Fuwang Peng

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.

Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes

HIV encephalitis (HIVE), the pathologic correlate of HIV‐associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation, and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV‐infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon‐γ inducible peptide‐10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the proinflammatory cytokines, IFN‐γ and TNF‐α, are also markedly increased in CNS tissues during HIV‐1 infection. In this study, we hypothesized that the interplay of host cytokines and HIV‐1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV‐1 and the proinflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF‐κB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV‐1 and cytokine‐mediated up‐regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.

Cocaine-mediated enhancement of virus replication in macrophages: Implications for human immunodeficiency virus-associated dementia

Injection drug use has been recognized as a major risk factor for acquired immunodeficiency syndrome (AIDS) from the outset of the epidemic. Cocaine, one of the most widely abused drugs in the United States, can both impair the functions of macrophages and CD4+ lymphocytes and also activate human immunodeficiency virus (HIV)-1 expression in these cells. Because the brain is the target organ for both cocaine and HIV, the objective of the present study was to explore the effects of cocaine on virus replication in macrophages, the target cells for the virus in the central nervous system (CNS). Cocaine markedly enhanced virus production in simian human immunodeficiency virus (SHIV)-infected monocyte-derived macrophages (MDMs) and in U1 cells, a chronically infected promonocytic cell line as monitored by enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Cocaine treatment also resulted in the activation of nuclear factor (NF)-κB and transcriptional activation of the HIV-LTR (long terminal repeat) gag-GFP (green fluorescent protein). Analyses of chemokines in cocaine-treated macrophages by real-time reverse transcriptase—polymerase chain reaction (RT-PCR) and Luminex assays suggested increased expression of interleukin (IL)-10, a cytokine that is known to promote HIV replication in MDMs. In addition to enhancing IL-10 expression, cocaine also caused an up-regulation of the macrophage activation marker, human leukocyte antigen (HLA)-DR, in MDMs. The synergistic effect of cocaine on virus replication and its enhancement of host activation markers suggest that cocaine functions at multiple pathways to accelerate HIV-associated dementia (HAD).

Cocaine-mediated Alteration in Tight Junction Protein Expression and Modulation of CCL2/CCR2 Axis Across the Blood-Brain Barrier: Implications for HIV-Dementia

One of the hallmark features underlying the pathogenesis of HIV encephalitis is the disruption of blood–brain barrier (BBB). Cocaine, often abused by HIV-infected patients, has been suggested to worsen the HIV-associated dementia (HAD) via unknown mechanisms. The objective of the present study was to explore the effects of cocaine on BBB permeability using human brain microvascular endothelial cells (HBMECs). Additionally, because the chemokine CCL2 and its receptor CCR2 play a crucial role in the recruitment of inflammatory cells into the central nervous system in HAD brains, we tested for the effect of cocaine in modulating the CCL2/CCR2 axis. Our findings suggest that exposure of HBMECs to cocaine correlated with the breakdown of ZO-1 tight junction protein and reorganization of the cytoskeleton resulting in stress fiber formation. Furthermore, cocaine also modulated upregulation of the CCL2/CCR2 axis in monocytes. These findings conform to the multifaceted effects of cocaine leading to accelerated progression of HIV-1 neuropathogenesis.

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.

TRPC channel-mediated neuroprotection by PDGF involves Pyk2/ERK/CREB pathway.

Platelet-derived growth factor-BB (PDGF) has been reported to provide tropic support for neurons in the central nervous system. The protective role of PDGF on dopaminergic neurons, especially in the context of HIV-associated dementia (HAD), however, remains largely unknown. Here, we show that exogenous PDGF was neuroprotective against toxicity induced by HIV-1 Tat in primary midbrain neurons. Furthermore, we report the involvement of transient receptor potential canonical (TRPC) channels in PDGF-mediated neuroprotection. TRPC channels are Ca2+-permeable, nonselective cation channels with a variety of physiological functions. Blocking TRPC channels with either a blocker or short-interfering RNAs (specific for TRPC 5 and 6) in primary neurons resulted in suppression of both PDGF-mediated neuroprotection as well as elevations in intracellular Ca2+. PDGF-mediated neuroprotection involved parallel but distinct ERK/CREB and PI3K/Akt pathways. TRPC channel blocking also resulted in suppression of PDGF-induced Pyk2/ERK/CREB activation, but not Akt activation. Relevance of these findings in vivo was further corroborated by intrastriatal injections of PDGF and HIV-1 Tat in mice. Administration of PDGF was able to rescue the dopaminergic neurons in the substantia nigra from Tat-induced neurotoxicity. This effect was attenuated by pre-treatment of mice with the TRP blocker, thus underscoring the novel role of TRPC channels in the neuroprotection mediated by PDGF.

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.

Platelet-derived Growth Factor-mediated Induction of the Synaptic Plasticity Gene Arc/Arg3.1

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc.

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 Synergistically Enhances IFN-γ-Induced Expression of CXCL10 in Blood-Derived Macrophages: Implications for HIV Dementia

There is increasing cumulative evidence that activated mononuclear phagocytes (macrophages/microglia) releasing inflammatory mediators in the CNS are a better correlate of HIV-associated dementia (HAD) than the actual viral load in the brain. Earlier studies on simian HIV/rhesus macaque model of NeuroAIDS confirmed that pathological changes in brains of macaques with encephalitis were associated with up-regulation of platelet-derived growth factor (PDGF) and the chemokine, CXCL10. Because the complex interplay of inflammatory mediators released by macrophages often leads to the induction of neurotoxins in HAD, we hypothesized that PDGF could interact with IFN-γ to modulate the expression of CXCL10 in these primary virus target cells. Although PDGF alone had no effect on the induction of CXCL10 in human macrophages, in conjunction with IFN-γ, it significantly augmented the expression of CXCL10 RNA & protein through transcriptional and posttranscriptional mechanisms. Signaling molecules, such as JAK and STATs, PI3K, MAPK, and NF-κB were found to play a role in the synergistic induction of CXCL10. Furthermore, PDGF via its activation of p38 MAPK was able to increase the stability of IFN-γ-induced CXCL10 mRNA. Understanding the mechanisms involved in the synergistic up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.