Rosmarinic acid reverses the deleterious effects of repetitive stress and tat protein.

Human immunodeficiency virus type 1 (HIV) has infected more than 40 million people worldwide and is associated with central nervous system (CNS) disruption in at least 30% of these persons. The use of highly active antiretroviral therapy (HAART) has significantly reduced the systemic immunopathology associated with HIV, but the occurrence of neurological disorders continues to be reported in notable numbers. The present study evaluated the potential of rosmarinic acid to reverse the detrimental effects of an intracerebral injection of the viral protein tat. Control and tat-injected rats were also subjected to repetitive restrain stress (RRS) for 28 days, 6 h per day, to investigate whether subsequent stress exposure would worsen the effects of tat. 14 days after the initiation of RRS, animals were treated with rosmarinic acid (10 mg/kg given intraperitoneally) daily until the end of the stress exposure period. We assessed locomotor activity and anxiety-like behavioral changes. We also measured plasma corticosterone levels and quantified the expression of mineralocorticoid receptors (MR), glucocorticoid receptors (GR) and brain-derived neurotrophic factor (BDNF) in the hippocampus. Rosmarinic acid attenuated anxiety-like behavior induced by tat and stress, reduced plasma corticosterone levels and increased the expression of hippocampal GR, MR and BDNF when compared to controls. These results suggest that rosmarinic acid may reverse the anxiogenic effect of HIV-1 viral protein tat and related stress through modulation of the hypothalamic-pituitary-adrenal axis and hippocampal neurotrophic factor levels.

Leucine-rich repeat kinase 2 modulates neuroinflammation and neurotoxicity in models of human immunodeficiency virus 1-associated neurocognitive disorders.

Leucine-rich repeat kinase 2 (LRRK2) is the single most common genetic cause of both familial and sporadic Parkinson's disease (PD), both of which share pathogenetic and neurologic similarities with human immunodeficiency virus 1 (HIV-1)-associated neurocognitive disorders (HAND). Pathologic LRRK2 activity may also contribute to neuroinflammation, because microglia lacking LRRK2 exposed to proinflammatory stimuli have attenuated responses. Because microglial activation is a hallmark of HIV-1 neuropathology, we have investigated the role of LRRK2 activation using in vitro and in vivo models of HAND. We hypothesize that LRRK2 is a key modulator of microglial inflammatory responses, which play a pathogenic role in both HAND and PD, and that these responses may cause or exacerbate neuronal damage in these diseases. The HIV-1 Tat protein is a potent neurotoxin produced during HAND that induces activation of primary microglia in culture and long-lasting neuroinflammation and neurotoxicity when injected into the CNS of mice. We found that LRRK2 inhibition attenuates Tat-induced pS935-LRRK2 expression, proinflammatory cytokine and chemokine expression, and phosphorylated p38 and Jun N-terminal kinase signaling in primary microglia. In our murine model, cortical Tat injection in LRRK2 knock-out (KO) mice results in significantly diminished neuronal damage, as assessed by microtubule-associated protein 2 (MAP2), class III ß-tubulin TUJ1, synapsin-1, VGluT, and cleaved caspase-3 immunostaining. Furthermore, Tat-injected LRRK2 KO animals have decreased infiltration of peripheral neutrophils, and the morphology of microglia from these mice were similar to that of vehicle-injected controls. We conclude that pathologic activation of LRRK2 regulates a significant component of the neuroinflammation associated with HAND.

PACAP27 is protective against tat-induced neurotoxicity.

Human immunodeficiency virus type-1 (HIV) infection of the central nervous system promotes neuronal injury and apoptosis that culminate in HIV-associated neurocognitive disorders (HAND). Viral proteins, such as transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanism remains unclear. To determine the effects of Tat, rat cortical neurons were exposed to nanomolar concentrations of Tat for various time points. Within a few hours, Tat induced the production of reactive oxygen species (ROS), and other indices of mitochondrial destabilization. In addition, we observed a significant induction of DNA double-strand breaks (DSBs) by Tat. We next investigated the neuroprotective activity of the pituitary adenylate cyclase-activating polypeptide 27 (PACAP27) against these cardinal features of Tat-induced neurodegeneration. PACAP27 (100 nM) inhibited all Tat-mediated toxic effects including DNA DSBs. Importantly, PACAP27 prevented the induction of neuronal loss induced by Tat. The neuroprotective effect of PACAP27 is correlated with its ability to release the anti-apoptotic chemokine CCL5. Our data support a mechanism of Tat neurotoxicity in which Tat induces mitochondrial destabilization, thus increasing the release of ROS, which causes DNA DSBs leading to cell death. PACAP27, through CCL5, mitigates the effects of Tat-induced neuronal dysfunction, suggesting that PACAP27 could be a new strategy for an adjunct therapy against HIV-associated neurocognitive disorders.

Hexokinase II and reperfusion injury: TAT-HK2 peptide impairs vascular function in Langendorff-perfused rat hearts.

RATIONALE: Mitochondrial-bound hexokinase II (HK2) was recently proposed to play a crucial role in the normal functioning of the beating heart and to be necessary to maintain mitochondrial membrane potential. However, our own studies confirmed that mitochondria from ischemic rat hearts were HK2-depleted, yet showed no indication of depolarization and responded normally to ADP. OBJECTIVE: To establish whether the human TAT-HK2 peptide used to dissociate mitochondrial-bound HKII in the Langendorff-perfused heart may exert its effects indirectly by impairing coronary function. METHODS AND RESULTS: Ischemic preconditioning was blocked in rat hearts perfused with 2.5 µmol/L TAT-HK2 before ischemia or at the onset of reperfusion. However, TAT-HK2 also decreased the phosphocreatine:ATP ratio that correlated with reduced rate pressure product and increased diastolic pressure. These effects were preceded by increased aortic pressure (Langendorff constant flow) or decreased coronary flow (Langendorff constant pressure), which was also observed, albeit less pronounced, at 200 nmol/L TAT-HK2 and was prevented by coperfusion with the NO-donor diethylamine NONOate. Mitochondria from TAT-HK2-perfused hearts showed no loss of bound HK2, unlike mitochondria from ischemic hearts where the expected loss was prevented by ischemic preconditioning. CONCLUSIONS: In the perfused rat heart, TAT-HK2 should be used with caution and careful attention to dosage because some of its effects may be mediated by vasoconstriction of the coronary vasculature rather than dissociation of HK2 from myocyte mitochondria.

HIV-1 Tat-induced cerebrovascular toxicity is enhanced in mice with amyloid deposits.

HIV-1-infected brains are characterized by elevated depositions of amyloid beta (Aß); however, the interactions between Aß and HIV-1 are poorly understood. In the present study, we administered specific HIV-1 protein Tat into the cerebral vasculature of 50-52-week-old double transgenic (B6C3-Tg) mice that express a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) and are characterized by increased Aß depositions in the brain. Exposure to Tat increased permeability across cerebral capillaries, enhanced disruption of zonula occludens (ZO)-1 tight junction protein, and elevated brain expression of matrix metalloproteinase-9 (MMP-9) in B6C3-Tg mice as compared with age-matched littermate controls. These changes were associated with increased leukocyte attachment and their transcapillary migration. The majority of Tat-induced effects were attenuated by treatment with a specific Rho inhibitor, hydroxyfasudil. The results of animal experiments were reproduced in cultured brain endothelial cells exposed to Aß and/or Tat. The present data indicate that increased brain levels of Aß can enhance vascular toxicity and proinflammatory responses induced by HIV-1 protein Tat.

A cationic TAT peptide inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells.

UNLABELLED: Abstract Purpose: To determine if a peptide, TAT-Cd(0), inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells. METHODS: TAT-Cd(0) and a control peptide, E(50,51)TAT-Cd(0), were added at various times throughout infection with the lacz-expressing hrR3 virus, and viral replication was measured by ß-galactosidase activity. Toxicity was assessed using a dye reduction assay. RESULTS: The CC(50) value for TAT-Cd(0) was ∼100 µM. In assays with peptide present at all times, TAT-Cd(0) was 150-fold more active than E(50,51)TAT-Cd(0) (EC(50) 0.2 vs. 30.0 µM). The EC(50) values of TAT-Cd(0) for entry inhibition, cell protection, virus inactivation, and inhibition of attachment were 0.1, 0.4, 9.5, and 3.0 µM, respectively. TAT-Cd(0) was less effective when added 1 h postinfection (EC(50) = 30.0 µM). CONCLUSIONS: TAT-Cd(0) is an effective inhibitor of Herpes simplex virus type 1 infection in human corneal epithelial cells and affects multiple steps before, or very early, in infection. The peptide has potential as an antiviral and further studies are warranted.

Small ruminant lentivirus Tat protein induces apoptosis in caprine cells in vitro by the intrinsic pathway.

The small ruminant lentiviruses, caprine arthritis-encephalitis virus (CAEV) and maedi visna virus (MVV) naturally cause inflammatory disease in goats and sheep, provoking chronic lesions in several different organs. We have previously demonstrated that in vitro infection of caprine cells by CAEV induces apoptosis through the intrinsic pathway (Rea-Boutrois, A., Pontini, G., Greenland, T., Mehlen, P., Chebloune, Y., Verdier, G. and Legras-Lachuer, C. 2008). In the present study, we used Tat deleted viruses and SLRV Tat-expression vectors to show that the SRLV Tat proteins are responsible for this apoptosis. We have also studied the activation of caspases-3, -8 and -9 by fluorescent assays in caprine cells expressing SRLV Tat proteins, and the effects of transfected dominant negative variants of these caspases, to show that Tat-associated apoptosis depends on activation of caspases-3 and -9, but not -8. A simultaneous disruption of mitochondrial membrane potential indicates an involvement of the mitochondrial pathway.

Clade-specific differences in neurotoxicity of human immunodeficiency virus-1 B and C Tat of human neurons: significance of dicysteine C30C31 motif.

OBJECTIVE: Human immunodeficiency virus-1 (HIV-1) causes mild to severe cognitive impairment and dementia. The transactivator viral protein, Tat, is implicated in neuronal death responsible for neurological deficits. Several clades of HIV-1 are unequally distributed globally, of which HIV-1 B and C together account for the majority of the viral infections. HIV-1-related neurological deficits appear to be most common in clade B, but not clade C prevalent areas. Whether clade-specific differences translate to varied neuropathogenesis is not known, and this uncertainty warrants an immediate investigation into neurotoxicity on human neurons of Tat derived from different viral clades METHODS: We used human fetal central nervous system progenitor cell-derived astrocytes and neurons to investigate effects of B- and C-Tat on neuronal cell death, chemokine secretion, oxidative stress, and mitochondrial membrane depolarization by direct and indirect damage to human neurons. We used isogenic variants of Tat to gain insights into the role of the dicysteine motif (C30C31) for neurotoxic potential of Tat RESULTS: Our results suggest clade-specific functional differences in Tat-induced apoptosis in primary human neurons. This study demonstrates that C-Tat is relatively less neurotoxic compared with B-Tat, probably as a result of alteration in the dicysteine motif within the neurotoxic region of B-Tat INTERPRETATION: This study provides important insights into differential neurotoxic properties of B- and C-Tat, and offers a basis for distinct differences in degree of HIV-1-associated neurological deficits observed in patients in India. Additional studies with patient samples are necessary to validate these findings.

Cargo-dependent cytotoxicity and delivery efficacy of cell-penetrating peptides: a comparative study.

The use of CPPs (cell-penetrating peptides) as delivery vectors for bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin, was discovered. Since then, several CPPs, including the widely used Tat (transactivator of transcription) peptide, have been developed and utilized to translocate a wide range of compounds across the plasma membrane of cells both in vivo and in vitro. Although the field has emerged as a possible future candidate for drug delivery, little attention has been given to the potential toxic side effects that these peptides might exhibit in cargo delivery. Also, no comprehensive study has been performed to evaluate the relative efficacy of single CPPs to convey different cargos. Therefore we selected three of the major CPPs, penetratin, Tat and transportan 10, and evaluated their ability to deliver commonly used cargos, including fluoresceinyl moiety, double-stranded DNA and proteins (i.e. avidin and streptavidin), and studied their effect on membrane integrity and cell viability. Our results demonstrate the unfeasibility to use the translocation efficacy of fluorescein moiety as a gauge for CPP efficiency, since the delivery properties are dependent on the cargo used. Furthermore, and no less importantly, the toxicity of CPPs depends heavily on peptide concentration, cargo molecule and coupling strategy.

Silencing the PTEN gene is protective against neuronal death induced by human immunodeficiency virus type 1 Tat.

Neurons are targets of toxicity induced by the human immunodeficiency virus (HIV)-1 protein Tat (transactivator of transcription). Exposure to Tat increases [Ca(2+)](i) in striatal neurons and activates multiple cell death pathways. In earlier studies the authors showed that Tat activated both caspase-3 and endonuclease-G, a caspase-independent effector of apoptosis, and that Tat-induced neurotoxicity was not attenuated by a caspase-3 inhibitor. Because Tat activates multiple, parallel death pathways, the authors attempted to reduce Tat-induced neurotoxicity by manipulating signaling pathways upstream of mitochondrial apoptotic events. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a negative regulator of Akt/PKB (protein kinase B) phosphorylation, was chosen as a target for silencing. Akt/PKB activity directs multiple downstream pathways mediated by GSK3beta, BAD, forkhead transcription factors, nuclear factor kappa B (NFkappaB), and others, in a manner that promotes proliferation and survival. Striatal neurons were nucleofected with short interfering RNA (siRNA) vectors targeting PTEN, or a negative-control siRNA. Although Tat(1-86) significantly increased the death of neurons transfected with control construct by 72 h, PTEN-silenced neurons were completely protected. These findings indicate that Akt is a critical intermediary in the direct neurotoxicity induced by HIV-1 Tat, and identify Akt regulation as a possible therapeutic strategy for Tat-induced neurotoxicity in HIV encephalitis (HIVE).

Nucleoside reverse transcriptase inhibitors and human immunodeficiency virus proteins cause axonal injury in human dorsal root ganglia cultures.

Distal symmetric polyneuropathy (DSP) has emerged as the most common complication of human immunodeficiency virus (HIV) infection, which is associated with neuronal injury in the dorsal root ganglion (DRG). With the advent of highly active antiretroviral therapy, especially nucleoside analogs, patients are living longer. Some of the antiretroviral drugs used to treat HIV infection have been associated with neuropathies. The pathogenesis of these neuropathies remains poorly understood. Utilizing a human fetal DRG model of predominantly nociceptive fibers, the authors investigated the effects of HIV gp120 and Tat(1-72), alone or in combination with nucleoside analogs on both morphological and ultra-structural changes in DRG neurons. Nucleoside analogs and HIV proteins both caused a significant decrease in the mean axonal length. However, ddI was the most potent, followed by ddC, d4T, and AZT. Despite the combined exposure to toxic dosages of HIV proteins and nucleoside analogs, there appeared to be a ceiling effect on the amount of axonal retraction, indicating that the proximal and distal axon are differentially regulated. In conclusion, both HIV proteins and nucleoside reverse transcriptase inhibitors (NRTIs) cause axonal damage by inducing mitochondrial injury and rearrangement of microtubules.

Ceftriaxone protects against the neurotoxicity of human immunodeficiency virus proteins.

Human immunodeficiency virus (HIV) proteins Tat and gp120 have been implicated in the pathogenesis of HIV dementia by various mechanisms, including down-regulation of excitatory amino acid transporter-2 (EAAT2), which is responsible for inactivation of synaptic glutamate. Recent work indicates that beta-lactam antibiotics are potent stimulators of EAAT2 expression. The authors treated mixed human fetal neuronal cultures with recombinant gp120 or Tat, in the presence or absence of ceftriaxone, and determined neurotoxicity by measuring mitochondrial membrane potential and neuronal cell death. Ceftriaxone produced dose-dependent attenuation of the neurotoxicity and neuronal cell death caused by both viral proteins. This study demonstrates that this class of drugs may have therapeutic efficacy in HIV dementia.

Human immunodeficiency virus type-1 protein Tat induces tumor necrosis factor-alpha-mediated neurotoxicity.

HIV-1 infection causes, with increasing prevalence, neurological disorders characterized in part by neuronal cell death. The HIV-1 protein Tat has been shown to be directly and indirectly neurotoxic. Here, we tested the hypothesis that a non-neurotoxic epitope of Tat can, through actions on immune cells, increase neuronal cell death. Tat(1-72) and a mutant Tat(1-72) lacking the neurotoxic epitope (Tat(Delta31-61)) concentration-dependently and markedly increased TNF-alpha production in macrophage-like differentiated human U937 and THP-1 cells, in mouse peritoneal macrophages and in mouse brain microglia. Tat(1-72) was but Tat(Delta31-61) was not neurotoxic when applied directly to neurons. Supernatants from U937 cells treated with either Tat(1-72) or Tat(Delta31-61) were neurotoxic and their immunoneutralization with an anti-TNF-alpha antibody decreased Tat(1-72)- and Tat(Delta31-61)-induced neurotoxicity. Together, these results demonstrate that the neurotoxic epitope of Tat(1-72) is different from the epitope that is indirectly neurotoxic following production of TNF-alpha from immune cells, and suggest that therapeutic interventions against TNF-alpha might be beneficial against HIV-1 associated neurological disorders.

Preventing HIV-1 Tat-induced neuronal apoptosis using antioxidant enzymes: mechanistic and therapeutic implications.

HIV-1 proteins, especially gp120 and Tat, elicit reactive oxygen species (ROS) and cause neuron apoptosis. We used antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1) and glutathione peroxidase (GPx1) to study signaling and neuroprotection from Tat-induced apoptosis. SOD1 converts superoxide to peroxide; GPx1 converts peroxide to water. Primary human neurons were transduced with SV40-derived vectors carrying SOD1 and GPx1, then HIV-1 Tat protein was added. Both SV(SOD1) and SV(GPx1) delivered substantial transgene expression. Tat decreased endogenous cellular, but not transduced, SOD1 and GPx1. Tat rapidly increased neuron [Ca(2+)](i), which effect was not altered by SV(SOD1) or SV(GPx1). However, both vectors together blocked Tat-induced [Ca(2+)](i) fluxes. Similarly, neither SV(SOD1) nor SV(GPx1) protected neurons from Tat-induced apoptosis, but both vectors together did. Tat therefore activates multiple signaling pathways, in one of which superoxide acts as an intermediate while the other utilizes peroxide. Gene delivery to protect neurons from Tat must therefore target both.

Flying in the face of resistance: antiviral-independent benefit of HIV protease inhibitors on T-cell survival.

Human immunodeficiency virus (HIV) infection results in excessive apoptosis of infected and uninfected cells, mediated by host and viral factors present in plasma. As HIV protease inhibitors (PIs) have intrinsic antiapoptotic properties, we questioned whether HIV PIs could block HIV-induced CD4+ T-cell death independent of their effects on HIV replication. We demonstrate that HIV PIs block the death of CD4+ T cells induced by HIV glycoprotein 120 (gp120), Vpr, and Tat, as well as host signals Fas ligand, tumor necrosis factor, and tumor necrosis factor-related apoptosis-inducing ligand. Using gp120/CXCR4 as a model, we show that the HIV PIs specifically block mitochondrial apoptosis signaling. Furthermore, HIV PIs inhibit CD4+ T-cell death induced by viruses with high-level resistance to PIs (P<0.01) and apoptosis induced by serum of HIV patients with known resistance to HIV PIs (P=0.01). Together, these results show that HIV PIs block CD4+ T-cell death and have a beneficial effect on CD4+ T-cell survival despite PI resistance.

HIV-tat induces formation of an LRP-PSD-95- NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes.

HIV infection of the central nervous system can result in neurologic dysfunction with devastating consequences in AIDS patients. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence that HIV can infect neurons. Here we show that the HIV-encoded protein tat triggers formation of a macromolecular complex involving the low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSD-95), N-methyl-d-aspartic acid (NMDA) receptors, and neuronal nitric oxide synthase (nNOS) at the neuronal plasma membrane, and that this complex leads to apoptosis in neurons negative as well as positive for NMDA receptors and also in astrocytes. Blockade of LRP-mediated tat uptake, NMDA receptor activation, or neuronal nitric oxide synthase significantly reduces ensuing neuronal apoptosis, suggesting that formation of this complex is an early step in tat toxicity. We also show that the inflammatory chemokine, CCL2, protects against tat toxicity and inhibits formation of the complex. These findings implicate the complex in HIV-induced neuronal apoptosis and suggest therapeutic targets for intervention in the pathogenesis of NeuroAIDS.

EGCG mitigates neurotoxicity mediated by HIV-1 proteins gp120 and Tat in the presence of IFN-gamma: role of JAK/STAT1 signaling and implications for HIV-associated dementia.

Human immunodeficiency virus (HIV)-1 infection of the central nervous system occurs in the vast majority of HIV-infected patients. HIV-associated dementia (HAD) represents the most severe form of HIV-related neuropsychiatric impairment and is associated with neuropathology involving HIV proteins and activation of proinflammatory cytokine circuits. Interferon-gamma (IFN-gamma) activates the JAK/STAT1 pathway, a key regulator of inflammatory and apoptotic signaling, and is elevated in HIV-1-infected brains progressing to HAD. Recent reports suggest green tea-derived (-)-epigallocatechin-3-gallate (EGCG) can attenuate neuronal damage mediated by this pathway in conditions such as brain ischemia. In order to investigate the therapeutic potential of EGCG to mitigate the neuronal damage characteristic of HAD, IFN-gamma was evaluated for its ability to enhance well-known neurotoxic properties of HIV-1 proteins gp120 and Tat in primary neurons and mice. Indeed, IFN-gamma enhanced the neurotoxicity of gp120 and Tat via increased JAK/STAT signaling. Additionally, primary neurons pretreated with a JAK1 inhibitor, or those derived from STAT1-deficient mice, were largely resistant to the IFN-gamma-enhanced neurotoxicity of gp120 and Tat. Moreover, EGCG treatment of primary neurons from normal mice reduced IFN-gamma-enhanced neurotoxicity of gp120 and Tat by inhibiting JAK/STAT1 pathway activation. EGCG was also found to mitigate the neurotoxic properties of HIV-1 proteins in the presence of IFN-gamma in vivo. Taken together, these data suggest EGCG attenuates the neurotoxicity of IFN-gamma augmented neuronal damage from HIV-1 proteins gp120 and Tat both in vitro and in vivo. Thus EGCG may represent a novel natural copound for the prevention and treatment of HAD.

Corneal toxicity of cell-penetrating peptides that inhibit Herpes simplex virus entry.

Cell-penetrating peptides (CPPs) inhibit Herpes simplex virus entry at low micromolar concentrations and may be useful either as prophylactic or therapeutic agents for herpetic keratitis. The aim of this study was to assess the in vitro and in vivo toxicity of three CPPs-EB, TAT-C, and HOM (penetratin)-for the cornea. Incubation of primary (HK320) or immortalized (THK320) human keratocytes with the EB peptide (up to 100 microM), bHOMd (up to 200 microM), or TAT-C (up to 400 microM) resulted in no evidence of toxicity using a formazan dye-reduction assay. Similar results were obtained with a human trabecular meshwork cell line (TM-1), primary human foreskin fibroblasts (DP-9), Vero, and HeLa cells with EB and TATC. The bHOMd peptide showed some toxicity in Vero and HeLa cells, with CC50 values of 70 and 93 microM, respectively. The EB peptide did not inhibit macromolecular synthesis in Vero cells at concentrations below 150 microM, although cell proliferation was blocked at concentrations of EB above 50 microM. In vivo toxicity was assessed by applying peptides in Dulbecco's modified Eagle's medium to the cornea 4 times daily for 7 d. At concentrations 1000 times the IC50 values, the EB and bHOM peptides showed no toxicity, whereas TAT-C caused some mild eyelid swelling. Some slight epithelial cell sloughing was seen with the bKLA peptide in vivo. These results suggest that these CPPs-and EB in particular-have a favorable toxicity profile, and that further development is warranted.

Inhibition of tumor necrosis factor-alpha signaling prevents human immunodeficiency virus-1 protein Tat and methamphetamine interaction.

Our previous studies demonstrated that the psychostimulant methamphetamine (MA) and the human immunodeficiency virus-1 (HIV-1) protein Tat interacted to cause enhanced dopaminergic neurotoxicity. The present study examined whether tumor necrosis factor-alpha (TNF-alpha) mediates the interaction between Tat and MA. In Sprague-Dawley rats, injections of Tat caused a small but significant increase in striatal TNF-alpha level, whereas MA resulted in no change. The increase in TNF-alpha induced by Tat + MA was not significantly different from that induced by Tat alone. Temporal analysis of TNF-alpha levels revealed a 50-fold increase 4 h after Tat administration. In C57BL/6 mice, Tat + MA induced a 50% decline in striatal dopamine levels, which was significantly attenuated in mice lacking both receptors for TNF-alpha. TNF-alpha synthesis inhibitors significantly attenuated Tat + MA neurotoxicity in hippocampal neuronal culture. The results suggest that Tat-induced elevation of TNF-alpha may predispose the dopaminergic terminals to subsequent damage by MA.

Interaction of HIV Tat and matrix metalloproteinase in HIV neuropathogenesis: a new host defense mechanism.

Tat, the HIV transactivating protein, and matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM) endopeptidases, have been implicated in the pathogenesis of HIV-associated dementia. However, the possibility that MMPs interact with viral proteins has remained unexplored. We therefore treated mixed human fetal neuronal cultures with recombinant Tat and select MMPs. Neurotoxicity was determined by measuring mitochondrial membrane potential and neuronal cell death. Previous studies have shown that Tat and MMP independently cause neurotoxicity. Surprisingly, we found the combination of Tat and MMP produced significant attenuation of neurotoxicity. To determine whether there was a physical interaction between Tat and MMP, we used protein electrophoresis and Western blot techniques, and found that MMP-1 can degrade Tat. This effect was blocked by MMP inhibitors. Furthermore, MMP-1 decreased Tat-mediated transactivation of the HIV long terminal repeat region, and this functionality was restored when MMP-1 activity was inhibited. These results suggest that the decrease in Tat-induced neurotoxicity and HIV transactivation is due to Tat's enzymatic cleavage by MMP-1. The direct interaction of human MMPs with viral proteins has now been demonstrated, with resultant modulation of Tat-mediated neurotoxicity and transactivation. This study elucidates a unique viral-host interaction that may serve as an innate host defense mechanism.