Cdk5/p35参与了慢性应激诱导的大鼠抑郁样行为

背景:对抑郁症发生机制的研究表明,抑郁症患者表现为海马以及其他脑边缘结构的体积减少和细胞丢失,抗抑郁剂可显著促进抑郁模型动物海马神经元发生,提示神经可塑性机制涉及了抑郁症的发生过程。周期素依赖性蛋白激酶-5(Cdk5)及其活性调节蛋白p35是保持成熟神经系统内边缘系统,尤其是海马中神经元具有潜在可塑性的关键激酶。Cdk5在中枢神经系统发育过程中起着至关重要的作用,主要与神经元迁移、轴突生长和神经递质释放有关,还参与细胞骨架形成、轴突导向、膜转运、突触功能和多巴胺信号转导等多种神经功能调控。然而,在某些病理性因素作用下,Cdk5激酶活性异常升高会引起神经元的过度死亡,导致一些神经退行性疾病的发生。Cdk5在调节神经元的生存方面发挥着重要的作用,那么在抑郁症损伤的海马神经元中,Cdk5是否也参与了神经元的生存过程,从而介导了抑郁症的发生尚未见到报道。目的:本研究将基于抑郁症神经可塑性的研究基础,探讨Cdk5/p35在抑郁症模型大鼠抑郁样行为中的作用,以期进一步阐明抑郁症发生的神经生物学机制,并为临床发现新的治疗抑郁症的有效手段奠定理论基础。方法:采用连续21d的慢性不可预见性的中等强度应激抑郁模型(CMS),以动物的体重变化、蔗糖水偏爱(sucrosep reference)和自发活动能力为指标,考察抑郁模型动物行为学变化;通过测定Cdk5特定底物-组蛋白H1被磷酸化的程度检测大鼠海马部位Cdk5激酶活性,用Westernblot的方法检测p35蛋白的表达;同时在应激过程中,采用微量注射的方法,分别在海马齿状回(DG),CA1及CA3亚区给予Cdk5激酶抑制剂(butyrolactone),观察抑制不同脑区Cdk5激酶对抑郁症模型动物体重,糖水偏爱、自发活动性等抑郁样行为的影响。此外,在慢性应激的同时,连续给予抗抑郁药venlafaxine和mirtazapine,考察抗抑郁剂的治疗作用对海马p35蛋白表达水平的影响。结果:慢性应激大鼠海马部位Cdk5激酶活性显著升高;Western blot结果表明应激大鼠海马DG区胞膜组分p35蛋白的表达水平明显上调,而胞浆p35蛋白表达水平显著降低;相关性分析结果表明海马Cdk5激酶活性与胞膜p35蛋白的表达水平呈明显正相关,而与胞浆p35蛋白的表达水平呈明显负相关;大鼠体重增加值,糖水偏爱与海马胞膜p35蛋白的表达水平呈显著负相关。上述结果表明海马Cdk5激酶活性升高参与了慢性应激诱导的大鼠抑郁样行为。行为学检测结果表明,海马DG区微注射Cdk5激酶抑制剂butyrolactone(50,100ng)可显著逆转慢性应激引起的大鼠抑郁样行为,而在CA1和CA3区微注射butyrolactone(100ng)则对大鼠的抑郁样行为没有明显影响,说明抑制Cdk5激酶活性改善大鼠抑郁样行为具有脑区特异性。在慢性应激实验中发现连续给予抗抑郁药venlafaxine(40mg·kg-1)和mirtazapine(20mg·kg-1)可明显降低DG区胞膜p35蛋白的表达水平,促进p35蛋白由胞膜转运至胞浆,而抗精神病药aripiprazole(5mg.kg-1)对慢性应激引起的DG区胞膜p35蛋白表达增加没有明显影响,说明降低胞膜p35蛋白的表达水平,抑制Cdk5激酶活性是抗抑郁药物特异性的。结论:本研究通过一系列实验证实了Cdk5/p35在慢性应激大鼠抑郁样行为中的作用,抑制Cdk5活性可有效逆转动物的抑郁样行为,抗抑郁剂在发挥治疗作用的同时可抑制Cdk5激酶的异常激活。因此,本研究结果为抑郁症的神经生物学机制研究和临床抗抑郁治疗药物研究开发提供了新的思路。

CYCLIN-DEPENDENT KINASE 5 IN THE DENTATE GYRUS MEDIATES DEPRESSIVE-LIKE BEHAVIOR IN RATS

Background:Depression is a serious psychiatric disorder that creates a major public health burden of disease and disability. It is quite widespread;approximately 16% of individuals in the United States have suffered from clinical depression during their lifetime. While antidepressants have been available for several decades,most of them are not totally effective. New approaches to clinically treating depression are thus needed,and the molecular and cellular mechanisms underlying the pathophysiology of depression could provide a new therapeutic target. Cyclin-dependent kinase 5 (Cdk5),is a proline-directed serine/threonine kinase that is highly active in the brain and is important for the development of the central nervous system. Cdk5 has been implicated in synaptic plasticity,which underlies learning and memory,as well as neuropsychiatric pathologies like depression and bipolar disorder. Recently,it also has been reported that inhibition of Cdk5 activity in the hippocampus facilitates extinction of learned contextual fear memory,suggesting that Cdk5 could be a potential target for treatment of emotional disorders. However,hippocampal Cdk5’s potential role in the development of depressive diseases has not been examined.Objectives:To investigate the possible roles that Cdk5/p35 might play in the development of depressive-like symptoms,using the chronic mild stress rat model. Methods:Two hundred and ninety-two male Sprague-Dawley rats (weighing 200-220 g at the start of the study) were individually housed under a constant temperature (23±s 2 degrees Celsius) and maintained on a 12 h light/dark cycle with free access to food and water. In order to examine the effects of Cdk5/p35 activity in hippocampus on depressive-like behavior,rats were subjected to the chronic mild stress procedure for 3 weeks. Subsequently,the depressive-like behaviors including sucrose preference,bodyweight increase and locomotor activity were examined;Cdk5 kinase activity was measured by an in vitro phosphorylation assay,and membrane and cytosolic p35 levels were detected by Western blot analysis. In order to investigate whether Cdk5 is functionally related to the depressive-like symptoms elicited by the CMS procedure,Buty,a Cdk5 inhibitor,was infused into DG,CA1 and CA3 subfield,respectively. Over the next 3 weeks,they received injections in DG,CA1 or CA3 on days 1,7,14 and 21,as detailed in a previous study. The control groups received intra-DG,CA1 or CA3 injection of saline containing 0.2% DMSO or different doses of Buty (25,50 and 100 ng). The chronic mild stress groups were given intra-DG,CA1 or CA3 injections of saline containing 0.2% DMSO and different doses of Buty (25,50 and 100 ng) during the 3 weeks of chronic mild stress. Each intracranial injection was administrated between 8.00-10.00 am,1 h prior to stress exposure to the stressor. The day after the last stress exposure,the sucrose preference test was performed and then the animals were decapitated. Subsequently,bilateral tissue punches from CA1,CA3 and DG (16 gauge) were obtained. Proteins were extracted for kinase activity measurement and Western blot assay. The subsequent experiment was designed to determine the role p35 translocation plays in the actions of antidepressants. Four groups of rats (n=8 per group) were used to examine effects of the antidepressant venlafaxine on DG p35 translocation using a 2×2 design with the between-subject factors of Group (Control or CMS) and Treatment (vehicle or venlafaxine). The other four groups of rats (n=8 per group) were used to examine the effects of the antidepressant mirtazapine on DG p35 translocation using a 2×2 design,with the between-subject factors Group (Control or CMS) and Treatment (vehicle or mirtazapine). Intraperitoneal drug or vehicle injections were performed for 21 consecutive days in both the control and CMS groups. The day after the last stress exposure,the sucrose preference test was performed and the animals were decapitated. Subsequently,bilateral DG tissue punches (16 gauge) were obtained. The cytoplasmic and membrane protein levels were measured by Western blot assay. Results:CMS increased hippocampal Cdk5 activity,which was accompanied by an upregulation of membrane p35,a Cdk5 activator,and a reduction of cytosolic p35 in the dentate gyrus (DG). Microinjection of a Cdk5 inhibitor,butyrolactone,in DG,but not in CA1 or CA3,reversed the depressive-like behaviors induced by CMS. A negative linear correlation was found between sucrose preference,bodyweight increase and the membrane p35 level in DG. Chronic treatment with antidepressants venlafaxine and mirtazapine,but not the antipsychotic aripiprazole,prevented the aberrant translocation of p35 induced by CMS in DG. Conclusion:Our results revealed that the development of depressive-like behavior was associated with increased Cdk5 activity through p35 translocation from the cytosol to the membrane in DG. These findings suggest that hippocampal Cdk5/p35 complex mediated the depressive-like behavior induced by CMS;thus,Cdk5 could be a novel target for therapeutic treatment of major depression.