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

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.

Effects of chronic HIV-1 Tat exposure in the CNS: heightened vulnerability of males versus females to changes in cell numbers, synaptic integrity, and behavior.

HIV-associated damage to the central nervous system results in cognitive and motor deficits. Anti-retroviral therapies reduce the severity of symptoms, yet the proportion of patients affected has remained the same or increased. Although approximately half of HIV-infected patients worldwide are women, the question of whether biological sex influences outcomes of HIV infection has received little attention. We explored this question for both behavioral and cellular/morphologic endpoints, using a transgenic mouse that inducibly expresses HIV-1 Tat in the brain. After 3 months of HIV-1 Tat exposure, both sexes showed similar reduced open field ambulation. Male Tat(+) mice also showed reduced forelimb grip strength and enhanced anxiety in a light-dark box assay. Tat(+) males did not improve over 12 weeks of repeated rotarod testing, indicating a motor memory deficit. Male mice also had more cellular deficits in the striatum. Neither sex showed a change in volume or total neuron numbers. Both had equally reduced oligodendroglial populations and equivalent microglial increases. However, astrogliosis and microglial nitrosative stress were higher in males. Dendrites on medium spiny neurons in male Tat(+) mice had fewer spines, and levels of excitatory and inhibitory pre- and post-synaptic proteins were disrupted. Our results predict sex as a determinant of HIV effects in brain. Increased behavioral deficits in males correlated with glial activation and synaptic damage, both of which are implicated in cognitive/motor impairments in patients. Tat produced by residually infected cells despite antiretroviral therapy may be an important determinant of the synaptodendritic instability and behavioral deficits accompanying chronic infection.

Ibudilast (AV411), and its AV1013 analog, reduce HIV-1 replication and neuronal death induced by HIV-1 and morphine.

OBJECTIVE: We explored the antiviral therapeutic potential of ibudilast (AV411, MN-166) and its amino analog, AV1013. METHODS: We analyzed whether Ibudilast, a nonselective cyclic AMP phosphodiesterase inhibitor that has been used clinically in Asia for bronchial asthma, poststroke dizziness, and ocular allergies, and AV1013, attenuate HIV-1 replication and the synergistic interactions seen with opiate abuse-HIV-1 comorbidity in neuronal death and inflammation. RESULTS: AV411 and AV1013 inhibited replication by HIV-1 in microglia and significantly suppressed Tat ± morphine-induced tumor necrosis factor-α and MIF production, the activation of the nuclear factor-kappa B subunit p65, and neuronal death. AV411 and AV1013 prevented HIV-1 replication, and attenuated tumor necrosis factor-α and MIF release at concentrations of 100  nmol/l and 1  µmol/l, which are likely achievable at clinical doses. More importantly, co-exposure with morphine did not negate the inhibitory actions of AV411. CONCLUSION: Collectively, our data suggest that AV411 and its amino analog, AV1013, may be useful neuroprotective agents counteracting neurotoxicity caused by infected and activated glia, and implicate them as potential therapies for the management of HIV-associated neurocognitive disorders in an opioid-abusing population.

A novel bivalent HIV-1 entry inhibitor reveals fundamental differences in CCR5-µ-opioid receptor interactions between human astroglia and microglia.

OBJECTIVE: We explored whether the opiate, morphine, affects the actions of maraviroc, as well as a recently synthesized bivalent derivative of maraviroc linked to an opioid antagonist, naltrexone, on HIV-1 entry in primary human glia. METHODS: HIV-1 entry was monitored in glia transiently transfected with an LTR construct containing a luciferase reporter gene under control of a promoter for the HIV-1 transactivator protein Tat. The effect of maraviroc and the bivalent ligand with or without morphine on CCR5 surface expression and cytokine release was also explored. RESULTS: Maraviroc inhibits HIV-1 entry into glial cells, whereas morphine negates the effects of maraviroc leading to a significant increase in viral entry. We also demonstrate that the maraviroc-containing bivalent ligand better inhibits R5-tropic viral entry in astrocytes than microglia compared to maraviroc when coadministered with morphine. Importantly, the inhibitory effects of the bivalent compound in astrocytes were not compromised by morphine. Exposure to maraviroc decreased the release of pro-inflammatory cytokines and restricted HIV-1-dependent increases in CCR5 expression in both astrocytes and microglia, whereas exposure to the bivalent had a similar effect in astrocytes but not in microglia. The CCR5-µ-opioid receptor (MOR) stoichiometric ratio varied among the two cell types with CCR5 expressed at much higher levels than MOR in microglia, which could explain the effectiveness of the bivalent ligand in astrocytes compared to microglia. CONCLUSION: A novel bivalent compound reveals fundamental differences in CCR5-MOR interactions and HIV-1 infectivity among glia, and has unique therapeutic potential in opiate abuse-HIV interactive comorbidity.

HIV-1 alters neural and glial progenitor cell dynamics in the central nervous system: coordinated response to opiates during maturation.

HIV-associated neurocognitive disorders (HANDs) are common sequelae of human immunodeficiency virus (HIV) infection, even when viral titers are well controlled by antiretroviral therapy. Evidence in patients and animal models suggests that neurologic deficits are increased during chronic opiate exposure. We have hypothesized that central nervous system (CNS) progenitor cells in both adult and developing CNS are affected by HIV infection and that opiates exacerbate these effects. To examine this question, neural progenitors were exposed to HIV-1 Tat(1-86) in the developing brain of inducible transgenic mice and in vitro. We examined whether Tat affected the proliferation or balance of progenitor populations expressing nestin, Sox2, and Olig2. Disease relevance was further tested by exposing human-derived progenitors to supernatant from HIV-1 infected monocytes. Studies concentrated on striatum, a region preferentially targeted by HIV and opiates. Results were similar among experimental paradigms. Tat or HIV exposure reduced the proliferation of undifferentiated (Sox2(+)) progenitors and oligodendroglial (Olig2(+)) progenitors. Coexposure to morphine exacerbated the effects of Tat or HIV-1(SF162) supernatant, but partially reversed HIV-1(IIIB) supernatant effects. Populations of Sox2(+) and Olig2(+) cells were also reduced by Tat exposure, although progenitor survival was unaffected. In rare instances, p24 immunolabeling was detected in viable human progenitors by confocal imaging. The vulnerability of progenitors is likely to distort the dynamic balance among neuron/glial populations as the brain matures, perhaps contributing to reports that neurologic disease is especially prevalent in pediatric HIV patients. Pediatric disease is atypical in developed regions but remains a serious concern in resource-limited areas where infection occurs commonly at birth and through breast feeding.

Opiate drug use and the pathophysiology of neuroAIDS.

Opiate abuse and HIV-1 have been described as interrelated epidemics, and even in the advent of combined anti-retroviral therapy, the additional abuse of opiates appears to result in greater neurologic and cognitive deficits. The central nervous system (CNS) is particularly vulnerable to interactive opiate-HIV-1 effects, in part because of the unique responses of microglia and astroglia. Although neurons are principally responsible for behavior and cognition, HIV-1 infection and replication in the brain is largely limited to microglia, while astroglia and perhaps glial progenitors can be latently infected. Thus, neuronal dysfunction and injury result from cellular and viral toxins originating from HIV-1 infected/exposed glia. Importantly, subsets of glial cells including oligodendrocytes, as well as neurons, express µ-opioid receptors and therefore can be direct targets for heroin and morphine (the major metabolite of heroin in the CNS), which preferentially activate µ-opioid receptors. This review highlights findings that neuroAIDS is a glially driven disease, and that opiate abuse may act at multiple glial-cell types to further compromise neuron function and survival. The ongoing, reactive cross-talk between opiate drug and HIV-1 co-exposed microglia and astroglia appears to exacerbate critical proinflammatory and excitotoxic events leading to neuron dysfunction, injury, and potentially death. Opiates enhance synaptodendritic damage and a loss of synaptic connectivity, which is viewed as the substrate of cognitive deficits. We especially emphasize that opioid signaling and interactions with HIV-1 are contextual, differing among cell types, and even within subsets of the same cell type. For example, astroglia even within a single brain region are heterogeneous in their expression of µ-, δ-, and κ-opioid receptors, as well as CXCR4 and CCR5, and Toll-like receptors. Thus, defining the distinct targets engaged by opiates in each cell type, and among brain regions, is critical to an understanding of how opiate abuse exacerbates neuroAIDS.

Morphine and gp120 toxic interactions in striatal neurons are dependent on HIV-1 strain.

A rigorously controlled, cell culture paradigm was used to assess the role of HIV-1 gp120 ± morphine in mediating opioid-HIV interactive toxicity in striatal neurons. Computerized time-lapse microscopy tracked the fate of individual neurons co-cultured with mixed-glia from mouse striata during opioid and gp120 exposure. Subpopulations of neurons and astroglia displayed µ-opioid receptor, CXCR4, and CCR5 immunoreactivity. While gp120 alone was or tended to be neurotoxic irrespective of whether X4-tropic gp120(IIIB), R5-tropic gp120(ADA), or dual-tropic gp120(MN) was administered, interactive toxicity with morphine differed depending on HIV-1 strain. For example, morphine only transiently exacerbated gp120(IIIB)-induced neuronal death; however, in combination with gp120(MN), morphine caused sustained increases in the rate of neuronal death compared to gp120(MN) alone that were prevented by naloxone. Alternatively, gp120(ADA) significantly increased the rate of neuron death, but gp120(ADA) toxicity was unaffected by morphine. The transient neurotoxic interactions between morphine and gp120(IIIB) were abrogated in the absence of glia suggesting that glia contribute significantly to the interactive pathology with chronic opiate abuse and neuroAIDS. To assess how mixed-glia might contribute to the neurotoxicity, the effects of morphine and/or gp120 on the production of reactive oxygen species (ROS) and on glutamate buffering were examined. All gp120 variants, and to a lesser extent morphine, increased ROS and/or decreased glutamate buffering, but together failed to show any interaction with morphine. Our findings indicate that HIV-1 strain-specific differences in gp120 are critical determinants in shaping both the timing and pattern of neurotoxic interactions with opioid drugs.

Toll-like receptor expression and activation in astroglia: differential regulation by HIV-1 Tat, gp120, and morphine.

In this study, we aimed to determine whether morphine alone or in combination with HIV-1 Tat or gp120 affects the expression of Toll-like receptors (TLRs) by astrocytes and to assess whether TLRs expressed by astrocytes function in the release of inflammatory mediators in vitro. TLR profiling by immunofluorescence microscopy, flow cytometry, in-cell westerns, and RT-PCR showed that subpopulations of astrocytes possessed TLR 2, TLR3, TLR4, and TLR9 antigenicity. Exposure to HIV-1 Tat, gp120, and/or morphine significantly altered the proportion of TLR-immunopositive and/or TLR expression by astroglia in a TLR-specific manner. Subsets of astroglia displayed significant increases in TLR2 with reciprocal decreases in TLR9 expression in response to Tat or gp120 ± morphine treatment. TLR9 expression was also significantly decreased by morphine alone. Exposing astrocytes to the TLR agonists LTA (TLR2), poly I:C (TLR3), LPS (TLR4) and unmethylated CpG ODN (TLR9) resulted in increased secretion of MCP-1/CCL2 and elevations in reactive oxygen species. TLR3 and TLR4 stimulation increased the secretion of TNF-α, IL-6, and RANTES/CCL5, while activation of TLR2 caused a significant increase in nitric oxide levels. The results suggest that HIV-1 proteins and/or opioid abuse disrupt the innate immune response of the central nervous system (CNS) which may lead to increased pathogenicity.

PTEN gene silencing prevents HIV-1 gp120(IIIB)-induced degeneration of striatal neurons.

To assess the role of the phosphatase and tensin homologue on chromosome 10 (PTEN) in mediating envelope glycoprotein 120 (gp120)-induced neurotoxicity in the striatum, PTEN was silenced using short interfering RNA (siRNA) vectors. PTEN activity directs multiple downstream pathways implicated in gp120-induced neuronal injury and death. PTEN is a negative regulator of Akt (protein kinase B) phosphorylation, but has also been shown to directly activate extrasynaptic NMDA receptors and dephosphorylate focal adhesion kinase. Rodent striatal neurons were nucleofected with green fluorescent protein (GFP)-expressing siRNA constructs to silence PTEN (PTENsi-GFP) or with negative-control (NCsi-GFP) vectors, and exposed to HIV-1 gp120(IIIB) using rigorously controlled, cell culture conditions including computerized time-lapse microscopy to track the fate of individual neurons following gp120 exposure. Immunofluorescence labeling showed that subpopulations of striatal neurons possess CXCR4 and CCR5 co-receptor immunoreactivity and that gp120(IIIB) was intrinsically neurotoxic to isolated striatal neurons. Importantly, PTENsi-GFP, but not control NCsi-GFP, constructs markedly decreased PTEN mRNA and protein levels and significantly attenuated gp120-induced death. These findings implicate PTEN as a critical factor in mediating the direct neurotoxic effects of HIV-1 gp120, and suggest that effectors downstream of PTEN such as Akt or other targets are potentially affected. The selective abatement of PTEN activity in neurons may represent a potential therapeutic strategy for the CNS complications of HIV-1.