Nathan Erdmann

HIV-1-Infected and/or Immune Activated Macrophages Regulate Astrocyte SDF-1 Production Through IL-1β

Stromal cell‐derived factor 1 alpha (SDF‐1α) and its receptor CXCR4 play important roles in the pathogenesis of human immunodeficiency virus type one (HIV‐1)‐associated dementia (HAD) by serving as a HIV‐1 co‐receptor and affecting cell migration, virus‐mediated neurotoxicity, and neurodegeneration. However, the underlying mechanisms regulating SDF‐1 production during disease are not completely understood. In this report we investigated the role of HIV‐1 infected and immune competent macrophage, the principal target cell and mediator of neuronal injury and death in HAD, in regulating SDF‐1 production by astrocytes. Our data demonstrated that astrocytes are the primary cell type expressing SDF‐1 in the brain. Immune‐activated or HIV‐1‐infected human monocyte‐derived‐macrophage (MDM) conditioned media (MCM) induced a substantial increase in SDF‐1 production by human astrocytes. This SDF‐1 production was directly dependent on MDM IL‐1β following both viral and immune activation. The MCM‐induced production of SDF‐1 was prevented by IL‐1β receptor antagonist (IL‐1Ra) and IL‐1β siRNA treatment of human MDM. These laboratory observations were confirmed in severe combined immunodeficient (SCID) mice with HIV‐1 encephalitis (HIVE). In these HIVE mice, reactive astrocytes showed a significant increase in SDF‐1 expression, as observed by immunocytochemical staining. Similarly, SDF‐1 mRNA levels were increased in the encephalitic region as measured by real time RT‐PCR, and correlated with IL‐1β mRNA expression. These observations provide direct evidence that IL‐1β, produced from HIV‐1‐infected and/or immune competent macrophage, induces production of SDF‐1 by astrocytes, and as such contribute to ongoing SDF‐1 mediated CNS regulation during HAD.

Calcium-permeable AMPA receptors containing Q/R-unedited GluR2 direct human neural progenitor cell differentiation to neurons

We identify calcium‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors on human neural progenitor cells (NPCs) and present a physiological role in neurogenesis. RNA editing of the GluR2 subunit at the Q/R site is responsible for making most AMPA receptors impermeable to calcium. Because a single‐point mutation could eliminate the need for editing at the Q/R site and Q/R‐unedited GluR2 exists during embryogenesis, the Q/R‐unedited GluR2 subunit presumably has some important actions early in development. Using calcium imaging, we found that NPCs contain calcium‐permeable AMPA receptors, whereas NPCs differentiated to neurons and astrocytes express calcium‐impermeable AMPA receptors. We utilized reverse‐transcription polymerase chain reaction and BbvI digestion to demonstrate that NPCs contain Q/R‐unedited GluR2, and differentiated cells contain Q/R‐edited GluR2 subunits. This is consistent with the observation that the nuclear enzyme responsible for Q/R‐editing, adenosine deaminase (ADAR2), is increased during differentiation. Activation of calcium‐permeable AMPA receptors induces NPCs to differentiate to the neuronal lineage and increases dendritic arbor formation in NPCs differentiated to neurons. AMPA‐induced differentiation of NPCs to neurons is abrogated by overexpression of ADAR2 in NPCs. This elucidates the role of AMPA receptors as inductors of neurogenesis and provides a possible explanation for why the Q/R editing process exists.—Whitney, N. P., Peng, H., Erdmann, N. B., Tian, C., Monaghan, D. T., Zheng, J. C. Calcium‐permeable AMPA receptors containing Q/R‐unedited GluR2 direct human neural progenitor cell differentiation to neurons. FASEB J. 22, 2888‐2900 (2008)

Glutamate production by HIV‐1 infected human macrophage is blocked by the inhibition of glutaminase

Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of human immunodeficiency virus (HIV) associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. The mechanism of glutamate regulation by HIV‐1 infection remains unclear. In this report, we investigated whether the enzyme glutaminase is responsible for glutamate generation by HIV‐1 infected monocyte‐derived macrophages. We tested the functionality of novel small molecule inhibitors designed to specifically block the activity of glutaminase. Glutaminase inhibitors were first characterized in a kinetic assay with crude glutaminase from rat brain revealing an uncompetitive mechanism of inhibition. The inhibitors were then tested in vitro for their ability to prevent glutamate generation by HIV‐infected macrophages, their effect upon macrophage viability, and HIV infection. To validate these findings, glutaminase specific siRNA was tested for its ability to prevent glutamate increase during infection. Our results show that both glutaminase specific small molecule inhibitors and glutaminase specific siRNA were effective at preventing increases in glutamate by HIV‐1 infected macrophage. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

In vitro Glutaminase Regulation and Mechanisms of Glutamate Generation in HIV-1 Infected Macrophage

Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV‐1‐associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV‐1 infection of cultured MP, a process dependent upon the glutamate‐generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage‐mediated neurotoxicity. Potential mechanisms of glutaminase‐mediated excitotoxicity including enzyme up‐regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV‐infected human primary macrophage revealed up‐regulation of the glutaminase isoform GAC, yet identified no changes in the kidney‐type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.

TRAIL-Mediated Apoptosis in HIV-1-Infected Macrophages Is Dependent on the Inhibition of Akt-1 Phosphorylation

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.

HIV‐infected macrophages mediate neuronal apoptosis through mitochondrial glutaminase

A significant number of patients infected with human immunodeficiency virus‐1 (HIV‐1) suffer cognitive impairment ranging from mild to severe HIV‐associated dementia (HAD), a result of neuronal degeneration in the basal ganglia, cerebral cortex and hippocampus. Mononuclear phagocyte dysfunction is thought to play an important role in the pathogenesis of HAD. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from over‐stimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondrial glutaminase in HIV‐infected macrophages is involved in converting glutamine to glutamate. Our results demonstrate that the concentration of glutamate in HIV‐1 infected conditioned media was dependent on glutamine dose, and HIV‐1 infected conditioned medium mediated glutamine‐dependent neurotoxicity. These results indicate HIV‐infection mediates neurotoxicity through glutamate production. In addition, glutamate‐mediated neurotoxicity correlated with caspase activation and neuronal cell cycle re‐activation. Inhibition of mitochondrial glutaminase diminished the HIV‐induced glutamate production, and attenuated NMDA over‐stimulation and subsequent neuronal apoptosis. These data implicate mitochondrial glutaminase in the induction of glutamate‐mediated neuronal apoptosis during HIV‐associated dementia, and provides a possible therapeutic strategy for HAD treatment.

Potentiation of Excitotoxicity in HIV-1 Associated Dementia and the Significance of Glutaminase

HIV-1 Associated Dementia (HAD) is a significant consequence of HIV infection. Although multiple inflammatory factors contribute to this chronic, progressive dementia, excitotoxic damage appears to be an underlying mechanism in the neurodegenerative process. Excitotoxicity is a cumulative effect of multiple processes occurring in the CNS during HAD. The overstimulation of glutamate receptors, an increased vulnerability of neurons, and disrupted astrocyte support each potentiate excitotoxic damage to neurons. Recent evidence suggests that poorly controlled generation of glutamate by phosphate-activated glutaminase may contribute to the neurotoxic state typical of HAD as well as other neurodegenerative disorders. Glutaminase converts glutamine, a widely available substrate throughout the CNS to glutamate. Inflammatory conditions may precipitate unregulated activity of glutaminase, a potentially important mechanism in HAD pathogenesis.

Cellular IAP1 regulates TRAIL-induced apoptosis in human fetal cortical neural progenitor cells

Neural stem/progenitor cells (NPCs) are present in the developing and adult central nervous system. NPC apoptosis is an important aspect of normal brain development. We show that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptor 2 is highly expressed on human NPCs derived from fetal cortex, yet TRAIL induces only minimal levels of apoptosis in NPCs. Caspase‐8 mRNA and protein, an important factor in the TRAIL‐mediated death pathway, is present at low levels in human NPCs. In contrast, inhibitors of apoptosis proteins (IAP), such as c‐IAP1, are highly expressed. The transcription inhibitor actinomycin D sensitized human NPCs to TRAIL‐induced apoptosis. Further, inhibition of cellular inhibitors of apoptosis protein 1 (c‐IAP1) expression by small interfering RNA (siRNA) increased TRAIL‐mediated caspase‐3 activation and apoptosis; thus, c‐IAP1 protects NPCs against TRAIL‐induced apoptosis and suppresses caspase‐3 activation. These findings illustrate the mechanisms for NPC resistance to apoptotic agonists such as TRAIL, and demonstrate a potentially important mechanism in CNS disease states.

The signaling and apoptotic effects of TNF-related apoptosis-inducing ligand in HIV-1 associated dementia

HIV-1 Associated Dementia (HAD) develops during progressive HIV-1 infection and is characterized by cognitive impairments, behavioral disorders and potential progressive motor abnormality. Abnormal inflammation within the central nervous system (CNS), activation of macrophage/microglia and involvement of proinflammatory cytokines have been suggested as primary factors in the pathogenesis of HAD. Impairment of neuronal function and neuronal cell death are believed to be the end pathophysiological result of HAD. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines, was suggested to participate in apoptotic cell death during HAD. As a death ligand, TRAIL was originally thought to target only tumor cells. TRAIL is not typically present in CNS; however, emerging data show that TRAIL can be induced by immune stimuli on macrophage and microglia, major disease effector cells during HAD. Upregulated TRAIL may then cause neuronal apoptosis through direct interaction with TRAIL receptors on neurons or through macrophage death-mediated release of neurotoxins. In this review, we summarize the pivotal role of TRAIL in HAD and TRAIL-initiated intracellular death cascades that culminate in neuronal apoptosis as observed in HAD.