Honghong Yao

Involvement of TRPC Channels in CCL2-Mediated Neuroprotection against Tat Toxicity

Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, plays a critical role in leukocyte recruitment and activation. In the present study, we identify an additional role for CCL2 that of neuroprotection against HIV-1 transactivator protein (Tat) toxicity in rat primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in CCL2-mediated neuroprotection. TRPC are Ca2+-permeable, nonselective cation channels with a variety of physiological functions. Blockage of TRPC channels resulted in suppression of both CCL2-mediated neuroprotection and intracellular Ca2+ elevations. Parallel but distinct extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) and Akt/nuclear factor κB (NF-κB) pathways were involved in the CCL2-mediated neuroprotection. Blocking TRPC channels and specific downregulation of TRPC channels 1 and 5 resulted in suppression of CCL2-induced ERK/CREB activation but not Akt/NF-κB activation. In vivo relevance of these findings was further corroborated in wild-type and CCR2 knock-out mice. In the wild-type but not CCR2 knock-out mice, exogenous CCL2 exerted neuroprotection against intrastriatal injection of HIV-1 Tat. These findings clearly demonstrate a novel role of TRPC channels in the protection of neurons against Tat through the CCL2/CCR2 axis.

Morphine enhances Tat-induced activation in murine microglia

There is increasing evidence that opiates accelerate the pathogenesis and progression of acquired immunodeficiency syndrome (AIDS), as well as the incidence of human immunodeficiency virus (HIV) encephalitis (HIVE), a condition characterized by inflammation, leukocyte infiltration, and microglial activation. The mechanisms, by which the HIV-1 transactivating protein Tat and opioids exacerbate microglial activation, however, are not fully understood. In the current study, we explored the effects of morphine and HIV-1 Tat1–72 on the activation of mouse BV-2 microglial cells and primary mouse microglia. Both morphine and Tat exposure caused up-regulation of the chemokine receptor CCR5, an effect blocked by the opioid receptor antagonist naltrexone. Morphine in combination with Tat also induced morphological changes in the BV-2 microglia from a quiescent to an activated morphology, with a dramatic increase in the expression of the microglial activation marker CD11b, as compared with cells exposed to either agent alone. In addition, the mRNA expression of inducible nitric oxide synthase (iNOS), CD40 ligand, Interferon-gamma-inducible protein 10 (IP-10), and the proinflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-6, which were elevated with Tat alone, were dramatically enhanced with Tat in the presence of morphine. In summary, these findings shed light on the cooperative effects of morphine and HIV-1 Tat on both microglial activation and HIV coreceptor up-regulation, effects that could result in exacerbated neuropathogenesis.

Cocaine and human immunodeficiency virus type 1 gp120 mediate neurotoxicity through overlapping signaling pathways.

Although it has been well documented that drugs of abuse such as cocaine cause enhanced progression of human immunodeficiency virus (HIV)-associated neuropathological disorders, the underlying mechanisms mediating these effects remain poorly understood. The present study demonstrated that exposure of rat primary neurons to both cocaine and gp120 resulted in increased cell toxicity compared to cells treated with either factor alone. The combinatorial toxicity of cocaine and gp120 was accompanied by an increase in both caspase-3 activity and expression of the proapoptotic protein Bax. Furthermore, increased neurotoxicity in the presence of both the agents was associated with a concomitant increase in the production of intracellular reactive oxygen species and loss of mitochondrial membrane potential. Increased neurotoxicity mediated by cocaine and gp120 was ameliorated by NADPH oxidase inhibitor apocynin, thus underscoring the role of oxidative stress in this cooperation. Signaling pathways including c-jun N-teminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)/ mitogen-activated protein kinases (MAPK), and nuclear factor (NF)-κB were also identified to be critical in the neurotoxicity induced by cocaine and gp120. These findings thus underscore the role of oxidative stress, mitochondrial and MAPK signal pathways in cocaine and HIV gp120-mediated neurotoxicity.

Cooperative induction of CXCL10 involves NADPH oxidase: Implications for HIV dementia.

With the increasing prevalence of HIV‐associated neurocognititve disorders (HAND), understanding the mechanisms by which HIV‐1 induces neuro‐inflammation and subsequent neuronal damage is important. The hallmark features of HIV‐encephalitis, the pathological correlate of HIV‐associated Dementia (HAD), are gliosis, oxidative stress, chemokine dysregulation, and neuronal damage/death. Since neurons are not infected by HIV‐1, the current thinking is that these cells are damaged indirectly by pro‐inflammatory chemokines released by activated glial cells. CXCL10 is a neurotoxic chemokine that is upregulated in astroglia activated by HIV‐1 Tat, IFN‐γ, and TNF‐α. In this study we have demonstrated that HIV‐1 Tat increases CXCL10 expression in IFN‐γ and TNF‐α stimulated human astrocytes via NADPH oxidase. We have shown that the treatment of astrocytes with a mixture of Tat and cytokines leads to a respiratory burst that is abrogated by apocynin, an NADPH oxidase inhibitor. Pretreatment of Tat, IFN‐γ, and TNF‐α stimulated astrocytes with apocynin also resulted in concomitant inhibition of CXCL10 expression. Additionally, apocynin was also able to reduce Tat and cytokine‐mediated activation of the corresponding signaling molecules Erk1/2, Jnk, and Akt with a decrease in activation and nuclear translocation of NF‐κB, important regulators of CXCL10 induction. Understanding the mechanisms involved in reducing both oxidative stress and the release of pro‐inflammatory agents could lead to the development of therapeutics aimed at decreasing neuro‐inflammation in patients suffering from HAD.

Platelet-derived growth factor protects neurons against gp120-mediated toxicity

The human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 has been implicated in mediating neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic effects of gp120 could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study the authors hypothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neurons against gp120-mediated apoptosis. SH-SY5Y cells treated with gp120 exhibited increased cell death when measured by lactate dehydrogenase (LDH) and deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, with concomitant loss of neurites and increased cell rounding. Pretreatment with PDGF-BB, however, reduced gp120-associated neurotoxicity and rescued the neurite outgrowth. Additionally, gp120-mediated activation of caspase-3 was also significantly reduced in cells pretreated with PDGF-BB. Antiapoptotic effects of PDGF-BB were also confirmed by monitoring levels of anti- and proapoptotic genes, Bcl-xL and Bax, respectively. Furthermore, PDGF-mediated protection against gp120 involved the phosphoinositide (PI) 3-kinase/Akt pathway. Taken together these findings lead us to suggest that PDGF-BB could be considered as a therapeutic agent that can mitigate gp120-mediated neurotoxicity in HAD.

HIV-1 Tat Co-Operates with IFN-γ and TNF-α to Increase CXCL10 in Human Astrocytes

HIV-associated neurological disorders (HAND) are estimated to affect 60% of the HIV infected population. HIV-encephalitis (HIVE), the pathological correlate of the most severe form of HAND is often characterized by glial activation, cytokine/chemokine dysregulation, and neuronal damage and loss. However, the severity of HIVE correlates better with glial activation rather than viral load. One of the characteristic features of HIVE is the increased amount of the neurotoxic chemokine, CXCL10. This chemokine can be released from astroglia activated with the pro-inflammatory cytokines IFN-γ and TNF-α, in conjunction with HIV-1 Tat, all of which are elevated in HIVE. In an effort to understand the pathogenesis of HAND, this study was aimed at exploring the regulation of CXCL10 by cellular and viral factors during astrocyte activation. Specifically, the data herein demonstrate that the combined actions of HIV-1 Tat and the pro-inflammatory cytokines, IFN-γ and TNF-α, result in the induction of CXCL10 at both the RNA and protein level. Furthermore, CXCL10 induction was found to be regulated transcriptionally by the activation of the p38, Jnk, and Akt signaling pathways and their downstream transcription factors, NF-κB and STAT-1α. Since CXCL10 levels are linked to disease severity, understanding its regulation could aid in the development of therapeutic intervention strategies for HAND.

Mechanisms of Platelet-derived growth factor- Mediated Neuroprotection: Implications in HIV Dementia

Platelet‐derived growth factor (PDGF) has been implicated in promoting survival and proliferation of immature neurons, and even protecting neurons from gp120‐induced cytotoxicity. However, the mechanisms involved in neuroprotection are not well understood. In the present study we demonstrate the role of phosphatidylinositol 3‐kinase (PI3K)/Akt signaling in PDGF‐mediated neuroprotection. Pharmacological inhibition of PI3K greatly reduced the ability of PDGF‐BB to block gp120 IIIB‐mediated apoptosis and cell death in human neuroblastoma cells. The role of Akt in PDGF‐mediated protection was further corroborated using a dominant‐negative mutant of Akt, which was able to block the protective effect of PDGF. We next sequentially examined the signals downstream of Akt in PDGF‐mediated protection in human neuroblastoma cells. In cells pretreated with PDGF prior to gp120 there was increased phosphorylation of both GSK‐3β and Bad, an effect that was inhibited by PI3‐kinase inhibitor. Nuclear translocation of NF‐κB, which lies downstream of GSK‐3β, however, remained unaffected in cells treated with PDGF. In addition to inducing phosphorylation of Bad, PDGF‐mediated protection also involved down‐regulation of the proapoptotic protein Bax. Furthermore, PDGF‐mediated protection also involved the inhibition of gp120‐induced release of mitochondrial cytochrome C. Our findings thus underscore the roles of both PI3K/Akt and Bcl family pathways in PDGF‐mediated neuroprotection.

PDGF-mediated protection of SH-SY5Y cells against Tat toxin involves regulation of extracellular glutamate and intracellular calcium

The human immunodeficiency virus (HIV-1) protein Tat has been implicated in mediating neuronal apoptosis, one of the hallmark features of HIV-associated dementia (HAD). Mitigation of the toxic effects of Tat could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study we demonstrated that Tat-induced neurotoxicity was abolished by NMDA antagonist-MK801, suggesting the role of glutamate in this process. Furthermore, we also found that pretreatment of SH-SY5Y cells with PDGF exerted protection against Tat toxicity by decreasing extracellular glutamate levels. We also demonstrated that extracellular calcium chelator EGTA was able to abolish PDGF-mediated neuroprotection, thereby underscoring the role of calcium signaling in PDGF-mediated neuroprotection. We also showed that Erk signaling pathway was critical for PDGF-mediated protection of cells. Additionally, blocking calcium entry with EGTA resulted in suppression of PDGF-induced Erk activation. These findings thus underscore the role of PDGF-mediated calcium signaling and Erk phosphorylation in the protection of cells against HIV Tat toxicity.

Molecular mechanism(s) involved in the synergistic induction of CXCL10 by human immunodeficiency virus type 1 Tat and interferon-γ in macrophages

Synergistic interactions between viral proteins and soluble host factors released from infected mononuclear phagocytes play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia (HAD). The chemokine CXCL10 has been found to be closely associated with the progression of HIV-1-related central nervous system (CNS) disease and its related neuropsychiatric impairment. In this report the authors demonstrate that the HIV-1 protein Tat can interact with the proinflammatory cytokine interferon (IFN)-γ to dramatically induce the expression of CXCL10 in macrophages. Synergistic induction of CXCL10 by both Tat and IFN-γ was susceptible to inhibition by the MEK1/2 inhibitor U0126 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. In addition, JAK/STAT pathway plays a major role in Tat/γ-mediated CXCL10 induction in macrophages because pre-treatment of stimulated macrophages with JAK inhibitor completely abrogated the synergistic induction of the chemokine. Functionality of the synergistically induced CXCL10 was further demonstrated by its chemotactic activity for peripheral blood lymphocytes. Taken together, these findings demonstrate that the cooperative interaction of Tat and IFN-γ results in enhanced chemokine expression, which in turn can amplify the inflammatory responses within the CNS of HAD patients by recruiting more lymphocytes in the brain.