Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.

Activated platelets form transient aggregates with monocytes in circulation and have a half-life of approximately 30–60 minutes. These complexes are increased in various inflammatory conditions and are an early marker of myocardial infarction. HIV-1 infection is associated with chronic inflammation, and increased CD16+ inflammatory monocytes have been observed in these individuals, probably as a result of increased interaction with platelets. However, narrow detection period and platelet activation during sample processing pose significant problems in detecting platelet-monocyte complexes (PMCs). A method was standardized addressing these difficulties, to enumerate PMCs involving CD16+ or CD16− monocytes in whole blood using flow cytometry. Blood collected from healthy individuals was treated with either collagen (for platelet activation) or LPS (for monocyte activation) and subsequently used to study effect of these treatments on PMC formation. This method was also validated for the ex vivo quantitation of PMCs in blood obtained from persons infected with HIV. The in vitro results demonstrated that platelet activation, but not monocyte activation, resulted in significant increase in PMC formation. There was a significant increase in CD16+ PMCs and platelet activation, in samples obtained from persons infected with HIV as compared to those without HIV infection. Furthermore, PMC percentages correlated positively with platelet activation. These findings improve the ability to detect PMCs and shed light on HIV pathogenesis.

Human immunodeficiency virus-associated neurological disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy. The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, "reversible" DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging human immunodeficiency virus (HIV)-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson's disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously, we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here, we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT V max , without changes in K m or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by total internal reflection fluorescence microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3␤. We conclude that Tat activates RyRs via a calcium-and calpain-mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3␤ inhibition as clinical therapeutic approaches for HAND. Finally, we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients and by demonstrating similar findings in Tat-expressing transgenic mice.

Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorder (HAND) is likely neuroinflammatory in origin, believed to be triggered by inflammatory and oxidative stress responses to cytokines and HIV protein gene products such as the HIV transactivator of transcription (Tat). Here we demonstrate increased messenger RNA for nuclear factor-kappa B (NF-κB) family member, transcription factor RelB, in the brain of doxycycline-induced Tat transgenic mice, and increased RelB synthesis in Tat-exposed microglial cells. Since genetic ablation of RelB in mice leads to multi-organ inflammation, we hypothesized that Tat-induced, newly synthesized RelB inhibits cytokine production by microglial cells, possibly through the formation of transcriptionally inactive RelB/RelA complexes. Indeed, tumor necrosis factor-alpha (TNFα) production in monocytes isolated from RelB deficient mice was significantly higher than in monocytes isolated from RelB expressing controls. Moreover, RelB overexpression in microglial cells inhibited Tat-induced TNFα synthesis in a manner that involved transcriptional repression of the TNFα promoter, and increased phosphorylation of RelA at serine 276, a prerequisite for increased RelB/RelA protein interactions. The Rel-homology-domain within RelB was necessary for this interaction. Overexpression of RelA itself, in turn, significantly increased TNFα promoter activity, an effect that was completely blocked by RelB overexpression. We conclude that RelB regulates TNFα cytokine synthesis by competitive interference binding with RelA, which leads to downregulation of TNFα production. Moreover, because Tat activates both RelB and TNFα in microglia, and because Tat induces inflammatory TNFα synthesis via NF-κB, we posit that RelB serves as a cryoprotective, anti-inflammatory, counter-regulatory mechanism for pathogenic NF-κB activation. These findings identify a novel regulatory pathway for controlling HIV-induced microglial activation and cytokine production that may have important therapeutic implications for the management of HAND.

B-cell malignancies are a common type of cancer. One approach to cancer therapy is to either increase oxidative stress or inhibit the stress response systems on which cancer cells rely. In this study, we combined non-toxic concentrations of Auranofin (AUR), an inhibitor of the thioredoxin (Trx) system, with non-toxic concentrations of buthionine-sulfoximine (BSO), a compound that reduces intracellular glutathione (GSH) levels, and investigated the effect of this drug combination on multiple pathways critical for malignant B-cell survival. AUR interacted synergistically with BSO at low concentrations to trigger death in multiple malignant B-cell lines and primary mantle cell lymphoma (MCL) cells. Additionally, there was less toxicity toward normal B-cells. Low AUR concentrations inhibited Trx reductase (TrxR) activity, an effect significantly increased by BSO co-treatment. TrxR over-expression partially reversed AUR+BSO toxicity. Interestingly, the combination of AUR+BSO inhibited NF-κB signaling. Moreover, synergistic cell death induced by this regimen was attenuated in cells over-expressing NF-κB proteins, arguing for a functional role for NF-κB inhibition in AUR+BSO-mediated cell death. Together, these findings suggest that AUR+BSO synergistically induce malignant B-cell death, a process mediated by dual inhibition of TrxR and NF-κB, and such an approach warrants further investigation in B-cell malignancies.