Primary Human Fetal Astrocytes Research Articles

Beta-2 adrenergic receptor agonism alters astrocyte phagocytic activity and has potential applications to psychiatric disease

Schizophrenia is a debilitating condition necessitating more efficacious therapies. Previous studies suggested that schizophrenia development is associated with aberrant synaptic pruning by glial cells. We pursued an interdisciplinary approach to understand whether therapeutic reduction in glial cell—specifically astrocytic—phagocytosis might benefit neuropsychiatric patients. We discovered that beta-2 adrenergic receptor (ADRB2) agonists reduced phagocytosis using a high-throughput, phenotypic screen of over 3200 compounds in primary human fetal astrocytes. We used protein interaction pathways analysis to associate ADRB2, to schizophrenia and endocytosis. We demonstrated that patients with a pediatric exposure to salmeterol, an ADRB2 agonist, had reduced in-patient psychiatry visits using a novel observational study in the electronic health record. We used a mouse model of inflammatory neurodegenerative disease and measured changes in proteins associated with endocytosis and vesicle-mediated transport after ADRB2 agonism. These results provide substantial rationale for clinical consideration of ADRB2 agonists as possible therapies for patients with schizophrenia.

MicroRNA-210 regulates the metabolic and inflammatory status of primary human astrocytes

BackgroundAstrocytes are the most numerous glial cell type with important roles in maintaining homeostasis and responding to diseases in the brain. Astrocyte function is subject to modulation by microRNAs (miRs), which are short nucleotide strands that regulate protein expression in a post-transcriptional manner. Understanding the miR expression profile of astrocytes in disease settings provides insight into the cellular stresses present in the microenvironment and may uncover pathways of therapeutic interest.MethodsLaser-capture microdissection was used to isolate human astrocytes surrounding stroke lesions and those from neurological control tissue. Astrocytic miR expression profiles were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Primary human fetal astrocytes were cultured under in vitro stress conditions and transfection of a miR mimic was used to better understand how altered levels of miR-210 affect astrocyte function. The astrocytic response to stress was studied using qPCR, enzyme-linked immunosorbent assays (ELISAs), measurement of released lactate, and Seahorse.ResultsHere, we measured miR expression levels in astrocytes around human ischemic stroke lesions and observed differential expression of miR-210 in chronic stroke astrocytes compared to astrocytes from neurological control tissue. We also identified increased expression of miR-210 in mouse white matter tissue around middle cerebral artery occlusion (MCAO) brain lesions. We aimed to understand the role of miR-210 in primary human fetal astrocytes by developing an in vitro assay of hypoxic, metabolic, and inflammatory stresses. A combination of hypoxic and inflammatory stresses was observed to upregulate miR-210 expression. Transfection with miR-210-mimic (210M) increased glycolysis, enhanced lactate export, and promoted an anti-inflammatory transcriptional and translational signature in astrocytes. Additionally, 210M transfection resulted in decreased expression of complement 3 (C3) and semaphorin 5b (Sema5b).ConclusionsWe conclude that miR-210 expression in human astrocytes is modulated in response to ischemic stroke disease and under in vitro stress conditions, supporting a role for miR-210 in the astrocytic response to disease conditions. Further, the anti-inflammatory and pro-glycolytic impact of miR-210 on astrocytes makes it a potential candidate for further research as a neuroprotective agent.

HIV infects astrocytes in vivo and egresses from the brain to the periphery.

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4–5.2% integrated HIV gag DNA and 2–7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.

Use of Primary Human Fetal Astrocytes and Tissue Explants as Ex Vivo Models to Study Zika Virus Infection of the Developing Brain.

Zika virus (ZIKV) infection during pregnancy can result in congenital Zika syndrome which is characterized by microcephaly and other neurodevelopmental disorders. In this chapter, we describe methods to model ex vivo ZIKV infection in astrocytes and tissue explants from human fetal brain. These cell- and tissue-based platforms have been useful to elucidate mechanisms of ZIKV persistence and might lead to important clues about virus-induced neuropathogenesis. In addition, these ex vivo model systems allow researchers to conduct drug discovery and development experiments in more representative settings of the developing human brain.

EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma.

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.

ZIKV Strains Differentially Affect Survival of Human Fetal Astrocytes versus Neurons and Traffic of ZIKV-Laden Endocytotic Compartments

Malformations of the fetal CNS, known as microcephaly, have been linked to Zika virus (ZIKV) infection. Here, the responses of mammalian and mosquito cell lines, in addition to primary human fetal astrocytes and neurons were studied following infection by ZIKV strains Brazil 2016 (ZIKV-BR), French Polynesia 2013 (ZIKV-FP), and Uganda #976 1947 (ZIKV-UG). Viral production, cell viability, infectivity rate, and mobility of endocytotic ZIKV-laden vesicles were compared. All cell types (SK-N-SH, Vero E6, C6/36, human fetal astrocytes and human fetal neurons) released productive virus. Among primary cells, astrocytes were more susceptible to ZIKV infection than neurons, released more progeny virus and tolerated higher virus loads than neurons. In general, the infection rate of ZIKV-UG strain was the highest. All ZIKV strains elicited differences in trafficking of ZIKV-laden endocytotic vesicles in the majority of cell types, including astrocytes and neurons, except in mosquito cells, where ZIKV infection failed to induce cell death. These results represent a thorough screening of cell viability, infection and production of three ZIKV strains in five different cell types and demonstrate that ZIKV affects vesicle mobility in all but mosquito cells.

Absence of LEDGF/p75 Expression in Astrocytes May Affect HIV-1 Integration Efficiency.

In spite of effective anti-retroviral therapy, HIV-1 infection may still lead to neurological impairment in patients. The underlying mechanism of neurodegeneration remains mysterious. HIV-1 does not infect neurons, but does infect microglia cells in the brain. It is controversial whether HIV-1 productively infects astrocytes, an abundant glial cell type in the brain. Thirty years of investigation have led to conflicting reports concerning the entry, infection, and production of progeny virions from astrocytes. New models from studies in primary human fetal astrocytes suggest phagocytosis of HIV-1 with little productive infection. The retroviral life cycle requires integration of the viral genome to the host genome. The host protein LEDGF/p75 is required for efficient HIV-1 integration. In the absence of LEDGF/p75, HIV-1 integration and infection efficiency is reduced ten fold. Differentiated astrocytes do not appear to express LEDGF/p75, which suggests these cells are disabled for efficient integration. Phagocytosis of HIV-1 virions and the lack of LEDGF/p75 expression in astrocytes suggest that this cell type is not efficiently infected in vivo.

Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869

Zika virus (ZIKV) is a neurotrophic flavivirus that is capable of infecting humans, leading to brain abnormalities during fetal development. The ZIKV infectivity in neural target cells remains poorly understood. Here, we found that ZIKV specifically infected glial fibrillary acidic protein- and S100B-positive primary human astrocytes derived from fetal brains. In contrast, neuron-specific Class III β-tubulin (TuJ1)-positive neurons in the astrocyte cultures and SOX2-positive neural progenitor cells derived from the fetal brains were less susceptible to ZIKV infection compared with astrocytes. The infected astrocytes released competent viral particles and manifested programmed cell death with a progressive cytopathic effect. Interestingly, ZIKV infection in human fetal astrocytes induced a significant increase of extracellular vesicles (EVs). Treatment with GW4869, a specific inhibitor of neutral sphingomyelinase-2, decreased EV levels, suppressed ZIKV propagation, and reduced the release of infectious virions in astrocytes. Therefore, ZIKV infects primary human fetal astrocytes and the infection can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869. Further investigation into sphingomyelin metabolism and EVs may provide insights to the therapeutic treatment of ZIKV infection.

Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells

There is significant need to develop physiologicallyrelevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20months invitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time invitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.

SummaryGrowing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.

HIV and drug abuse mediate astrocyte senescence in a β-catenin-dependent manner leading to neuronal toxicity.

SummaryEmerging evidence suggests that cell senescence plays an important role in aging‐associated diseases including neurodegenerative diseases. HIV leads to a spectrum of neurologic diseases collectively termed HIV‐associated neurocognitive disorders (HAND). Drug abuse, particularly methamphetamine (meth), is a frequently abused psychostimulant among HIV+ individuals and its abuse exacerbates HAND. The mechanism by which HIV and meth lead to brain cell dysregulation is not entirely clear. In this study, we evaluated the impact of HIV and meth on astrocyte senescence using in vitro and several animal models. Astrocytes constitute up to 50% of brain cells and play a pivotal role in marinating brain homeostasis. We show here that HIV and meth induce significant senescence of primary human fetal astrocytes, as evaluated by induction of senescence markers (β‐galactosidase and p16INK 4A), senescence‐associated morphologic changes, and cell cycle arrest. HIV‐ and meth‐mediated astrocyte senescence was also demonstrated in three small animal models (humanized mouse model of HIV/NSG‐huPBMCs, HIV‐transgenic rats, and in a meth administration rat model). Senescent astrocytes in turn mediated neuronal toxicity. Further, we show that β‐catenin, a pro‐survival/proliferation transcriptional co‐activator, is downregulated by HIV and meth in human astrocytes and this downregulation promotes astrocyte senescence while induction of β‐catenin blocks HIV‐ and meth‐mediated astrocyte senescence. These studies, for the first time, demonstrate that HIV and meth induce astrocyte senescence and implicate the β‐catenin pathway as potential therapeutic target to overcome astrocyte senescence.

HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity

Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors, which leads to HAND in HIV-1-infected AIDS patients.

AEG-1 promotes mesenchymal transition through the activation of Rho GTPases in human glioblastoma cells.

Despite growing evidence indicating that astrocyte elevated gene-1(AEG-1) plays pivotal roles in tumor progression in various types of human cancers including brain tumors; to date, its role in the regulation of mesenchymal transition is not clear in glioblastoma. In the present study, we investigated the contribution of AEG-1 to stress fiber formation and then the acquisition of mesenchymal characteristics of glioblastoma cells. Gain- and loss-of-function studies in normal immortalized primary human fetal astrocytes(IM-PHFAs) and glioblastoma cells revealed that overexpression of AEG-1 increased expression of mesenchymal markers including N-cadherin and two mesenchymal transition‑inducing transcription factors ZEB1 and Slug but decreased epithelial markers E-cadherin and ZO-1. In addition, knockdown of AEG-1 suppressed invasive ability and migration of glioblastoma cells. Overexpression of AEG-1 also induced stress fiber formation and activated the Rho GTPase signaling pathways in glioblastoma cells. Consistently, treatment with an RhoA inhibitor decreased AEG-1-mediated stress fiber formation in glioblastoma cells. Collectively, our findings suggest that AEG-1 promotes mesenchymal transition in glioblastoma through the regulation of the Rho signaling pathway, resulting in tumor invasion, a primary characteristic of malignant brain tumors.

Abstract 4775: Astrocyte elevated gene-1(AEG-1)as a potential diagnostic/prognostic marker for prostate cancer

Abstract AEG-1/MTDH/LYRIC was initially identified as a HIV-1-inducible gene in primary human fetal astrocytes. Astrocyte Elevated Gene-1 (AEG-1) is ubiquitously overexpressed in all or most cancers and plays a regulatory role in diverse and multiple processes of carcinogenesis. AEG1 is a multi-functional regulator of normal and abnormal physiology; it contributes to broad-spectrum resistance to various chemotherapeutics; and it was recently proposed as the first potential ‘pan-cancer’ gene However, despite its critical relevance to carcinogenesis, until now genetic polymorphisms for this important gene have not been studied for association with cancer susceptibility. For example, though studies have reported AEG-1 gene expression in animal models and tumor tissues, no studies have been reported in Caucasians on the AEG-1 gene polymorphisms in large samples to establish an association with cancers. We report a PCR-RFLP screening protocol for AEG-1 gene polymorphism which is cost effective, simple and reproducible. This study also shows the strong association of AEG-1 gene with prostate cancer risk and metastasis. DNA and RNA was isolated from 232 normal healthy age matched male Caucasians and 129 prostate cancer patients recruited in the study with an informed consent. AEG-1 gene polymorphism (rs2438211) was screened using PCR-RFLP method. Patients were compared with normal healthy controls and also within patient group comparisons were made between primary and metastasis cases. Genotype analysis was done using IBM SPSS 22 software and gene expression analysis was done using RT2 profiler PCR Array Data analysis version 3.5 (SABIOSCIENCES). A strong association of AEG-1 gene polymorphism with prostate cancer (p&amp;lt;0.0001) was observed. A high frequency of the mutant allele was observed in patients (27%) as compared to normal healthy controls (7.9%).This study also highlights the possible effect of AEG-1 interaction with other genes (p53, BCCIP) contributing to tumorigenesis. Citation Format: Radhika Gade Andavolu, Catherine Stafford, Patrick Herling, Isabella Bianco, Jean-Luc Cardenas, Henry Go, Svetlana Rubakovic, Murthy V. Andavolu. Astrocyte elevated gene-1(AEG-1)as a potential diagnostic/prognostic marker for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4775.

Metadherin regulates epithelial-mesenchymal transition in carcinoma.

Metadherin (MTDH) was first identified in primary human fetal astrocytes exposed to HIV-1 in 2002 and then recognized as an important oncogene mediating tumorigenesis, progression, invasiveness, and metastasis of carcinomas. Epithelial–mesenchymal transition (EMT) is a vital process in embryonic development, organ repair, and cancer progression. MTDH and EMT have also been proved to be related to the prognosis of patients with cancers. Recent studies reveal a relationship between MTDH overexpression and EMT in some malignancies. This review highlights the overexpression of MTDH and EMT in cancers and their correlations in clinical studies. Positive correlations have been established between MTDH and mesenchymal biomarkers, and negative correlations between MTDH and epithelial biomarkers have also been established. Furthermore, experiments reveal EMT regulated by MTDH, and some signal pathways have been established. Some anticancer drugs targeting MTDH and EMT are introduced in this review. Some perspectives concerning EMT regulation by MTDH are also presented in this review.

Dynasore disrupts trafficking of herpes simplex virus proteins.

Dynasore, a small-molecule inhibitor of the GTPase activity of dynamin, inhibits the entry of several viruses, including herpes simplex virus (HSV), but its impact on other steps in the viral life cycle has not been delineated. The current study was designed to test the hypothesis that dynamin is required for viral protein trafficking and thus has pleiotropic inhibitory effects on HSV infection. Dynasore inhibited HSV-1 and HSV-2 infection of human epithelial and neuronal cells, including primary genital tract cells and human fetal neurons and astrocytes. Similar results were obtained when cells were transfected with a plasmid expressing dominant negative dynamin. Kinetic studies demonstrated that dynasore reduced the number of viral capsids reaching the nuclear pore if added at the time of viral entry and that, when added as late as 8 h postentry, dynasore blocked the transport of newly synthesized viral proteins from the nucleus to the cytosol. Proximity ligation assays demonstrated that treatment with dynasore prevented the colocalization of VP5 and dynamin. This resulted in a reduction in the number of viral capsids isolated from sucrose gradients. Fewer capsids were observed by electron microscopy in dynasore-treated cells than in control-treated cells. There were also reductions in infectious progeny released into culture supernatants and in cell-to-cell spread. Together, these findings suggest that targeting dynamin-HSV interactions may provide a new strategy for HSV treatment and prevention. HSV infections remain a global health problem associated with significant morbidity, particularly in neonates and immunocompromised hosts, highlighting the need for novel approaches to treatment and prevention. The current studies indicate that dynamin plays a role in multiple steps in the viral life cycle and provides a new target for antiviral therapy. Dynasore, a small-molecule inhibitor of dynamin, has pleiotropic effects on HSV-1 and HSV-2 infection and impedes viral entry, trafficking of viral proteins, and capsid formation.

Characterization of the NT2-derived neuronal and astrocytic cell lines as alternative in vitro models for primary human neurons and astrocytes.

Primary human fetal neurons and astrocytes (HFNs and HFAs, respectively) provide relevant cell types with which to study in vitro the mechanisms involved in various human neurological diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. However, the limited availability of human fetal cells poses a significant problem for the study of these diseases when a human cell model system is required. Thus, generating a readily available alternative cell source with the essential features of human neurons and astrocytes is necessary. The human teratoma-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal and glial cells can be generated. Nevertheless, a direct comparison of NT2 neurons and primary HFNs in terms of their morphology physiological and chemical properties is still missing. This study directly compares NT2-derived neurons and primary HFNs using immunocytochemistry, confocal calcium imaging, high-performance liquid chromatography, and high-content analysis techniques. We investigated the morphological similarities and differences, levels of relevant amino acids, and internal calcium fluctuations in response to certain neurotransmitters/stimuli. We also compared NT2-derived astrocytes and HFAs. In most of the parameters tested, both neuronal and astrocytic cell types exhibited similarities to primary human fetal neurons and astrocytes. NT2-derived neurons and astrocytes are reliable in vitro tools and a renewable cell source that can serve as a valid alternative to HFNs/HFAs for mechanistic studies of neurological diseases.

Extracellular HIV-1 viral protein R affects astrocytic glyceraldehyde 3-phosphate dehydrogenase activity and neuronal survival

Extracellular human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a pleiotropic protein accomplishing several functions within the viral life cycle. While Vpr has been described extensively as an intracellular protein, very little is known about its role as an extracellular protein. In fact, HIV-1 Vpr has been detected in the blood, serum, and cerebrospinal fluid of HIV-1-infected patients, with concentrations increasingly higher in late-stage disease. To determine the role exogenous Vpr plays in HIV-associated central nervous system dysfunction, primary human fetal astrocytes were exposed to recombinant Vpr and a time- and dose-dependent decrease was demonstrated in two fundamental intracellular metabolites (adenosine-5'-triphosphate (ATP) and glutathione (GSH)). Additionally, exposure to exogenous Vpr led to increased caspase activity and secretion of proinflammatory cytokines IL-6 and IL-8 and chemoattractants, monocyte chemotactic protein-1, and migration inhibition factor. Extracellular Vpr also dampened the glycolytic pathway through impairment of glyceraldehyde 3-phosphate dehydrogenase activity, causing a decline in the levels of ATP. The reduction in intracellular ATP increased reactive oxygen species buildup, decreasing GSH concentrations, which affected several genes in the oxidative stress pathway. In addition, exposure of the SK-N-SH neuroblastoma cell line to conditioned medium from exogenous Vpr-treated astrocytes decreased synthesis of GSH, leading to their apoptosis. These observations point to a role that Vpr plays in altering astrocytic metabolism and indirectly affecting neuronal survival. We propose a model that may explain some of the neurological damage and therefore neurocognitive impairment observed during the course of HIV-1 disease.

The NRTIs Lamivudine, Stavudine and Zidovudine Have Reduced HIV-1 Inhibitory Activity in Astrocytes

HIV-1 establishes infection in astrocytes and macroage-lineage cells of the central nervous system (CNS). Certain antiretroviral drugs (ARVs) can penetrate the CNS, and are therefore often used in neurologically active combined antiretroviral therapy (Neuro-cART) regimens, but their relative activity in the different susceptible CNS cell populations is unknown. Here, we determined the HIV-1 inhibitory activity of CNS-penetrating ARVs in astrocytes and macrophage-lineage cells. Primary human fetal astrocytes (PFA) and the SVG human astrocyte cell line were used as in vitro models for astrocyte infection, and monocyte-derived macrophages (MDM) were used as an in vitro model for infection of macrophage-lineage cells. The CNS-penetrating ARVs tested were the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir (ABC), lamivudine (3TC), stavudine (d4T) and zidovudine (ZDV), the non-NRTIs efavirenz (EFV), etravirine (ETR) and nevirapine (NVP), and the integrase inhibitor raltegravir (RAL). Drug inhibition assays were performed using single-round HIV-1 entry assays with luciferase viruses pseudotyped with HIV-1 YU-2 envelope or vesicular stomatitis virus G protein (VSV-G). All the ARVs tested could effectively inhibit HIV-1 infection in macrophages, with EC90s below concentrations known to be achievable in the cerebral spinal fluid (CSF). Most of the ARVs had similar potency in astrocytes, however the NRTIs 3TC, d4T and ZDV had insufficient HIV-1 inhibitory activity in astrocytes, with EC90s 12-, 187- and 110-fold greater than achievable CSF concentrations, respectively. Our data suggest that 3TC, d4T and ZDV may not adequately target astrocyte infection in vivo, which has potential implications for their inclusion in Neuro-cART regimens.

Thrombin induces release of proinflammatory chemokines interleukin-8 and interferon-γ-induced protein-10 from cultured human fetal astrocytes

Thrombin is a multifunctional serine proteinase that induces a variety of responses from neural cells by cleavage of proteinase-activated receptors (PARs) including PAR1 and PAR4. Thrombin/PAR signaling has been implicated in the neuroinflammatory response that occurs in the brain following stroke and other central nervous system pathologies. The neuroinflammatory response involves astrocytes and results in induction of proinflammatory chemokines including interleukin-8 (IL-8 or CXCL8) and interferon-γ-induced protein-10 (IP-10 or CXCL10) in these cells. Astroctyes are known to express PARs, however the effect of thrombin on astrocytic chemokine secretion is unknown. Here we characterize the ability of thrombin to induce proliferation/metabolic activity and chemokine secretion in primary human fetal astrocytes. Thrombin induces dose-dependent astrocyte proliferation as well as release of both IL-8 and IP-10, but not IL-6 or the chemokine regulated and normal T cell expressed and secreted (RANTES). The chemokine responses were mimicked by PAR1, but not PAR4, activating peptides. Our data indicate that astrocytic chemokine release is part of the neuroinflammatory response triggered by the exposure of the central nervous system to thrombin.