RESUMEN
Besides having high potency and efficacy at the µ- (MOR) and other opioid receptor types, fentanyl has some affinity for some adrenergic receptor types, which may underlie its unique pathophysiological differences from typical opioids. To better understand the unique actions of fentanyl, we assessed the extent to which fentanyl alters striatal medium spiny neuronal (MSNs) activity via opioid or α1 adrenoceptors in dopamine type 1 or type 2 receptor- (D1 or D2) -expressing MSNs. In neuronal and mixed-glial co-cultures from the striatum, acute fentanyl (100 nM) exposure decreased the frequency of spontaneous action potentials. Overnight exposure of co-cultures to 100 nM fentanyl severely reduced the proportion of MSNs with spontaneous action potentials, which was unaffected by co-exposure to the opioid receptor antagonist naloxone (10 µM), but fully negated by co-administering the pan-α1 adrenoceptor inverse agonist prazosin (100 nM) and partially reversed by the selective α1A/C adrenoceptor antagonist RS 100329 (300 nM). Acute fentanyl (100 nM) exposure modestly reduced the frequency of action potentials and caused firing rate adaptations in D2, but not D1, MSNs. Prolonged (2-5 h) fentanyl (100 nM) application dramatically attenuated firing rates in both D1 and D2 MSNs. To identify possible cellular sites of α1 adrenoceptor action, α1 adrenoceptors were localized in subpopulations of striatal astroglia and neurons by immunocytochemistry, and Adra1a mRNA by in situ hybridization in astrocytes. Thus, sustained fentanyl exposure can inhibit striatal MSN activity via a non-opioid receptor-dependent pathway, that may be modulated via complex actions in α1 adrenoceptor-expressing striatal neurons and/or glia.
RESUMEN
Despite the advent of combination anti-retroviral therapy (cART), nearly half of people infected with HIV treated with cART still exhibit HIV-associated neurocognitive disorders (HAND). HAND can be worsened by co-morbid opioid use disorder. The basal ganglia are particularly vulnerable to HIV-1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co-exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co-exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety-like, novelty-seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5-hydroxyindoleacetic acid, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase-positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context-dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty-seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time-dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV-1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.
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Infecciones por VIH , VIH-1 , Humanos , Ratones , Animales , Morfina/farmacología , Conducta Exploratoria , VIH-1/metabolismo , Dopamina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Ratones Transgénicos , Analgésicos Opioides/farmacología , Ácido Homovanílico , Neurotransmisores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.
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Barrera Hematoencefálica , Fentanilo , VIH-1 , Ratones Transgénicos , Enfermedades Neuroinflamatorias , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/virología , Ratones , Fentanilo/farmacología , VIH-1/efectos de los fármacos , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Enfermedades Neuroinflamatorias/genética , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/virología , Infecciones por VIH/virología , Infecciones por VIH/genética , Infecciones por VIH/patología , Infecciones por VIH/tratamiento farmacológico , Modelos Animales de Enfermedad , Analgésicos Opioides/farmacología , Analgésicos Opioides/efectos adversos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/genética , Humanos , Encéfalo/efectos de los fármacos , Encéfalo/virología , Encéfalo/metabolismo , Encéfalo/patología , Trastornos Relacionados con Opioides/genética , Trastornos Relacionados con Opioides/patología , Trastornos Relacionados con Opioides/metabolismoRESUMEN
HIV-associated neurocognitive disorders (HAND) remain pervasive even with increased efficacy/use of antiretroviral therapies. Opioid use/abuse among HIV + individuals is documented to exacerbate CNS deficits. White matter (WM) alterations, including myelin pallor, and volume/structural alterations detected by diffusion tensor imaging are common observations in HIV + individuals, and studies in non-human primates suggest that WM may harbor virus. Using transgenic mice that express the HIV-1 Tat protein, we examined in vivo effects of 2-6 weeks of Tat and morphine exposure on WM using genomic and biochemical methods. RNA sequencing of striatal tissue at 2 weeks revealed robust changes in mRNAs associated with oligodendrocyte precursor populations and myelin integrity, including those for transferrin, the atypical oligodendrocyte marker N-myc downstream regulated 1 (Ndrg1), and myelin regulatory factor (Myrf/Mrf), an oligodendrocyte-specific transcription factor with a significant role in oligodendrocyte differentiation/maturation. Western blots conducted after 6-weeks exposure in 3 brain regions (striatum, corpus callosum, pre-frontal cortex) revealed regional differences in the effect of Tat and morphine on Myrf levels, and on levels of myelin basic protein (MBP), whose transcription is regulated by Myrf. Responses included individual and interactive effects. Although baseline and post-treatment levels of Myrf and MBP differed between brain regions, post-treatment MBP levels in striatum and pre-frontal cortex were compatible with changes in Myrf activity. Additionally, the Myrf regulatory ubiquitin ligase Fbxw7 was identified as a novel target in our model. These results suggest that Myrf and Fbxw7 contribute to altered myelin gene regulation in HIV.
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Infecciones por VIH , VIH-1 , Animales , Ratones , Imagen de Difusión Tensora , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Lóbulo Frontal/metabolismo , VIH-1/metabolismo , Ratones Transgénicos , Morfina , Factores de Transcripción/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The striatum is especially vulnerable to HIV-1 infection, with medium spiny neurons (MSNs) exhibiting marked synaptodendritic damage that can be exacerbated by opioid use disorder. Despite known structural defects in MSNs co-exposed to HIV-1 Tat and opioids, the pathophysiological sequelae of sustained HIV-1 exposure and acute comorbid effects of opioids on dopamine D1 and D2 receptor-expressing (D1 and D2) MSNs are unknown. To address this question, Drd1-tdTomato- or Drd2-eGFP-expressing reporter and conditional HIV-1 Tat transgenic mice were interbred. MSNs in ex vivo slices from male mice were assessed by whole-cell patch-clamp electrophysiology and filled with biocytin to explore the functional and structural effects of progressive Tat and acute morphine exposure. Although the excitability of both D1 and D2 MSNs increased following 48 h of Tat exposure, D1 MSN firing rates decreased below control (Tat-) levels following 2 weeks and 1 month of Tat exposure but returned to control levels after 2 months. D2 neurons continued to display Tat-dependent increases in excitability at 2 weeks, but also returned to control levels following 1 and 2 months of Tat induction. Acute morphine exposure increased D1 MSN excitability irrespective of the duration of Tat exposure, while D2 MSNs were variably affected. That D1 and D2 MSN excitability would return to control levels was unexpected since both subpopulations displayed significant synaptodendritic degeneration and pathologic phospho-tau-Thr205 accumulation following 2 months of Tat induction. Thus, despite frank morphologic damage, D1 and D2 MSNs uniquely adapt to sustained Tat and acute morphine insults.
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Dopamina , VIH-1 , Animales , Masculino , Ratones , Analgésicos Opioides/farmacología , Cuerpo Estriado/patología , VIH-1/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfina/farmacología , Neuronas/metabolismo , Receptores de Dopamina D1/metabolismoRESUMEN
Dynamic chloride (Cl-) regulation is critical for synaptic inhibition. In mature neurons, Cl- influx and extrusion are primarily controlled by ligand-gated anion channels (GABAA and glycine receptors) and the potassium chloride cotransporter K+-Cl- cotransporter 2 (KCC2), respectively. Here, we report for the first time, to our knowledge, a presence of a new source of Cl- influx in striatal neurons with properties similar to chloride voltage-gated channel 1 (ClC-1). Using whole cell patch-clamp recordings, we detected an outwardly rectifying voltage-dependent current that was impermeable to the large anion methanesulfonate (MsO-). The anionic current was sensitive to the ClC-1 inhibitor 9-anthracenecarboxylic acid (9-AC) and the nonspecific blocker phloretin. The mean fractions of anionic current inhibition by MsO-, 9-AC, and phloretin were not significantly different, indicating that anionic current was caused by active ClC-1-like channels. In addition, we found that Cl- current was not sensitive to the transmembrane protein 16A (TMEM16A; Ano1) inhibitor Ani9 and that the outward Cl- rectification was preserved even at a very high intracellular Ca2+ concentration (2 mM), indicating that TMEM16B (Ano2) did not contribute to the total current. Western blotting and immunohistochemical analyses confirmed the presence of ClC-1 channels in the striatum mainly localized to the somata of striatal neurons. Finally, we found that 9-AC decreased action potential firing frequencies and increased excitability in medium spiny neurons (MSNs) expressing dopamine type 1 (D1) and type 2 (D2) receptors in the brain slices, respectively. We conclude that ClC-1-like channels are preferentially located at the somata of MSNs, are functional, and can modulate neuronal excitability.
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Cloruros , Cuerpo Estriado , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Cuerpo Estriado/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp , Floretina/metabolismo , Floretina/farmacología , Receptores de Dopamina D2/metabolismoRESUMEN
Despite effective antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are found in nearly one-third of patients. Using a cellular co-culture system including neurons and human microglia infected with HIV (hµglia/HIV), we investigated the hypothesis that HIV-dependent neurological degeneration results from the periodic emergence of HIV from latency within microglial cells in response to neuronal damage or inflammatory signals. When a clonal hµglia/HIV population (HC69) expressing HIV, or HIV infected human primary and iPSC-derived microglial cells, were cultured for a short-term (24 h) with healthy neurons, HIV was silenced. The neuron-dependent induction of latency in HC69 cells was recapitulated using induced pluripotent stem cell (iPSC)-derived GABAergic cortical (iCort) and dopaminergic (iDopaNer), but not motor (iMotorNer), neurons. By contrast, damaged neurons induce HIV expression in latently infected microglial cells. After 48-72 h co-culture, low levels of HIV expression appear to damage neurons, which further enhances HIV expression. There was a marked reduction in intact dendrites staining for microtubule associated protein 2 (MAP2) in the neurons exposed to HIV-expressing microglial cells, indicating extensive dendritic pruning. To model neurotoxicity induced by methamphetamine (METH), we treated cells with nM levels of METH and suboptimal levels of poly (I:C), a TLR3 agonist that mimics the effects of the circulating bacterial rRNA found in HIV infected patients. This combination of agents potently induced HIV expression, with the METH effect mediated by the σ1 receptor (σ1R). In co-cultures of HC69 cells with iCort neurons, the combination of METH and poly(I:C) induced HIV expression and dendritic damage beyond levels seen using either agent alone, Thus, our results demonstrate that the cross-talk between healthy neurons and microglia modulates HIV expression, while HIV expression impairs this intrinsic molecular mechanism resulting in the excessive and uncontrolled stimulation of microglia-mediated neurotoxicity.
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Infecciones por VIH/metabolismo , VIH-1/patogenicidad , Microglía/virología , Neuronas/virología , Células Cultivadas , Técnicas de Cocultivo/métodos , Citocinas/metabolismo , VIH-1/genética , Humanos , Microglía/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Many persons infected with HIV-1 (PWH) and opioid-dependent individuals experience deficits in sociability that interfere with daily living. Sociability is regulated by the prefrontal cortico-hippocampal-amygdalar circuit. Within this circuit HIV-1 trans-activator of transcription (HIV-1 Tat) and opioids can increase dendritic pathology and alter neuronal firing. Changes in sociability are also associated with dysregulation of hypothalamic neuropeptides such as oxytocin or corticotropin releasing factor (CRF) in the prefrontal cortico-hippocampal-amygdalar circuit. Accordingly, we hypothesized that the interaction of HIV-1 Tat and morphine would impair inter-male social interactions and disrupt oxytocin and CRF within the PFC and associated circuitry. Male mice were exposed to HIV-1 Tat for 8 weeks and administered saline or escalating doses of morphine twice daily (s.c.) during the last 2 weeks of HIV-1 Tat exposure. Tat attenuated aggressive interactions with an unknown intruder, whereas morphine decreased both non-aggressive and aggressive social interactions in the resident-intruder test. However, there was no effect of Tat or morphine on non-reciprocal interactions in the social interaction and novelty tests. Tat, but not morphine, decreased oxytocin levels in the PFC and amygdala, whereas both Tat and morphine decreased the percentage of oxytocin-immunoreactive neurons in the hypothalamic paraventricular nucleus (PVN). In Tat(+) or morphine-exposed mice, regional levels of CRF and oxytocin correlated with alterations in behavior in the social interaction and novelty tests. Overall, decreased expression of oxytocin in the prefrontal cortico-hippocampal-amygdalar circuit is associated with morphine- and HIV-Tat-induced deficits in social behavior.
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VIH-1 , Morfina , Amígdala del Cerebelo/metabolismo , Animales , Masculino , Ratones , Morfina/farmacología , Oxitocina , Núcleo Hipotalámico Paraventricular/metabolismo , Corteza Prefrontal/metabolismo , Interacción Social , Transactivadores , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Approximately half of people infected with HIV (PWH) exhibit HIV-associated neuropathology (neuroHIV), even when receiving combined antiretroviral therapy. Opiate use is widespread in PWH and exacerbates neuroHIV. While neurons themselves are not infected, they incur sublethal damage and GABAergic disruption is selectively vulnerable to viral and inflammatory factors released by infected/affected glia. Here, we demonstrate diminished K+-Cl- cotransporter 2 (KCC2) levels in primary human neurons after exposure to HIV-1 or HIV-1 proteins ± morphine. Resulting disruption of GABAAR-mediated hyperpolarization/inhibition was shown using genetically-encoded voltage (Archon1) and calcium (GCaMP6f) indicators. The HIV proteins Tat (acting through NMDA receptors) and R5-gp120 (acting via CCR5) but not X4-tropic gp120 (acting via CXCR4), and morphine (acting through µ-opioid receptors) all induced KCC2 loss. We demonstrate that modifying KCC2 levels or function, or antagonizing NMDAR, CCR5 or MOR rescues KCC2 and GABAAR-mediated hyperpolarization/inhibition in HIV, Tat, or gp120 ± morphine-exposed neurons. Using an inducible, Tat-transgenic mouse neuroHIV model, we found that chronic exposure to Tat also reduces KCC2. Our results identify KCC2 as a novel therapeutic target for ameliorating the pathobiology of neuroHIV, including PWH exposed to opiates.
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Analgésicos Opioides/administración & dosificación , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/administración & dosificación , Morfina/administración & dosificación , Neuronas/efectos de los fármacos , Neuronas/virología , Simportadores/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Humanos , Masculino , Ratones Transgénicos , Células-Madre Neurales/efectos de los fármacos , Neuronas/metabolismo , Cotransportadores de K ClRESUMEN
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.
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Aminoácidos/metabolismo , Endocannabinoides/metabolismo , Mediadores de Inflamación/metabolismo , Metabolismo de los Lípidos/fisiología , Morfina/administración & dosificación , Neuroprotección/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesis , Analgésicos Opioides/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Mediadores de Inflamación/antagonistas & inhibidores , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroprotección/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Dimensión del Dolor/métodos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Myelin disruptions are frequently reported in human immunodeficiency virus (HIV)-infected individuals and can occur in the CNS very early in the disease process. Immature oligodendrocytes (OLs) are quite sensitive to toxic increases in [Ca2+ ]i caused by exposure to HIV-1 Tat (transactivator of transcription, a protein essential for HIV replication and gene expression), but sensitivity to Tat-induced [Ca2+ ]i is reduced in mature OLs. Tat exposure also increased the activity of Ca2+ /calmodulin-dependent kinase IIß (CaMKIIß), the major isoform of CaMKII expressed by OLs, in both immature and mature OLs. Since CaMKIIß is reported to interact with glycogen synthase kinase 3ß (GSK3ß), and GSK3ß activity is implicated in OL apoptosis as well as HIV neuropathology, we hypothesized that disparate effects of Tat on OL viability with maturity might be because of an altered balance of CaMKIIß-GSK3ß activities. Tat expression in vivo led to increased CaMKIIß and GSK3ß activity in multiple brain regions in transgenic mice. In vitro, immature murine OLs expressed higher levels of GSK3ß, but much lower levels of CaMKIIß, than did mature OLs. Exogenous Tat up-regulated GSK3ß activity in immature, but not mature, OLs. Tat-induced death of immature OLs was rescued by the GSK3ß inhibitors valproic acid or SB415286, supporting involvement of GSK3ß signaling. Pharmacologically inhibiting CaMKIIß increased GSK3ß activity in Tat-treated OLs, and genetically knocking down CaMKIIß promoted death in mature OL cultures treated with Tat. Together, these results suggest that the effects of Tat on OL viability are dependent on CaMKIIß-GSK3ß interactions, and that increasing CaMKIIß activity is a potential approach for limiting OL/myelin injury with HIV infection.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Infecciones por VIH/metabolismo , Oligodendroglía/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Supervivencia Celular , Infecciones por VIH/patología , VIH-1 , Ratones , Ratones Transgénicos , Oligodendroglía/patologíaRESUMEN
Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. Tat and morphine effects on the leakage of fluorescently labeled dextrans (10-, 40-, and 70-kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat- mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. Morphine exposure, and, to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within the striatum and significantly less dolutegravir within the hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.
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Fármacos Anti-VIH/farmacocinética , Barrera Hematoencefálica/efectos de los fármacos , VIH-1/genética , Morfina/efectos adversos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/virología , Permeabilidad Capilar , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/virología , Dextranos/farmacocinética , Didesoxinucleósidos/farmacocinética , Femenino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Infecciones por VIH/metabolismo , Infecciones por VIH/psicología , Infecciones por VIH/virología , VIH-1/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacocinética , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/virología , Lamivudine/farmacocinética , Ratones , Ratones Transgénicos , Modelos Biológicos , Trastornos Neurocognitivos/metabolismo , Trastornos Neurocognitivos/psicología , Trastornos Neurocognitivos/virología , Oxazinas , Piperazinas , Piridonas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans-activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a-tdTomato- or Drd2-eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200-300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light-dark transition task, a greater frequency of light-dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1.SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30-50% of HIV-infected individuals and synaptodendritic injury remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans-activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1.
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Conducta Animal/fisiología , Cuerpo Estriado/metabolismo , Locomoción/fisiología , Receptores de Dopamina D1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Espinas Dendríticas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Receptores de Dopamina D2 , Distribución Tisular , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
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.
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Analgésicos Opioides/farmacología , Regulación de la Expresión Génica/genética , Neuroglía/metabolismo , Alcaloides Opiáceos/farmacología , Receptores CCR5/deficiencia , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Complejo SIDA Demencia , Animales , Antagonistas de los Receptores CCR5/farmacología , Cuerpo Estriado/citología , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Maraviroc/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Neuroglía/efectos de los fármacos , Neuronas/fisiología , Alcaloides Opiáceos/metabolismo , Receptores CCR5/genética , Receptores Opioides mu/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The immunomodulatory prodrug 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), which acts as an agonist for sphingosine-1-phosphate (S1P) receptors (S1PR) when phosphorylated, is proposed as a novel pain therapeutic. In this study, we assessed FTY720-mediated antinociception in the radiant heat tail-flick test and in the chronic constriction injury (CCI) model of neuropathic pain in mice. FTY720 produced antinociception and antiallodynia, respectively, and these effects were dose-dependent and mimicked by the S1PR1-selective agonist CYM-5442. Repeated administration of FTY720 for 1 week produced tolerance to acute thermal antinociception, but not to antiallodynia in the CCI model. S1PR-stimulated [35S]GTPγS autoradiography revealed apparent desensitization of G protein activation by S1P or the S1PR1 agonist 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole (SEW-2871) throughout the brain. Similar results were seen in spinal cord membranes, whereby the Emax value of S1PR-stimulated [35S]GTPγS binding was greatly reduced in repeated FTY720-treated mice. These results suggest that S1PR1 is a primary target of FTY720 in alleviating both acute thermal nociception and chronic neuropathic nociception. Furthermore, the finding that tolerance develops to antinociception in the tail-flick test but not in chronic neuropathic pain suggests a differential mechanism of FTY720 action between these models. The observation that repeated FTY720 administration led to desensitized S1PR1 signaling throughout the central nervous system suggests the possibility that S1PR1 activation drives the acute thermal antinociceptive effects, whereas S1PR1 desensitization mediates the following: 1) tolerance to thermal antinociceptive actions of FTY720 and 2) the persistent antiallodynic effects of FTY720 in neuropathic pain by producing functional antagonism of pronociceptive S1PR1 signaling.
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Clorhidrato de Fingolimod/farmacología , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Péptidos Opioides/efectos de los fármacos , Receptores de Lisoesfingolípidos/metabolismo , Temperatura , Animales , Modelos Animales de Enfermedad , Clorhidrato de Fingolimod/uso terapéutico , Masculino , Ratones , Ratones Endogámicos ICR , Neuralgia/fisiopatología , Receptores de Lisoesfingolípidos/agonistas , NociceptinaRESUMEN
Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a "leaky" gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long-term morphine treatment (in vivo) but not upon direct exposure of glia to morphine (in vitro). LPS, a major bacterial product, also increased ATP-induced currents, as well as expression of P2X4, P2X7, IL6, IL-1ß mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS-induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine-induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine-related inflammation and constipation.-Bhave, S., Gade, A., Kang, M., Hauser, K. F., Dewey, W. L., Akbarali, H. I. Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.
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Conexina 43/metabolismo , Estreñimiento/inducido químicamente , Morfina/farmacología , Neuroglía/fisiología , Receptores Purinérgicos P2X/metabolismo , Transducción de Señal/efectos de los fármacos , Adenosina Trifosfato , Analgésicos Opioides/farmacología , Animales , Fenómenos Electrofisiológicos , Regulación de la Expresión Génica , Intestinos/efectos de los fármacos , Intestinos/fisiología , Lipopolisacáridos/toxicidad , Masculino , Potenciales de la Membrana , Ratones , ARN Mensajero , Receptores Purinérgicos P2X/genéticaRESUMEN
The HIV-1 regulatory protein, trans-activator of transcription (Tat), interacts with opioids to potentiate neuroinflammation and neurodegeneration within the CNS. These effects may involve the C-C chemokine receptor type 5 (CCR5); however, the behavioral contribution of CCR5 on Tat/opioid interactions is not known. Using a transgenic murine model that expresses HIV-1 Tat protein in a GFAP-regulated, doxycycline-inducible manner, we assessed morphine tolerance, dependence, and reward. To assess the influence of CCR5 on these effects, mice were pretreated with oral vehicle or the CCR5 antagonist, maraviroc, prior to morphine administration. We found that HIV-1 Tat expression significantly attenuated the antinociceptive potency of acute morphine (2-64â¯mg/kg, i.p.) in non-tolerant mice. Consistent with this, Tat attenuated withdrawal symptoms among morphine-tolerant mice. Pretreatment with maraviroc blocked the effects of Tat, reinstating morphine potency in non-tolerant mice and restoring withdrawal symptomology in morphine-tolerant mice. Twenty-four hours following morphine administration, HIV-1 Tat significantly potentiated (â¼3.5-fold) morphine-conditioned place preference and maraviroc further potentiated these effects (â¼5.7-fold). Maraviroc exerted no measurable behavioral effects on its own. Protein array analyses revealed only minor changes to cytokine profiles when morphine was administered acutely or repeatedly; however, 24â¯h post morphine administration, the expression of several cytokines was greatly increased, including endogenous CCR5 chemokine ligands (CCL3, CCL4, and CCL5), as well as CCL2. Tat further elevated levels of several cytokines and maraviroc pretreatment attenuated these effects. These data demonstrate that CCR5 mediates key aspects of HIV-1 Tat-induced alterations in the antinociceptive potency and rewarding properties of opioids.
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Analgésicos Opioides/farmacología , Tolerancia a Medicamentos/fisiología , Inflamación/metabolismo , Morfina/farmacología , Receptores CCR5/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Antagonistas de los Receptores CCR5/farmacología , Núcleo Caudado/metabolismo , Condicionamiento Operante/efectos de los fármacos , Citocinas/metabolismo , Inflamación/inducido químicamente , Inflamación/genética , Masculino , Maraviroc/farmacología , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Recompensa , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human immunodeficiency (HIV) infection results in neurocognitive deficits in about one half of infected individuals. Despite systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting central nervous system (CNS)-localized infection. Drug abuse exacerbates HIV-induced cognitive and pathological CNS changes. This study's purpose was to investigate the effects of the HIV-1 protein Tat and methamphetamine on factors affecting drug penetration across an in vitro BBB model. Factors affecting paracellular and transcellular flux in the presence of Tat and methamphetamine were examined. Transendothelial electrical resistance, ZO-1 expression, and lucifer yellow (a paracellular tracer) flux were aspects of paracellular processes that were examined. Additionally, effects on P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP-1) mRNA (via quantitative PCR [qPCR]) and protein (via immunoblotting) expression were measured; Pgp and MRP-1 are drug efflux proteins. Transporter function was examined after exposure of Tat with or without methamphetamine using the P-gp substrate rhodamine 123 and also using the dual P-gp/MRP-1 substrate and protease inhibitor atazanavir. Tat and methamphetamine elicit complex changes affecting transcellular and paracellular transport processes. Neither Tat nor methamphetamine significantly altered P-gp expression. However, Tat plus methamphetamine exposure significantly increased rhodamine 123 accumulation within brain endothelial cells, suggesting that treatment inhibited or impaired P-gp function. Intracellular accumulation of atazanavir was not significantly altered after Tat or methamphetamine exposure. Atazanavir accumulation was, however, significantly increased by simultaneous inhibition of P-gp and MRP. Collectively, our investigations indicate that Tat and methamphetamine alter aspects of BBB integrity without affecting net flux of paracellular compounds. Tat and methamphetamine may also affect several aspects of transcellular transport.
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Barrera Hematoencefálica/metabolismo , Metanfetamina/farmacología , Rodaminas/metabolismo , Migración Transendotelial y Transepitelial/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Sulfato de Atazanavir/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Línea Celular , Disfunción Cognitiva/virología , Infecciones por VIH/patología , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Rodaminas/farmacología , Migración Transendotelial y Transepitelial/fisiología , Proteína de la Zonula Occludens-1/biosíntesisRESUMEN
The secreted protein, YKL-40, has been proposed as a biomarker of a variety of human diseases characterized by ongoing inflammation, including chronic neurologic pathologies such as multiple sclerosis and Alzheimer's disease. However, inflammatory mediators and the molecular mechanism responsible for enhanced expression of YKL-40 remained elusive. Using several mouse models of inflammation, we now show that YKL-40 expression correlated with increased expression of both IL-1 and IL-6. Furthermore, IL-1 together with IL-6 or the IL-6 family cytokine, oncostatin M, synergistically upregulated YKL-40 expression in both primary human and mouse astrocytes in vitro. The robust cytokine-driven expression of YKL-40 in astrocytes required both STAT3 and NF-κB binding elements of the YKL-40 promoter. In addition, YKL-40 expression was enhanced by constitutively active STAT3 and inhibited by dominant-negative IκBα. Surprisingly, cytokine-driven expression of YKL-40 in astrocytes was independent of the p65 subunit of NF-κB and instead required subunits RelB and p50. Mechanistically, we show that IL-1-induced RelB/p50 complex formation was further promoted by oncostatin M and that these complexes directly bound to the YKL-40 promoter. Moreover, we found that expression of RelB was strongly upregulated during inflammation in vivo and by IL-1 in astrocytes in vitro. We propose that IL-1 and the IL-6 family of cytokines regulate YKL-40 expression during sterile inflammation via both STAT3 and RelB/p50 complexes. These results suggest that IL-1 may regulate the expression of specific anti-inflammatory genes in nonlymphoid tissues via the canonical activation of the RelB/p50 complexes.
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Adipoquinas/genética , Citocinas/farmacología , Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Lectinas/genética , Subunidad p50 de NF-kappa B/metabolismo , Factor de Transcripción ReIB/metabolismo , Adipoquinas/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Proteína 1 Similar a Quitinasa-3 , Citocinas/genética , Femenino , Glicoproteínas/metabolismo , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-1/genética , Interleucina-1/farmacología , Interleucina-6/genética , Interleucina-6/farmacología , Lectinas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Subunidad p50 de NF-kappa B/genética , Oncostatina M/farmacología , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción ReIB/genéticaRESUMEN
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, â¼60% 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.