Sanjay B. Maggirwar

Activated protein C inhibits tissue plasminogen activator–induced brain hemorrhage

Brain hemorrhage is a serious complication of tissue plasminogen activator (tPA) therapy for ischemic stroke. Here we report that activated protein C (APC), a plasma serine protease with systemic anticoagulant, anti-inflammatory and antiapoptotic activities, and direct vasculoprotective and neuroprotective activities, blocks tPA-mediated brain hemorrhage after transient brain ischemia and embolic stroke in rodents. We show that APC inhibits a pro-hemorrhagic tPA-induced, NF-κB–dependent matrix metalloproteinase-9 pathway in ischemic brain endothelium in vivo and in vitro by acting through protease-activated receptor 1. The present findings suggest that APC may improve thrombolytic therapy for stroke, in part, by reducing tPA-mediated hemorrhage.

HIV-1 Tat Induces Neuronal Death via Tumor Necrosis Factor-α and Activation of Non-N-methyl-d-aspartate Receptors by a NFκB-Independent Mechanism

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system may result in neuronal apoptosis in vulnerable brain regions, including cerebral cortex and basal ganglia. The mechanisms for neuronal loss are likely to be multifactorial and indirect, since HIV-1 productively infects brain-resident macrophages and microglia but does not cause cytolytic infection of neurons in the central nervous system. HIV-1 infection of macrophages and microglia leads to production and release of diffusible factors that result in neuronal cell death, including the HIV-1 regulatory protein Tat. We demonstrate in this report that recombinant Tat1–86 and Tat peptides containing the basic region induce neuronal apoptosis in approximately 50% of vulnerable neurons in both rat and human neuronal cultures, and this apoptotic cell death is mediated by release of the pro-inflammatory cytokine tumor necrosis factor α, and by activation of glutamate receptors of the non-N-methyl-d-aspartate subtype. Finally, we show that Tat-induced apoptosis of human neuronal cell cultures occurs in the absence of activation of the transcription factor NFκB. These findings further define cellular pathways activated by Tat, that dysregulate production of tumor necrosis factor α, and lead to activation of glutamate receptors and neuronal death during HIV-1 infection of the central nervous system.

HIV-1 Tat-Mediated Activation of Glycogen Synthase Kinase-3β Contributes to Tat-Mediated Neurotoxicity

Abstract : Human immunodeficiency virus type 1 (HIV‐1) Tat induces neuronal apoptosis. To examine the mechanism(s) that contribute to this process, we studied Tats effects on glycogen synthase kinase‐3β (GSK‐3β), an enzyme that has been implicated in the regulation of apoptosis. Addition of Tat to rat cerebellar granule neurons resulted in an increase in GSK‐3β activity, which was not associated with a change in protein expression and could be abolished by the addition of an inhibitor of GSK‐3β (lithium). Lithium also enhanced neuronal survival following exposure to Tat. Coprecipitation experiments revealed that Tat can associate with GSK‐3β, but direct addition of Tat to purified GSK‐3β had no effect on enzyme activity, suggesting that Tats effects might be mediated indirectly. As the activation of platelet activating factor (PAF) receptors is critical for the induction of neuronal death by several candidate HIV‐1 neurotoxins, we determined whether PAF can also activate GSK‐3β. Application of PAF to neuronal cultures activated GSK‐3β, and coincubation with lithium ameliorated PAF‐induced neuronal apoptosis. These findings are consistent with the existence of one or more pathways that can lead to GSK‐3β activation in neurons, and they suggest that the dysregulation of this enzyme could contribute to HIV‐induced neuronal apoptosis.

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.

Neuroprotective Activities of Sodium Valproate in a Murine Model of Human Immunodeficiency Virus-1 Encephalitis

Human immunodeficiency virus-1 (HIV-1) infection of the nervous system can result in neuroinflammatory events leading first to neuronal dysfunction then to cognitive and behavioral impairments in infected people. The multifaceted nature of the disease process, commonly called HIV-1-associated dementia (HAD), provides a number of adjunctive therapeutic opportunities. One proposed adjunctive therapy is sodium valproate (VPA), an anticonvulsant known to promote neurite outgrowth and increase β-catenin through inhibiting glycogen synthase kinase 3β activity and tau phosphorylation. We now show that VPA treatment of rat cortical neurons exposed to HIV-1 gp120 prevents resultant neurotoxic activities. This includes the induction of significant neurite outgrowth and microtubule-associated protein 2 (MAP-2) and neuron-specific nuclear protein (NeuN) antigens in affected neuronal cell bodies and processes. Similarly, VPA protects severe combined immunodeficient (SCID) mice against the neurodegeneration of HIV-1ADA infected monocyte-derived macrophages (MDMs). In SCID mice with HIV-1 MDM-induced encephalitis, VPA treatment significantly reduced neuronal phosphorylatedβ-catenin and tau without affecting HIV-1 replication or glial activation. We conclude that VPA protects neurons against HIV-1 infected MDM neurotoxicity, possibly through its effects on the phosphorylation of tau and β-catenin. The use of VPA as an adjuvant in treatment of human HAD is being pursued.

The Aryl Hydrocarbon Receptor Attenuates Tobacco Smoke-induced Cyclooxygenase-2 and Prostaglandin Production in Lung Fibroblasts through Regulation of the NF-κB Family Member RelB

Diseases such as chronic obstructive pulmonary disease and lung cancer caused by cigarette smoke affect millions of people worldwide. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that influences responses to certain environmental pollutants such as tobacco smoke. However, the physiological function(s) of the AhR is unknown. Herein we propose that the physiologic role of the AhR is to limit inflammation. We show that lung fibroblasts from AhR–/– mice produce a heightened inflammatory response to cigarette smoke, typified by increased levels of cyclooxygenase-2 (COX-2) and prostaglandins (PGs), when compared with wild type (AhR+/+) fibroblasts. This response was dependent on AhR expression as transient transfection of an AhR expression plasmid into AhR–/– fibroblasts significantly attenuated the smoke-induced COX-2 and PG production, confirming the anti-inflammatory role of the AhR. The AhR can interact with NF-κB. However, the heightened inflammatory response observed in AhR–/– fibroblasts was not the result of NF-κB (p50/p65) activation. Instead it was coupled with a loss of the NF-κB family member RelB in AhR–/– fibroblasts. Taken together, these studies provide compelling evidence that AhR expression limits proinflammatory COX-2 and PG production by maintaining RelB expression. The association between RelB and AhR may represent a new therapeutic and more selective target with which to combat inflammation-associated diseases.

PPAR-γ Ligands Repress TGFβ-Induced Myofibroblast Differentiation by Targeting the PI3K/Akt Pathway: Implications for Therapy of Fibrosis

Transforming growth factor beta (TGFβ) induced differentiation of human lung fibroblasts to myofibroblasts is a key event in the pathogenesis of pulmonary fibrosis. Although the typical TGFβ signaling pathway involves the Smad family of transcription factors, we have previously reported that peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands inhibit TGFβ-mediated differentiation of human lung fibroblasts to myofibroblasts via a Smad-independent pathway. TGFβ also activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway leading to phosphorylation of AktS473. Here, we report that PPAR-γ ligands, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and 15-deoxy-(12,14)-15d-prostaglandin J2 (15d-PGJ2), inhibit human myofibroblast differentiation of normal and idiopathic pulmonary fibrotic (IPF) fibroblasts, by blocking Akt phosphorylation at Ser473 by a PPAR-γ-independent mechanism. The PI3K inhibitor LY294002 and a dominant-negative inactive kinase-domain mutant of Akt both inhibited TGFβ-stimulated myofibroblast differentiation, as determined by Western blotting for α-smooth muscle actin and calponin. Prostaglandin A1 (PGA1), a structural analogue of 15d-PGJ2 with an electrophilic center, also reduced TGFβ-driven phosphorylation of Akt, while CAY10410, another analogue that lacks an electrophilic center, did not; implying that the activity of 15d-PGJ2 and CDDO is dependent on their electrophilic properties. PPAR-γ ligands inhibited TGFβ-induced Akt phosphorylation via both post-translational and post-transcriptional mechanisms. This inhibition is independent of MAPK-p38 and PTEN but is dependent on TGFβ-induced phosphorylation of FAK, a kinase that acts upstream of Akt. Thus, PPAR-γ ligands inhibit TGFβ signaling by affecting two pro-survival pathways that culminate in myofibroblast differentiation. Further studies of PPAR-γ ligands and small electrophilic molecules may lead to a new generation of anti-fibrotic therapeutics.

Bcl-2 Prevents CD95 (Fas/APO-1)-induced Degradation of Lamin B and Poly(ADP-ribose) Polymerase and Restores the NF-κB Signaling Pathway

In the study presented here, we investigated the possible interactions between CD95 (Fas/APO-1) and Bcl-2 by studying the effects of Bcl-2 on the modulation of cellular pathways activated by CD95 using HeLa cells as a model system. We report that stable expression of Bcl-2 in HeLa cells is associated with multiple phenotypic changes. Treatment of HeLa cells with anti-CD95 monoclonal antibody (mAb) resulted in preferential degradation of lamin B compared with lamins A and C. Significant lamin B degradation was detected as early as 1 h after anti-CD95 mAb treatment. In contrast, lamins A and C as well as actin remained unchanged until 4 h after treatment with anti-CD95 mAb, a time point that correlated with the period of DNA fragmentation. These results indicate that selective degradation of lamin B is an early cellular event in response to activation of the CD95 pathway and that it precedes DNA fragmentation. Overexpression of Bcl-2 resulted in prevention of lamin B degradation and DNA fragmentation into oligonucleosome fragments in response to the apoptotic signal by anti-CD95 mAb. In addition, in Bcl-2-overexpressing cells that were protected against apoptosis, anti-CD95 mAb-induced cleavage of poly(ADP-ribose) polymerase was completely blocked. Overexpression of Bcl-2 also resulted in restoration of the CD95-mediated signaling pathway involving activation of the transcription factor NF-κB (p50/RelA). These findings suggest that Bcl-2 prevents apoptosis in part by preventing the degradation of major nuclear polypeptides such as lamin B and poly(ADP-ribose) polymerase. In addition, our results demonstrate that CD95-mediated signaling involves activation of NF-κB (p50/RelA).

Activation of glycogen synthase kinase 3 beta (GSK‐3β) by platelet activating factor mediates migration and cell death in cerebellar granule neurons

Children with vertically acquired HIV‐1 can present with a rapidly progressive encephalopathy and neuronal apoptosis in the first 12–18 months of life. Furthermore, abnormal prenatal platelet activating factor (PAF) signalling may result in lissencephaly, a disorder of neuronal migration. PAF, produced from human immunodeficiency virus type 1 (HIV‐1) ‐infected brain‐resident macrophages, induces neuronal apoptosis in cultured cerebellar granule neurons (CGNs) in part by activating glycogen synthase kinase 3 beta (GSK‐3β). However, PAF can also inhibit migration of CGNs that are dispersed and allowed to reaggregate. Therefore, we investigated the biological effects following activation of GSK‐3β by PAF, and whether these effects were dependent on the culture conditions of the CGNs. We show here that activation of neuronal GSK‐3β by PAF is receptor‐specific, with similar kinetics of activation in both monolayer cultures of CGNs that have ceased to migrate and reaggregate cultures of CGNs that are actively migrating. However, PAF receptor activation in reaggregated CGNs inhibits neuronal migration and induces approximately half the level of neuronal apoptosis compared with PAF‐treated CGN cultures that have ceased to migrate. PAF‐mediated inhibition of neuronal migration in reaggregated CGNs or induction of apoptosis in CGNs that have ceased to migrate can be reversed by either PAF receptor antagonists, or the GSK‐3β inhibitors lithium or valproic acid, in a dose‐dependent manner. Abnormal PAF signalling that results in GSK‐3β overactivation may represent a common mechanism for pathological defects in neuronal migration in the prenatal period and neuronal apoptosis in the postnatal period.

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

BackgroundUnlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs.ResultsHere, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.ConclusionSince HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.