Escalating morphine dosing in HIV-1 Tat transgenic mice with sustained Tat exposure reveals an allostatic shift in neuroinflammatory regulation accompanied by increased neuroprotective non-endocannabinoid lipid signaling molecules and amino acids

BACKGROUND: Human immunodeficiency virus type-1 (HIV-1) and opiates cause long-term inflammatory insult to the central nervous system (CNS) and worsen disease progression and HIV-1-related neuropathology. The combination of these proinflammatory factors reflects a devastating problem as opioids have high abuse liability and continue to be prescribed for certain patients experiencing HIV-1-related pain. METHODS: Here, we examined the impact of chronic (3-month) HIV-1 transactivator of transcription (Tat) exposure to short-term (8-day), escalating morphine in HIV-1 Tat transgenic mice that express the HIV-1 Tat protein in a GFAP promoter-regulated, doxycycline (DOX)-inducible manner. In addition to assessing morphine-induced tolerance in nociceptive responses organized at spinal (i.e., tail-flick) and supraspinal (i.e., hot-plate) levels, we evaluated neuroinflammation via positron emission tomography (PET) imaging using the [¹⁸F]-PBR111 ligand, immunohistochemistry, and cytokine analyses. Further, we examined endocannabinoid (eCB) levels, related non-eCB lipids, and amino acids via mass spectrometry. RESULTS: Tat-expressing [Tat(+)] transgenic mice displayed antinociceptive tolerance in the tail withdrawal and hot-plate assays compared to control mice lacking Tat [Tat(-)]. This tolerance was accompanied by morphine-dependent increases in Iba-1 +/- 3-nitrotryosine immunoreactive microglia, and alterations in pro- and anti-inflammatory cytokines, and chemokines in the spinal cord and striatum, while increases in neuroinflammation were absent by PET imaging of [¹⁸F]-PBR111 uptake. Tat and morphine exposure differentially affected eCB levels, non-eCB lipids, and specific amino acids in a region-dependent manner. In the striatum, non-eCB lipids were significantly increased by short-term, escalating morphine exposure, including peroxisome proliferator activator receptor alpha (PPAR-alpha) ligands N-oleoyl ethanolamide (OEA) and N-palmitoyl ethanolamide (PEA), as well as the amino acids phenylalanine and proline. In the spinal cord, Tat exposure increased amino acids leucine and valine, while morphine decreased levels of tyrosine and valine but did not affect eCBs or non-eCB lipids. CONCLUSION: Overall results demonstrate that 3 months of Tat exposure increased morphine tolerance and potentially innate immune tolerance evidenced by reductions in specific cytokines (e.g., IL-1alpha, IL-12p40) and microglial reactivity. In contrast, short-term, escalating morphine exposure acted as a secondary stressor revealing an allostatic shift in CNS baseline inflammatory responsiveness from sustained Tat exposure

Effects of Opiates on Central Nervous System Immune Defense in Murine AIDS

Nearly half of human immunodeficiency virus type 1 (HIV-1) patients experience cognitive impairments. These deficits coincide with inflammation and neurodegeneration in the hippocampus and striatum, brain regions important in memory formation. Opioid abuse accelerates the onset of HIV-associated neurocognitive deficits (HAND) by modulating the inflammatory response of microglia and astrocytes. These glial cells initiate and maintain innate immune defense in the central nervous system (CNS). Using the LP-BM5 murine acquired immunodeficiency (MAIDS) model, we investigated the region- and cell-specific effects of morphine on CNS viral infection and immune defense. The LP-BM5 model offers an underutilized perspective in HIV study: it retains the context of a productive viral infection, virally-induced peripheral immunodeficiency, and regional differences in CNS viral entry and replication within a mouse model. To study chronic morphine’s effects in the LP-BM5 model, we infected 8-week-old male C57BL/6Ncr mice with 5x104 plaque-forming units of LP-BM5. Seven weeks after infection, mice were implanted with subcutaneous 25 mg morphine or placebo pellets. Using this model, we investigated the effects of one week of chronic morphine treatment on LP-BM5 infection and the glial immune response in three regions of interest in the CNS: the hippocampus, striatum, and frontal lobe. Chronic morphine impaired cognitive performance in LP-BM5-infected mice, correlating to increased LP-BM5 viral RNA expression in the hippocampus. However, morphine treatment decreased viral RNA in the striatum. Morphine suppressed expression of the proinflammatory markers CCL5 and inducible nitric oxide synthase in all regions studied without a proportionate increase in anti-inflammatory markers. The hippocampus and striatum exhibited critical differential changes in the type 1 interferon (IFN) response, which activates cells’ inherent antiviral capabilities. Morphine treatment increased RNA expression of IFN-α and IFN-β in the striatum. In hippocampus, morphine and LP-BM5 downregulated expression of multiple type 1 IFN-related genes. By inhibiting the hippocampal type 1 IFN response, morphine impedes cells’ ability to ward off viral infection and suppress replication. This work furthers our understanding of opiates’ region-specific effects on CNS antiviral defense, opening new potential avenues for treatment in morphine-potentiated HAND

Characterization of Thermoregulatory Efferents to the Paraventricular Nucleus of the Rat Hypothalamus

The paraventricular nucleus of the hypothalamus (PVN) is the gatekeeper to temperature-modulating hormones such as vasopressin. Indeed, it is in theory a key target region for thermoregulatory efferents of the preoptic area of the anterior hypothalamus (POAH). Although several retrograde labeling studies have identified a connection between the POAH and PVN, none have directly characterized the thermoregulatory nature of this interaction. We will identify the phenotype of these connections by injecting retrograde-labeling nanoprobes into the PVN of live tissue slices and quantified labeled POAH somas. These probes are designed for selective uptake by glutamate-releasing axon terminals, after which they diffuse rapidly to the neuron soma. Our results indicate that glutamatergic neurons do project from the POAH to the PVN. This technique will allow for further electrophysiologic studies to identify the thermosensitivity of neurons with a known connectivity and phenotype

Alterations In Osteopontin Modify Muscle Size In Females In Both Humans And Mice

PURPOSE: An osteopontin (OPN; SPP1) gene promoter polymorphism modifies disease severity in Duchenne muscular dystrophy, and we hypothesized that it might also modify muscle phenotypes in healthy volunteers. METHODS: Gene association studies were carried out for OPN (rs28357094) in the FAMuSS cohort (n = 752; mean ± SD age = 23.7 ± 5.7 yr). The phenotypes studied included muscle size (MRI), strength, and response to supervised resistance training. We also studied 147 young adults that had carried out a bout of eccentric elbow exercise (age = 24.0 ± 5.2 yr). Phenotypes analyzed included strength, soreness, and serum muscle enzymes. RESULTS: In the FAMuSS cohort, the G allele was associated with 17% increase in baseline upper arm muscle volume only in women (F = 26.32; P = 5.32 × 10), explaining 5% of population variance. In the eccentric damage cohort, weak associations of the G allele were seen in women with both baseline myoglobin and elevated creatine kinase. The sexually dimorphic effects of OPN on muscle were also seen in OPN-null mice. Five of seven muscle groups examined showed smaller size in OPN-null female mice, whereas two were smaller in male mice. The query of OPN gene transcription after experimental muscle damage in mice showed rapid induction within 12 h (100-fold increase from baseline), followed by sustained high-level expression through 16 d of regeneration before falling to back to baseline. CONCLUSION: OPN is a sexually dimorphic modifier of muscle size in normal humans and mice and responds to muscle damage. The OPN gene is known to be estrogen responsive, and this may explain the female-specific genotype effects in adult volunteers. Copyright © 2013 by the American College of Sports Medicine

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

Chronic HIV-1 Tat exposure alters anterior cingulate cortico-basal ganglia-thalamocortical synaptic circuitry, associated behavioral control, and immune regulation in male mice

HIV-1 selectively disrupts neuronal integrity within specific brain regions, reflecting differences in viral tropism and/or the regional differences in the vulnerability of distinct neuronal subpopulations within the CNS. Deficits in prefrontal cortex (PFC)-mediated executive function and the resultant loss of behavioral control are a particularly debilitating consequence of neuroHIV. To explore how HIV-1 disrupts executive function, we investigated the effects of 48 ​h, 2 and/or 8 weeks of HIV-1 Tat exposure on behavioral control, synaptic connectivity, and neuroimmune function in the anterior cingulate cortex (ACC) and associated cortico-basal ganglia (BG)-thalamocortical circuitry in adult, Tat transgenic male mice. HIV-1 Tat exposure increased novelty-exploration in response to novel food, flavor, and environmental stimuli, suggesting that Tat triggers increased novelty-exploration in situations of competing motivation (e.g., drive to feed or explore vs. fear of novel, brightly lit open areas). Furthermore, Tat induced adaptability in response to an environmental stressor and pre-attentive filtering deficits. The behavioral insufficiencies coincided with decreases in the inhibitory pre- and post-synaptic proteins, synaptotagmin 2 and gephyrin, respectively, in the ACC, and alterations in specific pro- and anti-inflammatory cytokines out of 23 assayed. The interaction of Tat exposure and the resultant time-dependent, selective alterations in CCL4, CXCL1, IL-12p40, and IL-17A levels in the PFC predicted significant decreases in adaptability. Tat decreased dendritic spine density and cortical VGLUT1 inputs, while increasing IL-1β, IL-6, CCL5, and CCL11 in the striatum. Alternatively, IL-1α, CCL5, and IL-13 were decreased in the mediodorsal thalamus despite the absence of synaptic changes. Thus, HIV-1 Tat appears to uniquely and systematically disrupt immune regulation and the inhibitory and excitatory synaptic balance throughout the ACC-BG-thalamocortical circuitry resulting in a loss of behavioral control

Alterations in osteopontin modify muscle size in females in both humans and mice.

PURPOSE: An osteopontin (OPN; SPP1) gene promoter polymorphism modifies disease severity in Duchenne muscular dystrophy, and we hypothesized that it might also modify muscle phenotypes in healthy volunteers. METHODS: Gene association studies were carried out for OPN (rs28357094) in the FAMuSS cohort (n = 752; mean ± SD age = 23.7 ± 5.7 yr). The phenotypes studied included muscle size (MRI), strength, and response to supervised resistance training. We also studied 147 young adults that had carried out a bout of eccentric elbow exercise (age = 24.0 ± 5.2 yr). Phenotypes analyzed included strength, soreness, and serum muscle enzymes. RESULTS: In the FAMuSS cohort, the G allele was associated with 17% increase in baseline upper arm muscle volume only in women (F = 26.32; P = 5.32 × 10), explaining 5% of population variance. In the eccentric damage cohort, weak associations of the G allele were seen in women with both baseline myoglobin and elevated creatine kinase. The sexually dimorphic effects of OPN on muscle were also seen in OPN-null mice. Five of seven muscle groups examined showed smaller size in OPN-null female mice, whereas two were smaller in male mice. The query of OPN gene transcription after experimental muscle damage in mice showed rapid induction within 12 h (100-fold increase from baseline), followed by sustained high-level expression through 16 d of regeneration before falling to back to baseline. CONCLUSION: OPN is a sexually dimorphic modifier of muscle size in normal humans and mice and responds to muscle damage. The OPN gene is known to be estrogen responsive, and this may explain the female-specific genotype effects in adult volunteers. Med Sci Sports Exerc 2013 Jun; 45(6):1060-8