Objective We previously examined the expression of specific C-terminal μ-opioid receptor (MOR) splice variants in human central nervous system cell types and HIV-infected brain tissue from subjects with neurocognitive impairment ± HIV encephalitis (HIVE). In the present study, we examined the N-terminal splice variant MOR-1K which mediates excitatory cellular signaling. Methods and Results We found segregation of expression ranging from undetectable to seemingly exclusive across nervous system cell types compared to the pool of C-terminal MOR splice variants using RT-PCR. Expression of MOR-1K mRNA was also increased in HIV-infected subjects with combined neurocognitive impairment and HIVE compared to the other groups. MOR-1K expression correlated with the level of subject neurocognitive impairment whereas the pool of C-terminal MOR splice variants did not. HIVE was also associated with increased expression of the inflammatory mediators MCP-1, MCP-2, and RANTES, but not the host HIV co-receptors CXCR4 and CCR5 or the CD4 receptor, using qRT-PCR. Network analysis of microarray data from these same subjects revealed filamin A (FLNA) as a possible interaction partner with MOR-1K, and FLNA gene expression was also found to be upregulated in HIVE using qRT-PCR. Overexpression of filamin A in HEK293 cells redistributed MOR-1K from intracellular compartments to the cell surface. Conclusion These results suggest that HIVE, and neurocognitive impairment depending on its severity, are associated with enhanced MOR-1K signaling through both increased expression and trafficking to the cell surface, which may alter the contribution of MOR receptor isoforms and exacerbate the effects of MOR activation in neuroAIDS.

The blood brain barrier (BBB) plays a critical role in the normal physiology of the central nervous system (CNS) by regulating what crosses from the periphery into the brain. Damage to the BBB or alterations in transport systems may mediate the pathogenesis of many CNS diseases, including HIV-associated CNS dysfunction. HIV-1 infection can result in neuropathologic changes in about one half of infected individuals and also can result in damage to the BBB. HIV-1 and the HIV-1 viral proteins, Tat and gp120, cause alterations in the integrity and function of the BBB through both paracellular and transcellular mechanisms. The current review discusses HIV and viral protein-mediated injury to the BBB with a focus on the effects on tight junction proteins, barrier permeability, and drug efflux proteins.

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Aging is associated with reduced liver function that may increase the risk for adverse drug reactions in older adults. We hypothesized that age-related changes to epigenetic regulation of genes involved in drug metabolism may contribute to this effect. We reviewed published epigenome-wide studies of human blood and identified the cytochrome P450 2E1 (CYP2E1) gene as a top locus exhibiting epigenetic changes with age. To investigate potential functional changes with age in the liver, the primary organ of drug metabolism, we obtained liver tissue from mice aged 4-32 months from the National Institute on Aging. We assayed global DNA methylation (5-methylcytosine, 5mC), hydroxymethylation (5hydroxymethylcytosine, 5hmC), and locus-specific 5mC and histone acetylation changes around mouse Cyp2e1. The mouse livers exhibit significant global decreases in 5mC and 5hmC with age. Furthermore, 5mC significantly increased with age at two regulatory regions of Cyp2e1 in tandem with decreases in its gene and protein expressions. H3K9ac levels also changed with age at both regulatory regions of Cyp2e1 investigated, while H3K27ac did not. To test if these epigenetic changes are associated with varying rates of drug metabolism, we assayed clearance of the CYP2E1-specific probe drug chlorzoxazone in microsome extracts from the same livers. CYP2E1 intrinsic clearance is associated with DNA methylation and H3K9ac levels at the Cyp2e1 locus but not with chronological age. This suggests that age-related epigenetic changes may influence rates of hepatic drug metabolism. In the future, epigenetic biomarkers could prove useful to guide dosing regimens in older adults.

HIV-1 infection results in blood-brain barrier (BBB) disruption, which acts as a rate-limiting step for HIV-1 entry into the CNS and for subsequent neuroinflammatory/neurotoxic actions. One mechanism by which HIV may destabilize the BBB involves actions of the HIV-1 regulatory protein, trans-activator of transcription (Tat). We utilized a conditional, Tat-expressing transgenic murine model to examine the influence of Tat1-86 expression on BBB integrity and to assess the relative numbers of phagocytic perivascular macrophages and microglia within the CNS in vivo. The effects of Tat exposure on sodium-fluorescein (Na-F; 0.376 kDa), horseradish peroxidase (HRP; 44 kDa), and Texas Red-labeled dextran (70 kDa) leakage into the brain were assessed in Tat-exposed (Tat+) and control (Tat−) mice. Exposure to HIV-1 Tat significantly increased both Na-F and HRP, but not the larger sized Texas Red-labeled dextran, confirming BBB breakdown and also suggesting the breach was limited to molecules <70 kDa. Additionally, at 5 d after Tat induction, Alexa Fluor® 488-labeled dextran was bilaterally infused into the lateral ventricles 5 d before the termination of the experiment. Within the caudate/putamen, Tat induction increased the proportion of dextran-labeled Iba-1+ phagocytic perivascular macrophages (~5-fold) and microglia (~3-fold) compared to Tat− mice. These data suggest that HIV-1 Tat exposure is sufficient to destabilize BBB integrity and to increase the presence of activated, phagocytic, perivascular macrophages and microglia in an in vivo model of neuroAIDS.

NLRP3 inflammasome has recently emerged as an attractive drug target for neurodegenerative disorders. In our continuing studies, a new chemical scaffold was designed as selective inhibitors of NLRP3 inflammasome. Initial characterization of the lead HL16 demonstrated improved however non-selective inhibition on the NLRP3 inflammasome. Structure-activity relationship studies of HL16 identified a new lead, 17 (YQ128), with an IC 50 of 0.30 ± 0.01 μM. Further studies from in vitro and in vivo models confirmed its selective inhibition on the NLRP3 inflammaome and its brain penetration. Furthermore, pharmacokinetic studies in rats at 20 mg/kg indicated extensive systemic clearance and tissue distribution, leading to a half-life of 6.6 hours. However, the oral bioavailability is estimated to be only 10%, which may reflect limited GI permeability and possibly high first-pass effects. Collectively, these findings strongly encourage development of more potent analogs with improved pharmacokinetic properties from this new chemical scaffold.

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.

With the current national opioid crisis, it is critical to examine the mechanisms underlying pathophysiologic interactions between human immunodeficiency virus (HIV) and opioids in the central nervous system (CNS). Recent advances in experimental models, methodology, and our understanding of disease processes at the molecular and cellular levels reveal opioid-HIV interactions with increasing clarity. However, despite the substantial new insight, the unique impact of opioids on the severity, progression, and prognosis of neuroHIV and HIV-associated neurocognitive disorders (HAND) are not fully understood. In this review, we explore, in detail, what is currently known about mechanisms underlying opioid interactions with HIV, with emphasis on individual HIV-1-expressed gene products at the molecular, cellular and systems levels. Furthermore, we review preclinical and clinical studies with a focus on key considerations when addressing questions of whether opioid-HIV interactive pathogenesis results in unique structural or functional deficits not seen with either disease alone. These considerations include, understanding the combined consequences of HIV-1 genetic variants, host variants, and μ-opioid receptor (MOR) and HIV chemokine co-receptor interactions on the comorbidity. Lastly, we present topics that need to be considered in the future to better understand the unique contributions of opioids to the pathophysiology of neuroHIV. Graphical Abstract Blood-brain barrier and the neurovascular unit. With HIV and opiate co-exposure (represented below the dotted line), there is breakdown of tight junction proteins and increased leakage of paracellular compounds into the brain. Despite this, opiate exposure selectively increases the expression of some efflux transporters, thereby restricting brain penetration of specific drugs.