Neuronal-Glial Interaction in the Treatment of Bipolar D

神经元-胶质细胞相互作用在双相 D 治疗中的作用

• 批准号: 7312910
• 负责人: HUSSEINI K MANJI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7312910
• 关键词: Neuronal; Glial; Interaction; Treatment; Bipolar

There is now compelling evidence that radial glial cells have the potential, not only to guide newly born neurons, but also to self-renew and to generate both neurons and astrocytes. Recent data has also shown that astrocytes increase the number of mature, functional synapses on central nervous system (CNS) neurons by sevenfold, demonstrating that CNS synapse numbers can be profoundly regulated by glia. Glial cells also play critical roles in regulating synaptic glutamate levels, CNS energy homeostasis, liberation of trophic factors, and form dynamic, complex synaptic networks with neurons. Nevertheless, the possibility of glial dysfunction in major psychiatric disorders has only recently received serious consideration due to the converging neuroimaging, postmortem morphometric and microarray studies, which have clearly revealed glial abnormalities in schizophrenia and mood disorders. To examine the effect of lithium (Li) on glia and neuron growth, we have established an astrocyte and neuronal primary culture system. We found that astrocytes, whose proliferation is increased by lithium, may indirectly (via liberation of factors from glial cells) regulate neuronal differentiation. Astrocytes may induce the pluripotent immature neuron to express an astrocytic phenotype. Next, we examined the alteration of cell signaling in astrocyte proliferation and neuronal differentiation to study the possible molecular mechanism of Li-induced action In addition, we examined whether Li affected growth of oligodendricytes, another type of glial cell. To investigate this more definitively, we began a series of in vitro and in vivo studies examining lithium's effects on oligodendrocytes. Chronic lithium treatment significantly increased the total number of oligodendrocytes in a dose-dependent manner, with a maximal effect observed with 1.0 mM lithium. To determine whether lithium affects BrdU incorporation, oligodendrocytes were treated with BrdU for 6 h in the absence or presence of lithium (1.0 mM). BrdU incorporation was determined by immunocytochemistry. BrdU-labeled cells were markedly increased by lithium treatment. O4 expression was examined by immunocytochemistry to further determine the cell phenotype of these BrdU-labeled cells. BrdU-positive cells were also O4+. Quantitatively, the percentage of BrdU-positive oligodendrocytes, as well as that of BrdU+O4+ cells, were significantly increased by the lithium treatment. Our data demonstrate for the first time that chronic lithium exerts a major effect on oligodendrocytes, increasing their proliferation. These observations raise the possibility that lithium may serve to correct abnormalities in white matter tracts, thereby restoring the functioning of critical circuits mediating affective, cognitive and motor symptom. These mechanisms may provide a potential target for improved long-term therapeutics for severe neuropsychiatric disorders. Recent evidence suggests that ATP acting via ionotropic (P2X) purinergic receptors might be involved in signaling between glial cells and within glial-neuronal networks. The P2X7 receptor, known as the cytolytic P2Z receptor, has been implicated in signaling between neuron and astrocytes, and has recently been postulated to represent a candidate gene for recurrent mood disorders. P2X7 receptors have been proposed as mediators of inflammation, and a potential role in neurodegeneration has been suggested. The P2X7 receptor shares 35-40% homology with other P2X receptors. It has two hydrophobic membrane-spanning domains and an extracellular loop, and forms transmembrane ion channels. Under normal conditions, extracellular nucleotides are present in only low concentrations. However, activated immune cells, such as lymphocytes, macrophages, microgli, and platelets, and dying cells may release high concentrations of different nucleotide di- and tri-phosphates into the extracellular space. Under inflammatory conditions, P2X7 receptor activation stimulates the induction of multiple cytokine pathways that may co-ordinate inflammatory responses, and triggers massive transmembrane ion fluxes (particularly influx of Ca2+ and Na+, and efflux of K+) and the formation of non-selective plasma membrane pores that result in cell death. In contrast to their neuronal counterpart, the function of P2X receptors in CNS glial cells is largely unknown. By Westen blot protein analysis, immunocytochemistry and immunohistochemistry, we examined expression of P2X7 receptors in astrocytes in vivo and in vitro and examined effect of lithium (Li) on the expression of P2X7 in astrocytes. We found that P2X7 receptor is expressed in astrocytes. P2X7 positive cells can be GFAP- and S100beta- positive, suggesting a colocalization of astrocyte proteins and P2X7 receptor in CNS. Moreover, we also found that Li (0.5-1.0 mM) resulted in significantly decreased expression of P2X7 receptor in cultured astrocytes. Finally, we also found chronic (5-day) Li-treatment significantly blocked ATP-induced influx of Ca2+ in astrocytes. Our data demonstrated that P2X7 is expressed in astrocytes, and its expression levels can be regulated by chronic treatment of lithium at therapeutically relevant concentrations. Considering P2X7 receptor?s role in the regulation of Ca2+ by ATP, this may be the molecular mechanism by which ATP-induced influx of Ca2+ can be blocked by lithium in astrocytes. Our data provide evidence showing that the P2X7 receptor in astrocytes may be a therapeutic target for mood disorder treatments Lithium has demonstrated to exert positive regulatory effects on cellular neurotrophic signaling, protecting neurons and astrocytes against a variety of insults. Lithium also has shown to robustly up-regulate the anti-apoptotic protein Bcl-2 expression in vitro and in vivo. Bcl-2 is localized to the ER and mitochondria and regulates calcium dynamics in these intracellular compartments. The ATP-gated calcium channel P2X7 is known to play a role in regulating calcium dynamics, and in glial and neuronal cytotoxicity. Therefore we sought to determine the role of Bcl-2 in lithium-mediated regulation on mitochondrial calcium uptake, utilizing Lenti-virus containing Bcl-2siRNA. Lithium dose-dependently increased Bcl-2 levels in astrocytes. BzATP (300?M) activated P2X7 ATP receptor and enhanced mitochondrial Ca2+ in cultured primary astrocytes. This effect was completely blocked by P2X7 antagonist ox-ATP, thus suggesting a P2X7- specific effect. We found that the mitochondrial calcium-uptake significantly increased during thapsigargin- (2uM) or P2X7-evoked intracellular calcium increase in lithium-treated astrocytes. This effect was abolished in Bcl-2 SiRNA lenti-virus infected cells compared to scrambled sequence infected cells (p<0.001). These results suggest an essential role for Bcl-2 in lithium-mediated increase in mitochondrial-Ca++ uptake in astrocytes. These findings might shed light on new mechanisms for the neuroprotective effect of lithium and lead to the development of novel, improved therapeutics.

现在有令人信服的证据表明，放射状胶质细胞不仅具有引导新生神经元的潜力，而且具有自我更新和生成神经元和星形胶质细胞的潜力。最近的数据还表明，星形胶质细胞使中枢神经系统（CNS）神经元上成熟的功能性突触数量增加了七倍，这表明中枢神经系统突触数量可以受到神经胶质细胞的深刻调节。胶质细胞在调节突触谷氨酸水平、中枢神经系统能量稳态、营养因子的释放以及与神经元形成动态、复杂的突触网络方面也发挥着关键作用。然而，由于神经影像学、死后形态测量和微阵列研究的融合，主要精神疾病中神经胶质功能障碍的可能性最近才得到认真考虑，这些研究清楚地揭示了精神分裂症和情绪障碍中的神经胶质异常。为了研究锂（Li）对神经胶质细胞和神经元生长的影响，我们建立了星形胶质细胞和神经元原代培养系统。 我们发现星形胶质细胞的增殖因锂而增加，可能间接（通过神经胶质细胞释放因子）调节神经元分化。星形胶质细胞可以诱导多能未成熟神经元表达星形胶质细胞表型。接下来，我们检查了星形胶质细胞增殖和神经元分化中细胞信号传导的改变，以研究锂诱导作用的可能分子机制。此外，我们检查了锂是否影响另一种神经胶质细胞少突胶质细胞的生长。为了更明确地研究这一点，我们开始了一系列体外和体内研究，检查锂对少突胶质细胞的影响。长期锂治疗以剂量依赖性方式显着增加了少突胶质细胞的总数，其中 1.0 mM 锂的效果最大。为了确定锂是否影响 BrdU 掺入，在不存在或存在锂 (1.0 mM) 的情况下用 BrdU 处理少突胶质细胞 6 小时。 BrdU 掺入通过免疫细胞化学测定。锂处理后，BrdU 标记的细胞数量显着增加。通过免疫细胞化学检查 O4 表达，以进一步确定这些 BrdU 标记细胞的细胞表型。 BrdU 阳性细胞也是 O4+。从数量上看，锂处理显着增加了 BrdU 阳性少突胶质细胞以及 BrdU+O4+ 细胞的百分比。我们的数据首次证明，长期锂对少突胶质细胞产生重大影响，增加其增殖。这些观察结果提出了锂可能有助于纠正白质束异常的可能性，从而恢复介导情感、认知和运动症状的关键回路的功能。这些机制可能为改善严重神经精神疾病的长期治疗提供潜在目标。 最近的证据表明，ATP 通过离子型 (P2X) 嘌呤能受体发挥作用，可能参与神经胶质细胞之间和神经胶质神经元网络内的信号传导。 P2X7 受体，也称为溶细胞性 P2Z 受体，与神经元和星形胶质细胞之间的信号传导有关，最近被认为是复发性情绪障碍的候选基因。 P2X7 受体已被认为是炎症介质，并且在神经退行性变中具有潜在作用。 P2X7 受体与其他 P2X 受体有 35-40% 的同源性。它具有两个疏水性跨膜结构域和一个细胞外环，并形成跨膜离子通道。在正常条件下，细胞外核苷酸仅以低浓度存在。然而，活化的免疫细胞，如淋巴细胞、巨噬细胞、小胶质细胞和血小板，以及垂死的细胞可能会向细胞外空间释放高浓度的不同核苷酸二磷酸和三磷酸。在炎症条件下，P2X7受体激活刺激多种细胞因子途径的诱导，这些途径可以协调炎症反应，并引发大量跨膜离子通量（特别是Ca2+和Na+的流入，以及K+的流出）和非选择性质膜的形成导致细胞死亡的毛孔。与神经元对应物相比，中枢神经系统胶质细胞中 P2X 受体的功能很大程度上未知。通过Westen blot蛋白分析、免疫细胞化学和免疫组织化学，我们检测了星形胶质细胞体内和体外P2X7受体的表达，并研究了锂（Li）对星形胶质细胞P2X7表达的影响。我们发现P2X7受体在星形胶质细胞中表达。 P2X7 阳性细胞可以是 GFAP 和 S100β 阳性，表明星形胶质细胞蛋白和 P2X7 受体在 CNS 中共定位。此外，我们还发现Li（0.5-1.0 mM）导致培养的星形胶质细胞中P2X7受体的表达显着降低。最后，我们还发现长期（5 天）锂治疗显着阻止了星形胶质细胞中 ATP 诱导的 Ca2+ 流入。我们的数据表明，P2X7 在星形胶质细胞中表达，其表达水平可以通过治疗相关浓度的锂的长期治疗来调节。考虑到P2X7受体在ATP调节Ca2+中的作用，这可能是星形胶质细胞中锂可以阻断ATP诱导的Ca2+内流的分子机制。我们的数据提供的证据表明星形胶质细胞中的 P2X7 受体可能是情绪障碍治疗的治疗靶点 锂已被证明对细胞神经营养信号产生积极的调节作用，保护神经元和星形胶质细胞免受各种损伤。锂还可以在体外和体内显着上调抗凋亡蛋白 Bcl-2 的表达。 Bcl-2 定位于 ER 和线粒体，调节这些细胞内区室中的钙动态。已知 ATP 门控钙通道 P2X7 在调节钙动力学以及神经胶质和神经元细胞毒性中发挥作用。因此，我们试图利用含有 Bcl-2siRNA 的慢病毒来确定 Bcl-2 在锂介导的线粒体钙吸收调节中的作用。锂剂量依赖性地增加星形胶质细胞中的 Bcl-2 水平。 BzATP (300?M) 激活 P2X7 ATP 受体并增强培养的原代星形胶质细胞中的线粒体 Ca2+。这种作用被P2X7拮抗剂ox-ATP完全阻断，因此表明P2X7特异性作用。我们发现，在经锂处理的星形胶质细胞中，毒胡萝卜素（2uM）或 P2X7 引起细胞内钙增加时，线粒体钙摄取显着增加。与乱序序列感染的细胞相比，这种效应在 Bcl-2 SiRNA 慢病毒感染的细胞中被消除 (p<0.001)。这些结果表明 Bcl-2 在锂介导的星形胶质细胞线粒体 Ca++ 摄取增加中发挥重要作用。这些发现可能揭示锂神经保护作用的新机制，并导致新型改进疗法的开发。