A direct and indirect mechanism for CCR5 in morphine and HIV-1 mediated neurodegeneration

A DIRECT AND INDIRECT MECHANISM FOR CCR5 IN OPIOID AND HIV-1 MEDIATED NEURODEGENERATION By Elizabeth M. Podhaizer, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Major Director: Kurt F. Hauser, Ph.D., Professor of Pharmacology & Toxicology Human immunodeficiency virus (HIV)-1 infection currently affects over 34 million people worldwide, and despite the use of cART, the prevalence of HIV-1 associated neurocognitive impairments (HAND) has not declined. Additionally, other co-morbid factors such as the abuse of injection drugs (i.e. heroin, morphine) increase both the frequency and the speed by which patients progress to AIDS. To begin to understand the mechanisms, we chose to examine a pathway, through CCR5, which may act as a convergence point for opioids and HIV-1 proteins. C-C chemokine receptor 5 (CCR5) is an immune receptor involved in physiological processes in the brain in addition to mediating neuroinflammatory signaling events, and it is a co-receptor for HIV-1. CCR5 interacts directly with gp120 to facilitate HIV-1 infection, and may interact indirectly with HIV-1 Tat through convergent signaling mechanisms. Additionally, CCR5 is modified by opioid responses, and so may be central to opioid-HIV-1 interactions that are seen in our model. We hypothesized that CCR5 would mediate the opioid-HIV-1 interaction. We examined both HIV-1 gp120 and HIV-1 Tat, both for interactions with opioids and modification by the CCR5 antagonist, maraviroc. HIV-1 gp120ADA was neurotoxic on its own, but showed no interactions with morphine. However, further probing revealed that morphine can in fact modify the neurotoxic effects of gp120, but that the response is dependent on gp120 strain. We did, however, find that morphine did enhance the neurotoxicity of Tat, which we’ve shown previously, as well as that inhibition of CCR5 can prevent this interactive effect. Additionally, use of CCR5 knockout glia or neurons modified the response and suggests that neurons and glia play different roles in the integration of opioid and HIV-1 signals. Sublethal effects of morphine and Tat were also dampened by maraviroc pretreatment or use of knockout cells, as was the secretion of chemokine ligands. Manipulation of CCR5 showed utility in preventing neurodegenerative effects both to HIV-1 proteins alone as well as to the interactive opioid-HIV-1 signaling responses and suggests that maraviroc, a cART therapeutic used to prevent viral entry, may also aid in reducing the chronic inflammatory state of the CNS that leads to the persistent neurocognitive complications

Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines

<p>Abstract</p> <p>Background</p> <p>Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice.</p> <p>Results</p> <p>Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in <it>Gli1</it>^-/-<it>2</it>^-/-iMEFs was severely reduced while <it>Gli2</it>^-/-<it>3</it>^-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both <it>Gli1</it>^-/-<it>2</it>^-/-and <it>Gli2</it>^-/-<it>3</it>^-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.</p> <p>Conclusion</p> <p>This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.</p

A central role for glial CCR5 in directing the neuropathological interactions of HIV-1 Tat and opiates

Abstract Background The collective cognitive and motor deficits known as HIV-associated neurocognitive disorders (HAND) remain high even among HIV+ individuals whose antiretroviral therapy is optimized. HAND is worsened in the context of opiate abuse. The mechanism of exacerbation remains unclear but likely involves chronic immune activation of glial cells resulting from persistent, low-level exposure to the virus and viral proteins. We tested whether signaling through C-C chemokine receptor type 5 (CCR5) contributes to neurotoxic interactions between HIV-1 transactivator of transcription (Tat) and opiates and explored potential mechanisms. Methods Neuronal survival was tracked in neuronal and glial co-cultures over 72 h of treatment with HIV-1 Tat ± morphine using cells from CCR5-deficient and wild-type mice exposed to the CCR5 antagonist maraviroc or exogenously-added BDNF (analyzed by repeated measures ANOVA). Intracellular calcium changes in response to Tat ± morphine ± maraviroc were assessed by ratiometric Fura-2 imaging (analyzed by repeated measures ANOVA). Release of brain-derived neurotrophic factor (BDNF) and its precursor proBDNF from CCR5-deficient and wild-type glia was measured by ELISA (analyzed by two-way ANOVA). Levels of CCR5 and μ-opioid receptor (MOR) were measured by immunoblotting (analyzed by Student’s t test). Results HIV-1 Tat induces neurotoxicity, which is greatly exacerbated by morphine in wild-type cultures expressing CCR5. Loss of CCR5 from glia (but not neurons) eliminated neurotoxicity due to Tat and morphine interactions. Unexpectedly, when CCR5 was lost from glia, morphine appeared to entirely protect neurons from Tat-induced toxicity. Maraviroc pre-treatment similarly eliminated neurotoxicity and attenuated neuronal increases in [Ca2+]i caused by Tat ± morphine. proBDNF/BDNF ratios were increased in conditioned media from Tat ± morphine-treated wild-type glia compared to CCR5-deficient glia. Exogenous BDNF treatments mimicked the pro-survival effect of glial CCR5 deficiency against Tat ± morphine. Conclusions Our results suggest a critical role for glial CCR5 in mediating neurotoxic effects of HIV-1 Tat and morphine interactions on neurons. A shift in the proBDNF/BDNF ratio that favors neurotrophic support may occur when glial CCR5 signaling is blocked. Some neuroprotection occurred only in the presence of morphine, suggesting that loss of CCR5 may fundamentally change signaling through the MOR in glia