Effect On Tau Phosphorylation And Spatial Memory Retention Induced By Single Injection Of Forskolin Into The Lateral Ventricle Of Rats

One of the major neuropathological features of Alzheimer's disease (AD) is the formation of large amounts of neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated tau. Tau is a kind of microtubule-associated proteins and hyperphosphorylated tau lose its biological functions, such as inducing the disassembly of microtubule, axonal transport deficit and the degeneration of neuron. However, hyperphosphrylated tau and spatial retention memory deficit-like animal model is very important for study of AD. Recently, our study has showed that Forskolin, the specific activator of cAMP dependent protein kinase (PKA), can induce tau hyperphosphorylation and spatial retention memory deficit in rats 24h after Forskolin injection into the Lateral Ventricle. In order to further investigate the effect of different concentration of Forskolin on tau phosphorylation and spatial retention memory and the effect of certain concentration of Forskolin on the duration of tau hyperphosphorylation and spatial memory, we injected different concentration of Forskolin (0μM, 20μM, 40μM, 80μM, 120μM, 160μM and 200μM) into the Lateral Ventricle and investgate the phosphorylation levels of tau by Western blot and immunocytochemistry and spatial memory retention by Morris Water Maze, respectively. The results are as following:1. Effect of different concentration of Forskolin on hippocampal tau phosphorylation and spatial memory retention of rats.40μl Forskolin with different concentration (0μM, 20μM, 40μM, 80μM, 120μM, 160μM and 200μM) was injected into the lateral ventricle of rats and 24h later injection of 20μM Forskolin induced significantly increased tau phosphorylation levels at Ser-396/404, ser-214 and Ser-198/199/202. The phosphorylated level of tau at these sites increased gradually from 20μM to 80μM and decreased gradually from 80μM to 200μM. The highest phosphorylation level of tau was induced by 80μM Forskolin and 200μM Foskolin had no effect on the level of tau phosphorylation at above sites. Results from Morris water maze showed that 80μM Forskolin induced obvious spatial retention memory deficit of rats but 200μM Forskolin has no effect on it. We detected the correlation between the phosphorylation of hippocampal tau at certain site with the record of spatial memory retention of rats. It was found that there existed positive correlation between tau phosphorylation at PHF-1 site(r=0.998, P<0.05)with the record of spatial memory retention and existed negative correlation at between tau phosphorylation at Tau-1 site(r=-0.997, P<0.05)with the record of spatial memory retention. There was no correlation between tau phosphorylation at PS214 site(r=0.91, P>0.05 ) with the record of spatial memory retention. The level of protein phosphatase-2A (PP2A) catalyze unit was recognized by the antibody R123d and the results showed 200μM Foskolin significantly elevated the level of R123d and 80μM Forsklin had no effect on it. The same changes also were observed at 48h after injection different concentration of Forskolin (0μM, 80μM and 200μM) into lateral ventricle of rats.2. Duration of phosphorylated tau and spatial retention memory deficit induced by injection of same concentration of Forskolin.Based on the results in the first part, we chose 80μM Forskolin to inject into the lateral ventricle. Results from western-blot/immunocytochemistry showed that the phosphorylation level of tau at Tau-1, PHF-1 and PS214 epitopes was significantly elevated at 24 h, 48 h and 72 h after single administration of Forskolin(P<0.05). The most significant elevation was seen at 48 h and it tended to recover at 72 h after injection(P<0.05). Results from Morris water maze also show the same direction. The correlation between them was also observed at the three observed time points, there is positive correlation at PHF-1 site(r=0.97, P<0.05); negative correlation at tau-1 site (r=-0.963, P<0.05); no correlation at PS214 site(r=0.705, P>0.05).In summary, different concentration of Forskolin has different effect on tau hyperphosphorylation and spatial retention memory deficit, 80μM Forskolin has the best effect on them; 80μM Forskolin can induce tau hyperphosphorylation and spatial retention memory deficit within a certain period of time. The level of tau phosphorylation in hippocampus has some correlation with the spatial memory deficit in rats.