David J. Volsky

PAGE: Parametric Analysis of Gene Set Enrichment

BackgroundGene set enrichment analysis (GSEA) is a microarray data analysis method that uses predefined gene sets and ranks of genes to identify significant biological changes in microarray data sets. GSEA is especially useful when gene expression changes in a given microarray data set is minimal or moderate.ResultsWe developed a modified gene set enrichment analysis method based on a parametric statistical analysis model. Compared with GSEA, the parametric analysis of gene set enrichment (PAGE) detected a larger number of significantly altered gene sets and their p-values were lower than the corresponding p-values calculated by GSEA. Because PAGE uses normal distribution for statistical inference, it requires less computation than GSEA, which needs repeated computation of the permutated data set. PAGE was able to detect significantly changed gene sets from microarray data irrespective of different Affymetrix probe level analysis methods or different microarray platforms. Comparison of two aged muscle microarray data sets at gene set level using PAGE revealed common biological themes better than comparison at individual gene level.ConclusionPAGE was statistically more sensitive and required much less computational effort than GSEA, it could identify significantly changed biological themes from microarray data irrespective of analysis methods or microarray platforms, and it was useful in comparison of multiple microarray data sets. We offer PAGE as a useful microarray analysis method.

Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH.

Neurodegeneration and dementia are common complications of AIDS caused by human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system. HIV-1 target cells in the brain include microglia, infiltrating macrophages and astrocytes, but rarely neurons. Astrocytes play an important role in the maintenance of the synaptic micro-environment and in neuronal signal transmission. To investigate potential changes in cellular gene expression associated with HIV-1 infection of astrocytes, we employed an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue and low-passage cells were infected with HIV-1. To identify genes that display both early and late expression modifications after HIV-1 infection and to avoid cloning genes displaying normal cell cycle fluctuations in astrocytes, RNAs were isolated and pooled from 6, 12, 24 h and 3 and 7 day uninfected and infected cells and used for RaSH. Temporal cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified, and hybridized by incubation of tester and driver PCR fragments. By subtracting temporal cDNAs derived from uninfected astrocytes from temporal cDNAs made from HIV-1 infected cells, genes displaying elevated expression in virus infected cells, termed astrocyte elevated genes (AEGs), were identified. Both known and novel AEGs, not reported in current DNA databases, are described that display early or late expression kinetics following HIV-1 infection or treatment with recombinant HIV-1 envelope glycoprotein (gp120). For selected AEGs, expression of their protein products was also tested by Western blotting and found to display elevated expression following HIV-1 infection. The comparable pattern of regulation of the AEGs following HIV-1 infection or gp120 treatment suggest that HIV-1 exposure of astrocytes, even in the absence of productive infection, can induce changes in cellular gene expression.

Mechanism of Ceftriaxone Induction of Excitatory Amino Acid Transporter-2 Expression and Glutamate Uptake in Primary Human Astrocytes

Glutamate is an essential neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters primarily expressed in astroglial cells. Dysfunction of EAAT2 is implicated in acute and chronic neurological disorders, including stroke/ischemia, temporal lobe epilepsy, amyotrophic lateral sclerosis, Alzheimer disease, human immunodeficiency virus 1-associated dementia, and growth of malignant gliomas. Ceftriaxone, one of the β-lactam antibiotics, is a stimulator of EAAT2 expression with neuroprotective effects in both in vitro and in vivo models based in part on its ability to inhibit neuronal cell death by glutamate excitotoxicity. Based on this consideration and its lack of toxicity, ceftriaxone has potential to manipulate glutamate transmission and ameliorate neurotoxicity. We investigated the mechanism by which ceftriaxone enhances EAAT2 expression in primary human fetal astrocytes (PHFA). Ceftriaxone elevated EAAT2 transcription in PHFA through the nuclear factor-κB (NF-κB) signaling pathway. The antibiotic promoted nuclear translocation of p65 and activation of NF-κB. The specific NF-κB binding site at the -272 position of the EAAT2 promoter was responsible for ceftriaxone-mediated EAAT2 induction. In addition, ceftriaxone increased glutamate uptake, a primary function of EAAT2, and EAAT2 small interference RNA completely inhibited ceftriaxone-induced glutamate uptake activity in PHFA. Taken together, our data indicate that ceftriaxone is a potent modulator of glutamate transport in PHFA through NF-κB-mediated EAAT2 promoter activation. These findings suggest a mechanism for ceftriaxone modulation of glutamate transport and for its potential effects on ameliorating specific neurodegenerative diseases through modulation of extracellular glutamate.

Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120.

L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V(max) for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-alpha (TNF-alpha) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-alpha production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease.

Insights into glutamate transport regulation in human astrocytes: Cloning of the promoter for excitatory amino acid transporter 2 (EAAT2)

Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy, Alzheimers disease, amyotrophic lateral sclerosis, Huntingtons disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid transporter-2 (EAAT2) is a major glutamate transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capable of modulating glutamate transport that could potentially inhibit, ameliorate, or prevent various neurodegenerative diseases.

Detection of HIV-1 DNA in Microglia/ Macrophages, Astrocytes and Neurons Isolated from Brain Tissue with HIV-1 Encephalitis by Laser Capture Microdissection

In HIV‐1 encephalitis, HIV‐1 replicates predominantly in macrophages and microglia. Astrocytes also carry HIV‐1, but the infection of oligodendrocytes and neurons is debated. In this study we examined the presence of HIV‐1 DNA in different brain cell types in 6 paraffin embedded, archival post‐mortem pediatric and adult brain tissues with HIV‐1 encephalitis by Laser Capture Microdissection (LCM). Sections from frontal cortex and basal ganglia were stained by immunohistochemistry for CD68 (microglia), GFAP (astrocytes), MAP2 (neurons), and p24 (HIV‐1 positive cells) and different cell types were microdissected by LCM. Individual cells or pools of same type of cells were lysed, the cell lysates were subjected to PCR using HIV‐1 gag SK38/SK39 primers, and presence of HIV‐1 DNAwas confirmed by Southern blotting. HIV‐1 gag DNAwas consistently detected by this procedure in the frontal cortex and basal ganglia in 1 to 20 p24 HIV‐1 capsid positive cells, and in pools of 50 to 100 microglia/macrophage cells, 100 to 200 astrocytes, and 100 to 200 neurons in HIV‐1 positive cases but not in HIV‐1 negative controls. These findings suggest that in addition to microglia, the infection of astro‐cytes and neurons by HIV‐1 may contribute to the development of HIV‐1 disease in the brain.

HIV-associated neurocognitive disorder — pathogenesis and prospects for treatment

In the past two decades, several advancements have improved the care of HIV-infected individuals. Most importantly, the development and deployment of combination antiretroviral therapy (CART) has resulted in a dramatic decline in the rate of deaths from AIDS, so that people living with HIV today have nearly normal life expectancies if treated with CART. The term HIV-associated neurocognitive disorder (HAND) has been used to describe the spectrum of neurocognitive dysfunction associated with HIV infection. HIV can enter the CNS during early stages of infection, and persistent CNS HIV infection and inflammation probably contribute to the development of HAND. The brain can subsequently serve as a sanctuary for ongoing HIV replication, even when systemic viral suppression has been achieved. HAND can remain in patients treated with CART, and its effects on survival, quality of life and everyday functioning make it an important unresolved issue. In this Review, we describe the epidemiology of HAND, the evolving concepts of its neuropathogenesis, novel insights from animal models, and new approaches to treatment. We also discuss how inflammation is sustained in chronic HIV infection. Moreover, we suggest that adjunctive therapies — treatments targeting CNS inflammation and other metabolic processes, including glutamate homeostasis, lipid and energy metabolism — are needed to reverse or improve HAND-related neurological dysfunction.

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: Potential role in neuropathogenesis

Neurodegeneration and dementia caused by human immunodeficiency virus type 1 (HIV-1) infection of the brain are common complications of acquired immunodeficiency syndrome (AIDS). Introduction of highly active antiretroviral therapy (HAART) reduced the incidence of HIV-1-associated dementia, but so far had no effect on the high frequency of milder neurological disorders caused by HIV-1. This indicates that some neuropathogenic processes persist during limited HIV-1 replication in the central nervous system (CNS). The authors are evaluating the hypothesis that interaction of HIV-1 with astrocytes, which bind HIV-1 but support limited productive HIV-1 infection, may contribute to these processes by disrupting astrocyte functions that are important for neuronal activity or survival. Using laser-capture microdissection on brain tissue samples from HIV-1-infected individuals, we found that HIV-1 DNA can be detected in up to 1% of cortical and basal ganglia astrocytes, thus confirming HIV-1 infection in astrocytes from symptomatic patients. Using rapid subtraction hybridization, the authors cloned and identified 25 messenger RNAs in primary human fetal astrocytes either up-regulated or down-regulated by native HIV-1 infection or exposure to gp120 in vitro. Extending this approach to gene microarray analysis using Affymetrix U133A/B gene chips, the authors determined that HIV-1 alters globally and significantly the overall program of gene expression in astrocytes, including changes in transcripts coding for cytokines, G-coupled protein receptors, transcription factors, and others. Focusing on a specific astrocyte function relevant to neuropathogenesis, the authors showed that exposure of astrocytes to HIV-1 or gp120 in vitro impairs the ability of the cells to transport l-glutamate and the authors related this defect to transcriptional inhibition of the EAAT2 glutamate transporter gene. These findings define new pathways through which HIV-1 may contribute to neuropathogenesis under conditions of limited virus replication in the brain.

Highly Productive Infection with Pseudotyped Human Immunodeficiency Virus Type 1 (HIV-1) Indicates No Intracellular Restrictions to HIV-1 Replication in Primary Human Astrocytes

ABSTRACT Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 μg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.

Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-α treatment

Genes displaying altered expression as a function of human immunodeficiency virus (HIV)-1 infection of cultured primary human fetal astrocytes (PHFA) were previously identified using a rapid subtraction hybridization (RaSH) method. This scheme identified both known and novel genes displaying elevated expression, astrocyte elevated genes (AEG), and decreased expression, astrocyte suppressed genes (ASG), in PHFA as a consequence of infection with HIV-1 or treatment with HIV-1 envelope glycoprotein (gp120). RaSH also identified both known and novel genes displaying enhanced (HR) or reduced (HS) expression in HIV-1 resistant versus HIV-1 susceptible human T-cell clones. In the present study, a customized microarray approach employing these RaSH-derived genes was used to distinguish overlapping gene expression changes occurring in PHFA as a function of treatment with HIV-1 and the neurotoxic agent tumor necrosis factor (TNF)-alpha. RaSH cDNAs were spotted (microarrayed) on nylon membranes and probed with temporally isolated reverse transcribed cDNAs from HIV-1-infected and TNF-alpha-treated PHFA. This strategy identified genes displaying parallel changes after TNF-alpha treatment as observed following HIV-1 infection. Confirmation of genuine differential expression was achieved by Northern blotting. These studies document that TNF-alpha can induce a set of corresponding changes in specific AEGs and ASGs as does HIV-1 infection in PHFA. Furthermore, this customized microarray approach with RaSH-derived clones represents an efficient and sensitive methodology for elucidating molecular changes in PHFA occurring as a consequence of treatment with pharmacological agents affecting astrocyte physiology.