Kurt F. Hauser, Ph.D. - US grants

Opiate drugs, as well as endogenous opioids (i.e., the body's own morphine- like substances), inhibit brain development in children. During development, components of endogenous opioid systems (i.e., endogenous opioids and opioid receptors) are present in high levels in many growing neural cells and tissues, and are likely to be the target of opiate drug action. By experimentally manipulating opioid-opioid receptor interactions it is possible to dramatically retard or accelerate neural and behavioral maturation. Despite the recognized importance of opiates in modifying growth, the mechanisms of their action are poorly understood, e.g., the specific developmental events that are influenced, the opioid-receptor containing targets, the critical periods of opioid sensitively, and the identity of the specific exogenous and endogenous opioids that influence maturation are not known. Our strategy is to explore the cellular and molecular mechanisms by which opiate drugs with abuse liability regulate the growth of the central nervous system. Our hypothesis is that opiates will selectively inhibit certain event(s) during neuro-ontogeny (e.g., cell proliferation or a selective aspect of cell differentiation) by acting through a specific opioid receptor (sub)type. To address these problems, organotypic (primary explant) cultures of the mouse cerebellum and spinal cord will be used as a model to directly assess the local role of opiates per se on the development of identified neurons in culture. Neuroblast proliferation will be examined in cerebellar explants using [3H]-thymidine impregnated Purkinje cells in cerebellar explants and motoneurons in explants of spinal cord. Specific aim 2 will directly effect neural growth in culture. Specific aim 3 will explore whether opiate drugs influence endogenous opioid gene and peptide expression by using Northern analysis and/or in situ hybridization, and immunocytochemistry. The opiate- dependent changes that are observed will be demonstrated to be specific at the level of the opioid receptor, i.e., dose-dependent, reversible by opioid antagonist, and stereospecific. The proposed studies will be the first to systematically explore each of these fundamental questions by directly assessing the cellular and molecular mechanisms of opiate action in vitro. Considering the ubiquity of opioid involvement in growth regulation, our results will have far- reaching significance towards understanding the basic mechanisms governing neuro-development. Moreover, these findings may provide the critical insight needed to design therapies to prevent\correct developmental abnormalities in offspring resulting from maternal drug abuse.

Opiate drugs affect nervous system development by disrupting endogenous opioid systems (endogenous opioids and opioid receptors). Opiates can profoundly inhibit growth. Despite wide-spread licit and illicit use, the developmental mechanisms of opiate action are not understood. This proposal examines the developmental neurobiology of opiate drugs with abuse liability. Interrelated cell and molecular biological experiments will primarily use primary cultures of highly purified populations of cells from the mouse cerebellum and cerebral cortex to explore how opiates directly regulate brain growth. Growth, and opioid receptor and gene expression, will be studied in identified populations of dividing neuroblasts (cerebellar external granular layer cells) and glia (i.e., astrocytes, oligodendrocytes, and microglia) and their progenitors, in vitro, and to a limited extent in vivo. Alterations in growth will be demonstrated to be opioid receptor specific by showing that they are dose-dependent, antagonist-reversible, and stereospecific. Specific aim 1 will determine the effects of opiates on the generation (proliferation and survival) of identified populations of neural cells. Specific aim 2 will identify which opioid receptor types mediate growth. Growth will be manipulated using prototypic opioid receptor agonists and antagonists for both traditional mu (mu), delta-, and kappa- as well as non-traditional "immature', opioid receptor types and subtypes. Opioid receptors will be localized in developing neural cells using (i) a wipe-count method and radioligand autoradiography, (ii) fluorescent-labeled ligands and scanning laser confocal microscopy, and (iii) in situ hybridization for opioid receptor mRNA. Specific aim 3 will determine whether dividing cells intrinsically express opioids by examining opioid gene expression in identified cells in culture using in situ hybridization and immunocytochemistry. The production of opioids by dividing cells is proposed as an autocrine/paracrine mechanism of growth regulation, and is likely to be an important site for opiate drug action. Determining the direct mechanisms of action is crucial towards understanding the etiology of opiate-dependent abnormalities in neural and behavioral development. Our hypothesis is that opiates inhibit nervous system maturation by affecting the production of neurons and/or glia. The results will obtain a unified picture of how opiate regulate growth, correlating developmental effects with cellular sites of opioid gene and receptor expression. This study will have far- reaching significance towards understanding the basic mechanisms by which opiates regulate nervous system growth, and perhaps towards designing therapeutic interventions to prevent/correct developmental abnormalities in children resulting from maternal drug abuse.

DESCRIPTION: (Applicant's Abstract) Opiate drugs alter the pathogenesis of AIDS. The progression to AIDS dementia in HIV-1-positive individuals may be more rapid in opiate drug abusers than non-abusers. Brain regions with a high number/density of opioid receptors, such as the striaturn and hippocampus, display increased viral loads and are preferentially susceptible to HIV infection. Although HIV itself propagates in microglia and astroglia, HIV-1 protein "virotoxins," including gp120 and Tat, are subsequently released and cause the degeneration of neighboring neurons and glia. Despite evidence of an enhanced vulnerability of neurons and glia to HIV following opiate exposure, the reasons for the susceptibility are not understood since opioids can be neuroprotective or promote apoptosis. Our laboratory and others discovered that phenotypically distinct subsets of glia express mu opioid receptors, and chronic exposure to opiate drugs of abuse destabilize ion homeostasis and cytokine production in neurons and/or glia. Our preliminary data indicate that opiates exacerbate the toxicity to HIV virotoxin-exposed striatal neurons and destabilize Ca 2+ in astroglia. We predict the preferential susceptibility of HIV-1 afflicted individuals to opiates may result from the selective vulnerability of p opioid receptor-expressing neuronsandglia. Our hypothesis is that opiates will directly modify the toxic effects of HIV-1 proteins in opioid receptor-expressing neurons and astroglia. To test the hypothesis, we will explore the role of mu receptors using pharmacologic and genetic (mu receptor knockout mice) strategies. Aim 1 will assess the effects of opiate drugs on the pathogenesis of HIV-1 virotoxin-exposed (gp l20 or Tat) mu receptor-expressing striatal neurons, astroglia, and microglia. Aim 2 will identify morphine-induced alterations in viability, dendritic pathology, reactive astrogliosis, and microglial infiltration/activation in the striata of mice stereotaxically injected with gp120/Tat viral proteins in vivo. Aim 3 will determine whether mu opiates alter gpl20/Tat-induced destabilization of intracellular Ca 2+ and cytokine production. Our goal is to determine the mechanisms by which opioids contribute to the pathobiology of HIV infection in the central nervous system, and to identify interactive events that could be targeted for therapeutic intervention.

DESCRIPTION: (Applicant's Abstract) Opioids are fundamental in the pathogenesis of AIDS. The progression to AIDS dementia in HlV-1-positive individuals may be more rapid in opiate drug abusers than those abstaining from opiates. Brain regions with a high number/density of opioid receptors, such as the striatum and hippocampus, display increased viral loads and are preferentially susceptible to HIV infection. Although HIV itself propagates in microglia and astroglia, HIV-1 protein ' aboutvirotoxins," including gp120 and Tat, are subsequently released and cause the degeneration of neighboring neurons and glia. Despite evidence of an enhanced vulnerability of neurons and glia to HIV following opiate exposure, the reasons for the susceptibility are not understood. Our laboratory and others discovered that phenotypically distinct subsets of astroglia and microglia express about, 6, and/or K opioid receptors, and chronic exposure to opiate drugs of abuse destabiiize ion homeostasis and increase oxidative stress in neurons and glia similarly to HIV proteins. Our preliminary data indicate that opiates exacerbate the toxicity to HIV virotoxin-exposed striatal neurons. We predict the preferential susceptibi,'ity of HIV-1 afflicted individuals to opiates may result from the selective vulnerability of opioid receptor-expressing neurons and astroglia. Ourhypothesis is that opiates exacerbate HIV-1 toxicityin neurons and glia by fuffher stressing already jeoparclized [Ca2+]; and free radical homeostatic systems. To test this hypothesis, aim 1 will systematically assess the interactive role of ,u, 6, and K receptor activation on gp120/Tat-induced toxicity in mouse and human neurons and astroglia in vitro. Aim 2 will determine whether about opiates and gp120/Tat virotoxins, which both destabilize intracellular Ca2+ and increase reactive oxygen species, act through similar pathways by blocking putative toxic signaling events. Aim 3 will identify morphine-induced alterations in toxicity in striatal neurons and glia in vivo in mice stereotaxically injected with gp1201Tat viral proteins. Our goal is to determine the mechanisms by which opioids contribute to the pathobiology of HIV infection in the central nervous system, and to identify the underlying signaling pathways that could be targeted for therapeutic intervention.

Opiate abuse potentiates the neuropathogenesis of HIV by synergistically increasing dendritic pathology (varicosity formation, beading, fragmentation, pruning), while promoting additive dendritic spine losses (plasticity). Behavioral deficits in spatial and non-spatial memory tasks are accompanied by synaptic losses and dendritic pathology preceding neuron death, suggesting that neuronal injury and reduced synaptic connectivity underlie the ability of opioids to aggravate HIV-associated neurological disorders (HAND). We have found that phenotypically distinct subpopulations of hippocampal CA1 interneurons appear to be highly sensitive to Tat ± opiates, and disruptions to these interneuron subpopulations may contribute, in part, to pyramidal cell dysfunction/injury. These findings represent a fundamental shift in our understanding of opioid drug action and propel the grant in novel directions. We hypothesize that opiates and HIV-induced hippocampal behavioral dysfunction is caused by disruptions to synaptic function and organization in vulnerable neuronal subpopulations that disrupt specific neural networks within the hippocampus. Aim 1 will characterize the neurophysiologic events underlying opioid and HIV-dependent neuronal dysfunction and injury in hippocampal CA1 pyramidal cells in whole-cell, patch-clamp recordings of CA1 pyramidal cells. Alterations in long-term potentiation and depression will be explored, as will deficits in subthreshold postsynaptic potentials in response to opiates and Tat. During patch-clamp recordings, neurons will be biocytin-filled and subsequently analyzed via 3D-reconstruction for dendritic pathology and spine density. Aim 2 will determine how vulnerable subsets of MOR-expressing CA1 interneurons exacerbate opiate and HIV-1-induced hippocampal dysfunction, neuronal injury, and disrupt network function. Tat tg mice will be crossed with MORfloxed;VGAT-Cre mice, which lack MOR+ CA1 interneurons. We will also determine how Tat and opiates affect synaptic processing and network function in CA1 by examining the integration of synaptic inputs by imaging genetically encoded-voltage indicators (GEVIs) selectively expressed in CA1 pyramidal neurons. Aim 3 will identify the neurophysiologic mechanisms underlying opiate and infectious HIV-1/HIV protein-induced neuronal dysfunction and injury in human hippocampal neurons. Opiate and Tat-induced neurophysiological and structural deficits will be correlated with deficits seen in Tat mice assessed in Aims 1 and 2. Our long-term goal is to define the mechanisms by which opiate drug abuse exacerbates neurodegenerative and functional defects, and to identify key underlying events that could be targeted therapeutically.

DESCRIPTION (provided by applicant): The goal of the Program Project is to understand how opiate drugs exacerbate the effects of HIV in the CNS. To achieve this goal, we will address the following hypothesis: Opiate drugs, through selective actions at f-opioid receptors (MOR), exacerbate the pathogenesis of HIV-1 by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Inflammatory processes and viral products released by human immunodeficiency virus type one (HIV-1) infected cells cause widespread metabolic derangement, immune dysregulation, disruption of neuron-glial relationships, and neuronal dysfunction, which contribute to HIV encephalitis and perhaps dementia. Opiate drugs, such as heroin and oxycodone, intrinsically disrupt CNS function, by interfering with endogenous opioid and neuroimmune function. CNS disruption and neurotoxicity are exacerbated when opiates are combined with HIV. Project 1 will examine neurodegenerative mechanisms in opioid and HIV protein-exposed cell cultures and mice. Neuron death and non-lethal effects (e.g., neuritic pruning will be examined in + MOR-expressing populations of striatal neurons in vitro. Signaling between premitochondrial pathways involving PTEN and Akt will be examined for both caspase-3 dependent and independent cell death using pharmacological, transfection (silencing/overexpression vectors) and genetic strategies (MOR-knockout and PTEN-deficient mice). Project 2 will explore opioid and HIV disruption of astroglial function (e.g. Ca2+, oxyradical production, and EAAT1/2 glutamate transporters) and the release of inflammatory cytokines (e.g. TNFa, IL-6) in vitro and in vivo, and will identify aspects of astroglial destabilization that contribute to pathological changes in neurons. Project 3 will examine opioid-HIV-induced changes in free radical production, redox-sensitive intracellular signaling pathways (e.g., proteasome activity/immunoproteasome subunit expression, NFkappaB, MAP kinase) and inflammatory signaling in microglia. Last, studies using conditional transgenic mice expressing Tat or gp120 and severe combined immunodeficient (SCID) mice injected with HIV-infected human monocytes will assess opioid-HIV effects in neurons, astroglia, and microglia in vivo. The Projects are supported by an administrative core (Core A), and a transgenic/SCID mouse core (Core B) and recombinant viral protein core (Core B), and common findings, e.g., on neuronal dysfunction/death will be integrated in Project 1.

DESCRIPTION (provided by applicant): The progression to AIDS dementia in HIV-positive individuals appears to be markedly accelerated in opiate drug abusers. In the CNS, HIV infects microglia, and to a lesser extent astroglia, increasing the production of oxyradicals, pro-inflammatory cytokines, and the release of HIV-1 proteins such as gp120 and Tat, which can cause injury and even death in bystander neurons. Opioid drugs of abuse can synergistically increase each of the above pathophysiological effects of HIV through direct actions on glia. We have discovered that fractalkine, which is a key regulator of microglial directed toxicity to neurons, is completely protective against the accelerated HIV-1 neurotoxicity caused by morphine co-exposure. By contrast, blocking fractalkine receptors (CX3CR1) completely mimics the neurotoxic effects of combined morphine and Tat. This suggests that morphine-HIV in combination selectively attenuate fractalkine levels or signaling via CX3CR1. Studies are proposed that hypothesize that morphine induces collateral damage in HIV encephalitis by disrupting fractalkine-CX3CR1 interactions and enhancing microglial mediated killing of neurons. These studies will unambiguously determine the specific cellular origin(s), target(s), and causal glia-neuron (or vice versa) directed signaling underlying fractalkine-mediated neuroprotection following opioid HIV Tat/gp120 exposure. In addition, the expression, release, and processing of fractalkine, the expression of CX3CR1 and key regulators of fractalkine function (ADAM10, ADAM17, and cathepsin S) will be explored in neurons, microglia, and astroglia following morphine HIV-1 Tat and gp120. Lastly, the potential neuroprotective effects of fractalkine and fractalkine "sheddase" inhibitors will be assessed in a novel Tat transgenic mouse model of HIV encephalitis co-exposed to morphine. These experiments represent a high risk/high reward strategy appropriate for the R21 mechanism with the goal of understanding how fractalkine prevents toxic neuron-glial interactions in opioid drug-HIV comorbidity. PUBLIC HEALTH RELEVANCE: Emerging evidence indicates that opioid drug abuse blocks the actions of a neuroprotective factor (termed fractalkine) normally present on the surface of neurons. We discovered that when we add fractalkine to lethal concentrations of morphine and HIV proteins it completely blocked neuronal injury and death in cell culture. The proposal will examine whether opioids cause neurons to lose fractalkine and/or whether opioids cause immune cells to ignore the protective fractalkine signal, and determine whether fractalkine could be used therapeutically.

DESCRIPTION (provided by applicant): A K02 independent Scientist Award will enable the candidate to obtain maximum protected time at Virginia Commonwealth University (VCU) to pursue NIDA funded research on substance abuse-HIV-1 comorbidity, acquire advanced cross-disciplinary expertise, and to mentor student and faculty scientists in this area. Drug abuse and HIV-1 are interlinked epidemics with horrific consequences. To address this problem, the candidate directs grant P01 DA19398, "Opiate drug abuse and CNS vulnerability to HIV", is Principal Investigator (PI) on grant R0I DA18633, "Mechanisms of opiate drug-HIV: lnduced neurodegeneration", is a co-l on R01 DA024461, "Glial progenitors as targets of HIV/opiate interactions", is a co-l on a pending NIDA R03 grant to study opioid drug-hepatitis C virus (HCV) interactive pathology, and is a consultant on numerous other projects. The applicant has published seminal studies demonstrating: that opioids can directly affect CNS maturation;the cellular basis of opioid receptor and function in astrocytes and oligodendrocytes;and that opioids intrinsically exacerbate the pathogenesis of neuroAIDS-identifying both intra- and intercellular pathologic mechanisms in neurons and multiple glial types. The applicant has established worldwide collaborations that continue to optimize approaches to opioid abuse-HIV-1 comorbidity, has mentored highly successful pre- and postdoctoral, and basic and clinical faculty;including training in the responsible conduct of research (RCR), has co-directed a NIDA training grant, and organized local and international conferences promoting substance abuse research. A K02 award will enable the candidate to: (1) pursue the goals of current grants, while developing cutting-edge approaches to substance abuse (e.g., self-administration paradigms), HlV-1 (humanized SCID mouse model), and molecular neurovirology;(2) to merge basic and clinical approaches through translational research with the VCU HIV/AIDS Center and joint VCU/Johns Hopkins Univ. NIDA CTN;while continuing to mentor students/early career scientists (including under-represented individuals and an emphasis on RCR). He was recruited to VCU by the Dept. of Pharmacology and Toxicology to pursue these goals and receives tremendous support for this endeavor. PUBLIC HEALTH RELEVANCE: Opioid (heroin) abuse and HIV/AIDS are interrelated epidemics. Not only does drug abuse spread HIV, but also the candidate discovered that opioids intrinsically promote the neurodegenerative effects of HIV. The candidate respectfully requests this award to devote more time to pursue research into the mechanisms underlying opioid drug abuse-HIV interactions to indentify new therapies for opioid abuse and neuroAIDS.

DESCRIPTION: A K02 independent Scientist Award will enable the candidate to obtain maximally protected time at Virginia Commonwealth University (VCU) to pursue NIDA funded research on substance abuse-HIV-1 comorbidity, acquire advanced cross-disciplinary expertise, and to mentor student and faculty scientists in this area. Drug abuse and HIV-1 are interlinked epidemics with horrific consequences. To address this problem, the candidate directs grants R01 DA018633, Mechanisms of opiate drug-HIV-induced neurodegeneration and R01 DA033200 Regulation of HIV and opiate-directed synaptodendritic injury/death in striatum, and is a multi-Pl on R01 DA034231 Glial origins of HIV and opiate-driven synaptodendritic injury, and is a co-l on other grants and consultant on numerous other projects. The applicant has published seminal studies demonstrating: that opioids can directly affect CNS maturation; the cellular basis of opioid receptor and function in astrocytes and oligodendrocytes; and that opioids intrinsically exacerbate the pathogenesis of neuroAIDS-identifying both intra- and intercellular pathologic mechanisms in neurons and multiple glial types. The applicant has established worldwide collaborations that continue to optimize approaches to opioid abuse-HIV-1 comorbidity, has mentored highly successful pre- and postdoctoral, and basic and clinical faculty; including training in the responsible conduct of research (RCR), has co-directed a NIDA training grant, and organized local and international conferences promoting substance abuse research. A K02 award will enable the candidate to: (1) pursue the goals of current grants, while developing cutting-edge approaches to substance abuse (e.g., whole-cell patch-clamp electrophysiology) to assess the trigger events underlying opiate and HIV interactive synaptodendritic injury) and better models of substance abuse (self-administration). (2) To merge basic and clinical approaches through translational research using engineered HIV proviral vectors as a novel source non-infectious HIV/virotoxins for studying electrophysiological effects in bystander neurons and to adopt CRISPRs (clustered regularly interspaced short palindromic repeats), a genome editing strategy, with the goal of completely eliminating or adding receptor genes (e.g., OPRM1). The candidate will continue to mentor students/early career scientists (including under-represented individuals and with an emphasis on RCR). He was recruited to VCU by the Dept. of Pharmacology and Toxicology to pursue these goals and receives tremendous support for this endeavor.

DESCRIPTION (provided by applicant): Drug abuse and HIV/AIDS are interlinked epidemics because of increased spread of HIV-1 through needle sharing and through the exchange of sex for drugs. During the past decade, we have proposed that, besides peripheral immune dysregulation, opiates exacerbate the pathological effects of HIV in the CNS though direct actions on ¿-opioid receptor expressing (MOR) neurons and glia. The CNS may be preferentially vulnerable to opiate and HIV-1 interactions because of the complexity and interrelatedness of MOR action in neurons, astroglia, microglia and endothelium. We recently reported that convergent, neurotoxic effects of Tat or gp120 and morphine are largely due to actions of MOR+ glia. Our in vivo work supports a central role for glia in sublethal/lethal synaptodendritic injury. Morphine worsens structural and functional irregularities in hippocampus and striatum of Tat-expressing mice, including spine losses and synaptodendritic injury. Neurotoxic interactions with morphine and gp120 are HIV strain specific. Medium from monocytes exposed to live or UV-inactivated R5-tropic HIV-1SF162 virions is toxic to neurons; this is significantly attenuated by the CCR5 antagonist maraviroc. We hypothesize that opiate-induced exacerbation of HIV toxicity is regulated through differential CCR5 signaling between microglia, astroglia, and neurons, which can injure or protect neurons depending on context. We propose that opiates and Tat or HIV interact downstream of TNF¿ to enhance inflammatory chemokine release from astroglia. In particular, convergent effects of opiates and Tat/HIV appear to dysregulate CCL5/CCR5 signaling, creating a milieu that promotes microglial motility and spiraling microglial activation. Our hypotheses are tested in 3 aims that examine individual roles of astroglia and microglia in driving neurotoxic opiate-HIV interactions. Aims 1 & 2 use murine models for both: a) long-term, repeated imaging of cell death and sublethal neurodegenerative changes; and b) in vivo studies to detect population changes, neuron pathology, and behavioral deficits. Models include mice with cell-specific Cre-lox ablation of MOR, and inducible Tat transgenic mice crossed with CCL5-/- or CCR-/- mice. In Aim 3, human glia and neurons are used to study neurodegenerative changes due to HIV and opiate effects related to CCL5- CCR5 signaling in an infectious model with multiple strains of HIV. Infectious/non-infectious effects of CCR5 activation are sorted using live and inactivated virus, and mutant viral strains. The CCR5 pathway is especially relevant to HIV. CCR5 is an HIV co-receptor for M-tropic strains; CCR5 polymorphisms affect disease susceptibility; the CCR5 antagonist maraviroc slows HIV progression clinically. Studies are essential for informing therapeutic strategies. By defining how each glial type contributes to CCL5/CCR5 dysregulation, judicious cell- and pathway-specific measures to prevent the neurotoxic sequelae of opiate-HIV co-exposure can be designed.

DESCRIPTION (provided by applicant): The neurotoxic consequences of opiate drug and HIV-1 interactions on striatal neurons and on the underlying intracellular signaling pathways (autophagy, ER-stress/unfolded protein responses (UPR), apoptosis) resulting in sublethal injury and death will be explored. Accumulating evidence indicates that opioid drug abuse per se directly exacerbates the neuropathology of HIV-1. We have found that (1) opioid drugs exacerbate neuronal injury and death through independent actions on ?-opioid receptor (MOR) expressing neurons, astroglia and microglia; and (2) that neuronal death is preceded by non-lethal changes in synaptodendritic pathology that are presumably reversible. Opiate abuse potentiates the neuropathogenesis of HIV largely/exclusively by synergistically disrupting glial function and dendritic pathology. We hypothesize that opioid-HIV interactive increases in neuron death are preceded by cumulative insults to synaptic organization and function that originate in glia, are non-lethal, and assumed reversible. These reductions in neuronal function likely underlie cognitive impairment in neuroAIDS and represent an important therapeutic target for chronic drug abuse-HIV comorbidity. Aim 1 will examine opioid and HIV-1-dependent (Tat, gp120, and live virus) sublethal neuronal dysfunction and/or death as a continuum using multiple in vitro models including primary human dissociated neuron, astroglia, and microglial cultures. It is likely that apoptosis, autophagy, and ER-stress/UPR are operative in sublethal injury as well as during neuron death, and these pathways are evaluated and tested for potentially reversible effects. Aim 2 will identify the extent to which apoptotic, autophagic, and ER-stress/UPR events are associated with opiate-HIV-induced neuronal dysfunction and/or death in vivo. Our ability to visualize/manipulate apoptotic, autophagic, and UPR events in living, cultured neurons will garner new insight into the pathogenesis of opioid abuse/HIV comorbidity and will reveal novel strategies to reverse neuron injury. Our labs were the first to show that opioids, in large part via glial intermediates, exacerbate HIV-induced CNS neuroimmune responses and neuronal injury. Our long-term goal is to define the mechanisms by which opiate drug use or abuse contributes to neurodegeneration accompanying HIVE, and to identify signaling pathways that could be targeted for therapeutic intervention.

DESCRIPTION: Drug abuse directly contributes to one-third of all HIV-1 infections in the United States. While epidemiological data have demonstrated that opioid abuse is a risk factor for HIV-1 infection and progression to AIDS, accumulating evidence reveals possible synergistic interactions between the mu opioid (MOR) and CCR5 chemokine receptors in this pathologic process. Therefore, a thorough understanding of the neural pathways likely involved in opioid enhancement of HIV-1 infection is essential. Our hypothesis is that bivalent ligands containing both a MOR antagonist and a CCR5 antagonist may serve as chemical probes to study the interaction of these two major receptors with respect to HIV-1 infection enhanced by opioid abuse. The oligomerization of opioid receptors and CCR5 uniquely affects immune cell function and their molecular interactions may underlie their apparently synergistic effects in the CNS. Bivalent ligands have been shown to be powerful molecular tools for characterization of G-protein coupled receptor (GPCR) protein-protein interactions, to interfere with normal function related to these interactions, or even to treat diseases by targeting such interactions. We believe a ligand of this kind may not only serve as a pharmacological probe to help clarify the mechanism of opioid abuse-enhanced HIV-1 infection and understand the neuropathogenesis of dementia due to drug abuse and HIV-1 infection, but may also be therapeutic in repressing this enhanced HIV-1 infection. Therefore, the long-term goals of this project are to elucidate the molecular mechanism of opioid-enhanced HIV-1 infection by using such bivalent ligands, and to explore the potential application of these ligands for the treatment of opioid abuse and HIV-1 infection-related dementia. The specific aims of this proposal are to: 1) characterize the bivalent ligands in cellular binding and functional assays, at both acute and chronic conditions; 2) examine the efficacy of bivalent ligands in blocking HIV-1 entry and infectivity via CCR5 and MOR-CCR5 interactions; and 3) study the pathogenesis of neuroAIDS by applying the bivalent ligand probes.

? DESCRIPTION (provided by applicant): Chronic pain diminishes the quality of life for millions of patients, but currently used classes of analgesics possess varied efficacy and are associated with a variety of untoward side effects. Thus, novel targets to treat chronic pain and development of new drugs that have better efficacy and/or fewer side effects than existing pharmacotherapies are greatly needed. A particularly promising target is the sphingosine-1-phosphate (S1P) receptor system, which mediates CNS neuromodulatory functions. FTY720-phosphate, the active metabolite of FTY720 (FTY; fingolimod), approved by the FDA for treatment of relapsing multiple sclerosis, acts as an agonist at four of the five S1P receptors (S1P1, 3, 4, 5). Interestingly, studies have demonstrated that FTY and other S1P receptor (S1PR) agonists produce antinociception in acute thermal rodent pain models and these effects are blocked by central administration of an S1P1-selective antagonist. Moreover, FTY reverses hyperalgesic states in rodent neuropathic pain models. However, it is unclear whether S1P1 or other S1PR subtypes mediate these effects and their site(s) of action. Thus, the overarching hypothesis of this application is that the S1P1 receptor represents a novel and promising target for the treatment of neuropathic pain. Here, we will test whether S1P1 receptors in the CNS mediate anti-hyperalgesic effects in a mouse neuropathic pain model, using a combination of pharmacological and gene targeting approaches. Therefore, the Specific Aims are to: 1) Determine the role of S1P1Rs in alleviation of neuropathic pain by S1PR ligands; 2) Determine the role of FTY-induced S1PR adaptation in FTY-mediated reversal of neuropathic pain; and 3) Determine the role of S1P and S1P1 receptors in spinal glia in CCI-induced neuropathic pain and its reversal by FTY. The studies proposed herein will establish whether FTY and selective S1PR ligands reverse pain-related behavior in the mouse CCI neuropathic pain model, whether S1P1 receptors in the nervous system mediate these actions and the specific cell types involved in the response. In order to be useful in treating chronic pain, the drug must retain its effectiveness during prolonged treatment. Thus, evidence supporting a role of S1P1 in specific cell types to reduce neuropathic pain without tolerance or motor impairment will provide proof of principle that S1P1 receptors are a viable target to treat neuropathic pain and possibly other chronic pain-related disorders.

Project Summary Drug abuse directly contributes to one-third of all HIV infections in the United States. Epidemiological data have demonstrated that opioid abuse is a significant risk factor for HIV infection and progression to AIDS while accumulating evidence reveals possible synergistic interactions between the mu opioid (MOR) and CCR5 chemokine receptors in this pathologic process. Therefore, a thorough understanding of the neural pathways likely involved in opioid enhancement of HIV infection is essential. The putative dimerization of the mu opioid and CCR5 receptors uniquely affects immune cell function and their molecular interactions may underlie their apparently synergistic effects in the CNS. Bivalent ligands have been shown to be powerful molecular tools for characterization of G-protein coupled receptor (GPCR) protein-protein interactions, to interfere with normal function related to these interactions, or even to treat diseases by targeting such interactions. As a proof-of- concept, our recently developed bivalent ligand carrying MOR-CCR5 dual antagonist pharmacophores has shown significantly higher inhibitory effect on HIV-1 invasion in human macrophages and astrocytes compared to a simple mixture of the two antagonists. Our hypothesis is that bivalent ligands containing both an MOR antagonist and a CCR5 antagonist may serve as chemical probes to study the interaction of these receptors with respect to HIV infection enhanced by opioid abuse. We believe a ligand of this kind may serve as a pharmacological probe to help clarify the molecular mechanism of opioid abuse enhanced HIV infection, and help establish this evolving protein-protein interaction model as a potential target for therapeutic intervention in opioid abuse enhanced neuroAIDS. The specific aims of this proposal are to: 1) design and synthesize novel bivalent ligands containing both an MOR antagonist and a CCR5 antagonist as dual pharmacophores by applying crystal structures of ligand bound MOR and CCR5 proteins, molecular modeling, and chemical synthesis; 2) characterize these bivalent ligands with receptor binding and functional assays; and 3) examine the relative efficacy of bivalent ligands in blocking HIV entry and infectivity via CCR5, and CCR5-MOR interactions. We believe such an effort will build the foundation to understand such a complicated biological process, define a novel therapeutic target to treat neuroAIDS, and facilitate future treatment development for the disease.

Program Director/Principal Investigator (Last, First, Middle): Hauser, Kurt F. & Knapp, Pamela E. HIV-associated neurocognitive disorder (HAND) remains a serious clinical problem even in patients receiving cART, and HIV-infected persons who abuse opiates are at greater risk for HAND. Opiates can act directly via µ-opiate receptors (MOR) on glia to amplify secondary neurotoxic effects. The concept that damage to oligodendroglia (OLs) adds to neurologic deficits in HIV is largely unexplored, even though white matter deficits occur quite early after infection. Importantly, our experimental work shows that OLs are vulnerable to viral protein or HIV exposure. A 7 d co-exposure of transgenic mice to HIV-1 Tat ± morphine damages OLs, which exhibit cytoarchitectural/ultrastructural anomalies, and elevated caspase-3 and TUNEL expression, the latter indicating OL death (69; Fig 1). OLs are the only CNS cell type that die in situ in response to acute Tat and morphine coexposure. In vitro, Tat kills developing OLs, while mature OLs instead show a loss of myelin membrane; both outcomes are related to Ca2+- and GluR-mediated mechanisms (84). OL damage, in addition to vascular/blood brain barrier damage, would contribute to myelin pallor consistently noted in imaging studies. Our central hypothesis is that HIV-1 Tat interacts with opiates to directly injure MOR-expressing OLs. Since immature OLs express MOR and are preferentially vulnerable to HIV/Tat, we predict that myelin repair processes are especially vulnerable. Herein, we propose a comprehensive morphological and functional assessment of the time course of OL and myelin damage caused by acute and chronic (?3 month) HIV/Tat and morphine coexposure. Opiate exposure is intermittent to better model the exposure of injection drug users (IDUs) who contract HIV. Studies are done in both sexes, as myelination is influenced by sex steroids. The central hypothesis is tested in 3 related aims. Aim 1 develops a thorough picture of opiate-related OL and myelin damage in situ using an HIV-1 Tat model. Function is assessed using electrophysiology; damage to white matter tracts and OLs is assessed by histology, high resolution DT-MRI and tractography/connectivity, stereology, and electron microscopy. Vascular changes as assessed by blood-brain barrier leakiness are monitored histologically and correlated with areas of demyelination. In Aim 2, MOR is deleted from OLs to test whether opiate interactions occur via direct actions on OLs in vivo; the direct effects of morphine ± HIV, Tat and gp120 on MOR-expressing, human OLs are compared in vitro. Aim 3 tests structural and functional recovery after morphine is withdrawn. These comprehensive studies answer critical questions about OL/myelin vulnerability and the potential for their recovery after combined HIV/Tat and opiate insults. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page

Opioid drug abuse exacerbates pathological and behavioral/cognitive deficits of neuroAIDS. HIV-1-associated neurocognitive disorders (HAND) remain evident in nearly half the individuals infected. HIV does not uniformly target the brain. Some brain regions, such as the basal ganglia, a region critical for drug reward and highly enriched in µ opioid receptors (MOR), are greatly affected by HIV-1, while other areas are less affected. Within the basal ganglia, e.g., HIV, Tat and gp120 causes losses in the synaptodendritic complexity of striatal medium spiny neurons (MSNs) and morphine exacerbates these effects; however, despite the pronounced damage to some MSNs, other striatal MSNs appear unaffected. We discovered that dopamine D2 receptor (Drd2) expressing MSNs (D2 MSNs) showed significantly greater structural and functional vulnerability to Tat ± morphine than dopamine D1 receptor (Drd1a) expressing MSNs (D1 MSNs) at 14 d following Tat induction when anxiety-like and learning/memory deficits, but not motor disorders (which occur later). Moreover, despite enhanced overall susceptibility, some D2 MSNs were unaffected suggesting that additional phenotypic differences, e.g., expression of MOR (which differs among D2 MSNs), also contribute to Tat and morphine- induced injury in D2 MSNs. Based on this and other evidence, we hypothesize that phenotypically distinct MSN subtypes are selectively vulnerable to Tat and morphine coexposure and that the structural and functional deficits in specific neural circuits underlie specific behavioral dysfunctions. To address this hypothesis, the following specific aims are proposed in both male and female mice. Aim 1 will examine the nature and timing of synaptodendritic injury in striatal D2 ± MOR MSN subgroups after short (14 d) and prolonged (2 month) Tat and morphine exposure. Drd2-eGFP-MOR-mCherry and Tat interbred mice will be exposed to morphine/Tat for 14 d (when cognitive, but not motor, deficits are evident) or 2 months (when cognitive and motor deficits are present). Aim 2 will examine the nature and timing of delayed D1 ± MOR MSN synaptodendritic injury in Tat tg;Drd1a-tdTomato reporter mice. In Aims 1 and 2, cognitive and motor performance will be correlated with the electrophysiologic and morphologic (3D reconstruction of synapses/dendrites and stereology) findings in D1 ± MOR and D2 ± MOR MSNs. Aim 3 will address the question of why specific MSN subtypes are more vulnerable to HIV Tat ± morphine, which will provide considerable insight into fundamental mechanisms underlying the interactive toxicity. Aim 3 will use in vitro approaches to examine the extent to which dopaminergic, glutaminergic and BDNF (TrkB, p75NTR) receptor signaling might selectively rescue synaptodendritic injury and dysfunction in HIV, Tat, or gp120-exposed D1 ± MOR and D2 ± MOR MSN subtypes. Specific excitotoxic pathways (e.g., Na+ influx via NMDA, ATP depletion, Ca2+ overload), synaptodendritic injury, and survival will be examined to gauge the role of dopaminergic, glutamatergic, and BDNF signaling in mediating MSN subtype vulnerability following HIV and opiate exposure.

Injection drug use increases the probability of contracting HIV, and opioids accelerate the progression of HIV-1 infection through immune suppression and direct CNS actions. Opioid receptors are widely expressed on immune cells and glia, and opioids can modulate chemokine signaling. CCR5 plays a critical role in HIV infection as a co-receptor for entry of CCR5-preferring strains. CCR5 levels in general, and in microglia/CNS macrophages, have been correlated with severity of HIV neurologic disease. Individuals homozygous for the CCR5?32 mutation resist infection, and the CCR5 antagonist maraviroc (MVC) is used clinically to inhibit HIV entry. Results from the last funding period using non-infective paradigms reveal that CCR5 may also promote HIV-related neuropathology apart from its role in enhancing HIV infection. For example, blockade of CCR5 with MVC increases synaptic plasticity, reverses Tat-induced reductions in antinocicetion, tolerance, and morphine dependence, and enhances survival of Tat-treated striatal neurons. CCR5 overactivation may be a pivotal factor in the ability of opiates to amplify HIV neuropathogenesis, and heterologous interactions between MOR and CCR5 likely underlie many CNS deficits in HAND. CCR5 and MOR interactions in the context of HIV are likely fundamentally different among CNS regions due to distinct glial/neuronal distributions of CCR5 and MOR, their ligands, and effector coupling. Critical to this proposal, MVC rescued Tat-induced deficits in a spatial learning/memory task involving hippocampal function (Barnes maze), and hippocampal damage is involved in learning/memory deficits in HAND patients, irrespective of cART. To explore the hypothesis that aberrant CCR5-MOR interactions underlie HIV-induced hippocampal deficits, we will systematically interrogate those interactions in the context of HIV/Tat and opiate exposure. In vivo and ex vivo studies in HIV-1 Tat transgenic mice are complemented by in vitro studies using human neurons exposed to Tat, gp120, and HIV. Three interrelated Aims test hypotheses in male and female mice. Aim 1 tests CCR5 impact on the dynamics of HIV-1/Tat and MOR interactions in the hippocampus, using functional assessments of behavior (spatial and non-spatial tasks) and neurophysiology (LTP and membrane properties in ex vivo slices). Aim 2 tests how CCR5 activation or blockade (genetic, MVC) alters HIV-1/Tat and MOR interactions, using Tat, gp120, and HIV in murine or human co-cultures of hippocampal neurons and glia. Neuron dysfunction (optical physiology), [Ca2+]i homeostasis, synaptodendritic injury, and death, as well as underlying inflammation and signaling pathways involved (bioplex/MSD multiplex) are explored. Aim 3 examines heterologous interactions between MOR and CCR5, assessing receptor dynamics and activity by [3H]naloxone or [3H]maraviroc-stimulated [35S]GTP?S autoradiography in hippocampal sections, and concentration-effect binding curves in membranes from brain and cultured neurons/glia. TrkB and p75NTR roles in regulating outcomes tested pharmacologically; protection by altering the proBDNF/mBDNF balance (tPA to increase proBDNF cleavage) assessed in all Aims.