Daniela Salvemini - US grants

DESCRIPTION (provided by applicant): Opiate/narcotic analgesics, typified by morphine sulfate, are the most effective analgesics for treating acute and chronic severe pain, but their clinical utility is often hampered by the development of analgesic tolerance and painful hypersensitivity to both innocuous and noxious stimuli. The mechanisms by which chronic opiate exposure induce hyperalgesia and antinociceptive tolerance are unclear but neuroimmune activation, cellular apoptosis and oxidative/nitrative stress in the spinal cord have been proposed, Ceramide is a sphingolipid signaling molecule with powerful proapoptotic and proinflammatory properties and may also contribute to oxidative/nitrative stress. Ceramide is generated from de novo synthesis coordinated by serine palmitosyltransferase and ceramide synthase and/or by enzymatic hydrolysis of sphingomyelin by sphingomyelinases (SMases). Using a well established murine model, our preliminary experiments revealed that repeated administration of morphine increased the levels of ceramide in the dorsal horn of the lumbar segment of the spinal cord and that its inhibition by fumonisin B1, an inhibitor of ceramide synthase, attenuated the development of antinociceptive tolerance. These events were associated with inhibition of apoptosis and oxidative/nitrative stress in dorsal horn tissues. Furthermore, inhibition of ceramide synthesis by D609 and myriocin, inhibitors of SMAse/sphingomyelin synthase and serine palmitoyltransferase respectively blocked antinociceptive tolerance. Together, these findings support the central thesis of this exploratory proposal: increased formation of ceramide in the spinal cord is an important pathway in the development of morphine-induced hyperalgesia and antinociceptive tolerance. To address this novel hypothesis, we propose a comprehensive experimental strategy employing molecular, bio-analytical, biochemical, pharmacological and genetic approaches. Two Specific Aims will test our hypothesis. In Specific Aim 1, we will demonstrate by pharmacologic and genetic approaches that inhibition of the increased formation of ceramide blocks the development of morphine- induced hyperalgesia and antinociceptive tolerance thus identifying the predominant enzymatic pathway responsible for its biosynthesis. In Specific Aim 2, we will elucidate the molecular and biochemical mechanisms whereby ceramide modulates hyperalgesia and antinociceptive tolerance. Specifically, we will determine ceramide's effects on three biochemical pathways within spinal tissue: (a) neuroimmune activation, (b) oxidative/nitrative stress and (c) apoptosis. Successful validation of our hypothesis will define for the first time the important role of ceramide in morphine-induced hyperalgesia and antinociceptive tolerance providing the scientific foundation towards the development of inhibitors of ceramide biosynthesis as adjunct to opiates for the management of chronic pain, in particular for those patients who require long-term opioid treatment for pain relief. PUBLIC HEALTH RELEVANCE Opioid drugs such as morphine are the most effective analgesics for treating severe chronic pain, but their pain-relieving action is often diminished during chronic administration, necessitating dose escalation that reduces quality of life for the patient. Our studies will determine for the first time that inhibition of ceramide biosynthesis, with novel agents, restores the pain-relieving action of morphine. The broader implications of our findings may open a new frontier in chronic pain management thus alleviating the socioeconomic consequences it causes.

DESCRIPTION (provided by applicant): Opiates like morphine sulfate are the most effective analgesics for treating acute and chronic severe pain, but their use is limited by development of tolerance and hypersensitivity to innocuous and noxious stimuli. The mechanisms of such opiate-induced hyperalgesia and antinociceptive tolerance are unclear. Peroxynitrite (ONOO-) produced from superoxide (O2.-) and nitric oxide (.NO), is a potent pro-inflammatory and pro- apoptotic reactive nitrogen species now implicated in hyperalgesia. Our central HYPOTHESIS is that endogenous ONOO- is the proximal molecule in a cascade of signaling events leading to morphine-induced hyperalgesia and antinociceptive tolerance. In mice we showed that repeated morphine administration leads to nitrotyrosine (NT, a marker of ONOO-) formation. The role of ONOO- was proven by showing that co- administering morphine with inhibitors of .NO synthase, scavengers of O2.- or novel ONOO- decomposition catalysts attenuated NT formation and prevented antinociceptive tolerance. These associative and causal links involving ONOO- and antinociceptive tolerance coincided with: 1) post-translational nitration of MnSOD, a glutamate transporter (GLT-1), and glutamine synthase (GS);2) increased formation of pro-inflammatory cytokines;and 3) oxidative DNA damage and activation of the nuclear factor poly(ADP-ribose) polymerase. Inhibiting ONOO- attenuated these changes. Finally, results with systemic administration of ONOO- decomposition catalysts were confirmed by intrathechal dosing, thus supporting its role at the level of the spinal cord. Three Specific Aims will test our hypothesis. Specific Aim 1 will define the associative and causal link between spinal ONOO- and the development of morphine-induced hypersensitivity and antinociceptive tolerance. Specific Aim 2 will identify, using pharmacological and genetic approaches, the contribution of the NADPH oxidase as source of O2.- and thus, de novo ONOO- formation during the development of morphine hypersensitivity and antinociceptive tolerance. Specific Aim 3 will elucidate the molecular and biochemical mechanisms whereby ONOO- modulates hyperalgesia and antinociceptive tolerance. Results from these studies will define the important role of ONOO- in the development of antinociceptive tolerance providing the rationale towards the development of ONOO- decomposition catalysts as adjuncts to opiates for the management of chronic pain. Project Narrative: Chronic pain affects approximately 86 million Americans and cost at least $100 billion annually in medical expenses and reduced work productivity. PUBLIC HEALTH RELEVANCE Opioid drugs such as morphine are the most effective analgesics for treating severe chronic pain, but its pain-relieving action is often diminished during chronic administration, necessitating dose escalation that reduces quality of life for the patient. Our studies will determine for the first time that removal of peroxynitrite with novel agents, restores the pain-relieving action of morphine opening a new frontier in chronic pain management thereby improving the associated socioeconomic consequences.

DESCRIPTION (provided by applicant): One third of Americans suffer from some form of chronic pain, (30% being resistant to analgesic therapy), making it a significant health problem with serious economic impact (estimated cost of approximately $100 billion annually). Chronic pain associated with complex regional pain syndromes is particularly difficult to manage. Chronic pain associated with inflammatory diseases such as rheumatoid arthritis is often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Current drug regimens are marginally effective and often display unacceptable side effects. Over the past decade, our multidisciplinary team has produced experimental results which clearly implicate the overproduction of peroxynitrite as a key mediator of inflammatory and chronic pain states in addition to the development of morphine-induced hyperalgesia and antinociceptive tolerance. Thus, the direct scavenging of this neurotoxic entity by small molecule drugs which act in enzyme-like catalytic fashion provides an unconventional approach to a completely novel analgesic strategy. The overriding goal is a broadly effective treatment for chronic pain associated with inflammatory diseases. Thus, a series of metal-charge-shielded metalloporphyrin and porphyrinoids with membrane penetration properties will be synthesized and screened as peroxynitrite decomposition catalysts. Pharmacokinetic and bioavailability studies will follow. Chemical entities displaying the highest catalytic activity with drug like properties will then be tested for analgesic efficacy and potency in 2 well established animal models of inflammation and arthritis While the main focus our research objective is in the transition of acute to chronic pain and therapeutic intervention to alleviate the chronic pain state, successful identification of orally active peroxynitrite decomposition catalysts will address numerous chronic disease states known to be driven by nitroxidative stress including diabetes, atherosclerosis and Parkinson's disease thus having major impact upon the quality of life for these patient populations. We have also shown that prototype catalysts act in a highly synergistic manner with subtherapeutic levels of selective COXII inhibitors, non-selective COX-I inhibitors, steroids, and methotrexate. Thus, through this approach we may be able to greatly lower the dosages of these important drugs for effective treatment of pain without or with greatly diminished side- effects. PUBLIC HEALTH RELEVANCE: The currently used drug regimens for the treatment of chronic pain associated with inflammatory diseases and arthritis, which encompass a number of varied mechanisms of action, are marginally effective and often display unacceptable side effects. Thus, a broadly effective treatment for chronic pain based upon the catalytic decomposition of the key neurotoxic species, peroxynitrite, would have wide-ranging beneficial effects for patients. In addition, the identification of orally active peroxynitrite decomposition catalysts will address numerous chronic disease states known to be driven by nitroxidative stress including diabetes, atherosclerosis and Parkinson's disease thus having major impact upon the quality of life for these patient populations.

DESCRIPTION (provided by applicant): Chemotherapy-induced peripheral neuropathy (CIPN) accompanied by chronic neuropathic pain is the major dose-limiting toxicity of widely used antitumoral agents in the taxane (e.g., paclitaxel), platinum-complex (e.g., oxaliplatin), vinca alkaloids (e.g., vincristine) & proteasome-inhibitor (e.g., bortezomib) classes.1-3 Thus, CIPN is one of most common causes of dose reduction & discontinuation of what is otherwise a life-saving therapy.2-7 Addressing this major public health issue by identifying therapeutic targets with immediate potential translation to the clinic is of paramount significance. We have identified A3 adenosine receptor (A3AR) agonism as a new viable therapeutic strategy for treating or reversing CIPN (Appendix 1 & ref8). Noteworthy, the selective A3AR agonists IB-MECA & its 2-chloro analogue (Cl-IBMECA) are in advanced clinical trials as antiinflammatory & antitumor agents.9,10 This proposal highlights a multidisciplinary research plan that builds upon our preliminary data to explore the breadth of A3AR agonist applicability in CIPN while investigating underlying protective mechanism(s) of action. Using IB-MECA, three Specific Aims will test our central hypothesis: A3AR agonists are effective therapeutics in CIPN caused by chemotherapeutics with distinct antitumor mechanisms of action (paclitaxel, oxaliplatin & bortezomib) with beneficial effects exerted at the level of the peripheral sensory afferent (PSA) neuron &/or spinal cord. In Aim 1, we will test if 1) IB-MECA blocks & reverses neuropathic pain, 2) the effects of IB-MECA are specific to an A3AR mediated mechanism using pharmacological & genetic knockout approaches, 3) potential central & peripheral site(s) of action underlie IB-MECA's action & 4) IB-MECA prevents chemotherapy-evoked degeneration of intraepidermal nerve fibers & primary afferent spontaneous discharge. In Aim 2, we will investigate the mechanism(s) whereby IB-MECA attenuates neuropathic pain through mitoprotective effects in PSA. Finally, in Aim 3, we will investigate if IB-MECA's effects include attenuating neuroinflammation &/or the dysregulation of glutamate homeostasis in the spinal cord, processes known to be essential to central sensitization. We will focus on NF?B & MAPK (ERK1/2, p38) signaling & glial-derived pro (TNF?, IL1?, & IL6)/anti (IL10)-inflammatory cytokines, as well as, the effects on the expression & activities of spinal glutamate transporters (neuronal & glial) & glial glutamine synthetase. If our hypothesis holds true, the outcome of our results are anticipated to provide the pharmacological rationale for proof-of-concept for the use of selective A3AR agonists as a new approach in CIPN. From a translational perspective, this could conceivably lead to a fast track investigation of IB-MECA for CIPN. This exciting possibility underscores the immediate clinical impact that our research proposal may have in this critical & unmet medical setting. Given the breadth of disorders impacted by A3AR agonists understanding their mechanism-based effects has far-reaching basic science & clinical implications.

? DESCRIPTION (provided by applicant): Opioid use for the long-term treatment of chronic pain is limited by relatively poor efficacy & the emergence of adaptive CNS changes that result in analgesic tolerance, increase pain (opioid-induced hypersensitivity, OIH) & physical dependence that offset analgesia, pose a health burden & create community abuse liability.1-9 We now implicate for the first time opioid-induced dysfunction in adenosine neuromodulation via the A3 GPCR subtype, adenosine receptor (A3AR) & its control by adenosine kinase (AdK) in analgesic tolerance, OIH, & dependence. This proposal highlights a multidisciplinary research plan aimed at exploring the contribution of the AdK-to-A3AR axis & the breadth of A3AR agonist applicability while investigating its underlying protective mechanism(s) & sites of action in the CNS. Noteworthy, A3AR agonists, such as IB-MECA & its 2-chloro analogue (Cl-IB-MECA), have advanced to Phase II/III clinical trials as novel anti-inflammatory & anticancer agents with good safety profiles.10-13 In Aim 1, we will investigate the temporal & cellular expression of AdK (& its enzymatic activity) & A3AR in SC glia & neurons (immunofluorescence & genetic/proteomic analysis). In parallel, purine nucleoside concentrations in SC & CSF (from lumbar puncture) will be measured by targeted metabolic approaches. We will (1) characterize the pharmacological profile of A3AR agonists via dose- response curves & time course studies as well as effect of gender on A3AR effects & (2) examine the contribution of the SC as a site of action. As a corollary, we will explore the clinical generalization of findings by testing oxycodon & A3AR agonists & examine the contribution of the rostral ventromedial medulla (RVM), as an additional site of action. In Aim 2, to gather a mechanistic understanding of how A3AR agonism confers protection, we will begin our initial exploration in signaling pathways engaged at the level of the SC dorsal horn. To this end, we will examine using proteomic analysis if the beneficial effects of A3AR agonists are driven, at least in part, by inhibiting the GSK3ß & P2X7R-inflammasome pathways known to govern IL-1ß & neuroinflammation. We will also evaluate if these effects are associated with a switch from pro-inflammatory to anti-inflammatory states with increased IL-10 expression & function. We expect our results to provide a robust scientific foundation for a new translational effort in the treatment of opioid's unwanted side effects that counter-regulate opioid analgesia based upon selective A3AR-targeted therapies, while evaluating the potential for translational impact with A3AR agonists already in clinical trials. Selective activation of A3AR would not only transform current approaches to chronic pain, but may benefit other disorders driven by deregulation of adenosine homeostasis (i.e., drug of abuse pathologies).14 This project is a perfect fit for the CEBRA program as it meets its objectives by testing a highly novel & significant hypothesis for which there is scant information & if confirmed, would have substantial impact on current thinking in this field.

Cognitive impairment (chemobrain) is a common neurotoxicity associated with chemotherapy treatment that is estimated to affect >50% of patients.1 However, little is known about the mechanisms underlying CICI, and there have been no FDA-approved preventive or curative interventions. It is therefore imperative that we understand the underlying causes of this serious adverse drug reaction and identify novel therapeutic approaches with the potential for rapid translation to the clinic. Our preliminary data identify a key mechanism driving CICI based on CNS alterations of adenosine-dependent metabolic regulation and a novel target for therapeutic intervention - the A3 adenosine receptor (AR) subtype (A3AR). Therefore, our proposal directly responds to PAR-16-275: Serious Adverse Drug Reaction Research. Extracellular adenosine and its signaling at ARs are regulated by ectonucleotidases and adenosine kinase (ADK). Our preliminary results in mouse models of chemotherapy (cisplatin and doxorubicin)-induced cognitive impairment (CICI) reveal that chemotherapy altered the expression of these enzymes in centers of cognitive function, including the prefrontal cortex (PFC) and hippocampus, and produced morphological abnormalities in the brain (e.g., in white matter organization, dendritic arborization and spine density). Mechanistically, we found that chemotherapy led to mitochondrial dysfunction, oxidative and nitrative stress (nitroxidative stress) and neuroinflammation in CNS. Pilot data suggest that chemotherapy engaged the NLRP3 inflammasome, which is critical in IL1? formation.2 Noteworthy, supplementing adenosine signaling with highly selective, A3AR agonists significantly attenuated CICI without any loss in locomotor activity. This is highly exciting since A3AR agonists are already in advanced clinical trials as anticancer agents with a good safety profile. The mechanisms underpinning the beneficial effects of A3AR agonists are not known. We hypothesize that: chemotherapy disrupts adenosine homeostasis leading to mitochondrial dysfunction and NLRP3- driven neuroinflammation that culminate in cognitive impairment; supplementing adenosine signaling with selective A3AR agonists provides an effective approach for the management of CICI. This proposal uses a multidisciplinary research plan to explore the applicability of A3AR agonists in CICI while investigating underlying protective mechanism(s). Two Specific Aims will test our hypothesis. In Aim 1, we will test the hypothesis that chemotherapy causes the dysregulation of adenosine metabolism and loss of adenosine signaling at A3AR leading to CICI. In Aim 2, we will investigate the mode of action underlying the beneficial effects of A3AR agonists in preserving cognitive function. Our results are anticipated to provide new molecular insights that will advance our understanding of how CICI develops by establishing the specific role of the adenosine-A3AR axis. These studies are predicted to lead to expedited ?proof of concept? studies opening the door to a new translational effort in the treatment of CICI to fulfill this highly unmet medical need.

Opioid use for chronic pain is limited by antinociceptive tolerance, opioid-induced hyperalgesia (OIH), physical dependence and addiction.1-3 The triggering mechanisms remain elusive. Our published work4,5 and preliminary data suggest for the first time that dysregulation of sphingolipid metabolism in the central nervous system (CNS) leading to exaggerated production of sphingosine 1-phosphate (S1P) and activation of an astrocyte-based S1P receptor subtype 1 (S1PR1) signaling pathway is central to these processes. Oral administration of CNS penetrant S1PR1 competitive and functional antagonists, including FTY720 (Gilenya®),6 blocked morphine and oxycodone-induced antinociceptive tolerance and OIH in rodents of both genders, as well as morphine-induced dependence and reward. FTY720 is already FDA-approved6 and therefore, rapid clinical translation of the expected finding is very feasible. We also discovered that sustained opioid-unwanted actions do not develop in conditional S1PR1-knockout mice lacking S1PR1 in spinal astrocytes and that the beneficial effects of S1PR1-targeted agents are attenuated by at least 90% in conditional S1PR1-knockdown mice of both genders, which lack one S1pr1 allele in spinal astrocytes when compared to their littermate controls. These results unravel the importance of astrocyte-based S1PR1 signaling and suggest that astrocytes are a cellular target for anti-S1PR1 activity. Increased levels of S1P in CNS were associated with increased 1) astrocyte reactivity, 2) expression of the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (critical in IL1? formation and signaling)7 and 3) formation of inflammatory/ neuroexcitatory cytokines. Blocking S1PR1 inhibited these processes. In contrast, IL10 (an important anti- inflammatory and neuroprotective cytokine) increased significantly. Intrathecal delivery of a neutralizing anti- IL10 antibody blocked the effects of S1PR1 antagonists suggesting that their beneficial effects are driven by an endogenous IL10 pathway. A multidisciplinary plan builds on our strong preliminary data to test our hypothesis: An astrocyte-based SphK1/S1P/S1PR1 signaling pathway driven by NLRP3-induced neuroinflammation in the CNS underlies the development of morphine-induced antinociceptive tolerance/OIH and reward. Targeting S1PR1 provides a novel approach for therapeutic intervention. Three aims will test our hypothesis: 1) Establish S1PR1 as a molecular target for sustained opioid intervention, 2) Examine opioid-induced alterations of sphingolipid metabolism and SphK1/S1P/S1PR1 signaling and 3) Determine molecular and biochemical pathways engaged downstream of S1PR1 activation. Impact: Our results will unravel a previously unrecognized role for an astrocyte based SphK1/S1P/S1PR1- signaling pathway in sustained opioid use and will provide the foundation for clinical evaluation of S1PR1- targeted therapeutics as adjunct to opioids.

Neuropathic pain conditions are exceedingly difficult to treat.1-4 Novel non-narcotic analgesics are desperately needed. Our receptomic and unbiased transcriptomic approaches identified the orphan G-protein coupled receptor (oGPCR), GPR160, as a major oGPCR whose transcript is significantly increased in the dorsal horn of the spinal cord (DH-SC) ipsilateral to nerve injury, in a model of traumatic nerve-injury induced neuropathic pain caused by constriction of the sciatic nerve in rats (CCI).5 No changes were found in the dorsal root ganglia. The role of GPR160 signaling in nociceptive processing is not known. Small molecule inhibitors of GPR160 are not yet available. Intrathecal (i.th.) injections of Gpr160 mRNA-targeted small interfering RNA (siGpr160) or neutralizing GPR160 antibody significantly blocked and reversed CCI-induced mechano- allodynia with no observable side-effects. Similar findings were obtained in another model of traumatic nerve injury (spared nerve injury model)6 and in a model of chemotherapy-induced neuropathic pain,7-10 underscoring generalization of findings. GPR160 inhibition had no effect in acute pain (hot plate/tail flick). Based on the existing literature anti-GPR160 approaches are unlikely to interfere with chemotherapies or promote cancer growth.11-13 We also de-orphanized GPR160 by identifying cocaine- and amphetamine-regulated transcript peptide (CARTp)14-16 as a ligand. CARTp is so-named because its level is increased following exposure to cocaine and amphetamine and other substances of abuse (i.e. morphine).16 Existing literature suggests that CARTp signaling activates protein kinase A (PKA),17,18 extracellular signal-regulated kinase (ERK)16-22 and cAMP-response element binding protein (CREB) pathways.18,23 These pathways are crucial to persistent pain sensitization.24,25 A recent study has linked CARTp-induced PKA/ERK/CREB to a cyclic AMP-independent, pertussis-toxin sensitive G?i/o-linked receptor.18 Guided by strong preliminary data, the following hypothesis will be tested: ?CARTp/GPR160 signaling in the spinal cord is essential for the development and maintenance of neuropathic pain states?. Two Specific Aims will be pursued in male and female rodents across multiple models of neuropathic pain states. A multidisciplinary approach will be carried out in independent labs that have extensive experience in GPCR signaling and pain, using in vitro and in vivo studies that include genetic, behavioral, biochemical, molecular and immunohistochemical tests as well as targeted mass spectrometry. Proposed studies in Aim 1 will validate GPR160 as a non-opioid receptor target for therapeutic intervention in neuropathic pain whereas proposed studies in Aim 2 will characterize GPR160 coupling and downstream molecular signaling pathways. Impact: Discovery and validation of novel candidate targets for development of non-addictive therapeutics will have a huge impact in the treatment of chronic pain patients. Our discovery and proposed studies will provide foundational insights of CARTp/GPR160 signaling in the central nervous system and will validate GPR160 as a target for therapeutic intervention.

The proposed research supplements the HEAL grant Discovery and validation of a novel orphan GPCR as a target for therapeutic intervention in neuropathic pain (R01NS113257). Neuropathic pain conditions are exceedingly difficult to treat1-4 and novel non-narcotic analgesics are desperately needed. Our recent work led to the discovery that activation of the orphan G protein-coupled receptor 160 (oGPCR; GPR160) in the spinal cord contributes to the development of neuropathic pain states5. Since there are no small molecule antagonists of GPR160, contribution of GPR160 signaling was unraveled using genetic and immunopharmacological approaches. Blocking GPR160 blocks and reverses neuropathic pain with no effect in acute pain settings, suggesting that GPR160 is important in the transition from acute to chronic pain. We also de-orphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a ligand.5 Blocking endogenous CARTp signaling in the spinal cord attenuates neuropathic pain, whereas intrathecal injection of CARTp evokes painful hypersensitivity in rodents through GPR160-dependent extracellular signal-regulated kinase (ERK) and cyclic AMP response element-binding pathways (CREB). Our findings are the first to de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target with non-opioid based small molecule GPR160 antagonists, and provide insights to its signaling pathways.5 GPR160 has never been isolated and biochemically characterized and studies to understand the direct interaction of CARTp with the purified protein have never been done.5 Here, we propose to isolate and biochemically characterize GPR160, currently identified as one of the Understudied Druggable Genome targets, and to establish methods for biochemical characterization of GPR160 interaction with CARTp activator. We will miniaturize and optimize biochemical assay and scale up protein production for future high throughput biochemical screening to identify potent inhibitors of GPR160 activation. Specifically, we will develop GPR160 expression system(s) and purification protocol(s) and will characterize/optimize stability and solubility of the receptor for biochemical studies using a nanodisc approach. We will characterize direct interaction of GPR160 and CARTp using purified proteins and complementary binding assays such as fluorescence polarization (FP) with fluorescein-labeled (FAM-) CARTp and surface plasmon resonance (SPR) using unlabeled CARTp. These studies are critical for defining the molecular mechanism of CARTp/GPR160 interactions and initiating large-scale screens for new inhibitors to develop novel therapeutics. Impact: Discovery and development of small molecule GPR160 antagonists as non-addictive analgesics is anticipated to have a huge impact on the treatment of chronic pain patients.

Deficits in participation-level performance of specific cognitive tasks are one of the primary cognitive impairments reported after a mild TBI.3 There are no FDA-approved preventive or curative interventions. Mitochondrial dysfunction4-10 and neuroinflammation11-15 in the central nervous system (CNS) are postulated as underlying causes of cognitive impairment. Our preliminary data suggests that dysregulation of adenosine metabolism and loss of signaling at the A3 adenosine receptor (AR) subtype (A3AR) is key to these processes. Extracellular adenosine is regulated by ectonucleotidases and adenosine kinase and preliminary results in a mouse model of mild-TBI (close head weight drop)2,16 revealed that TBI altered the expression of these enzymes in the prefrontal cortex (PFC) and hippocampus. This was associated with time dependent development of memory and learning deficits [Novel Object/Place Recognition (NOPRT) and T-maze tests]. Supplementing adenosine signaling with highly selective, orally bioavailable, CNS penetrant A3AR agonists given therapeutically significantly attenuated TBI-induced memory and learning deficits 4 weeks after injury without any confounding influence on locomotor activity. Moreover, pilot data now links A3AR agonism to the inhibition of TBI-induced oxidative/nitrative stress (nitroxidative stress), neuroinflammation and activation of nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. Our findings are very exciting since A3AR agonists (e.g., IB-MECA) are already in advanced clinical trials for other indications with a good safety profile.17,18 Two Specific Aims will test our hypothesis: dysregulation of adenosinergic signaling in the brain contributes to TBI-induced cognitive impairment; restoring adenosinergic signaling with highly selective A3AR agonists is an effective strategy for therapeutic intervention. A multidisciplinary approach is used in male and female mice. Pharmacological and genetic studies as well as time course and dose responses with selective A3AR agonists are proposed. Studies will also include A3AR antagonists and A3AR knockout mice to confirm selectivity of response. In Aim 1, we will examine whether a decrease in adenosine signaling at the A3AR contributes to TBI-induced cognitive deficits. In Aim 2, we will 1) examine whether restoring A3AR signaling with A3AR agonists ameliorates TBI-induced cognitive deficits, 2) validate A3AR as a target for therapeutic intervention with selective A3AR agonists and 3) investigate their mechanism of action by examining their impact on the mitochondrial dysfunction and neuroinflammatory processes that are intimately linked to the development of cognitive deficits. Our multidisciplinary research plan is anticipated to provide evidence for the applicability of highly selective A3AR agonists to preserve cognitive function following TBI while investigating their underlying protective mechanism(s).