Shongshan Fan

Glycogen Synthase Kinase 3β-Mediated Apoptosis of Primary Cortical Astrocytes Involves Inhibition of Nuclear Factor κB Signaling

ABSTRACT Recent studies have revealed a positive correlation between astrocyte apoptosis and rapid disease progression in persons with neurodegenerative diseases. Glycogen synthase kinase 3β (GSK-3β) is a molecular regulator of cell fate in the central nervous system and a target of the phosphatidylinositol 3-kinase (PI-3K) pathway. We have therefore examined the role of the PI-3K pathway, and of GSK-3β, in regulating astrocyte survival. Our studies indicate that inhibition of PI-3K leads to apoptosis in primary cortical astrocytes. Furthermore, overexpression of a constitutively active GSK-3β mutant (S9A) is sufficient to cause astrocyte apoptosis, whereas an enzymatically inactive GSK-3β mutant (K85M) has no effect. In light of reports on the interplay between GSK-3β and nuclear factor κB (NF-κB), and because of the antiapoptotic activity of NF-κB, we examined the effect of GSK-3β overexpression on NF-κB activation. These experiments revealed strong inhibition of NF-κB activation in astrocytes upon overexpression of the S9A, but not the K85M, mutant of GSK-3β. This was accompanied by stabilization of the NF-κB-inhibitory protein, IκBα and down-regulation of IκB kinase (IKK) activity. These findings therefore implicate GSK-3β as a regulator of NF-κB activation in astrocytes and suggest that the pro-apoptotic effects of GSK-3β may be mediated at least in part through the inhibition of NF-κB pathway.

Methamphetamine Causes Mitrochondrial Oxidative Damage in Human T Lymphocytes Leading to Functional Impairment

Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.

Inhibition of Mixed Lineage Kinase 3 Prevents HIV-1 Tat-Mediated Neurotoxicity and Monocyte Activation

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.

Infection of Human Immunodeficiency Virus and Intracellular Viral Tat Protein Exert a Pro-survival Effect in a Human Microglial Cell Line

Abstract The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4+ T lymphocytes is well studied and typically results in virally induced cytolysis. In contrast, relatively little is known concerning the interplay between HIV-1 and microglia. Recent findings suggest that, counter-intuitively, HIV-1 infection may extend the lifespan of microglia. We developed a novel cell line model system to confirm and mechanistically study this phenomenon. We found that transduction of a human microglial cell line with an HIV-1 vector results in a powerful cytoprotective effect following apoptotic challenge. This effect was reproduced by ectopic expression of a single virus-encoded protein, Tat. Subsequent studies showed that the pro-survival effects of intracellular Tat could be attributed to activation of the PI-3-kinase (PI3K)/Akt pathway in the microglial cell line. Furthermore, we found that expression of Tat led to decreased expression of PTEN, a negative regulator of the PI-3-K pathway. Consistent with this, decreased p53 activity and increased E2F activity were observed. Based on these findings, a model of possible regulatory circuits that intracellular Tat and HIV-1 infection engage during the cytoprotective event in microglia has been suggested. We propose that the expression of Tat may enable HIV-1 infected microglia to survive throughout the course of infection, leading to persistent HIV-1 production and infection in the central nervous system.

Dyad of CD40/CD40 Ligand Fosters Neuroinflammation at the Blood–Brain Barrier and Is Regulated via JNK Signaling: Implications for HIV-1 Encephalitis

Human immunodeficiency virus 1 (HIV-1) infection may result in activation of peripheral monocytes followed by their infiltration into the CNS, where the release of proinflammatory mediators causes neurologic disease. Previously, we detected high levels of soluble CD40 ligand (CD40L) in CSF and plasma of HIV-infected patients with cognitive impairment. We now show that CD40, a receptor for CD40L, is highly expressed in brain endothelial cells of patients affected by HIV-1 encephalitis (HIVE), suggesting an important role for the CD40/CD40L dyad in regulating blood–brain barrier (BBB) functions. This concept was further supported by in vitro experiments. Exposure of primary human brain microvascular endothelial cells (BMVECs) to CD40L upregulated the expression of adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, which caused a fourfold increase in monocyte adhesion to BMVECs and stimulated migration across an in vitro BBB model. Investigations into the intracellular signaling pathways that govern these events revealed that cJUN-N-terminal kinase (JNK) is critical to CD40 activation in the BMVECs. CD40L induced activation of mixed-lineage-kinase-3 and JNK, leading to the subsequent activation of cJUN/AP-1 (activating-protein-1). JNK inhibition in the BMVECs prevented CD40L-mediated induction of adhesion molecules, monocyte adhesion, and transendothelial migration. These new findings support the concept that the CD40/CD40L dyad plays an important role in HIVE neuroinflammation.

Human immunodeficiency virus-encoded Tat activates glycogen synthase kinase-3β to antagonize nuclear factor-κB survival pathway in neurons

The pathogenesis of human immunodeficiency virus type 1 (HIV‐1)‐associated dementia is mediated by neuronal dysfunction and death, brought about by the action of soluble neurotoxic factors that are released by virally infected macrophages and microglia. Paradoxically, many candidate HIV‐1 neurotoxins also possess the ability to activate nuclear factor‐kappa B (NF‐κB), which has a potent pro‐survival effect in primary neurons. The present study explored this conundrum and investigated why NF‐κB might fail to protect neurons that are exposed to candidate HIV‐1 neurotoxins. Here, we evaluated the ability of virus‐depleted conditioned medium produced by HIV‐1‐infected human macrophages (HIV‐MCMs) to modulate NF‐κB activity in neurons. We demonstrated that HIV‐MCMs inhibit the normal signaling pathways that lead to NF‐κB activation in neurons. This inhibitory effect of HIV‐MCM is dependent upon the presence of HIV‐1 Tat, which activates glycogen synthase kinase (GSK)‐3β in neurons. Activation of GSK‐3β, in turn, results in modification of the NF‐κB subunit RelA at serine 468, thereby regulating the physical interaction of RelA with histone deacetylase‐3 corepressor molecules. Furthermore, neutralization of Tat or inhibition of GSK‐3β activity prevents neuronal apoptosis induced by HIV‐MCM. We conclude that HIV‐1 Tat may compromise neuronal function and fate by interfering with normal survival pathways subserved by NF‐κB. These findings may have important therapeutic implications for the management of HIV‐1‐associated dementia.

Attenuation of HIV-1 replication in macrophages by cannabinoid receptor 2 agonists

Infiltrating monocytes and macrophages play a crucial role in the progression of HIV‐1 infection in the CNS. Previous studies showed that activation of the CB2 can attenuate inflammatory responses and affect HIV‐1 infectivity in T cells and microglia. Here, we report that CB2 agonists can also act as immunomodulators on HIV‐1‐infected macrophages. First, our findings indicated the presence of elevated levels of CB2 expression on monocytes/macrophages in perivascular cuffs of postmortem HIV‐1 encephalitic cases. In vitro analysis by FACS of primary human monocytes revealed a step‐wise increase in CB2 surface expression in monocytes, MDMs, and HIV‐1‐infected MDMs. We next tested the notion that up‐regulation of CB2 may allow for the use of synthetic CB2 agonist to limit HIV‐1 infection. Two commercially available CB2 agonists, JWH133 and GP1a, and a resorcinol‐based CB2 agonist, O‐1966, were evaluated. Results from measurements of HIV‐1 RT activity in the culture media of 7 day‐infected cells showed a significant decrease in RT activity when the CB2 agonist was present. Furthermore, CB2 activation also partially inhibited the expression of HIV‐1 pol. CB2 agonists did not modulate surface expression of CXCR4 or CCR5 detected by FACS. We speculate that these findings indicate that prevention of viral entry is not a central mechanism for CB2‐mediated suppression in viral replication. However, CB2 may affect the HIV‐1 replication machinery. Results from a single‐round infection with the pseudotyped virus revealed a marked decrease in HIV‐1 LTR activation by the CB2 ligands. Together, these results indicate that CB2 may offer a means to limit HIV‐1 infection in macrophages.

Involvement of the p53 and p73 transcription factors in neuroAIDS.

HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.

Activation of adenosine A2A receptor protects sympathetic neurons against nerve growth factor withdrawal.

Adenosine mediates a range of effects in the central nervous system (CNS), including the promotion of neuronal survival, but its actions on sympathetic neurons are less well characterized. We therefore sought to understand the role of endogenous adenosine in contributing to the survival of neurotrophin‐dependent sympathetic neurons. Rat superior cervical ganglion (SCG) cultures were maintained in the continuous presence of nerve growth factor (NGF) and then exposed to adenosine deaminase (ADA), to deplete endogenous adenosine. This resulted in a marked increase in cellular apoptosis, to a level that approximated the effect of NGF withdrawal. Furthermore, the addition of exogenous adenosine to NGF‐deprived SCG neurons resulted in enhanced cell survival. Analysis of adenosine receptor (AR) subtypes on these neurons, using real‐time RT‐PCR and receptor binding analyses, revealed that the A2A receptor was the major subtype present. Accordingly, the A2A receptor agonist CGS21680 significantly reduced both ADA‐induced and NGF‐withdrawal‐induced neuronal apoptosis, whereas the A1 receptor agonist R‐PIA had no such effect. The survival‐promoting effect of CGS21680 was eliminated when cells were coincubated with a molar excess of an A2A receptor antagonist. Finally, follow‐up experiments revealed that CGS21680 prevented the induction of early apoptotic events, such as changes in mitochondrial integrity and caspase activation, and that it also triggered an increase in ERK activation, which was essential for neurotrophin‐independent cell survival. Taken together, these findings provide evidence that endogenous adenosine may be important in mediating protection of sympathetic neurons and that it may act via the A2A receptor subtype.

Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction

We and others have demonstrated that stimulants such as methamphetamine (METH) exerts immunosuppressive effects on the host’s innate and adaptive immune systems and has profound immunological implications. Evaluation of the mechanisms responsible for T-cell immune dysregulation may lead to ways of regulating immune homeostasis during stimulant use. Here we evaluated the effects of METH on T cell cycle entry and progression following activation. Kinetic analyses of cell cycle progression of T-cell subsets exposed to METH demonstrated protracted G1/S phase transition and differentially regulated genes responsible for cell cycle regulation. This result was supported by in vivo studies where mice exposed to METH had altered G1 cell cycle phase and impaired T-cell proliferation. In addition, T cells subsets exposed to METH had significant decreased expression of cyclin E, CDK2 and transcription factor E2F1 expression. Overall, our results indicate that METH exposure results in altered T cell cycle entry and progression. Our findings suggest that disruption of cell cycle machinery due to METH may limit T-cell proliferation essential for mounting an effective adaptive immune response and thus may strongly contribute to deleterious effect on immune system.