Validation of a new large-pore channel as a novel target for neuropathic pain

项目总结 神经病理性疼痛是一种使人虚弱的慢性疼痛综合征，影响着约2500万美国人。估计 每年直接医疗费用和生产力损失造成的总经济负担约为2500亿美元。此外， 人们严重依赖阿片类药物的处方，尽管它们的长期治疗能力很差。 控制疼痛。这导致了阿片类药物过量和成瘾的危机。根据疾控中心的数据，187人 在美国，每天都有人死于阿片类药物过量。因此，确定新的治疗靶点势在必行 安全的疼痛治疗，无滥用责任。小胶质细胞增多症和神经炎症已成为关键驱动因素 神经病理性疼痛。作为对周围神经损伤的反应，脊髓中的小胶质细胞被激活并 经历了扩散。三磷酸腺苷是最重要的小胶质细胞激活剂之一。在刺激的时候 嘌呤能受体，小胶质细胞释放神经调节剂，导致脊髓神经元敏化 导致慢性疼痛的回路。尽管它在小胶质细胞激活中起着核心作用，但其来源和释放 ATP的作用机制尚不清楚，尤其是在神经损伤后。这项提议的理由是 阻断脊髓损伤后过度自分泌/旁分泌三磷酸腺苷的来源将提供 靶向靶向单个下游嘌呤能受体的有效策略 神经性疼痛。体积调节阴离子通道(Vrac)是大孔通道的一员 超家族及其通道活性在小胶质细胞中已被观察到，使其成为ATP的一个很好的候选者 放手。然而，30多年来，vrac的分子身份一直是个谜。通过基因组- 在宽RNAi屏幕上，我们确定SWELL1(又名LRRC8A)是vrac唯一重要的亚基。我们的 初步数据显示，1)依赖Swell1的vrac直接渗透到ATP，并可被激活 BV2小胶质细胞中的炎性介质1-磷酸鞘氨醇；2)小胶质细胞特异性的Swell1基因敲除 慢性缩窄性损伤(CCI)小鼠脊髓细胞外ATP增强减少 神经病理性疼痛模型；3)重要的是，突变小鼠也表现出脊髓小胶质细胞减少， CCI后神经元过度活动和神经病理性疼痛样行为。基于这些发现，我们 假设含有Swell1的vrac从小胶质细胞释放ATP，并介导自分泌和 旁分泌嘌呤能信号驱动脊髓小胶质细胞激活、神经炎症和疼痛 神经损伤后的超敏反应。我们已经组建了一个协同调查团队，并将使用 测试这一假设的创新和多学科方法，并将验证Swell1渠道作为 神经病理性疼痛的新靶点通过遗传(目标1)、机制(目标2)和药理学(目标3) 学习。我们的研究将为验证和潜在地翻译小说Swell1-产生重要的知识 针对神经性疼痛治疗的靶向治疗，避免阿片类药物的陷阱。

PROJECT SUMMARY Neuropathic pain is a debilitating chronic pain syndrome, affecting ~25 million Americans. The estimated total economic burden of direct health care cost and lost productivity is ~$250 billion each year. Moreover, there has been a heavy-dependence on the prescription of opioids despite their poor long-term ability to manage pain. This contributes to the crisis of opioid overdoes and addictions. According to CDC, 187 people die every day in the U.S. from opioid overdoses. It is thus imperative to identify new therapeutic targets for safe pain treatment without abuse liability. Microgliosis and neuroinflammation have emerged as key drivers of neuropathic pain. In response to peripheral nerve injury, microglia in the spinal cord are activated and undergo proliferation. ATP is one of the most prominent activators of microglia. Upon stimulation of purinergic receptors, microglia release neuromodulators, which cause sensitization of the spinal neuronal circuits leading to chronic pain. Despite its central role in microglial activation, the source and the release mechanism of ATP remain poorly understood, especially after nerve injury. The rationale for this proposal is that blocking the source of exaggerated autocrine/paracrine ATP in the spinal cord after injury will provide an effective alternative to current strategies of targeting individual downstream purinergic receptors in neuropathic pain. Volume-Regulated Anion Channel (VRAC) is a member of the large-pore channel superfamily and its channel activity has been observed in microglia, making it a good candidate for ATP release. However, the molecular identity of VRAC was a mystery for over 3 decades. Through a genome- wide RNAi screen, we identified SWELL1 (aka, LRRC8A) as the only essential subunit of VRAC. Our preliminary data showed that 1) Swell1-dependent VRAC directly permeates ATP and can be activated by inflammatory mediator sphingosine 1-phosphate in BV2 microglia; 2) Microglia-specific Swell1 knockout mice showed reduced extracellular ATP enhancement in the spinal cord in a chronic constriction injury (CCI) model of neuropathic pain; 3) Importantly, the mutant mice also exhibited decreased spinal microgliosis, neuronal hyperactivity, and neuropathic pain-like behaviors after CCI. Based on these findings, we hypothesize that Swell1-containing VRAC releases ATP from microglia and mediates autocrine and paracrine purinergic signaling driving spinal microglia activation, neuroinflammation, and pain hypersensitivity after nerve injury. We have assembled a synergistic team of investigators and will use innovative and multidisciplinary approaches to test this hypothesis and will validate the Swell1 channel as a new target for neuropathic pain through genetic (Aim 1), mechanistic (Aim 2), and pharmacological (Aim 3) studies. Our study will generate important knowledge for validating and potentially translating novel Swell1- targeting therapies for neuropathic pain treatment and avoid the pitfalls of opioids.