Patrick Ronaldson - US grants

DESCRIPTION (provided by applicant): Hypoxia/reoxygenation (H/R) is a component of several brain diseases such as traumatic brain injury, acute respiratory syndrome, obstructive sleep apnea, high altitude cerebral edema, acute mountain sickness, cardiac arrest and ischemic stroke. The objective of current stroke therapy is to restore perfusion to ischemic brain; however, considerable brain cellular damage and BBB dysfunction occurs when blood flow/oxygen supply is re-established. Therefore, there is a critical need for development of novel treatment strategies that can rescue salvageable brain tissue from damage and/or protect BBB integrity during H/R. In this grant, we will test the hypothesis that organic anion transporting polypeptides (Oatps) and multidrug resistance proteins (Mrps), two families of endogenous BBB transporters, can be targeted for treatment of H/R. Two specific aims will test this hypothesis. Aim 1: To investigate CNS drug delivery mediated by Oatps during H/R. In this aim, we will focus on Oatp1a4, the primary drug transporting Oatp at the rodent BBB. We will investigate, in vivo, Oatp1a4 mediated transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins) (Aim 1A). We will then correlate changes in Oatp1a4-mediated statin transport with indices of neuroprotective and antioxidant efficacy (Aim 1B). Transforming growth factor (TGF-ß) signaling regulates Oatp1a4 expression/activity. Therefore, we will evaluate effects of dorsomorphin and SB431542, two TGF-ß receptor inhibitors, on BBB functional expression of Oatp1a4 to evaluate targeting of this pathway for control of CNS drug delivery (Aim 1C). Aim 2: To evaluate if pharmacological targeting of Mrps at the BBB protects BBB integrity following H/R. Glutathione (GSH), a critical CNS antioxidant, is a substrate for Mrp1, Mrp2, and Mrp4. Therefore, we will study in vivo H/R-induced changes in expression/activity of these Mrps at the BBB (Aim 2A). We will then examine changes in GSH and its oxidized form GSH disulfide (GSSG) at the BBB resulting from alterations in GSH transport systems and enzymes (Aim 2B). We will evaluate regulation of Mrps and GSH synthetic/metabolic enzymes at the BBB by nuclear factor erythroid 2-related factor 2 (Nrf2) signaling (Aim 2C). Since Nrf2 signaling is activated by oxidative stress, our studies will be conducted in the presence and absence of the reactive oxygen species (ROS) scavenger TEMPOL (i.e., 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) to determine if therapeutic targeting of oxidative stress can control BBB Mrp-mediated transport. Our goal in this grant is to facilitate discovery of novel approaches for treating diseases with an H/R component by therapeutic targeting of endogenous transporters at the BBB.

PROJECT SUMMARY Over the past several years, we have studied changes in critical blood-brain barrier (BBB) tight junction (TJ) proteins (i.e., claudin-5, occludin) in response to diseases or drugs. Our work has shown that claudin-5 and occludin expression and trafficking is modulated by pain (i.e., TJ dysregulation), an effect that increases brain uptake of opioids such as codeine. Our preliminary data show that acetaminophen (APAP) can modulate expression of claudin-5 at the BBB and increase paracellular permeability (i.e., ?leak?). Leak has previously been shown to occur with low dose, acute cocaine administration as well as methamphetamine. Both APAP and pain target transforming growth factor- ? (TGF-?) signaling, a pathway that controls TJ expression and BBB integrity. In this grant, we hypothesize that APAP, by itself and in the setting of pain, can increase BBB ?leak? and enhance CNS opioid delivery. These studies are highly significant because they will uncover mechanisms of altered opioid antinociception and adverse drug events that can occur in individuals who abuse or misuse opioids or APAP. Additionally, APAP is taken with other centrally acting drugs where ?APAP leak? can lead to therapeutic toxicity. Aim 1: To investigate, in vivo, changes in TJ protein expression and trafficking at the BBB following APAP administration. We will study how APAP alters CNS effects and adverse events of opioids. We will investigate the time course of changes in transmembrane TJ protein (i.e., claudin-5, occludin) expression and trafficking and the dose-response relationship of APAP on changes in TJ protein complexes in male and female Sprague-Dawley rats (Aim 1A). We will then study the temporal relationship between CNS opioid delivery, opioid-associated antinociception, respiratory depression, and opioid-associated reward behavior (Aim 1B). Since we have shown that transforming growth factor- ? (TGF-?) signaling regulates BBB integrity, we will study the role of this pathway on claudin-5 and occludin expression and trafficking (Aim 1C). Aim 2: To examine involvement of APAP on BBB integrity in pain. Using established in vivo pain models (i.e., l-carrageenan-induced acute inflammatory pain, chronic pain spinal nerve ligation), we will study the time course of APAP effects on TJ protein expression/trafficking (i.e., claudin-5, occludin) as well as on CNS opioid uptake (Aim 2A). We will demonstrate effects of single versus multiple doses of APAP on TGF-? signaling pathways in both pain models (Aim 2B). We will study how changes in CNS opioid delivery affect opioid antinociception, respiratory depression, and opioid-associated reward behavior (Aim 2C). Since we have shown that APAP increases functional expression of the critical opioid transporter P-glycoprotein, we will examine the contribution of claudin-5/occludin modulation and P-gp changes to CNS opioid delivery (Aim 2D). Our group is uniquely positioned to provide a mechanistic explanation (i.e., signaling, activity, trafficking) for adverse drug events in individuals who abuse/misuse prescription pain drugs.

PROJECT SUMMARY Ischemic stroke is a significant public health concern in the United States. Current therapeutic approaches for stroke involve thrombolytic therapy with recombinant tissue plasminogen activator (r-tPA) or endovascular treatments; however, many patients still experience disability. The goal of improving post-stroke outcomes requires novel neuroprotective drugs for stroke treatment. While many such compounds have been identified in preclinical stroke studies, none of these have been successfully translated to the clinic. In contrast, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (i.e., statins) are routinely given to stroke patients due to an inherent ability of these drugs to improve post-stroke functional outcomes. In this grant, we will test the mechanistic hypothesis that neuroprotection from statins results from transport across the blood-brain barrier (BBB) that is mediated by the critical uptake transporter organic anion transporting polypeptide 1a4 (Oatp1a4). This hypothesis will be tested by two aims. Aim 1: To investigate CNS delivery of statins mediated by Oatp1a4 in ischemic stroke. To evaluate the role of Oatp1a4 in CNS drug delivery during stroke, we will investigate Oatp1a4-mediated statin transport at the BBB using the transient middle cerebral artery occlusion (tMCAO) model. In these studies, age-matched male and female rats will be subjected to tMCAO for 90 min (Aim 1A). We will then demonstrate that Oatp1a4- mediated statin delivery improves both biomarkers of neuroprotection and BBB integrity (Aim 1B). We will also perform neurocognitive, sensorimotor, and motor performance studies (i.e., functional neurological tests) in animals administered statins and subjected to tMCAO with reperfusion times of up to 21 days (Aim 1C). In all studies, statins will be administered intravenously either at the time of reperfusion or after 2 h of reperfusion to show that early administration of neuroprotective drugs can improve post-stroke outcomes. Aim 2: Transforming Growth Factor-b (TGF-b) signaling can be targeted to control Oatp1a4- mediated CNS statin delivery in ischemic stroke. In these experiments, we will perform dose-response studies and multiple-dosing experiments in age-matched male and female Sprague-Dawley rats using the TGF- b/ALK1 agonist bone morphogenetic protein (BMP)-9 and the TGF-b/ALK5 antagonist SB431542. We will study the activation of specific Smad proteins that control TGF-b signaling in brain microvascular endothelial cells (Aim 2A). We will also determine the time course of Oatp1a4 expression and activity changes following BMP-9 or SB431542 treatment and their effects on CNS delivery of statins (Aim 2B). Additionally, we will measure indices of neuroprotection, markers of BBB protection, and neurological outcomes in rats subjected to tMCAO, administered BMP-9 or SB431542, and injected intravenously with a statin (Aim 2C). Overall, these studies are clinically relevant because they will demonstrate the effective BBB transport mechanisms are required to confer effectiveness of neuroprotective drugs in stroke.