【作者】 张聪；

【导师】 徐江平； 张汉霆；

【作者基本信息】 南方医科大学 ， 药理学， 2014， 博士

【摘要】 研究目的：磷酸二酯酶(phosphodiesterases, PDEs)是体内唯一负责水解第二信使环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)的酶类。磷酸二酯酶4(phosphodiesterases4, PDE4)是PDEs家族成员之一,PDE4抑制剂的研究主要集中在呼吸系统和中枢神经系统疾病上,在临床上主要用于治疗慢性阻塞性肺疾病(COPD)、哮喘及银屑病。近年来,越来越多的研究表明,PDE4可以通过多种途径改善神经系统疾病,例如抑郁症、老年痴呆、脑卒中等。相应的抑制剂能够通过调控细胞内cAMP水平从而改善疾病所致的抑郁症状和学习记忆障碍。但是几乎所有的非选择性抑制剂都会产生严重的胃肠道反应,限制了该类药物的临床使用。PDE4家族包含20多种亚亚型(由于mRNA初级产物经酶的不同剪接形成,亦称为剪接变异体),分布在细胞不同的区域从而发挥不同的作用。因此本课题的研究目的是利用慢病毒介导的PDE4DmiRNA沉默长链PDE4D的表达,在小鼠慢性不可预知性温和刺激引起的抑郁症模型和Aβ42海马DG区微量注射诱导的认知损伤模型上阐明长链PDE4D(尤其是PDE4D4/5)对小鼠抑郁症状和学习记忆障碍的调节作用。课题分为三大部分：第一部分是构建并鉴定携带PDE4DmiRNA的慢病毒载体。第二部分是初步观察慢病毒介导的PDE4DmiRNA对慢性不可预知性温和刺激引起的小鼠抑郁行为的调节作用,检测海马组织cAMP通路的水平。第三部分是研究慢病毒介导的PDE4DmiRNA对正常小鼠学习记忆能力的影响,并进一步阐明慢病毒介导的PDE4DmiRNA对Aβ42诱导的学习记忆损伤模型中小鼠认知行为的调节作用及其对cAMP信号通路和促炎因子的影响。该研究为进一步阐明长链PDE4D在抑郁症和认知障碍疾病中的作用提供实验依据。研究方法：(1)首先构建能转录产生PDE4D4miRNA的重组质粒,与慢病毒包装质粒共转染293T细胞产生高滴度慢病毒。体外感染培养的293T细胞检测其转染效率。采用脑立体定位技术在小鼠海马DG区微量注射慢病毒介导的PDE4DmiRNA引起目的基因表达沉默,14天后取海马组织检测PDE4DmiRNA对PDE4A/B/D以及PDE4D3/4/5的mRNA水平的影响。(2)采用慢性不可预知性温和刺激使小鼠接受应激67天,诱导产生小鼠抑郁样症状,糖水偏爱实验检测小鼠抑郁程度。抑郁模型建立后,采用脑立体定位技术在小鼠海马DG区微量注射慢病毒,14天后进行行为学实验。强迫游泳实验,悬尾实验和糖水偏爱实验反应小鼠抑郁状态；高架十字迷宫实验和旷场实验反应小鼠的焦虑状态；高效液相质谱联用技术检测小鼠海马中cAMP水平；PCR技术和Western Blot技术分别检测了小鼠海马cAMP-PKA-CREB通路的变化。(3)采用Morris水迷宫实验,新物体识别实验和跳台实验评价小鼠的学习记忆能力。小鼠双侧海马DG区注射老化的Aβ42(0.5μg/side)诱导小鼠学习记忆损伤,24小时后注射慢病毒在同一位点,14天后采用水迷宫和新物体识别用来评价小鼠的学习记忆能力；高效液相质谱联用技术检测小鼠海马中cAMP水平；PCR技术和Western Blot技术分别检测了小鼠海马cAMP-PKA-CREB通路以及炎症因子的变化。研究结果：(1)慢病毒转染293T细胞72小时后,在荧光显微镜下观察绿色荧光蛋白的表达。无论携带阴性对照还是PDE4D基因序列的慢病毒均表现出强烈的荧光。在动物体内实验中亦发现了相同的结果。而且,PDE4DmiRNA能显著降低PDE4D, PDE4D4和PDE4D5的表达水平,而对PDE4A/B和PDE4D3的表达没有显著性影响。(2)在小鼠经历慢性不可预知性温和刺激过程中,应激小鼠在糖水偏爱实验中的糖水偏爱度和小鼠体重与非应激组小鼠相比均明显降低。给予慢病毒治疗后,CUMS组小鼠在强迫游泳实验和悬尾实验中,不动时间显著降低,且糖水偏爱率依然处于低态,微量注射慢病毒后能够逆转CUMS引起的不动时间的减少,提高糖水偏爱率,改善小鼠抑郁样症状。同时慢病毒逆转CUMS引起的海马cAMP水平的下降,增强海马pCREB蛋白表达,增加BDNF mRNA水平,而不影响CREB的mRNA水平。(3)小鼠持续给予rolipram或单次注射慢病毒14天后,rolipram显著性减少小鼠到达平台的潜伏期,增加跳台实验中的逃避潜伏期,起到增强记忆的作用。而且慢病毒介导的PDE4DmiRNA对学习记忆的增强作用不如rolipram效果明显。慢病毒介导的PDE4DmiRNA可以逆转Aβ42诱导的小鼠认知障碍。研究发现小鼠双侧海马DG区注射老化的Aβ42(0.5μg/side)可以显著减少小鼠水迷宫探索实中在平台象限的停留时间和穿越次数,新物体实验训练24小时后,Aβ42小鼠的识别指数与对照组相比显著降低,说明Aβ42诱导小鼠产生学习记忆损伤。微量注射慢病毒后小鼠探索实验中平台象限的停留时间和穿越次数以及物体识别实验中识别指数均显著增加,表明慢病毒介导的PDE4DmiRNA能够逆转Aβ42引起的认知障碍,同时可以增强海马pCREB和BDNF蛋白表达,减少IL-1β, TNF-α和NF-κB的水平。结论：本课题从提高抑制PDE4靶点的特异性为出发点,构建了慢病毒介导的发夹结构的PDE4DmiRNA,特异性抑制PDE4D4/5的表达,微量注射小鼠海马DG区后,慢病毒可以与宿主细胞整合并持续高表达,且PCR技术验证了miRNA的沉默效率,为目的基因的沉默提供了可靠的实验依据。继而我们发现,长链PDE4D表达降低可以增加海马内第二信使cAMP水平,激活cAMP/PKA/CREB信号通路,进一步增加下游BDNF蛋白的表达,同时减少凋亡因子和促炎症因子的产生,从而起到逆转慢性不可预知性刺激引起的小鼠抑郁样症状和改善Aβ42诱导的小鼠学习记忆损伤的作用。这些结果为治疗抑郁症和AD引起的抑郁症状和学习记忆障碍提供了新的治疗靶点,有望通过抑制PDE4D4/5亚亚型来对抗抑郁症状和认知损伤。同时为阐明PDE4D亚亚型的情绪和认知调节作用提供了可靠的实验依据,也为开发PDE4亚型抑制剂提供了理论基础。更多还原

【Abstract】 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.更多还原