Effect Of Escitalalopram On AD-Like Tau Hyperphosphorylation And Its Mechanism

Part 1Escitalopram Ameliorates Forskolin-Induced Tau Hyperphosphorylation in HEK293/tau441 CellsBackground:More than 60% of the AD patients have depression symptoms. Senile depression is also one of the risk factors of AD. Antidepressant drugs have been used in AD patients with depressive symptoms. However, whether new antidepressant drugs can ameliorate tau hyperphosphorylation, one of the important pathological characteristics of AD, are still unclear, and there has no report about its underlying possible mechanism.Methods:To investigate the effect of escitalopram on tau hyperphosphoryaltion, we treated the HEK293/tau441 cells with 4 μM of forskolin for 2 h. Then we treated the cells with different dose of escitalopram for 22 h. We measured the phosphorylation level of tau and the level of phosphorylation-associate enzyme by Western blotting.Results:Escitalopram could protect tau from hyperphosphorylation induced by pharmacological activation of protein kinase A (PKA) in vitro dose dependently. Similarly, fluoxetine could also decrease forskolin-induced tau hyperphosphorylation. However, R-citalopram had no effect on tau phosphorylation. Compared with escitalopram treatment alone, the co-culture with R-citalopram and escitalopram partially increased the level of the phosphorylated tau. Escitalopram could increase the level of serine-9-phosphorylated GSK-3β (inactive form) and the phosphorylation level of Akt at Ser473 (active form), while had no effect on the level of Tyrosine-307-phosphorylated PP-2A. Unexpectedly,5-HT1A agonist 8-OH-DPAT did not decrease forskolin-induced tau hyperphophorylation.Conclusions:Escitalopram could protect forskolin-induced tau hyperphosphorylation at multiple AD-related sites, and the mechanism involves inactivation of GSK-3β.Our findings suggest that escitalopram could be a promising therapeutic target for AD-like tau hyperphosphorylation, this may support a potential effective role of antidepressants, at least of the SSRI class, in the prevention of dementia associated with depression in patients.Part 2Escitalopram ameliorates tau hyperphosphorylation and spatial memory deficits induced by protein kinase A activation in SD ratsBackground:Protein kinase A (PKA) plays a crucial role in tau hyperphosphorylation, and clinical and preclinical studies have demonstrated the therapeutic potential of selective serotonin reuptake inhibitor (SSRI) antidepressants in Alzheimer’s disease (AD) patients and AD animal models. However, the role of escitalopram in PKA-induced tau hyperphosphorylation and spatial memory deficits is unknown.Methods:To investigate the effects of escitalopram on forskolin-induced behavioral changes, we first measured the spatial memory retention of the rats using the Morris water maze, and then we examined the depression-and anxiety-like behaviors of the rats using the sucrose consumption test and the open field test. We measured the phosphorylation level of tau at different epitopes and the level of phosphorylation-associate kinases and phosphatases, such as GSK-3β, protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2 and MAP kinase kinase-1/2 in hippocampal extracts by Western blotting.Results:We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3p\We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). We did not detect any significant change in the activation of protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2 and MAP kinase kinase-1/2 in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin.Conclusions:Escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.Part 3Escitalopram alleviates Alzheimer’s disease-type tau pathologies and cognitive deficits in the P301L tau transgenic pR5 model of tauopathyBackground:Escitalopram, a kind of selective serotonin reuptake inhibitor (SSRI) antidepressant, has been previously reported to ameliorate tau hyperphosphorylation and spatial memory deficits induced by protein kinase A activation in SD rats. In this study, we determined whether escitlopram also has beneficial effects in the P301L mouse, which accumulates hyperphosphorylated tau.Methods:Mice were intraperitoneal injected with either escitalopram (10 mg/kg) or saline for 4 weeks, and a battery of behavioral tests were conducted before tissue collection and biochemical analyses of brain tissue with Western blot.Results:Wild-type (WT) mice statistically outperformed transgenic (TG) saline mice in the Morris water maze, while treatment with escitalopram partly improved performance of transgenic mice in the Morris water maze. Other behavioral changes were not evident. Beneficial changes in brain biochemistry were evident in escitalopram-treated mice for several proteins. Compared with WT mice, the level of tau phosphorylation was significantly increased, while escitalopram treatment significantly reduced tau phosphorylation. The TG mice had significantly less pSer9-GSK3β and pSer473-Akt than wild-type controls, and for each protein, escitalopram treatment significantly reversed these changes.Conclusions:These data suggest that escitalopram is a potential treatment not only for Alzheimer’s disease, but also for other tau hyperphosphorylattion associated neurodegenerative diseases.Part 4Escitalopram attenuates β-amyloid-induced tau hyperphosphorylation in primary hippocampal neurons through the 5-HT1A receptor mediated Akt/GSK-3β pathwayBackground:Tau hyperphosphorylation is an important pathological feature of Alzheimer’s disease (AD). Therefore, influencing tau hyperphosphorylation could be a promising approach for the preventative treatment of AD. This study aimed to investigate whether escitalopram, an antidepressant from the SSRI class used to treat depression, could inhibit amyloid-β (Aβ) induced tau hyperphosphorylation in vitro and to explore the underlying mechanisms in primary hippocampal neurons.Methods:Effects of escitalopram were measured in primary hippocampal neurons prepared from fetal brains of rats. Western blot and immunofluorescence staining were used.Results:Escitalopram decreased Aβ1-42--induced tau hyperphosphorylation in hippocampal neurons, fluoxetine also decreased Aβ1-42-induced tau hyperphosphorylation while R-citalopram not. In addition, escitalopram activated the Akt/GSK-3β pathway, and inhibition of PI3K blocked the attenuation of tau hyperphosphorylation induced by escitalopram. Moreover, the 5-HT1A receptor agonist 8-OH-DPAT also activated the Akt/GSK-3β pathway and significantly decreased Aβ1-42-induced tau hyperphosphorylation, whereas the 5-HT1A receptor antagonist WAY-100635 significantly blocked the activation of Akt/GSK-3β pathway induced by escitalopram and eliminated the effects of escitalopram on tau hyperphosphorylation. Furthermore, escitalopram improved Aβ1-42 induced impairment of neurite outgrowth and spine density; escitalopram also reversed Aβ1-42 induced alterations of synaptic proteins.Conclusions:These findings provide new mechanisms of action for the antidepressant escitalopram by attenuating tau hyperphosphorylation. Overall, our findings indicate that escitalopram may have potential as a therapeutic strategy for the tau hyperphosphorylation associated disease, including AD.