Role Of Lentivirus-mediated PDE4DmiRNA On Depression And Cognitive Impairement

Objective:Phosphodiesterase (PDE) is an enzyme responsible for the hydrolysis of second messenger cAMP and cGMP in various biological systems, particularly in the central nervous system (CNS). Phosphodiesterase4(PDE4) is a member of the PDE superfamily and its inhibitors are mainly used for the treatment of chronic obstructive pulmonary disease, asthma and psoriasis. In the CNS, an increasing number of researches have shown that PDE4plays a key role in neurodegenerative diseases (e.g. Alzheimer’s disease), mental disorders (such as depression and schizophrenia) and stroke as well. Previous research showed the selectively PDE4inhibitor (rolipram) can improve neurodegenerative disorders by regulating the level of intracellular cAMP. However, the serious adversed reaction hinders its clinical application because of the lack of selectivity for PDE4subtype and splice variants. The PDE4family consists of four subtypes (PDE4A-D) and more than20splice variants, and distributes in the distinct microdomains of the cell. PDE4D is one of the subtypes and enriched in the CNS. It was reported that this subtype of PDE4appears to be involved in the process of neurological diseases. So far, its roles in depression and cognitive impairment are only beginning to be unveiled and the possible mechanisms are still need to be investigated in detail. In current project, we constructed the lentiviral vector containing a specific microRNA/miRNA-mir hairpin structure (4DshR) and confirmed the efficiency of gene knockdown. And then, we investigated whether long-form PDE4D knockdown by lentivirus can reverse memory impairment caused by Aβ1-42(Aβ42) and improve depressive-like behaviours induced by chronic unpredictable mild stress (CUMS) in mice. Furthermore, the influence of cAMP/PKA/CREB/BDNF signal pathway was also determined. The content of this thesis includes three parts:first, we constructed lentivirus vector containing a specific miRNA with hairpin structure and identified its silencing efficiency. We then investigated the role of PDE4DmiRNA on depressive behaviors by forced swimming test and tail suspension test in mice suffered from chronic unpredictable mild stress and the possible involvement of signaling cascades includes the cAMP-PKA-CREB pathway. Finally, we investigated the role of lentivirus-mediated PDE4DmiRNA on memory by MWM test, and the novel object recognition test in normal mice, lentivirus-mediated PDE4DmiRNA against Aβ42-induced cognitive impairment and changes in cAMP-PKA-CREB pathway and proinflammatory cytokines were also clarified. The studies further elucidate the role of long-form PDE4D subtype in regulation of depression and cognitive disorders.Methods:(1) Stereotactic technique was used to perform microinjection of lentivirus-mediated PDE4DmiRNA in the DG region of hippocampus. Hippocampal tissue was dissected for the detection of mRNA levels of PDE4A/PDE4B/PDE4D and PDE4D3/4/5.(2) Fourteen days after the microinjection of lentivirus-mediated PDE4DmiRNA in the DG region of hippocampus, depressive-like behaviors were evaluated by the forced swimming test, tail suspension test and sucrose preference test in mice. Chronic unpredictable mild stress was used to establish the model of depression. Animals received CUMS displayed decreased body weight and sucrose preference. The forced swimming test, tail suspension test and sucrose preference test were used to evaluate the depressive-like behaviors in mice. High performance liquid chromatography mass spectrometry (HPLC-MS/MS) was used to detect the level of cAMP of hippocampus in mice. RT-PCR and Western Bloting were used to detect the changes of related mRNA and proteins in mice, respectively.(3) Stereotactic technique was used to perform microinjection of lentivirus-mediated PDE4DmiRNA in the DG region of hippocampus. After fourteen days, the ability of learning and memory was measured by Morris water maze test and novel object recognition test. We examined whether long-form PDE4D knockdown by lentiviral reversed memory impairment caused by Aβ42in mice using the Morris water maze (MWM) and novelty object recognition tests. Western blotting analysis was used to assess protein levels of cAMP response element-binding protein (CREB, unphosphorylated and phosphorylated [pCREB]), brain-derived neurotrophic factor (BDNF), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nuclear factor-KB (NF-κB) to explore the neurochemical mechanisms.Results:(1) Seventy-two hours after lentivirus transfection in293T cells, the expression of green fluorescent protein (GFP) was observed by the fluorescence microscope in the hippocampi whether carried negative control or PDE4DmiRNA lentivirus vector, and the GFP expression shows strong fluorescence. Microinfusions of lentiviruses resulted in downregulated expression of PDE4D, PDE4D4and PDE4D5without changing the expression of PDE4A/B and PDE4D3.(2) We found that chronic unpredictable mild stress decreased body weight and sucrose preference rate in the sucrose preference compared with non-stressed mice and induced depressive-like symtoms, and also decreased immobility time in the forced swimming test and the tail suspension test, PDE4DmiRNA reversed CUMS induced depressive-like symptoms and cAMP decline. Meanwhile, CUMS procedure decreased the level of cAMP and reduced the expression of pCREB protein, and BDNF mRNA levels without affecting the CREB mRNA level which was reversed by lentivirus-mediated PDE4DmiRNA.(3) Rrolipram significantly reduced the latency to reach the platform in MWM, and increased escape latency in passive avoidance test, that suggest the function of learning and memory enhancement. Lentivirus-mediated PDE4DmiRNA also significantly reduced the latency to reach the platform in water maze test and increased the escape latency in passive avoidance test. Aggregated AP42(0.5μg/side) bilaterally infused into the dentate gyrus decreased cAMP levels, and produced memory deficits in the MWM and object recognition tests. Microinfusions of lentiviruses resulted in downregulated expression of PDE4D4and4D5proteins and reversed Aβ42-induced cAMP decline and memory deficits. Treatment also concomitantly increased pCREB and BDNF and reduced IL-1β, TNF-α, and NF-κB (p65) in the hippocampus of Aβ42-challenged mice. These results suggest that long-form PDE4D knockdown may offer a promising treatment for memory loss associated with Alzheimer’s disease (AD).Conclusion:PDE4inhibitors have mainly focused on the treatment of respiratory and central nervous system diseases. But almost all of the non-selective PDE4inhibitors have severe nausea and vomiting and other gastrointestinal side effects, which limited the clinical use of these drugs. Many PDE4D splice variants distribute in the emetic central, including last Polar Regions and the solitary tract nucleus, it may be one of the explanations that why nausea and vomiting occurred when administrated with rolipram. Therefore, we constructed a lentiviral vectors for PDE4DmiRNA which specifically inhibited the expression of PDE4D4/5and improved the target specificity of PDE4D inhibitor. We found that reduced expression of long PDE4D could increase the level of cAMP in the hippocampus and activate cAMP/PKA/CREB signaling pathway, phosphorylated CREB further increased the expression of BDNF, and reduced the production of apoptotic factors and proinflammatory cytokines. These results provide a new target for the treatment of depression and memory disorders such as AD, and also provided a theoretical basis for the development of inhibitors of PDE4isoforms.