In situ analysis of neuronal injury and neuroinflammation during HIV-1 infection.

BACKGROUND: Since the introduction of combination antiretroviral therapy (cART) the brain has become an important human immunodeficiency virus (HIV) reservoir due to the relatively low penetration of many drugs utilized in cART into the central nervous system (CNS). Given the inherent limitations of directly assessing acute HIV infection in the brains of people living with HIV (PLWH), animal models, such as humanized mouse models, offer the most effective means of studying the effects of different viral strains and their impact on HIV infection in the CNS. To evaluate CNS pathology during HIV-1 infection in the humanized bone marrow/liver/thymus (BLT) mouse model, a histological analysis was conducted on five CNS regions, including the frontal cortex, hippocampus, striatum, cerebellum, and spinal cord, to delineate the neuronal (MAP2ab, NeuN) and neuroinflammatory (GFAP, Iba-1) changes induced by two viral strains after 2 weeks and 8 weeks post-infection. RESULTS: Findings reveal HIV-infected human cells in the brain of HIV-infected BLT mice, demonstrating HIV neuroinvasion. Further, both viral strains, HIV-1JR-CSF and HIV-1CH040, induced neuronal injury and astrogliosis across all CNS regions following HIV infection at both time points, as demonstrated by decreases in MAP2ab and increases in GFAP fluorescence signal, respectively. Importantly, infection with HIV-1JR-CSF had more prominent effects on neuronal health in specific CNS regions compared to HIV-1CH040 infection, with decreasing number of NeuN+ neurons, specifically in the frontal cortex. On the other hand, infection with HIV-1CH040 demonstrated more prominent effects on neuroinflammation, assessed by an increase in GFAP signal and/or an increase in number of Iba-1+ microglia, across CNS regions. CONCLUSION: These findings demonstrate that CNS pathology is widespread during acute HIV infection. However, neuronal loss and the magnitude of neuroinflammation in the CNS is strain dependent indicating that strains of HIV cause differential CNS pathologies.

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-α) 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-1α, 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.

Endocannabinoids exert CB1 receptor-mediated neuroprotective effects in models of neuronal damage induced by HIV-1 Tat protein.

In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease that specifically targets the brain and causes HIV-1-associated neurocognitive disorders (HAND). Endocannabinoids (eCBs) elicit neuroprotective and anti-inflammatory actions in several central nervous system (CNS) disease models, but their effects in HAND remain unknown. HIV-1 does not infect neurons, but produces viral toxins, such as transactivator of transcription (Tat), that disrupt neuronal calcium equilibrium and give rise to synaptodendritic injuries and cell death, the former being highly correlated with HAND. Consequently, we tested whether the eCBs N-arachidonoylethanolamine (anandamide/AEA) and 2-arachidonoyl-glycerol (2-AG) offer neuroprotective actions in a neuronal culture model. Specifically, we examined the neuroprotective actions of these eCBs on Tat excitotoxicity in primary cultures of prefrontal cortex neurons (PFC), and whether cannabinoid receptors mediate this neuroprotection. Tat-induced excitotoxicity was reflected by increased intracellular calcium levels, synaptodendritic damage, neuronal excitability, and neuronal death. Further, upregulation of cannabinoid 1 receptor (CB1R) protein levels was noted in the presence of HIV-1 Tat. The direct application of AEA and 2-AG reduced excitotoxic levels of intracellular calcium and promoted neuronal survival following Tat exposure, which was prevented by the CB1R antagonist rimonabant, but not by the CB2R antagonist AM630. Overall, our findings indicate that eCBs protect PFC neurons from Tat excitotoxicity in vitro via a CB1R-related mechanism. Thus, the eCB system possesses promising targets for treatment of neurodegenerative disorders associated with HIV-1 infection.

Oligodendrocytes Are Targets of HIV-1 Tat: NMDA and AMPA Receptor-Mediated Effects on Survival and Development.

Myelin pallor in HIV(+) individuals can occur very early during the disease process. While myelin damage might partly originate from HIV-induced vascular changes, the timing suggests that myelin and/or oligodendrocytes (OLs) may be directly affected. Histological (Golgi-Kopsch, electron microscopy) and biochemical studies have revealed an increased occurrence of abnormal OL/myelin morphology and dysregulated myelin protein expression in transgenic mice expressing the HIV-1 transactivator of transcription (Tat) protein. This suggests that viral proteins by themselves might cause OL injury. Since Tat interacts with NMDARs, we hypothesized that activation of NMDARs and subsequent disruption of cytoplasmic Ca(2+) ([Ca(2+)]i) homeostasis might be one cause of white matter injury after HIV infection. In culture, HIV-1 Tat caused concentration-dependent death of immature OLs, while more mature OLs remained alive but had reduced myelin-like membranes. Tat also induced [Ca(2+)]i increases and Thr-287 autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase II ß (CaMKIIß) in OLs. Tat-induced [Ca(2+)]i was attenuated by the NMDAR antagonist MK801, and also by the AMPA/kainate receptor antagonist CNQX. Importantly, both MK801 and CNQX blocked Tat-induced death of immature OLs, but only MK801 reversed Tat effects on myelin-like membranes. These results suggest that OLs can be direct targets of HIV proteins released from infected cells. Although viability and membrane production are both affected by glutamatergic receptor-mediated Ca(2+) influx, and possibly the ensuing CaMKIIß activation, the roles of AMPARs and NMDARs appear to be different and dependent on the stage of OL differentiation. SIGNIFICANCE STATEMENT: Over 33 million individuals are currently infected by HIV. Among these individuals, ∼60% develop HIV-associated neurocognitive disorders. Myelin damage and white matter injury have been frequently reported in HIV patients but not extensively studied. Clinical studies using combined antiretroviral therapy (cART) together with adjunctive "anti-inflammatory" drugs show no improvement over cART alone, suggesting existence of injury mechanisms in addition to inflammation. In our studies, oligodendrocytes exhibited rapid increases in intracellular Ca(2+) level upon HIV-1 transactivator of transcription (Tat) exposure. Thus, immature and mature oligodendrocytes can be direct targets of Tat. Since ionotropic glutamate receptor antagonists can partially or fully reverse the detrimental effects of Tat, glutamate receptors could be a potential therapeutic target for white matter damage in HIV patients.

Morphine Tolerance and Physical Dependence Are Altered in Conditional HIV-1 Tat Transgenic Mice.

Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1-related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)-inducible manner [Tat(+)], their Tat(-) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(-)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(-)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence.

HIV-1 Tat causes cognitive deficits and selective loss of parvalbumin, somatostatin, and neuronal nitric oxide synthase expressing hippocampal CA1 interneuron subpopulations.

Memory deficits are characteristic of HIV-associated neurocognitive disorders (HAND) and co-occur with hippocampal pathology. The HIV-1 transactivator of transcription (Tat), a regulatory protein, plays a significant role in these events, but the cellular mechanisms involved are poorly understood. Within the hippocampus, diverse populations of interneurons form complex networks; even subtle disruptions can drastically alter synaptic output, resulting in behavioral dysfunction. We hypothesized that HIV-1 Tat would impair cognitive behavior and injure specific hippocampal interneuron subtypes. Male transgenic mice that inducibly expressed HIV-1 Tat (or non-expressing controls) were assessed for cognitive behavior or had hippocampal CA1 subregions evaluated via interneuron subpopulation markers. Tat exposure decreased spatial memory in a Barnes maze and mnemonic performance in a novel object recognition test. Tat reduced the percentage of neurons expressing neuronal nitric oxide synthase (nNOS) without neuropeptide Y immunoreactivity in the stratum pyramidale and the stratum radiatum, parvalbumin in the stratum pyramidale, and somatostatin in the stratum oriens, which are consistent with reductions in interneuron-specific interneuron type 3 (IS3), bistratified, and oriens-lacunosum-moleculare interneurons, respectively. The findings reveal that an interconnected ensemble of CA1 nNOS-expressing interneurons, the IS3 cells, as well as subpopulations of parvalbumin- and somatostatin-expressing interneurons are preferentially vulnerable to HIV-1 Tat. Importantly, the susceptible interneurons form a microcircuit thought to be involved in feedback inhibition of CA1 pyramidal cells and gating of CA1 pyramidal cell inputs. The identification of vulnerable CA1 hippocampal interneurons may provide novel insight into the basic mechanisms underlying key functional and neurobehavioral deficits associated with HAND.

Asymmetry of the endogenous opioid system in the human anterior cingulate: a putative molecular basis for lateralization of emotions and pain.

Lateralization of the processing of positive and negative emotions and pain suggests an asymmetric distribution of the neurotransmitter systems regulating these functions between the left and right brain hemispheres. By virtue of their ability to selectively mediate euphoria, dysphoria, and pain, the µ-, δ-, and κ-opioid receptors and their endogenous ligands may subserve these lateralized functions. We addressed this hypothesis by comparing the levels of the opioid receptors and peptides in the left and right anterior cingulate cortex (ACC), a key area for emotion and pain processing. Opioid mRNAs and peptides and 5 "classical" neurotransmitters were analyzed in postmortem tissues from 20 human subjects. Leu-enkephalin-Arg (LER) and Met-enkephalin-Arg-Phe, preferential δ-/µ- and κ-/µ-opioid agonists, demonstrated marked lateralization to the left and right ACC, respectively. Dynorphin B (Dyn B) strongly correlated with LER in the left, but not in the right ACC suggesting different mechanisms of the conversion of this κ-opioid agonist to δ-/µ-opioid ligand in the 2 hemispheres; in the right ACC, Dyn B may be cleaved by PACE4, a proprotein convertase regulating left-right asymmetry formation. These findings suggest that region-specific lateralization of neuronal networks expressing opioid peptides underlies in part lateralization of higher functions, including positive and negative emotions and pain in the human brain.

Effects of HIV-1 Tat on enteric neuropathogenesis.

The gastrointestinal (GI) tract presents a major site of immune modulation by HIV, resulting in significant morbidity. Most GI processes affected during HIV infection are regulated by the enteric nervous system. HIV has been identified in GI histologic specimens in up to 40% of patients, and the presence of viral proteins, including the trans-activator of transcription (Tat), has been reported in the gut indicating that HIV itself may be an indirect gut pathogen. Little is known of how Tat affects the enteric nervous system. Here we investigated the effects of the Tat protein on enteric neuronal excitability, proinflammatory cytokine release, and its overall effect on GI motility. Direct application of Tat (100 nm) increased the number of action potentials and reduced the threshold for action potential initiation in isolated myenteric neurons. This effect persisted in neurons pretreated with Tat for 3 d (19 of 20) and in neurons isolated from Tat(+) (Tat-expressing) transgenic mice. Tat increased sodium channel isoforms Nav1.7 and Nav1.8 levels. This increase was accompanied by an increase in sodium current density and a leftward shift in the sodium channel activation voltage. RANTES, IL-6, and IL-1ß, but not TNF-α, were enhanced by Tat. Intestinal transit and cecal water content were also significantly higher in Tat(+) transgenic mice than Tat(-) littermates (controls). Together, these findings show that Tat has a direct and persistent effect on enteric neuronal excitability, and together with its effect on proinflammatory cytokines, regulates gut motility, thereby contributing to GI dysmotilities reported in HIV patients.

Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²âº overload.

Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the µ-opioid receptor antagonist CTAP and were not observed in neurons cultured from µ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via µ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of µ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

Cannabinoid receptor 1 positive allosteric modulator ZCZ011 shows differential effects on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

The cannabinoid receptor type 1 (CB1R) is a promising therapeutic target for various neurodegenerative diseases, including HIV-1-associated neurocognitive disorder (HAND). However, the therapeutic potential of CB1R by direct activation is limited due to its psychoactive side effects. Therefore, research has focused on indirectly activating the CB1R by utilizing positive allosteric modulators (PAMs). Studies have shown that CB1R PAMs (ZCZ011 and GAT211) are effective in mouse models of Huntington's disease and neuropathic pain, and hence, we assess the therapeutic potential of ZCZ011 in a well-established mouse model of neuroHIV. The current study investigates the effect of chronic ZCZ011 treatment (14 days) on various behavioral paradigms and the endocannabinoid system in HIV-1 Tat transgenic female and male mice. Chronic ZCZ011 treatment (10 mg/kg) did not alter body mass, locomotor activity, or anxiety-like behavior regardless of sex or genotype. However, differential effects were noted in hot plate latency, motor coordination, and recognition memory in female mice only, with ZCZ011 treatment increasing hot plate latency and improving motor coordination and recognition memory. Only minor effects or no alterations were seen in the endocannabinoid system and related lipids except in the cerebellum, where the effect of ZCZ011 was more pronounced in female mice. Moreover, AEA and PEA levels in the cerebellum were positively correlated with improved motor coordination in female mice. In summary, these findings indicate that chronic ZCZ011 treatment has differential effects on antinociception, motor coordination, and memory, based on sex and HIV-1 Tat expression, making CB1R PAMs potential treatment options for HAND without the psychoactive side effects.

Effects of acute Δ9-tetrahydrocannabinol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

Cannabis use is highly prevalent especially among people living with HIV (PLWH). Activation of the anti-inflammatory and neuroprotective endocannabinoid system by phytocannabinoids, i.e. Δ9-tetrahydrocannabinol (THC), has been proposed to reduce HIV symptoms. However, THC's effects on HIV-related memory deficits are unclear. Using HIV-1 Tat transgenic mice, the current study investigates acute THC effects on various behavioral outcomes and the endocannabinoid system. For the rodent tetrad model, THC doses (1, 3, 10 mg/kg) induced known antinociceptive effects, with Tat induction increasing antinociceptive THC effects at 3 and 10 mg/kg doses. Only minor or no effects were noted for acute THC on body temperature, locomotor activity, and coordination. Increased anxiety-like behavior was found for females compared to males, but acute THC had no effect on anxiety. Object recognition memory was diminished by acute THC in Tat(-) females but not Tat(+) females, without affecting males. The endocannabinoid system and related lipids were not affected by acute THC, except for THC-induced decreases in CB1R protein expression levels in the spinal cord of Tat(-) mice. Female sex and Tat induction was associated with elevated 2-AG, AEA, AA, CB1R, CB2R, FAAH and/or MAGL expression in various brain regions. Further, AEA levels in the prefrontal cortex of Tat(+) females were negatively associated with object recognition memory. Overall, findings indicate that acute THC exerts differential effects on antinociception and memory, dependent on sex and HIV Tat expression, potentially in relation to an altered endocannabinoid system, which may be of relevance in view of potential cannabis-based treatment options for PLWH.

A helping HAND: therapeutic potential of MAGL inhibition against HIV-1-associated neuroinflammation.

Background: Human immunodeficiency virus (HIV) affects nearly 40 million people globally, with roughly 80% of all people living with HIV receiving antiretroviral therapy. Antiretroviral treatment suppresses viral load in peripheral tissues but does not effectively penetrate the blood-brain barrier. Thus, viral reservoirs persist in the central nervous system and continue to produce low levels of inflammatory factors and early viral proteins, including the transactivator of transcription (Tat). HIV Tat is known to contribute to chronic neuroinflammation and synaptodendritic damage, which is associated with the development of cognitive, motor, and/or mood problems, collectively known as HIV-associated neurocognitive disorders (HAND). Cannabinoid anti-inflammatory effects are well documented, but therapeutic utility of cannabis remains limited due to its psychotropic effects, including alterations within brain regions encoding reward processing and motivation, such as the nucleus accumbens. Alternatively, inhibiting monoacylglycerol lipase (MAGL) has demonstrated therapeutic potential through interactions with the endocannabinoid system. Methods: The present study utilized a reward-related operant behavioral task to quantify motivated behavior in female Tat transgenic mice treated with vehicle or MAGL inhibitor MJN110 (1 mg/kg). Brain tissue was collected to assess dendritic injury and neuroinflammatory profiles, including dendritic microtubule-associated protein (MAP2ab) intensity, microglia density, microglia morphology, astrocyte density, astrocytic interleukin-1ß (IL-1ß) colocalization, and various lipid mediators. Results: No significant behavioral differences were observed; however, MJN110 protected against Tat-induced dendritic injury by significantly upregulating MAP2ab intensity in the nucleus accumbens and in the infralimbic cortex of Tat(+) mice. No or only minor effects were noted for Iba-1+ microglia density and/or microglia morphology. Further, Tat increased GFAP+ astrocyte density in the infralimbic cortex and GFAP+ astrocytic IL-1ß colocalization in the nucleus accumbens, with MJN110 significantly reducing these measures in Tat(+) subjects. Lastly, selected HETE-related inflammatory lipid mediators in the striatum were downregulated by chronic MJN110 treatment. Conclusions: These findings demonstrate anti-inflammatory and neuroprotective properties of MJN110 without cannabimimetic behavioral effects and suggest a promising alternative to cannabis for managing neuroinflammation.

Effects of acute cannabidiol on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

Background: Some evidence suggests that cannabidiol (CBD) has potential to help alleviate HIV symptoms due to its antioxidant and anti-inflammatory properties. Here we examined acute CBD effects on various behaviors and the endocannabinoid system in HIV Tat transgenic mice. Methods: Tat transgenic mice (female/male) were injected with CBD (3, 10, 30 mg/kg) and assessed for antinociception, activity, coordination, anxiety-like behavior, and recognition memory. Brains were taken to quantify endocannabinoids, cannabinoid receptors, and cannabinoid catabolic enzymes. Additionally, CBD and metabolite 7-hydroxy-CBD were quantified in the plasma and cortex. Results: Tat decreased supraspinal-related nociception and locomotion. CBD and sex had little to no effects on any of the behavioral measures. For the endocannabinoid system male sex was associated with elevated concentration of the proinflammatory metabolite arachidonic acid in various CNS regions, including the cerebellum that also showed higher FAAH expression levels for Tat(+) males. GPR55 expression levels in the striatum and cerebellum were higher for females compared to males. CBD metabolism was altered by sex and Tat expression. Conclusion: Findings indicate that acute CBD effects are not altered by HIV Tat, and acute CBD has no to minimal effects on behavior and the endocannabinoid system.

Acute Effects of Monoacylglycerol Lipase Inhibitor ABX1431 on Neuronal Hyperexcitability, Nociception, Locomotion, and the Endocannabinoid System in HIV-1 Tat Male Mice.

Background: Evidence suggests that monoacylglycerol lipase (MAGL) inhibitors can potentially treat HIV symptoms by increasing the concentration of 2-arachidonoylglycerol (2-AG). We examined a selective MAGL inhibitor ABX1431 in the context of neuroHIV. Methods: To assess the effects of ABX1431, we conducted in vitro and in vivo studies. In vitro calcium imaging on frontal cortex neuronal cultures was performed to evaluate the role of ABX1431 (10, 30, 100 nM) on transactivator of transcription (Tat)-induced neuronal hyperexcitability. Following in vitro experiments, in vivo experiments were performed using Tat transgenic male mice. Mice were treated with 4 mg/kg ABX1431 and assessed for antinociception using tail-flick and hot plate assays followed by locomotor activity. After the behavioral experiments, their brains were harvested to quantify endocannabinoids (eCB) and related lipids through mass spectrometry, and cannabinoid type-1 and -2 receptors (CB1R and CB2R) were quantified through western blot. Results: In vitro studies revealed that adding Tat directly to the neuronal cultures significantly increased intracellular calcium concentration, which ABX1431 completely reversed at all concentrations. Preincubating the cultures with CB1R and CB2R antagonists showed that ABX1431 exhibited its effects partially through CB1R. In vivo studies demonstrated that acute ABX1431 increased overall total distance traveled and speed of mice regardless of their genotype. Mass spectrometry and western blot analyses revealed differential effects on the eCB system based on Tat expression. The 2-AG levels were significantly upregulated following ABX1431 treatment in the striatum and spinal cord. Arachidonic acid (AA) was also upregulated in the striatum of vehicle-treated Tat(+) mice. No changes were noted in CB1R expression levels; however, CB2R levels were increased in ABX1431-treated Tat(-) mice only. Conclusion: Findings indicate that ABX1431 has potential neuroprotective effects in vitro partially mediated through CB1R. Acute treatment of ABX1431 in vivo shows antinociceptive effects, and seems to alter locomotor activity, with upregulating 2-AG levels in the striatum and spinal cord.

Role of adrenal glucocorticoid signaling in prefrontal cortex gene expression and acute behavioral responses to ethanol.

BACKGROUND: Glucocorticoid hormones modulate acute and chronic behavioral and molecular responses to drugs of abuse including psychostimulants and opioids. There is growing evidence that glucocorticoids might also modulate behavioral responses to ethanol ( EtOH ). Acute EtOH activates the hypothalamic-pituitary-adrenal axis, causing the release of adrenal glucocorticoid hormones. Our prior genomic studies suggest that glucocorticoids play a role in regulating gene expression in the prefrontal cortex (PFC) of DBA2/J (D2) mice following acute EtOH administration. However, few studies have analyzed the role of glucocorticoid signaling in behavioral responses to acute EtOH . Such work could be significant, given the predictive value for the level of response to acute EtOH in the risk for alcoholism. METHODS: We studied whether the glucocorticoid receptor (GR) antagonist, RU-486, or adrenalectomy (ADX) altered male D2 mouse behavioral responses to acute (locomotor activation, anxiolysis, or loss-of-righting reflex [LORR]) or repeated (sensitization) EtOH treatment. Whole-genome microarray analysis and bioinformatics approaches were used to identify PFC candidate genes possibly responsible for altered behavioral responses to EtOH following ADX. RESULTS: ADX and RU-486 both impaired acute EtOH (2 g/kg)-induced locomotor activation in D2 mice without affecting basal locomotor activity. However, neither ADX nor RU-486 altered the initiation of EtOH sensitization (locomotor activation or jump counts), EtOH -induced anxiolysis, or LORR. ADX mice showed microarray gene expression changes in PFC that significantly overlapped with acute EtOH -responsive gene sets derived by our prior microarray studies. Q-rtPCR analysis verified that ADX decreased PFC expression of Fkbp5 while significantly increasing Gpr6 expression. In addition, high-dose RU-486 pretreatment blunted EtOH -induced Fkbp5 expression. CONCLUSIONS: Our studies suggest that EtOH 's activation of adrenal glucocorticoid release and subsequent GR activation may partially modulate EtOH 's acute locomotor activation in male D2 mice. Furthermore, because adrenal glucocorticoid basal tone regulated PFC gene expression, including a significant set of acute EtOH -responsive genes, this suggests that glucocorticoid-regulated PFC gene expression may be an important factor modulating acute behavioral responses to EtOH .

The link between chronic cocaine use, B cell perturbations, and blunted immune recovery in HIV-infected individuals on suppressive ART.

Background: We recently reveal that anti-CD4 autoantibodies contribute to blunted CD4+ T cell reconstitution in HIV+ individuals on antiretroviral therapy (ART). Cocaine use is common among HIV+ individuals and is associated with accelerated disease progression. However, the mechanisms underlying cocaine-induced immune perturbations remain obscure. Methods: We evaluated plasma levels of anti-CD4 IgG and markers of microbial translocation, as well as B-cell gene expression profiles and activation in HIV+ chronic cocaine users and non-users on suppressive ART, as well as uninfected controls. Plasma purified anti-CD4 IgGs were assessed for antibody-dependent cytotoxicity (ADCC). Results: HIV+ cocaine users had increased plasma levels of anti-CD4 IgGs, lipopolysaccharide (LPS), and soluble CD14 (sCD14) versus non-users. An inverse correlation was observed in cocaine users, but not non-drug users. Anti-CD4 IgGs from HIV+ cocaine users mediated CD4+ T cell death through ADCC in vitro. B cells from HIV+ cocaine users exhibited activation signaling pathways and activation (cycling and TLR4 expression) related to microbial translocation versus non-users. Conclusions: This study improves our understanding of cocaine associated B cell perturbations and immune failure and the new appreciation for autoreactive B cells as novel therapeutic targets.

HIV-1 coinfection and morphine coexposure severely dysregulate hepatitis C virus-induced hepatic proinflammatory cytokine release and free radical production: increased pathogenesis coincides with uncoordinated host defenses.

Coinfection with human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) is a global problem that is more prevalent in injection drug users because they have a higher risk for acquiring both viruses. The roles of inflammatory cytokines and oxidative stress were examined in HIV-1- and HCV-coinfected human hepatic cells. Morphine (the bioactive product of heroin), HIV-1 Tat and the MN strain gp120 (gp120(MN)) proteins, and X4 HIV-1(LAI/IIIB) and R5 HIV-1(SF162) isolates were used to study the mechanisms of disease progression in HCV (JFH1)-infected Huh7.5.1 cell populations. HCV increased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release and augmented production of reactive oxygen species (ROS), nitric oxide (NO), and 3-nitrotyrosine (3-NT) in Huh7.5.1 cells. Morphine preferentially affected R5-tropic, but not X4-tropic, HIV-1 interactions with Huh7.5.1 cells. HIV-1 proteins or isolates increased cytokine release in HCV-infected cells, while adding morphine to coinfected cells caused complex imbalances, significantly disrupting cytokine secretion depending on the cytokine, morphine concentration, exposure duration, and particular pathogen involved. Production of ROS, NO, and 3-NT increased significantly in HCV- and HIV-1-coexposed cells while exposure to morphine further increased ROS. The proteasome inhibitor MG132 significantly decreased oxyradicals, cytokine levels, and HCV protein levels. Our findings indicate that hepatic inflammation is increased by combined exposure to HCV and HIV-1, that the ubiquitin-proteasome system and NF-κB contribute to key aspects of the response, and that morphine further exacerbates the disruption of host defenses. The results suggest that opioid abuse and HIV-1 coinfection each further accelerate HCV-mediated liver disease by dysregulating immune defenses.

Differential expression and HIV-1 regulation of µ-opioid receptor splice variants across human central nervous system cell types.

The µ-opioid receptor (MOR) is known to undergo extensive alternative splicing as numerous splice variants of MOR have been identified. However, the functional significance of MOR variants, as well as how splice variants other than MOR-1 might differentially regulate human immunodeficiency virus type-1 (HIV-1) pathogenesis in the central nervous system (CNS), or elsewhere, has largely been ignored. Our findings suggest that there are specific differences in the MOR variant expression profile among CNS cell types, and that the expression levels of these variants are differentially regulated by HIV-1. While MOR-1A mRNA was detected in astroglia, microglia, and neurons, MOR-1 and MOR-1X were only found in astroglia. Expression of the various forms of MOR along with the chimeric G protein qi5 in HEK-293T cells resulted in differences in calcium/NFAT signaling with morphine treatment, suggesting that MOR variant expression might underlie functional differences in MOR-effector coupling and intracellular signaling across different cell types. Furthermore, the data suggest that the expression of MOR-1 and other MOR variants may also be differentially regulated in the brains of HIV-infected subjects with varying levels of neurocognitive impairment. Overall, the results reveal an unexpected finding that MOR-1 may not be the predominant form of MOR expressed by some CNS cell types and that other splice variants of MOR-1, with possible differing functions, may contribute to the diversity of MOR-related processes in the CNS.

Morphine potentiates neurodegenerative effects of HIV-1 Tat through actions at µ-opioid receptor-expressing glia.

Individuals infected with human immunodeficiency virus-1 who abuse opiates can have a higher incidence of virus-associated neuropathology. Human immunodeficiency virus does not infect neurons, but viral proteins such as transactivator of transcription and glycoprotein 120, originating from infected glia, are neurotoxic. Moreover, functional changes in glial cells that enhance inflammation and reduce trophic support are increasingly implicated in human immunodeficiency virus neuropathology. In previous studies, co-exposure with morphine enhanced transactivator of transcription neurotoxicity towards cultured striatal neurons. Since those cultures contained µ-opioid receptor-expressing astroglia and microglia, and since glia are the principal site of infection in the central nervous system, we hypothesized that morphine synergy might be glially mediated. A 60 hour, repeated measures paradigm and multiple co-culture models were used to investigate the cellular basis for opiate-enhanced human immunodeficiency virus neurotoxicity. Morphine co-exposure significantly enhanced transactivator of transcription-induced neuron death when glia were present. Synergistic effects of morphine on transactivator of transcription neurotoxicity were greatest with neuron-glia contact, but also occurred to a lesser extent with glial conditioned medium. Importantly, synergy was lost if glia, but not neurons, lacked µ-opioid receptors, indicating that opiate interactions with human immunodeficiency virus converge at the level of µ-opioid receptor-expressing glia. Morphine enhanced transactivator of transcription-induced inflammatory effectors released by glia, elevating reactive oxygen species, increasing 3-nitrotyrosine production by microglia, and reducing the ability of glia to buffer glutamate. But neuron survival was reduced even more with glial contact than with exposure to conditioned medium, suggesting that noxious elements associated with cell contact augment the toxicity due to soluble factors. Similar morphine-transactivator of transcription synergy was also observed in studies with the clade C sequence of HIV-1 transactivator of transcription, which did not cause neuron death unless morphine was present. Several paradoxical observations related to opiate effects were noted when µ-opioid receptors were specifically ablated from either glia or neurons. This suggests that µ-opioid receptor loss in isolated cell types can fundamentally distort cell-to-cell signalling, revealing opponent processes that may exist in individual cell types. Our findings show the critical role of glia in orchestrating neurotoxic interactions of morphine and transactivator of transcription, and support the emerging concept that combined exposure to opiates and human immunodeficiency virus drives enhanced pathology within the central nervous system.

Oral Enrichment of Streptococcus and its Role in Systemic Inflammation Related to Monocyte Activation in Humans with Cocaine Use Disorder.

Cocaine use is commonly associated with increased chronic systemic inflammation. However, the drivers for cocaine use-mediated systemic inflammation are not fully understood. In the current study, we recruited individuals with cocaine use disorder and healthy individuals who did not use cocaine and collected paired saliva and blood samples. The saliva samples were used to assess the oral microbiome, and the plasma samples were evaluated for 33 cytokines and chemokines. Cocaine users exhibited decreased saliva microbial diversities compared to non-users. Streptococcus was the only increased genus in the saliva from cocaine users, whereas several genera were decreased in cocaine users compared to non-users. Notably, cocaine users exhibited increased plasma levels of several monocyte activation markers, including monocyte chemoattractant protein (MCP)-4, macrophage inflammatory protein (MIP)-3α, macrophage-derived chemokine (MDC), and thymus and activation-regulated chemokine (TARC), all of which were correlated with increased saliva levels of three Streptococcus species. Furthermore, treatment with Streptococcus or its lipoteichoic acid preferentially activated primary human monocytes to produce proinflammatory cytokines and chemokines, such as MIP-3α and TARC, in vitro compared to controls. However, monocytes failed to produce these chemokines after exposure to cocaine or cocaine plus bacteria compared to medium or bacteria alone. This study revealed that chronic cocaine use-associated inflammation in the blood may result from increased oral Streptococcus and its effects on myeloid cell activation, but does not result from cocaine directly.