Involvement of the p53 and p73 transcription factors in neuroAIDS

HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.     

HIV-1 Tat protein promotes neuronal dysfunction through disruption of microRNAs

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.     

Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated Neuronal dysfunction

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3′-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.     

HIV-1 Tat increases BAG3 via NF-κB signaling to induce autophagy during HIV-associated neurocognitive disorder

The human immunodeficiency virus-1 (HIV-1) regulatory protein Tat plays an important role during HIV-1-associated neurocognitive disorders (HAND) by inducing neuronal autophagy. In this study, we used immunohistochemistry, immunofluorescence, western blot, qRT-PCR, and RNA interference to elucidate the involvement of Bcl-2-associated athanogene 3 (BAG3) in the pathogenesis of HIV-1 Tat-induced autophagy during HAND. We found that BAG3 expression is elevated in astrocytes in frontal cortex of macaques infected with simian immunodeficiency virus-human immunodeficiency chimeric virus (SHIV). In addition, in human primary glioblastoma cells (U87), HIV-1 Tat upregulated BAG3 in an NF-κB-dependent manner to induce autophagy. Importantly, suppression of BAG3 or inhibition of NF-κB activity reversed the HIV-1 Tat-induced autophagy. These results indicate that HIV-1 Tat induces autophagy by upregulating BAG3 via NF-κB signaling, which suggests BAG3 and NF-κB could potentially serve as novel targets for HAND therapies.     

MiRNA-891a-5p mediates HIV-1 Tat and KSHV Orf-K1 synergistic induction of angiogenesis by activating NF-κB signaling

Co-infection with HIV-1 and Kaposi''s sarcoma-associated herpesvirus (KSHV) is the cause of aggressive AIDS-related Kaposi''s sarcoma (AIDS-KS) characterized by abnormal angiogenesis. The impact of HIV-1 and KSHV interaction on the pathogenesis and extensive angiogenesis of AIDS-KS remains unclear. Here, we explored the synergistic effect of HIV-1 Tat and KSHV oncogene Orf-K1 on angiogenesis. Our results showed that soluble Tat or ectopic expression of Tat enhanced K1-induced cell proliferation, microtubule formation and angiogenesis in chorioallantoic membrane and nude mice models. Mechanistic studies revealed that Tat promoted K1-induced angiogenesis by enhancing NF-κB signaling. Mechanistically, we showed that Tat synergized with K1 to induce the expression of miR-891a-5p, which directly targeted IκBα 3′ untranslated region, leading to NF-κB activation. Consequently, inhibition of miR-891a-5p increased IκBα level, prevented nuclear translocation of NF-κB p65 and ultimately suppressed the synergistic effect of Tat- and K1-induced angiogenesis. Our results illustrate that, by targeting IκBα to activate the NF-κB pathway, miR-891a-5p mediates Tat and K1 synergistic induction of angiogenesis. Therefore, the miR-891a-5p/NF-κB pathway is important in the pathogenesis of AIDS-KS, which could be an attractive therapeutic target for AIDS-KS.     

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.     

MiR-217 is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation by down-regulation of SIRT1

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and may contribute to the development and progression of many infective diseases including human immunodeficiency virus 1 (HIV-1) infection. The Tat protein is fundamental to viral gene expression. In this study, our goal was to investigate the regulation of a specific miRNA (known as miR-217) in multinuclear activation of galactosidase indicator (MAGI) cells and explore the mechanisms by which miR-217 influenced Tat-induced HIV-1 transactivation through down-regulation of SIRT1 expression. We showed that miR-217 was up-regulated when Tat was expressed in multinuclear activation of galactosidase indicator cells. Forced expression of "miR-217 mimics" increased Tat-induced LTR transactivation. In addition, miR-217 significantly inhibited SIRT1 protein expression by acting on the 3'-UTR of the SIRT1 mRNA. In turn, the decrease in SIRT1 protein abundance provoked by miR-217 affected two important types of downstream signaling molecules that were regulated by Tat. Lower expression of SIRT1 caused by miR-217 enhanced Tat-induced phosphorylation of IKK and p65-NFkB and also exacerbated the loss of AMPK phosphorylation triggered by Tat. Our results uncover previously unknown links between Tat and a specific host cell miRNA that targets SIRT1. We also demonstrate that this regulatory mechanism impinges on p65-NFkB and AMPK signaling: two important host cell pathways that influence HIV-1 pathogenesis. Our results also suggest that strategies to augment SIRT1 protein expression by down-regulation of miR-217 may have therapeutic benefits to prevent HIV-1 replication.     

Rho-kinase inhibitor hydroxyfasudil protects against HIV-1 Tat-induced dysfunction of tight junction and neprilysin/Aβ transfer receptor expression in mouse brain microvessels

HIV-1 transactivator protein (Tat) induces tight junction (TJ) dysfunction and amyloid-beta (Aβ) clearance dysfunction, contributing to the development and progression of HIV-1-associated neurocognitive disorder (HAND). The Rho/ROCK signaling pathway has protective effects on neurodegenerative disease. However, the underlying mechanisms of whether Rho/ROCK protects against HIV-1 Tat-caused dysfunction of TJ and neprilysin (NEP)/Aβ transfer receptor expression have not been elucidated. C57BL/6 mice were administered sterile saline (i.p., 100 μL) or Rho-kinase inhibitor hydroxyfasudil (HF) (i.p., 10 mg/kg) or HIV-1 Tat (i.v., 100 μg/kg) or HF 30 min before being exposed to HIV-1 Tat once a day for seven consecutive days. Evans Blue (EB) leakage was detected via spectrophotometer and brain slides in mouse brains. The protein and mRNA levels of zonula occludens-1 (ZO-1), occludin, NEP, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in mouse brain microvessels were, respectively, analyzed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Exposure of the mice to HIV-1 Tat increased the amount of EB leakage, EB fluorescence intensity, blood–brain barrier (BBB) permeability, as well as the RAGE protein and mRNA levels, and decreased the protein and mRNA levels of ZO-1, occludin, NEP, and LRP1 in mouse brain microvessels. However, these effects were weakened by Rho-kinase inhibitor HF. Taken together, these results provide information that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-induced dysfunction of TJ and NEP/Aβ transfer receptor expression in the C57BL/6 mouse brain. These findings shed some light on potentiality of inhibiting Rho/Rock signaling pathway in handling HAND.

     

Resistance of chimpanzee T cells to human immunodeficiency virus type 1 Tat-enhanced oxidative stress and apoptosis.

CD4+ T-cell depletion in AIDS patients involves induction of apoptosis in human immunodeficiency virus (HIV)-infected and noninfected T cells. The HIV type 1 (HIV-1)-transactivating protein Tat enhances apoptosis and activation-induced cell death (AICD) of human T cells. This effect is mediated by the CD95 (APO-1/Fas) receptor-CD95 ligand (CD95L) system and may be linked to the induction of oxidative stress by Tat. Here we show that HIV-1 Tat-induced oxidative stress is necessary for sensitized AICD in T cells caused by CD95L expression. Tat-enhanced apoptosis and CD95L expression in T cells are inhibited by neutralizing anti-Tat antibodies, antioxidants, and the Tat inhibitor Ro24-7429. Chimpanzees infected with HIV-1 show viral replication resembling early infection in humans but do not show T-cell depletion or progression towards AIDS. The cause for this discrepancy is unknown. Here we show that unlike Tat-treated T cells in humans, Tat-treated chimpanzee T cells do not show downregulation of manganese superoxide dismutase or signs of oxidative stress. Chimpanzee T cells are also resistant to Tat-enhanced apoptosis, AICD, and CD95L upregulation.     

HIV-1 Tat inhibits NGF-induced Egr-1 transcriptional activity and consequent p35 expression in neural cells

Infection with HIV-1 causes degeneration of neurons leading to motor and cognitive dysfunction in AIDS patients. One of the key viral regulatory proteins, Tat, which is released by infected cells, can be taken up by various uninfected cells including neurons and by dysregulating several biological events induces cell injury and death. In earlier studies, we demonstrated that treatment of neuronal cells with Tat affects the nerve growth factor (NGF) signaling pathway involving MAPK/ERK. Here we demonstrate that a decrease in the level of Egr-1, one of the targets for MAPK, by Tat has a negative impact on the level of p35 expression in NGF-treated neural cells. Further, we demonstrate a reduced level of Egr-1 association with the p35 promoter sequence in NGF-treated cells expressing Tat. As p35, by associating with Cdk5, phosphorylates several neuronal proteins including neurofilaments and plays a role in neuronal differentiation and survival, we examined kinase activity of p35 complexes obtained from cells expressing Tat. Results from H1 kinase assays showed reduced activity of the p35 complex from Tat-expressing cells in comparison to that from control cells. Accordingly, the level of phosphorylated neurofilaments was diminished in Tat-expressing cells. Similarly, treatment of PC12 cells with Tat protein or supernatant from HIV-1 infected cells decreased kinase activity of p35 in these cells. These observations ascribe a role for Tat in altering p35 expression and its activity that affects phosphorylation of proteins involved in neuronal cell survival.     

Human immunodeficiency virus-1 Tat protein and methamphetamine interact synergistically to impair striatal dopaminergic function

The human immunodeficiency virus (HIV)-1 transactivating protein Tat may be pathogenically relevant in HIV-1-induced neuronal injury. The abuse of methamphetamine (MA), which is associated with behaviors that may transmit HIV-1, may damage dopaminergic afferents to the striatum. Since Tat and MA share common mechanisms of injury, we examined whether co-exposure to these toxins would lead to enhanced dopaminergic toxicity. Animals were treated with either saline, a threshold dose of MA, a threshold concentration of Tat injected directly into the striatum, or striatal injections of Tat followed by exposure to MA. Threshold was defined as the highest concentration of toxin that would not result in a significant loss of striatal dopamine levels. One week later, MA-treated animals demonstrated a 7% decline in striatal dopamine levels while Tat-treated animals showed an 8% reduction. Exposure to both MA + Tat caused an almost 65% reduction in striatal dopamine. This same treatment caused a 56% reduction in the binding capacity to the dopamine transporter. Using human fetal neurons, enhanced toxicity was also observed when cells were exposed to both Tat and MA. Mitochondrial membrane potential was disrupted and could be prevented by treatment with antioxidants. This study demonstrates that the HIV-1 'virotoxin' Tat enhances MA-induced striatal damage and suggests that HIV-1-infected individuals who abuse MA may be at increased risk of basal ganglia dysfunction.     

DNM3OS regulates GAPDH expression and influences the molecular pathogenesis of Huntington’s disease

Emerging studies have suggested that dysregulated long non-coding RNAs (lncRNAs) are associated with the pathogenesis of neurodegenerative diseases (NDD) including Huntington's disease (HD); however, the pathophysiological mechanism by which lncRNA dysregulation participates in HD remains to be elucidated. Here, we aim to analyse the expression of lncRNA-DNM3OS and identify the possible DNM3OS/miR-196b-5p/GAPDH pathway. PC12 cells induced by rat pheochromocytoma expressing HD gene exon 1 fragment with either 23 or 74 polyglutamine repeats fused to the green fluorescent protein (GFP) were cultured. Our results show that GAPDH and DNM3OS were upregulated in HD PC12 cells, downregulation of which lead to inhibition of aggregate formation accompanied by a decreased apoptosis rate and increased relative ROS levels and cell viability. Moreover, upregulated DNM3OS decreased the expression of miR-196b-5p by sponging, and GAPDH was a direct target of miR-196b-5p, playing an important pathogenic role in the formation of aggregates in the HD cell model. Our study uncovers a novel DNM3OS/miR-196b-5p/GAPDH pathway involved in the molecular pathogenesis of HD, which may offer a potential therapeutic strategy for HD.