Slow and persistent synaptic inhibition is mediated by metabotropic GABA B receptors (GABA B Rs). GABA B Rs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABA B Rs 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 GABA B Rs 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 GABA B Rs. These sorting events are paralleled by a reduction in GABA B R-dependent activation of inwardly rectifying K + channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABA B R2 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 GABA B Rs 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 GABA B Rs 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.he availability of neurotransmitter receptors, a major determinant of synaptic efficacy, is regulated by coordinated mechanisms of intracellular trafficking that deliver newly synthesized receptors to the plasma membrane and remove them for storage, recycling, or degradation (1). The molecular mechanisms controlling the availability of GABA B receptors (GABA B Rs), which are central players in the modulation of excitatory and inhibitory synaptic activity, are unclear.GABA B Rs mediate slow and prolonged inhibitory synaptic signals (2, 3). Consistent with these roles, modifications in the function of GABA B Rs are implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, cognition, and addictive mechanisms to drugs of abuse (3-7). GABA B Rs are members of the G protein-coupled receptor (GPCR) superfamily and are obligatory heteromers composed of two related subunits, namely GABA B R1 and GABA B R2 (3, 8). GABA B R1 binds agonist with high affinity, whereas GABA B R2 mediates coupling to Gαi (9, 10). GABA B Rs are located in GABA-ergic and glutamatergic pre-and postsynaptic terminals, but their distribution does not coincide with the active zone, postsynaptic density, or inhibitory postsynaptic specializations. Rather, they are perisynaptic receptors activated by GABA spillover (3, 11). Stimulation of GABA B Rs decreases the levels of cAMP, inhibits neurotr...

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.

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. c-aminobutyric acid (GABA) type B metabotropic receptors (GABA B Rs) 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 GABA B R biosynthesis has not been thoroughly explored. Here, we show that GABA B1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dyneindependent mechanisms control dendritic ER transport. GABA B Rs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABA B Rs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles.

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.

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.

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.

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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.