bind its secretase, and this induced APP processing and dissection byβ- andγ-secretase, and increased Aβlevel.ConclusionTaken together, we have found in the present study that elevation of plasma Hcy can increase the expression of PS1, enhance theγ-secretase activity; and induce the APP phosphorylation, increase the ability and affinity for APP to bind its secretase and increase the Aβproduction. Simultaneous supplement of folate and vit-B12 restores partially the plasma Hcy level, and thus attenuates the abnormal hyperphosphorylation of APP, and decrease the Aβproduction and accumulation.