The potassium channel Kv1.3 in perinatal brain injury

项目摘要/摘要 许多早产和围产期脑损伤的幸存者患有长期的神经后遗症。 这些新生儿迫切需要早期有效和安全的神经保护干预。我们的小组是 对一种名为Kv1.3(KCNA3)的电压门控钾通道的药理学感兴趣，它发挥着一种 通过调节膜电位影响细胞内在免疫细胞激活中的重要作用 钙信号转导等机制。我们的小组先前发现Kv1.3是促炎症所必需的 小胶质细胞的状态，并提供证据支持Kv1.3作为阿尔茨海默病的治疗靶点 和成人中风。最近，我们将我们的研究扩展到新生儿脂多糖的小鼠模型- 致敏的缺氧缺血性脑损伤(LPS-HI)中，单核巨噬细胞(MPS)的激活， 包括小胶质细胞、单核细胞和巨噬细胞)起着关键的病理作用。这种模式复制了一种主要的 围产期脑损伤的一种形式，围产期感染/炎症使大脑对随后的HI敏感 侮辱和加重脑损伤。我们发现Kv1.3基因敲除或选择性药物抑制 Kv1.3可减轻内毒素-HI脑损伤。令人惊讶的是，虽然Kv1.3的RNA和蛋白质水平在 从LPS-HI脑中分离的MPS，全细胞膜片钳未能检测到显著的质膜Kv1.3 (Pm-Kv1.3)MP细胞表面的通道活动。一个合乎逻辑的推论是，一个Kv1.3的细胞内池， 例如在一些肿瘤细胞系中描述的线粒体中的Kv1.3被激活。事实上，我们最近的数据 内毒素缺血组MPS线粒体Kv1.3(mito-Kv1.3)增加。这一发现标志着 新生儿内毒素-缺氧模型与成人内毒素-缺氧模型中MP激活机制的重要差异 中风、阿尔茨海默氏症和内毒素注射，因为在后者，PM-Kv1.3的活性显著上调。我们的 因此，假说是MPS的促炎激活，对内毒素-HI的神经毒性作用至关重要， 需要Kv1.3，主要贡献来自mito-Kv1.3。为了验证这一假设，我们将解决三个目标。 在目标1中，我们将使用定向删除方法来区分Kv1.3在 侵袭性单核细胞中有胞质小胶质细胞和Kv1.3。这样的决心将有助于理解动态 涉及单核细胞-小胶质细胞相互作用和神经元损伤的神经免疫机制，这方面的研究很少 在新生儿的大脑中已知。鉴于我们关于mito-Kv1.3的新发现，在目标2中，我们将验证mito-Kv1.3。 Kv1.3可作为治疗内毒素-HI脑损伤的潜在靶点。药理工具能够 选择性靶向PM-Kv1.3和MITO-Kv1.3将测试其在减轻内毒素脑损伤中的疗效。 嗨，模特。在目标3中，我们将进一步验证MITO-Kv1.3通过调节线粒体膜的假说 潜能，促进线粒体代谢状态的重新编程，以驱动功能极化。我们的目标是 揭示mito-Kv1.3和MP激活状态之间的机制联系，并设计有前景的新的 减轻围产期脑损伤的治疗方法。

Project Summary/Abstract Many survivors of premature birth and perinatal brain injury suffer from long-term neurological sequelae. These newborns urgently need early effective and safe interventions for neuroprotection. Our group is interested in the pharmacology of a voltage-gated potassium channel called Kv1.3 (KCNA3), which plays an important role in immune cell activation by modulating membrane potential to influence intracellular mechanisms such as Ca2+ signaling. Our group previously found that Kv1.3 is required for the pro-inflammatory state of microglia, and has provided evidence to support Kv1.3 as a therapeutic target for Alzheimer's disease and adult stroke. Recently we extended our study to a mouse model of neonatal lipopolysaccharides- sensitized hypoxic-ischemic brain injury (LPS-HI), in which activation of mononuclear phagocytes (MPs, which include microglia, monocytes, and macrophages) plays a key pathological role. This model replicates a major form of perinatal brain injury in which perinatal infection/inflammation sensitizes the brain to subsequent HI insult and augments brain injury. We showed that Kv1.3 knockout or selective pharmacological inhibition of Kv1.3 mitigates the LPS-HI brain injury. Surprisingly, while Kv1.3 RNA and protein levels were increased in MPs isolated from LPS-HI brains, whole-cell patch-clamp failed to detect significant plasma membrane Kv1.3 (PM-Kv1.3) channel activity on the MP cell surface. A logical inference is that an intracellular pool of Kv1.3, such as Kv1.3 in mitochondria, described in some tumor cell lines, is activated instead. Indeed, our recent data show increased mitochondrial Kv1.3 (mito-Kv1.3) in MPs isolated from LPS-HI brains. This discovery marks an important difference in MP activation mechanisms between the neonatal LPS-HI model and the adult models of stroke, Alzheimer's, and LPS injection, as in the latter the PM-Kv1.3 activity is significantly upregulated. Our hypothesis, therefore, is that pro-inflammatory activation of MPs, critical for neurotoxic actions in LPS-HI, requires Kv1.3, with a major contribution from mito-Kv1.3. To test this hypothesis, we will address three Aims. In Aim 1 we will use a targeted deletion approach to distinguish the respective contributions of Kv1.3 in residential microglia and Kv1.3 in invading monocytes. Such a determination will help understand dynamic neuroimmune mechanisms involving monocyte-microglia interactions and neuronal injury, about which little is known in neonatal brains. In view of our novel findings regarding mito-Kv1.3, in Aim 2 we will validate mito- Kv1.3 as a potential therapeutic target for LPS-HI brain injury. Pharmacological tools that are able to selectively target PM-Kv1.3 and mito-Kv1.3 will be tested for their efficacy in mitigating brain injury in the LPS- HI model. In Aim 3, we will further test the hypothesis that mito-Kv1.3, via regulating mitochondrial membrane potential, facilitates reprogramming of mitochondrial metabolic state to drive functional polarization. Our goal is to uncover the mechanistic link between mito-Kv1.3 and MP activation state, and design promising new therapeutic approaches to mitigate perinatal brain injury.