Morphine disrupts macrophage functions even during HIV infection.

HIV-associated neurocognitive impairment (HIV-NCI) is a debilitating comorbidity that reduces quality of life in 15-40% of people with HIV (PWH) taking antiretroviral therapy (ART). Opioid use has been shown to increase neurocognitive deficits in PWH. Monocyte-derived macrophages (MDMs) harbor HIV in the CNS even in PWH on ART. We hypothesized that morphine (MOR), a metabolite of heroin, further dysregulates functional processes in MDMs to increase neuropathogenesis. We found that, in uninfected and HIV-infected primary human MDMs, MOR activates these cells by increasing phagocytosis and up-regulating reactive oxygen species. Effects of MOR on phagocytosis were dependent on µ-opioid receptor activity and were mediated, in part, by inhibited lysosomal degradation of phagocytized substrates. All results persisted when cells were treated with both MOR and a commonly prescribed ART cocktail, suggesting minimal impact of ART during opioid exposure. We then performed mass spectrometry in HIV-infected MDMs treated with or without MOR to determine proteomic changes that suggest additional mechanisms by which opioids affect macrophage homeostasis. Using downstream pathway analyses, we found that MOR dysregulates ER quality control and extracellular matrix invasion. Our data indicate that MOR enhances inflammatory functions and impacts additional cellular processes in HIV-infected MDMs to potentially increases neuropathogenesis in PWH using opioids.

Treatment with buprenorphine prior to EcoHIV infection of mice prevents the development of neurocognitive impairment.

Approximately 15-40% of people living with HIV develop HIV-associated neurocognitive disorders, HAND, despite successful antiretroviral therapy. There are no therapies to treat these disorders. HIV enters the CNS early after infection, in part by transmigration of infected monocytes. Currently, there is a major opioid epidemic in the United States. Opioid use disorder in the context of HIV infection is important because studies show that opioids exacerbate HIV-mediated neuroinflammation that may contribute to more severe cognitive deficits. Buprenorphine is an opioid derivate commonly prescribed for opiate agonist treatment. We used the EcoHIV mouse model to study the effects of buprenorphine on cognitive impairment and to correlate these with monocyte migration into the CNS. We show that buprenorphine treatment prior to mouse EcoHIV infection prevents the development of cognitive impairment, in part, by decreased accumulation of monocytes in the brain. We propose that buprenorphine has a novel therapeutic benefit of limiting the development of neurocognitive impairment in HIV-infected opioid abusers as well as in nonabusers, in addition to decreasing the use of harmful opioids. Buprenorphine may also be used in combination with HIV prevention strategies such as pre-exposure prophylaxis because of its safety profile.

The Effects of Opioids on HIV Neuropathogenesis.

HIV associated neurocognitive disorders (HAND) are a group of neurological deficits that affect approximately half of people living with HIV (PLWH) despite effective antiretroviral therapy (ART). There are currently no reliable molecular biomarkers or treatments for HAND. Given the national opioid epidemic, as well as illegal and prescription use of opioid drugs among PLWH, it is critical to characterize the molecular interactions between HIV and opioids in cells of the CNS. It is also important to study the role of opioid substitution therapies in the context of HIV and CNS damage in vitro and in vivo. A major mechanism contributing to HIV neuropathogenesis is chronic, low-level inflammation in the CNS. HIV enters the brain within 4-8 days after peripheral infection and establishes CNS reservoirs, even in the context of ART, that are difficult to identify and eliminate. Infected cells, including monocytes, macrophages, and microglia, produce chemokines, cytokines, neurotoxic mediators, and viral proteins that contribute to chronic inflammation and ongoing neuronal damage. Opioids have been shown to impact these immune cells through a variety of molecular mechanisms, including opioid receptor binding and cross desensitization with chemokine receptors. The effects of opioid use on cognitive outcomes in individuals with HAND in clinical studies is variable, and thus multiple biological mechanisms are likely to contribute to the complex relationship between opioids and HIV in the CNS. In this review, we will examine what is known about both HIV and opioid mediated neuropathogenesis, and discuss key molecular processes that may be impacted by HIV and opioids in the context of neuroinflammation and CNS damage. We will also assess what is known about the effects of ART on these processes, and highlight areas of study that should be addressed in the context of ART.

Frontline Science: Buprenorphine decreases CCL2-mediated migration of CD14+ CD16+ monocytes.

HIV infection of the CNS causes neuroinflammation and damage that contributes to the development of HIV-associated neurocognitive disorders (HAND) in greater than 50% of HIV-infected individuals, despite antiretroviral therapy (ART). Opioid abuse is a major risk factor for HIV infection. It has been shown that opioids can contribute to increased HIV CNS pathogenesis, in part, by modulating the function of immune cells. HIV enters the CNS within two weeks after peripheral infection by transmigration of infected monocytes across the blood brain barrier (BBB). CD14+ CD16+ monocytes are a mature subpopulation that is increased in number in the peripheral blood of HIV-infected people. Mature monocytes can be productively infected with HIV, and they transmigrate preferentially across the BBB in response to CCL2, a chemokine elevated in the CNS and CSF of HIV-infected people even with ART. Buprenorphine, an opioid derivate, is an opioid replacement therapy for heroin addiction. It is a partial agonist of µ-opioid receptor and full antagonist of κ-opioid receptor. The effects of buprenorphine on CCL2-mediated CD14+ CD16+ monocytes transmigration across the BBB, a critical mechanism that promotes neuroinflammation and HAND, have not been characterized. We showed for the first time that buprenorphine decreases several steps of CCL2-mediated human mature monocyte transmigration. We propose that buprenorphine treatment in the context of HIV infection could serve a dual purpose, to treat opioid addiction and also to reduce neuroinflammation. Additionally, buprenorphine may be used as a treatment for HAND not only in the context of opioid abuse.

HIV-Tat regulates macrophage gene expression in the context of neuroAIDS.

Despite the success of cART, greater than 50% of HIV infected people develop cognitive and motor deficits termed HIV-associated neurocognitive disorders (HAND). Macrophages are the major cell type infected in the CNS. Unlike for T cells, the virus does not kill macrophages and these long-lived cells may become HIV reservoirs in the brain. They produce cytokines/chemokines and viral proteins that promote inflammation and neuronal damage, playing a key role in HIV neuropathogenesis. HIV Tat is the transactivator of transcription that is essential for replication and transcriptional regulation of the virus and is the first protein to be produced after HIV infection. Even with successful cART, Tat is produced by infected cells. In this study we examined the role of the HIV Tat protein in the regulation of gene expression in human macrophages. Using THP-1 cells, a human monocyte/macrophage cell line, and their infection with lentivirus, we generated stable cell lines that express Tat-Flag. We performed ChIP-seq analysis of these cells and found 66 association sites of Tat in promoter or coding regions. Among these are C5, CRLF2/TSLPR, BDNF, and APBA1/Mint1, genes associated with inflammation/damage. We confirmed the association of Tat with these sequences by ChIP assay and expression of these genes in our THP-1 cell lines by qRT-PCR. We found that HIV Tat increased expression of C5, APBA1, and BDNF, and decreased CRLF2. The K50A Tat-mutation dysregulated expression of these genes without affecting the binding of the Tat complex to their gene sequences. Our data suggest that HIV Tat, produced by macrophage HIV reservoirs in the brain despite successful cART, contributes to neuropathogenesis in HIV-infected people.

Opioids and Opioid Maintenance Therapies: Their Impact on Monocyte-Mediated HIV Neuropathogenesis.

BACKGROUND: HIV-1 enters the CNS within two weeks after peripheral infection and results in chronic neuroinflammation that leads to HIV associated neurocognitive disorders (HAND) in more than 50% of infected people. HIV enters the CNS by transmigration of infected monocytes across the blood brain barrier. Intravenous drug abuse is a major risk factor for HIV-1 infection, and opioids have been shown to alter the progression and severity of HAND. Methadone and buprenorphine are opioid derivates that are used as opioid maintenance therapies. They are commonly used to treat opioid dependency in HIV infected substance abusers, but their effects on monocyte migration relevant to the development of cognitive impairment are not well characterized. CONCLUSION: Here, we will discuss the effects of opioids and opioid maintenance therapies on the inflammatory functions of monocytes and macrophages that are related to the development of neuroinflammation in the context of HIV infection.

Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors.

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles.

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway.

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.

Endoplasmic reticulum sorting and kinesin-1 command the targeting of axonal GABAB receptors.

In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABA(B)Rs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABA(B)Rs are obligatory heteromers constituted by two subunits, GABA(B)R1 and GABA(B)R2. GABA(B)R1a and GABA(B)R1b are the most abundant subunit variants. GABA(B)R1b is located in the somatodendritic domain whereas GABA(B)R1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABA(B)R1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABA(B)Rs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABA(B)R1a operate in a pre-Golgi compartment. In the axon GABA(B)R1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABA(B)R1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABA(B)Rs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.

Neuronal protein trafficking: emerging consequences of endoplasmic reticulum dynamics.

The highly polarized morphology and complex geometry of neurons is determined to a great extent by the structural and functional organization of the secretory pathway. It is intuitive to propose that the spatial arrangement of secretory organelles and their dynamic behavior impinge on protein trafficking and neuronal function, but these phenomena and their consequences are not well delineated. Here we analyze the architecture and motility of the archetypal endoplasmic reticulum (ER), and their relationship to the microtubule cytoskeleton and post-translational modifications of tubulin. We also review the dynamics of the ER in axons, dendrites and spines, and discuss the role of ER dynamics on protein mobility and trafficking in neurons.

Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.

Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.