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.

PROJECT NARRATIVE 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. It also contributes to an alarming epidemic of opioid addictions and overdoes. Therefore, there is an urgent need to identify new therapeutic targets for pain treatment without abuse liability. This study aims to validate a new large-pore Swell1 ion channel as a novel target for neuropathic pain through genetic, mechanistic, and pharmacological studies.