Long-Jun Wu - US grants

? DESCRIPTION (provided by applicant): Microglia are principal immune cells in the central nervous system. After peripheral nerve injury, spinal microglia participate in the development of neuropathic pain by transforming from resting to reactive states. However, no evidence directly addresses the microglial role in the initiation or maintenance of neuropathic pain. Recently developed transgenic mice (CX3CR1CreER/+:R26iDTR/+) enable us to ablate CX3CR1-positive cells including microglia in a controllable fashion. Our pilot experiments found that depletion of CX3CR1-positive cells completely reversed the neuropathic pain, while selective microglia ablation partially reduced pain hypersensitivities after peripheral nerve injury. Based on these exciting results, we hypothesize that resident microglia in the spinal cord are critical i the initiation but not maintenance of neuropathic pain. We will test this hypothesis by determining: (1) the function of CX3CR1-positive cells in the initiation and maintenance of neuropathic pain; (2) the role of resident microglia and peripheral macrophages in neuropathic pain; and (3) the spinal and brain microglia in neuropathic pain. The results obtained from this proposal will pinpoint the temporal and spatial resolution of microglia/macrophages in the development of neuropathic pain. The current proposal is the first attempt to use microglial ablation approaches to study neuropathic pain, with the aim of evaluating microglia as a potential therapeutic target for the treatment of neuropathic pain. This study will advance our understanding of microglial mechanism of neuropathic pain. The mechanism may also serve as a common model to address the role of microglia in the pathogenesis of various neurological diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's diseases, ischemic stroke, and epilepsy.

PROJECT SUMMARY/ABSTRACT: Electrical brain stimulation (EBS) is a FDA-approved neuromodulation therapy applied to several neurological disorders. However, the molecular basis of its efficacy remains unclear. Here we propose investigation of a glial mechanism for EBS mediated by astrocytes-derived extracellular vesicles (EVs). We recently discovered from both in vitro and in vivo experiments that electrical stimulation affects the release of EVs from astrocytes. In this proposal we will address two questions: 1) what is the molecular mechanism of electrical stimulation induced EVs release; 2) what is the biological function of the EVs released under electrical stimulations. Our exploratory research plan includes the following three steps: First - molecular characterization of astrocytic EVs (Aim1). In this aim we start from primary cultured astrocytes and systematically evaluate the effect of electrical stimulation parameters on EV cargos; Second - molecular mechanisms of how stimulation affects astrocytic EVs (Aim 2). In this aim we apply a high-resolution imaging technique to primary cultured astrocytes and focus on the trafficking of intracellular vesicles, including vesicle fusion to or budding off the plasma membrane. Third - functional characterizations of astrocytic EVs (Aim 3). In this aim, we will subject purified EVs collected in step 1 to both in vitro and in vivo functional testing. Focusing on neuronal activities as readout, we will first examine EV functions on primary cultured neurons; then we use in vivo animal models combined with 2-photon microscope technology to examine how EVs affect neuronal activities in both short- and long-term periods, and also how EVs affect animal behavior. In summary, our findings will help guide optimization of stimulation with next-generation EBS devices, with the ultimate goal of enhancing efficacy and treatments for patients with neurological disorders.

Project Summary Despite considerable research over the past 30 years, there is still no established effective treatment to improve recovery following spinal cord injury (SCI). In part, this reflects incomplete understanding of the complex secondary pathobiological mechanisms involved. The aim of our research is to understand the cellular and molecular mechanisms responsible for post-injury neuroinflammation in order to allow future development of novel therapies. The voltage-gated proton channel Hv1 is a newly discovered ion channel, highly expressed in resting microglia of the brain. Under pathological conditions, microglial Hv1 is required for NADPH oxidase (NOX)-dependent generation of ROS (reactive oxygen species) by providing charge compensation for exported electrons and relieving intracellular acidosis. Thus, Hv1 is a unique target for controlling multiple NOX activities and ROS production. However, neither the precise signaling mechanisms underlying this finding nor critical role of Hv1 in the pathophysiology of SCI are fully understood. Based on our preliminary data, we will test the hypothesis that microglial Hv1 functions as a key mechanism in neuroinflammation, through altered NOX2/ROS/IFN-? signaling that modulates microglia-astrocyte interaction, thus affecting long-term neurological outcomes after SCI. We will use systemic or microglial Hv1 KO, microglial NOX2 KO transgenic mice and in vivo and in vitro innovatively technologies to determine the mechanisms of SCI-triggered Hv1 elevation on post-injury neuroinflammation. Aim 1 will determine the function and mechanisms of the Hv1 in neuroinflammation after SCI. Multiple quantitative assessments of microglia-mediated neuroinflammation will be combined with genetic or pharmacological intervention targeting Hv1 to test the hypothesis that SCI-induced microglial Hv1 activation mediates detrimental neuroinflammation and functional deficits through altered microglial NOX2/ROS signaling. Aim 2 will elucidate the role of microglial NOX2 in post-injury neuroinflammation. We will utilize genetic intervention to delete Hv1-dependent up-regulation of NOX2 in microglia, and evaluate the effects on microglial NOX2 coupling to Hv1 on neuroinflammation after SCI. Aim 3 will identify critical role of Hv1/NOX2-derived ROS/IFN-? in SCI-chronic neuroinflammation through microglia-astrocyte interaction. Complimentary cellular, molecular, and genetic approaches will be used to test the hypothesis that Hv1/NOX2-mediated microglial ROS activates pro-inflammatory astrocytes resulting in secreting IFN? that in turn reinforces microglial inflammation, thus contributes to astrocytes dysfunction and neuronal damage. Our study will be the first to implicate microglial Hv1/NOX2/ROS/IFN-? signaling in the pathophysiology of SCI, leading to novel treatment approaches for SCI. Given the proposed roles for Hv1 in other inflammatory models, Hv1 signaling represents a generic mechanism relevant to other neuroinflammatory states.

Neuromyelitis optica (NMO) is a severe, relapsing IgG-mediated autoimmune disease targeting the central nervous system (CNS), inducing inflammation and preferential demyelination of optic nerve and spinal cord. Most patients experience severe impairments. IgG autoantibodies specific for the astrocytic aquaporin-4 (AQP4) water channel are the primary cause of the disease pathophysiology. However, little is known about the mechanisms driving NMO lesion progression following the binding of IgG to the astrocyte membrane on entering the CNS. Our proposed project will investigate the potential contribution of microglia, the resident immune cell of the CNS, to the evolving NMO lesion. We have developed an informative mouse model of NMO. NMO-IgG is infused intrathecally. Our preliminary results show significant motor dysfunction, astrocyte activation, and a unique pattern of early microglial convergence on astrocytes. Prevention of microglial activity suppressed development of motor dysfunction. In sum, these data clearly indicate astrocyte-microglia communication as an early event after NMO-IgG enters the CNS. Aim 1, will investigate the mechanisms underlying astrocyte-microglia crosstalk; Aim 2, will assess the contribution of microglia to NMO pathogenesis, and Aim 3 will utilize novel genetic tools to manipulate microglial activity as a potential therapeutic approach to NMO management. The research we propose represents the first attempt to investigate the specific contribution of microglia to NMO pathogenesis. The results should clarify the importance of astrocyte-microglia crosstalk and its underlying mechanisms in NMO. The study will not only improve understanding of neuroimmune interaction in NMO but will potentially establish that microglia are a pertinent target for NMO therapy.