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.

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.

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.

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.

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.

Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage.

Microglia are the resident antigen-presenting cells within the central nervous system (CNS), and they serve immune-like functions in protecting the brain against injury and invading pathogens. By contrast, activated microglia can secrete numerous reactants that damage neurons. The pathogenesis of various neurodegenerative diseases has been associated with microglial activation, but the signaling pathways that program a neuronally protective or destructive phenotype in microglia are not known. To increase the understanding of microglial activation, microarray analysis was used to profile the transcriptome of BV-2 microglial cells after activation. Microglia were activated by lipopolysaccharide, the HIV neurotoxic protein TAT, and dopamine quinone, each of which has been linked to dopamine neuronal damage. We identified 210 of 9882 genes whose expression was differentially regulated by all activators (116 increased and 94 decreased in expression). Gene ontology analysis assigned up-regulated genes to a number of specific biological processes and molecular functions, including immune response, inflammation, and cytokine/chemokine activity. Genes down-regulated in expression contribute to conditions that are permissive of microglial migration, lowered adhesion to matrix, lessened phagocytosis, and reduction in receptors that oppose chemotaxis and inflammation. These results elaborate a broad profile of microglial genes whose expression is altered by conditions associated with both neurodegenerative diseases and microglial activation.

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.

HIV tat and neurotoxicity.

HIV tat is the transactivator of HIV-1, supporting efficient viral replication by stabilizing the transcription of viral genes. Tat can be released from HIV-infected cells and alter several functions in uninfected cells. In the brain, tat induces neuronal dysfunction/toxicity, even though neurons cannot be directly infected with HIV, resulting in CNS pathology, such as the dementia and encephalitis associated with NeuroAIDS. This review discusses the most recent data addressing tat-induced neurotoxicity and integrates these new findings in the context of NeuroAIDS.

MH2 domain of Smad3 reduces HIV-1 Tat-induction of cytokine secretion.

HIV-1 infection of the central nervous system (CNS) is associated with dysregulation of several important cytokines and chemokines, which are involved in inflammatory process. Earlier studies ascribed a critical role for Tat, a potent viral transcription activator, in this process by enhancing the expression of several immunomodulators including TGFbeta and MCP-1. Investigation of signaling pathways which are controlled by these cytokines led to identification of MH2 domain of Smad3, the downstream activator of TGFbeta pathway, as a modulator of MCP-1 promoter activity. The level of MCP-1 is increased in AIDS patients with neurologic problems, through recruitment of inflammatory cells, which can contribute to neuropathogenesis of AIDS. Therefore, we attempted to investigate the effect of MH2 on expression of MCP-1 and other immunolmodulators in CNS cells. By employing an adenovirus expression vector, we demonstrated that MH2 can decrease the levels of Tat-induced activation of MCP-1 and several other cytokines and chemokines in astrocytic cells. In addition, we showed that MH2 significantly reduced the activity of cytokines produced by cultures of adenovirus-MH2 transduced cells as measured by the transmigration of human PBMC cells. Thus, MH2 domain of Smad3 is a potential agent that may be developed as an inhibitor for the cytokine-mediated inflammatory responses in the brain and may have the potential to prevent transmigration of HIV-1-infected monocytes across the blood brain barrier in AIDS patients.

Methamphetamine and human immunodeficiency virus protein Tat synergize to destroy dopaminergic terminals in the rat striatum.

Dysfunction of the dopaminergic system accompanied by loss of dopamine in the striatum is a major feature of human immunodeficiency virus-1-associated dementia. Previous studies have shown that human immunodeficiency virus-1-associated dementia patients with a history of drug abuse have rapid neurological progression, prominent psychomotor slowing, more severe encephalitis and more severe dendritic and neuronal damage in the frontal cortex compared with human immunodeficiency virus-1-associated dementia patients without a history of drug abuse. In a previous study, we showed that methamphetamine and human immunodeficiency virus-1 protein Tat interact to produce a synergistic decline in dopamine levels in the rat striatum. The present study was carried out to understand the underlying cause for the loss of dopamine. Male Sprague-Dawley rats were administered saline, methamphetamine, Tat or Tat followed by methamphetamine 24 h later. Two and seven days later the animals were killed and tissue sections from striatum were processed for silver staining to examine terminal degeneration while sections from striatum and substantia nigra were processed for tyrosine hydroxylase immunoreactivity. Striatal tissue was also analyzed by Western blotting for tyrosine hydroxylase protein levels. Compared with controls, methamphetamine+Tat-treated animals showed extensive silver staining and loss of tyrosine hydroxylase immunoreactivity and protein levels in the ipsilateral striatum. There was no apparent loss of tyrosine hydroxylase in the substantia nigra. Markers for oxidative stress were significantly increased in striatal synaptosomes from Tat+methamphetamine group compared with controls. The results indicate that methamphetamine and Tat interact to produce an enhanced injury to dopaminergic nerve terminals in the striatum with sparing of the substantia nigra by a mechanism involving oxidative stress. These findings suggest a possible mode of interaction between methamphetamine and human immunodeficiency virus-1 infection to produce enhanced dopaminergic neurotoxicity in human immunodeficiency virus-1 infected/methamphetamine-abusing patients.

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.

HIV-1 Tat neurotoxicity in primary cultures of rat midbrain fetal neurons: changes in dopamine transporter binding and immunoreactivity.

HIV-1 neurotoxic proteins (Tat, gp120) are believed to play a major role in pathogenesis of dementia in a significant portion of the AIDS patient population. Dopaminergic systems appear to be particularly important in HIV-associated dementia. In the current studies, we determined that primary cell cultures prepared from the midbrain of 18-day-old rat fetuses are sensitive to Tat neurotoxicity and investigated the possible effects of Tat on DAT-specific ligand binding and DAT immunoreactivity in rat fetal midbrain cultures. We found that Tat neurotoxicity was associated with a significant decrease in [3H]WIN 35428 binding. Immunostaining of cell cultures with antibodies recognizing the C-end epitope of DAT did not reveal significant changes in DAT immunoreactivity. The results of this study implicate involvement of monoamine transmission systems in HIV-associated dementia.

Cocaine-mediated enhancement of Tat toxicity in rat hippocampal cell cultures: the role of oxidative stress and D1 dopamine receptor.

It is becoming widely accepted that psychoactive drugs can significantly alter the progression of neuropathological changes in the HIV-infected brain. The use of cocaine can aggravate the neurotoxic effects of HIV-1 proteins such as HIV-1 transactivating protein Tat and virus' envelope protein gp120. HIV-1 Tat is believed to play an important role in pathogenesis of HIV dementia (HAD). Tat is neurotoxic and a constantly growing body of evidence suggests that the toxic effects of Tat are oxidative stress-dependent. The current study reports that recombinant Tat 1-72 triggered mitochondrial depolarization, increased intracellular production of reactive oxygen species (ROS) and protein oxidation, and caused neuronal degeneration in primary hippocampal rat cell cultures. A 10 microM dose of the antioxidant Trolox, the water-soluble analog of Vitamin E, ameliorated increased intracellular ROS production and prevented cell viability decline in Tat-treated cell cultures. This fact demonstrates that Tat-induced changes in neuronal oxidative status play an important role in the mechanism of Tat neurotoxicity. While non-toxic by itself, a physiologically relevant dose of cocaine (1.5 microM) significantly enhanced Tat-induced oxidative stress and neurotoxicity in rat hippocampal cell cultures. The antioxidant Trolox significantly improved the survival of neurons exposed to the combination of 50 nM Tat and 1.5 microM cocaine but did not provide complete protection. The specific D1 dopamine receptor antagonist SCH 23390 (10 microM) did not affect Tat toxicity, but did suppress cocaine-mediated potentiation of Tat toxicity. Our results demonstrate that cocaine-mediated potentiation of Tat neurotoxicity may be related to its ability to augment Tat-induced oxidative stress.

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.

Synaptic transport of human immunodeficiency virus-Tat protein causes neurotoxicity and gliosis in rat brain.

Neurodegeneration, synaptic alterations, and gliosis are prominent features of human immunodeficiency virus (HIV) encephalitis, but HIV encephalitis is distinct from other viral encephalitides because neurodegeneration occurs in uninfected neurons at anatomical sites that are often distant from the site of viral replication. The HIV protein Tat is both neurotoxic and proinflammatory; however, its contribution to HIV-related synaptic dysfunction remains unknown. To determine the consequences of continuous Tat production in brain, we genetically engineered rat C6 glioma cells to stably produce Tat and stereotaxically infused these cells into the rat striatum or hippocampus. We discovered that HIV-Tat protein could be transported along anatomical pathways from the dentate gyrus to the CA3/4 region and from the striatum to the substantia nigra, resulting in behavioral abnormalities, neurotoxicity, and reactive gliosis. This demonstrates a unique neuronal transport property of a viral protein and establishes a mechanism for neuroglial dysfunction at sites distant from that of viral replication. Tat may thus be an important participant in brain dysfunction in HIV dementia.

Gonadal steroids differentially modulate neurotoxicity of HIV and cocaine: testosterone and ICI 182,780 sensitive mechanism.

BACKGROUND: HIV Associated Dementia (HAD) is a common complication of human immunodeficiency virus (HIV) infection that erodes the quality of life for patients and burdens health care providers. Intravenous drug use is a major route of HIV transmission, and drug use is associated with increased HAD. Specific proteins released as a consequence of HIV infection (e.g., gp120, the HIV envelope protein and Tat, the nuclear transactivating protein) have been implicated in the pathogenesis of HAD. In primary cultures of human fetal brain tissue, subtoxic doses of gp120 and Tat are capable of interacting with a physiologically relevant dose of cocaine, to produce a significant synergistic neurotoxicity. Using this model system, the neuroprotective potential of gonadal steroids was investigated. RESULTS: 17beta-Estradiol (17beta-E2), but not 17alpha-estradiol (17alpha-E2), was protective against this combined neurotoxicity. Progesterone (PROG) afforded limited neuroprotection, as did dihydrotestosterone (DHT). The efficacy of 5alpha-testosterone (T)-mediated neuroprotection was robust, similar to that provided by 17beta-E2. In the presence of the specific estrogen receptor (ER) antagonist, ICI-182,780, T's neuroprotection was completely blocked. Thus, T acts through the ER to provide neuroprotection against HIV proteins and cocaine. Interestingly, cholesterol also demonstrated concentration-dependent neuroprotection, possibly attributable to cholesterol's serving as a steroid hormone precursor in neurons. CONCLUSION: Collectively, the present data indicate that cocaine has a robust interaction with the HIV proteins gp120 and Tat that produces severe neurotoxicity, and this toxicity can be blocked through pretreatment with ER agonists.

Characterisation of cell-penetrating peptide-mediated peptide delivery.

Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. However, examination of cellular toxicity showed that antennapedia

Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI.

Gene therapy aimed at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. Polyethylenimine (PEI) has been utilized for gene delivery to the airways. In this study, we describe a new modification of PEI, in which an oligopeptide related to the protein transduction domain of HIV-1 TAT was covalently coupled to 25 kDa PEI (PEI) through a heterobifunctional polyethylenglycol (PEG) spacer resulting in a TAT-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection was observed for small (approximately 90 nm) polyplexes as well as significantly improved stability against polyanions, Alveofact, bronchial alveolar lining fluid and DNase. To determine polyplex toxicity in vitro, MTT assays were performed and, for in vivo testing, the mice bronchial alveolar lavage was investigated for total cell counts, quantity of neutrophils, total protein and TNF-alpha concentration. All parameters suggest significantly lower toxicity for TAT-PEG-PEI. Transfection efficiencies of both PEI and TAT-PEG-PEI polyplexes with DNA were studied under in vitro conditions (A549) and in mice after intratracheal instillation. While luciferase expression in A549 cells was much lower for TAT-PEG-PEI (0.2 ng/mg protein) than for PEI (2 ng/mg), significantly higher transfection efficiencies for TAT-PEG-PEI were detected in mice. Reporter gene expression was distributed through bronchial and alveolar tissue. Thus, TAT-PEG-PEI represents a new approach to non-viral gene carriers for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under in vivo conditions.

A soluble factor produced by macrophages mediates the neurotoxic effects of HIV-1 Tat in vitro.

OBJECTIVES: There is now a strong consensus that the neurotoxic properties of HIV-1 are likely to be mediated by an indirect mechanism in which neurones are damaged by infected mononuclear cells. The aim of this study was to determine the ability of HIV-1 Tat to induce neurotoxic properties in a murine macrophage cell line RAW264.7. DESIGN: Simple culture systems using dissociated neurones may not provide the appropriate microenvironment in which to observe the complex cell-cell interactions that occur in the brain. We have therefore developed a more physiological model in which rat organotypic hippocampal slices are co-cultured with the murine macrophage cell line RAW264.7. Effects of Tat were studied by using a stable Tat expressing RAW264.7 cell line or by addition of recombinant Tat protein to co-cultures. METHODS: Organotypic hippocampal slices prepared from 8-10 day rat pups were grown on membrane inserts that were placed into six-well plates on which RAW264.7 cells were growing as an adherent monolayer. Cell death in the slices was assessed using propidium iodide. Specific astrocytic (glial fibrillary acidophilic protein; GFAP) and neuronal (microtubule-associated protein; MAP2) markers were visualized by immunocytochemistry. RESULTS: RAW264.7 cells that either expressed or were exposed to HIV-1 Tat protein, produced a soluble factor that caused profound degeneration in brain slice cultures involving loss of both glial cells and neurones. By contrast treatment of slice cultures with Tat in the absence of RAW264.7 cells was not neurotoxic. CONCLUSIONS: The neurotoxic properties previously attributed to HIV-1 Tat are likely to be mediated via induction of macrophage derived soluble factor(s).