Jadwiga Turchan

Perturbation of sphingolipid metabolism and ceramide production in HIV-dementia

Infection by the human immunodeficiency virus type 1 (HIV‐1) often results in neurological dysfunction including HIV dementia (HIVD). Alterations in cytokine and redox balance are thought to play important roles in the pathogenesis of HIVD, but the specific mechanisms underlying neuronal dysfunction and death are unknown. Activation of cytokine receptors and oxidative stress can induce the production of ceramide from membrane sphingomyelin, and recent findings suggest that ceramide is an important mediator of a form of programmed cell death called apoptosis. We now report that levels of ceramide, sphingomyelin, and hydroxynonenal (HNE) are significantly increased in brain tissues and cerebrospinal fluid of HIVD patients. Exposure of cultured neurons to the neurotoxic HIV proteins gp120 and Tat resulted in increased cellular levels of sphingomyelin, ceramide, and HNE. The ceramide precursor palmitoyl‐CoA sensitized neurons to Tat and gp120 toxicity, whereas an inhibitor of ceramide production reduced Tat and gp120‐induced increases of ceramide and HNE and protected the neurons from Tat and gp120‐induced death. These results suggest that HIV‐1 infection may promote a lipid imbalance in neural cells, resulting in an overproduction of ceramide and consequent cellular dysfunction and death.

Molecular basis for interactions of HIV and drugs of abuse.

Summary: In certain populations around the world, the HIV pandemic is being driven by drug‐abusing populations. Mounting evidence suggests that these patient populations have accelerated and more severe neurocognitive dysfunction compared with non‐drug‐abusing HIV‐infected populations. Because most drugs of abuse are central nervous system stimulants, it stands to reason that these drugs may synergize with neurotoxic substances released during the course of HIV infection. Clinical and laboratory evidence suggests that the dopaminergic systems are most vulnerable to such combined neurotoxicity. Identifying common mechanisms of neuronal injury is critical to developing therapeutic strategies for drug‐abusing HIV‐infected populations. This article reviews 1) the current evidence for neurodegeneration in the setting of combined HIV infection and use of methamphetamine, cocaine, heroin or alcohol; 2) the proposed underlying mechanisms involved in this combined neurotoxicity; and 3) future directions for research. This article also suggests therapeutic approaches based on our current understanding of the neuropathogenesis of dementia due to HIV infection and drugs of abuse.

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine

BackgroundHuman immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness.To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat.ResultsAcute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors.ConclusionOur observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

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.

Novel markers of oxidative stress in actively progressive HIV dementia

Oxidative stress leads to the production of reactive oxygen species that can attack lipid membranes resulting in cellular dysfunction and death. Cellular redox state is closely linked to ceramide, sphingomyelin, and 4-hydroxynonenal (HNE) levels. We describe data showing increased levels of these oxidative stress markers in HIV encephalitis. In addition, actively progressing HIV dementia is associated with increases in HNE and ceramide, while inactive HIV dementia is associated with increases in sphingomyelin. These markers may be useful for distinguishing between different clinical phenotypes of HIV dementia.

Matrix Metalloproteinase 1 Interacts with Neuronal Integrins and Stimulates Dephosphorylation of Akt

Several studies have demonstrated that matrix metalloproteinases (MMPs) are cytotoxic. The responsible mechanisms, however, are not well understood. MMPs may promote cytotoxicity through their ability to disrupt or degrade matrix proteins that support cell survival, and MMPs may also cleave substrates to generate molecules that stimulate cell death. In addition, MMPs may themselves act on cell surface receptors that affect cell survival. Among such receptors is the α2β1 integrin, a complex that has previously been linked to leukocyte death. In the present study we show that human neurons express α2β1 and that pro-MMP-1 interacts with this integrin complex. We also show that stimulation of neuronal cultures with MMP-1 is associated with a rapid reduction in the phosphorylation of Akt, a kinase that can influence caspase activity and cell survival. Moreover, MMP-1-associated dephosphorylation of Akt is inhibited by a blocking antibody to the α2 integrin, but not by batimastat, an inhibitor of MMP-1 enzymatic activity. Such dephosphorylation is also stimulated by a catalytic mutant of pro-MMP-1. Additional studies show that MMP-1 causes neuronal death, which is significantly diminished by both a general caspase inhibitor and anti-α2 but not by batimastat. Together, these results suggest that MMP-1 can stimulate dephosphorylation of Akt and neuronal death through a non-proteolytic mechanism that involves changes in integrin signaling.

Neuroprotective Therapy for HIV Dementia

Despite the development and use of effective antiretroviral therapy, HIV dementia persists and has important socio-economic consequences. Significant progress has been made in our current understanding of the neuropathogenesis of HIV infection, and it is clear that adjunctive neuroprotective therapy in addition to antiretroviral therapy are necessary for prevention and treatment of this entity. In this manuscript, we discuss the rationale and the pathophysiological mechanisms that support the development of neuroprotective therapy for HIV dementia. We review all the placebo controlled clinical trials conducted to date with neuromodulatory/neuroprotective therapy in patients with HIV dementia and discuss their outcomes. We also provide a thorough review of potential new treatments for HIV dementia based on the experimental literature. We hope that this manuscript will serve as an important guide for future approaches for clinical trials and drug development for patients with HIV dementia.

Effects of apolipoprotein E on the human immunodeficiency virus protein Tat in neuronal cultures and synaptosomes

Human immunodeficiency virus type 1 (HIV‐1)‐associated dementia is observed in 20–30% of patients with acquired immunodeficiency syndrome (AIDS). The ϵ4 allele of the apolipoprotein E (APOE) gene currently is thought to play a role as a risk factor for the development of HIV dementia. The HIV protein Tat is neurotoxic and binds to the same receptor as apoE, the low‐density lipoprotein receptor‐related protein (LRP). In this study, we investigated the role apoE plays in Tat toxicity. Synaptosomes from wild‐type mice treated with Tat had increased reactive oxygen species (ROS), increased lipid and protein oxidation, and decreased mitochondrial membrane potential. Synaptosomes from APOE‐knockout mice also had increased ROS, increased protein oxidation, and decreased mitochondrial membrane potential, but to a significantly lesser degree. Treatment of synaptosomes with heparinase and Tat increased Tat‐induced oxidative stress, consistent with the notion of Tat requiring interaction with neuronal membranes to induce oxidative damage. Human lipidated apoE3 greatly protected neurons from Tat‐induced toxicity, whereas human lipidated apoE4 showed no protection. We demonstrated that human apoE3 has antioxidant properties against Tat‐induced toxicity. Taken together, the data suggest that murine apoE and human apoE4 act similarly and do not protect the cell from Tat‐induced toxicity. This would allow excess Tat to remain outside the cell and interact with synaptosomal membranes, leading to oxidative stress and neurotoxicity, which could contribute to dementia associated with HIV. We show that the antioxidant properties of apoE3 greatly outweigh the competition for clearance in deterring Tat‐induced oxidative stress.

Selective Isolation and Purification of Tat Protein via Affinity Membrane Separation

This work deals with the separation of Tat protein from a complex fermentation broth using an affinity membrane system. Tat is a regulatory protein that is critical for HIV‐1 replication and thus a potential candidate for vaccine and drug development. Furthermore, Tat can facilitate transport of exogenous molecules across cell membranes and is implicated in pathogenesis of HIV dementia. Affinity membranes were prepared through coupling of avidin within a 4‐stack membrane construct. Tat (naturally biotinylated) accessibility in the bacterial lysate feed was influenced by the presence of RNAse, protein concentration, and ionic strength. Enhanced accessibility translated to a marked increase in the overall product yield per pass. The purity of the membrane‐isolated Tat was compared to that prepared via packed column chromatography through SDS‐PAGE, Western blot, activity assay, and neurotoxicity studies. Tat protein produced via membrane separation yielded primarily monomeric forms of the oligopeptide sequence, whereas column chromatography produced predominately polymeric forms of Tat. These differences resulted in changes in the neurotoxicity and cellular uptake of the two preparations.

Protection of human cerebral neurons from neurodegenerative insults by gene delivery of soluble tumor necrosis factor p75 receptor.

Apoptosis plays an important role in neuronal cell death in both chronic and acute human neurodegenerative diseases, including amyotrophic lateral sclerosis, Huntington’s disease, cerebral ischemia, and human immunodeficiency virus (HIV) encephalopathy. We evaluated the ability of the extracellular binding domain of a dimeric tumor necrosis factor receptor (p75TNFR) to prevent neurotoxicity and death of human fetal cerebral neurons that were exposed in vitro to toxic agents known to be implicated in human neurological disorders, including tumor necrosis factor (TNFα) and the HIV proteins Tat and gp120. The extracellular domain of p75TNFR is capable of binding and neutralizing both soluble and transmembrane-anchored TNFα. We efficiently transduced human neurons using adenoviral vectors expressing p75TNFR (Ad.p75TNFR) or a control gene (lacZ). Treatment of control cultures with the toxic agents TNFα, TNFα plus actinomycin D, or Tat and gp120, induced neurotoxic alterations and apoptotic death of neurons. By contrast, transduction of neurons with Ad.p75TNFR prevented apoptosis and cell death due to these agents. We conclude that viral vector transfer of the p75TNFR gene efficiently protects human neurons from TNFα-, Tat- or gp120-induced apoptosis and cell death. These results suggest that p75TNFR transduction of neurons by viral vectors could be therapeutically useful in the treatment of many human neurodegenerative diseases.