Investigation of Mitochondrial Function in Bipolar Disor

情绪障碍传统上被认为是神经化学障碍，但新的证据表明，中枢神经系统的结构可塑性和细胞弹性受损。最近的临床前和临床研究表明，参与调节细胞生存和死亡的信号通路是情绪稳定剂作用的长期靶点。锂和丙戊酸盐是治疗双相情感障碍的常见情绪稳定剂，并间接显示 调节细胞通路，包括CREB、BDNF、Bcl-2和MAP激酶，这些作用可能刺激它们的一些延迟的长期有益作用。具体地说，锂和丙戊酸盐在体内和体外上调Bcl-2，通过阻断线粒体的凋亡程序来防止神经细胞死亡。最近有报道称阿尔茨海默氏症和帕金森氏症患者存在线粒体功能障碍，目前尚不清楚双相情感障碍患者是否也存在类似的线粒体异常 这项正在进行的研究的目的是调查情绪稳定剂对线粒体功能的各个方面的影响。大鼠原代皮质神经元和人神经母细胞瘤SH-SY5Y细胞用丙戊酸盐和锂治疗相关浓度的慢性处理。Western Blot分析测定线粒体蛋白水平。用氧图系统测量呼吸活动。蛋白质印迹分析显示 锂和丙戊酸盐处理的细胞线粒体部分上调孔蛋白(一种电压依赖的阴离子选择通道)和抗凋亡蛋白Bcl2和Bclx/L。氧图测量表明，锂和丙戊酸盐以时间和剂量依赖的方式增加呼吸频率。推测Bclxl和Bcl2可能通过影响其他外膜蛋白的性质来维持其通过复杂阴离子的能力。最近有报道说， BCL-XL在体外可与孔蛋白相互作用，调节其门控特性。也有研究表明，细胞过度表达Bcl2会导致线粒体体积和结构的复杂性增加。 由于Bcl2是细胞存活的关键调节因子，我们使用siRNAs(小干扰RNAs)来降低bcl2的水平，并确定这种操纵对情绪稳定剂的生化效应的影响。SiRNA是一种小的双链RNA，长度为21-22个核苷酸。它们触发同源RNA的降解，并诱导哺乳动物细胞中特定基因表达的关闭。在这项研究中，我们通过使用siRNA敲除技术，研究了Bcl-2作为线粒体功能调节因子的作用。Bcl2siRNA/pSilencer显著下调了人神经母细胞瘤SH-SY5Y细胞中Bcl2基因的表达，使Bcl2蛋白水平降低约40%。用3种不同的染料研究了丙戊酸锂(VPA)对神经细胞线粒体数、线粒体膜电位、线粒体氧化和自由基(ROS)产生的影响。在线粒体膜电位不变的情况下，Mitotracker绿在脂质环境中会变成荧光；JC-1与线粒体膜电位成正比积累；Mitotracker红一旦进入线粒体就会氧化成荧光产物，2‘，7’-二氯荧光素二乙酸酯(H2DCFDA)用于通过染色ROS来评估氧化应激。共聚焦显微镜观察显示，慢性锂处理可使SH-SY5Y细胞线粒体膜电位升高。SH-SY5Y细胞中Bcl2基因的下调伴随着线粒体氧化的明显减少和Bcl2蛋白水平的显著降低。这些结果表明，情绪稳定剂通过上调Bcl2来增强线粒体的功能；鉴于线粒体在调节短期和长期可塑性中的关键作用，这些作用可能是治疗效果的关键。 这些新的结果表明，锂和丙戊酸盐可能通过对线粒体蛋白和线粒体功能的影响而发挥其对神经可塑性和细胞复原力的长期影响。

Mood disorders have traditionally been considered to be neurochemical disorders, but new evidence demonstrates impairments of structural plasticity and cellular resilience in the central nervous system. Recent preclinical and clinical studies have shown that signaling pathways involved in regulating cell survival and death are long-term targets for the actions of mood stabilizers. Lithium and valproate are common mood stabilizers for bipolar disorder and are shown to indirectly regulate cell pathways, including CREB, BDNF, Bcl-2, and MAP kinases, effects which may stimulate some of their delayed long-term beneficial effects. Specifically, lithium and valproate upregulate Bcl-2 in vivo and in vitro, preventing neuronal cell death through blocking the mitochondrial program for apoptosis. Mitochondrial dysfunction has been recently reported in Alzheimers' and Parkinson's diseases and it is not clear if similar mitochondrial abnormalities occur in bipolar disorders.The purpose of this ongoing study is to investigate the effects of mood stabilizers on various aspects of mitochondrial function. Rat primary cortical neurons and human neuroblastoma SH-SY5Y cells were treated chronically with valproate and lithium at therapeutically relevant concentrations. Western Blot analysis was used to determine mitochondrial protein levels. The Oxygraph system was used to measure the respiratory activity. Western blot analysis showed upregulation of porin (a voltage dependent anion selective channel) and anti-apoptotic proteins Bcl-2 and Bcl-x/L in the mitochondrial fraction of cells treated with lithium and valproate. Oxygraph measurements demonstrated that lithium and valproate increased respiratory rate both time- and dose-dependently. It is postulated Bcl-xL and Bcl-2 may act to influence the properties of other outer membrane proteins to maintain their ability to pass complex anions. Recently it was reported that Bcl-xL can interact with porin and regulate its gating properties in vitro. It has also been shown that Bcl-2 overexpressing cells result in an increased mitochondrial volume and structural complexity. Since Bcl-2 is a key regulator of cell survival, we have used SiRNAs (small interfering RNAs) to knock down bcl-2 levels and determine the consequences of this manipulation on the biochemical effects of mood stabiliziers. SiRNAs are small double-strand RNAs, 21-22 nucleotides in length. They trigger the degradation of cognate RNA and induce the shut-down of a specific gene expression in mammalian cells. In this study we investigated the role of Bcl-2 as a regulator of mitochondrial function, through the use of siRNA knockdown techniques. Bcl-2 siRNA/pSilencer significantly knocked down Bcl-2 gene expression, resulting in ~40% reductions in Bcl-2 protein levels in human neuroblastoma SH-SY5Y cells. 3 different dyes have been used to study the effects of lithium and valproate (VPA) on quantity of mitochondria, mitochondrial membrane potential, mitochondrial oxidation and generation of free radicals (ROS) in neuronal cells. Mitotracker green becomes fluorescent in the lipid environment of mitochondria regardless of the membrane potential; JC-1 accumulates in proportion to the mitochondrial membrane potential; Mitotracker red oxidizes to a fluorescent product once inside the mitochondria and 2',7'-dichlorofluorescin diacetate (H2DCFDA) for assessing oxidative stress by staining ROS. Confocal microscope study showed that chronic lithium treatment increased mitochondrial membrane potential of the SH-SY5Y cells. The knockdown of Bcl-2 gene in SH-SY5Y cells was accompanied by clear reductions in mitochondrial oxidation and significantly reduced Bcl-2-protein levels. These results suggest that mood stabilizers enhance mitochondrial function through Bcl-2 upregulation; in view of the critical role of mitochondria in regulating short & long-term plasticity, these effects may be criitical to the therapeutic effects. These novel results suggest that lithium and valproate may exert some of their long-term effects on neuroplasticity and cellular resilience via hitherto underappreciated effects on mitochondrial proteins and mitochondrial function