Phosphodiesterase-4(PDE4)and Subtypes In The Regulation Of Cognitive Dysfunction And Neuroinflammation Induced By Amyloid-β Peptides (Aβ)

Objective:Alzheimer's disease (AD) is a kind neurodegenerative diseases characterized by the P-amyloid plaques and neurofibrillary tangles as the important marker and associated with chronic neuroinflammation and cognitive dysfunction. By inhibiting neurotoxicity induced by P-amyloid may be theoretical basis to develop the new drugs which to slow down the nerve damage and the process of Alzheimer's disease. Phosphodiesterase-4 (PDE4), an enzyme that specifically catalyzes the hydrolysis of cyclic AMP (cAMP), plays a crucial role in a variety of signal transduction pathway in brain function. More and more study has revealed that the inhibition of PDE4 mediated cAMP/PKA/CREB signal transduction pathway upregulation reverses the cognitive impairment caused by many factors and inflammatory diseases. Therefore, the main purpose of this study was to clarify the pathological role of Aβand also demonstrated whether different fragments:Aβ1-42 or Aβ25-35 induced the memory deficits with role of neuroinflammation and the reduction of pCREB signaling. We also used the PDE4 specific inhibitor rolipram to demonstrated whether the reversed effect on memory deficits induced by Aβassociated with anti-inflammatory response. In order to further clarify whether neuroinflammation plays important role on memory deficits, we used TNF-a antibody to block the neuroinflammation induced by Aβand to test if the cognitive dysfunction associated with the reduction of the neuroinflammation. In order to clarify the relationship of the memory deficits between the neuroinflammation and the inhibition of pCREB signaling induced by Aβ, we used the PKA blocker (H89) to block the activation effect of rolipram on the PKA/CREB signaling to see the blocking pCREB-mediated signaling pathway can block the PDE4 inhibitor on cognitive function and anti-inflammation effects. Secondly, to further clarify the regulation of the PDE4 subtypes on the learning and memory, we used the PDE4A knockout mice to demonstrated the role of PDE4A on the memory and the neurogenesis. And we also used the PDE4A,B and D subtype gene knockout mice to further clarify the different regulation on the cognitive dysfunction and neuroinflammation induced by Aβ. Therefore, this study further enriches the regulation of PDE4 and its subtypes in the AD and also provides a theoretical basis for developing the highly selective and efficient PDE4 or subtypes inhibitors with the role of anti-inflammaion effect.Methods:(1) CA1 or DG subregions of hippocampus were injected with "aging" Aβ1-42 (0.4,1.6μg/side),10 days later the locomotor activity were tested in order to record the line crossing and rears.24 hours later, mice were individually placed in the center of the box containing two identical objects located in the two diagonal corners. The cumulative time spent in exploring each object was recorded during a 5-min period. 24 hours after training, mice were tested for memory using same procedure except that one of the familiar objects was replaced with a novel object. The time of exploration of each object (Tf and Tn for familiar and novel objects, respectively) was recorded for determination of the recognition index (RI=Tn/Tn+Tf).24 h after the novel object recognition test, the training of the step-down passive avoidance were conducted.24 h after the training using the same procedure except the no shocks were delivered, the step-down latency was recorded, with an upper cut-off time of 300 s.24 h after all the behavioral test, the animals were sacrificed and the pCREB> CREB,BDNF,NF-κB p65,IL-1βin the hippocampus were detected. Moreover, to verify the accuracy of the injected position in the CA1 and DG subregions, we used the GFP-labeled lentiviruses to inject into the CA1 and DG subregions according to the different coordinates,14 days later the virus expression in the CA1 and DG subregions was observed by immunohistochemistry.(2) First, we used the reverse sequence of Aβ1-42 and blank solvent as the control, the aged Aβ1-42 (0.1,0.4,1.6μg/side) were injected into the CA1 subregion of hippocampus. The Rolipram (0.5mg/kg) or Vehicle were administrated 24 h after the surhery.10 days later, the behavioral test of the locomotor activity, the novel object recognition test, the step down passive avoidance and the Morris water maze test were conducted in order.24 h after all the behavioral test, the animals were sacrificed and the pCREB,CREB,IL-1β,TNF-α,NF-κB p65,COX-2,IL-10,SOD and the Aβ1-42 levels in the hippocampus and cortex were detected by western bloting. And PDE4A, B, D subtype expression in the hippocampus also were examined in order to clarify the different effect of Aβ1-42 on the different PDE4 subtypes. Secondly, in order to verify the dose-effect of the rolipram (0.1,0.25,0.5 mg/kg) on the memory deficits induced by Aβ25-35 (10μg/side) in the SD rats, the behavioral test of the Morris water maze test, the passive avoidance test were conducted in order from the day 12.24 h after the last behavioral test, the animals were sacrificed and the pCREB,NF-κB p65,Bcl-2 and the Bax in the hippocampus were detected by western bloting. Finally, in order to verify whether the memory deficits induced by Aβis associated with neuroinflammation and also to clarify the important regulation of pCREB signaling on the memory and neuroinflammation, we microinjected the aged Aβ1-42 into the CA1 subregion of the hippocampus (0.4μg/side) and to tested whether the TNF-αantibody could block the neuroinflammation and memory deficits. And we also used the PKA blocker of the H89 to test whether this blocker could block the effects of the anti-inflammatory and cognitive enhancement when the pCREB signaling was blocked. In this study,10 days after the surgery all the animals were tested for the locomotor activity,the object recognition test,the step down passive avoidance task and the Morris water maze test.24 h after the last behavioral test, the animals were sacrificed and the pCREB,CREB,NF-κB p65 and the TNF-αin the hippocampus were detected by western bloting.(3) We first used the PDE4A subtype knockout mice comparing with wild-type on the learning and memory changes and its mechanism. The locomotor activity, the object recognition test and step down passive avoidance test were conducted in order. The pCREB, CREB and BDNF were detected respectively in the hippocampus, cortex and striatum by the method of Western bloting and the neurogenesis of hippocampus was tested by immunohistochemistry. Second, in order to demonstrated the regulation of PDE4D on the memory deficits and neuroinflammation induced by the Aβ1-42, we microinjected the aged Aβ1-42 (0.4μg/side) into the CA1 subregion of hippocampus in the PDE4D-/-mice.10 days after the surgery, the locomotor activity, the object recognition test and step down passive avoidance test and the Morris water maze were tested in order. And then the pCREB, CREB, NF-κB p65, TNF-α, IL-1β, COX-2, IL-10 in the hippocampus were detected by Western bloting. Finally, in order to clarify the regulation of PDE4A, B and D subtypes on the mempry deficits and neuroinflammation induced by Aβ1-42, the PDE4A, B and D knockout mice were used and the DG subregion of hippocampus was microinjected the aged Aβ1-42 (0.4μg/side).10 days after the surgery, the locomotor activity, the object recognition test and step down passive avoidance test were tested in order. And then the pCREB, BDNF, NF-κB p65 and COX-2 in the hippocampus were detected by Western bloting.Results:(1) Our preliminary data shown that the microinjected position accurately into the CA1 and DG subregions of the hippocampus. The treatment of Aβ1-42 did not changed the locomotor activity between all groups. The recognition index were significantly decreased by Aβ1-42 microinjected into the CA1 or DG subregions of hippocampus. For the step-down passive avoidance test, two doses of Aβ1-42 injected into the CA1 subregion significantly reduced the escape latency (retention), and only the dose 1.6μg/side injected into the DG subregion of hippocampus significantly reduced escape latency (retention). Consistent with the behavior test, Aβ1-42 injected into the CA1 subregion significantly decreased the expression of pCREB and BDNF in the hippocampus. Only the dose of 1.6μg/side microinjected into the DG subregion significantly reduced the expression of pCREB in the hippocampus expect for the expression of BDNF was significantly decreased by the both dose. All treatments did not effect the expression of CREB between all groups. Two doses of Aβ1-42 significantly increased NF-κB p65 and IL-1βexpression in the CA1 and DG subregions of hippocampus.(2) We found that the Aβ1-42 (0.1,0.4 and 1.6μg/side) dose-dependently dexreased the cognitive function in the object recognition test, the step down passive avoidance test and the Morris water maze test, and the rolipram (0.5 mg/kg) reversed these effects. All the treatments had no effect on the locomotor activity. The Aβ1-42 also dose-dependently increased the expression of the IL-1β,TNF-α,NF-κB p65, COX-2 levels and the rolipram significantly reduced the release of these inflammation-related factors. And the Aβ1-42 also dose-dependently inhibited the phosphorylation of CREB, this was reversed by the rolipram. And we also found that Aβ1-42 dose-dependently slightly induced the formation of IL-10, and rolipram also played regulatory role on the IL-10. Our study also found that rolipram had no effect on the the Aβ1-42 itself. In order to clarify which PDE4 subtypes were impacted by Aβ1-42, we found that Aβ1-42 significantly increased PDE4A5,PDE4A?,PDE4B1,PDE4B2,PDE4B3,PDE4B4,PDE4D4,PDE4D5 levels. We also found that microinfusions of Aβ25-35 into bilateral CA1 subregions (10μg/side) impaired learning ability and long-term spatial memory in the Morris water maze test, it also decreased long-term memory in the passive avoidance test. Levels of NF-κB p65 and Bax were increased while levels of pCREB and Bcl-2 were decreased in the hippocampus in rats treated with Aβ25-35. These effect of Aβ25-35 were reversed by repeated treatment with rolipram in a dose dependent manner. The further study also demonstrated that TNF-αantibody blocked the inflammatory response induced by Aβ1-42 and only slightly alleviated the learning and memory deficits. While the TNF-αslightly increased the Aβ1-42-induced learning and memory impairments. We also found that the PKA inhibitor of H89 not only blocked the cognitive enhancing effect of rolipram and also the anti-inflammatory effects.(3) Mice deficient in PDE4A display significant memory enhancing effect comparied with WT mice. But the locomotor activity was not changed by PDE4A subtype knock out. The PDE4A knockout also increased the expression of pCREB and BDNF in the hippocampus expect for the cortex and striatum. The proliferation and survival of newborn cells were increased significantly in the hippocampus of PDE4A knockout mice compared with WT mice. The PDE4D knockout mice also reversed the memory deficits downregulation of pCREB signaling induced by Aβ1-42 microinjected into the CA1 subregion of the hippocampus. Although the mice deficit in PDE4D did not significantly decreased the expression of the NF-κB p65, TNF-α, IL-1κinduced by Aβ1-42, the PDE4D knockout displayed inhibited effect on COX-2 overexpression and slightly increased effect on IL-10. Further studies also showed that PDE4A and D subtypes knockout reversed the memory deficits induced by microinjected Aβ1-42 into the DG subregion of hippocampus except for PDE4B knockout mice. Consistent with behavioral test, the PDE4A and PDE4D knockout mice reversed the pCREB/BDNF signaling except for PDE4B knockout mice. The PDE4A and PDE4D knockout mice decreased the expression of COX-2 induced by Aβ1-42 except for the NF-κB p65. However, PDE4B knockout mice displayed the downregulation effect on the expression of NF-κB p65 except for the COX-2.Conclusion:Microinjected Aκ1-42 or 25-35 into the CA1 and (or) DG subregions of the hippocampus produced the inflammatory responses, apoptosis, memory deficits and downregulation of pCREB/BDNF signaling. These provide a new theoretical basis which help to find the new drugs targets focus on the anti-inflammatory and accompanied upregulaition of pCREB-mediated signaling in Alzheimer's disease. PDE4 inhibitor (rolipram) reversed the memory deficits and neuroinflammation induced by Aβand different PDE4 subtypes were increased by Aβ. PDE4 may be play important role on the regulation on the memory deficits and neuroinflammation in AD. We also extended these findings to the demonstration that the protective effect of PDE4 against Aβinsult may be partly attributed to the blockade of inflammatory responses and apoptosis in the hippocampus. And we also demonstrated that PDE4 inhibitor may be through pCREB signaling to reversed the memory deficits and inflammation induced by Aβ. Our study also firstly demonstrated that PDE4A subtype plays important role on the memory and neurogenesis. Further study also clarified that the PDE4A and D subtypes kncokout reversed the memory deficits and downregulation of pCREB signaling except for the neuroinflammation induced by Aβ. Although PDE4B subtype knockout did not reversed the memory deficits and downregulation of pCREB signaling, PDE4B knockout mice displayed anti-inflammatory effect. The regulate effect of PDE4A,B and D subtypes on COX-2 may be contribuate to the different regulation on neuroinflammation. These demonstrated that COX-2 may be play an important role on the progress of anti-inflammatory effect.