Open Access

RETRACTED ARTICLE: Increased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in brain of HIV-1 transgenic rats

  • Jagadeesh Sridhara Rao1Email author,
  • Hyung-Wook Kim1,
  • Matthew Kellom1,
  • Dede Greenstein2,
  • Mei Chen1,
  • Andrew David Kraft3,
  • Gaylia Jean Harry3,
  • Stanley Isaac Rapoport1 and
  • Mireille Basselin1
Journal of Neuroinflammation20118:101

DOI: 10.1186/1742-2094-8-101

Received: 9 March 2011

Accepted: 16 August 2011

Published: 16 August 2011

The Erratum to this article has been published in Journal of Neuroinflammation 2012 9:19
The Retraction Note to this article has been published in Journal of Neuroinflammation 2017 14:95

Abstract

Background

Cognitive impairment has been reported in human immune deficiency virus-1- (HIV-1-) infected patients as well as in HIV-1 transgenic (Tg) rats. This impairment has been linked to neuroinflammation, disturbed brain arachidonic acid (AA) metabolism, and synapto-dendritic injury. We recently reported upregulated brain AA metabolism in 7- to 9-month-old HIV-1 Tg rats. We hypothesized that these HIV-1 Tg rats also would show upregulated brain inflammatory and AA cascade markers and a deficit of synaptic proteins.

Methods

We measured protein and mRNA levels of markers of neuroinflammation and the AA cascade, as well as pro-apoptotic factors and synaptic proteins, in brains from 7- to 9-month-old HIV-1 Tg and control rats.

Results

Compared with control brain, HIV-1 Tg rat brain showed immunoreactivity to glycoprotein 120 and tat HIV-1 viral proteins, and significantly higher protein and mRNA levels of (1) the inflammatory cytokines interleukin-1β and tumor necrosis factor α, (2) the activated microglial/macrophage marker CD11b, (3) AA cascade enzymes: AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2)-IVA, secretory sPLA2-IIA, cyclooxygenase (COX)-2, membrane prostaglandin E2 synthase, 5-lipoxygenase (LOX) and 15-LOX, cytochrome p450 epoxygenase, and (4) transcription factor NF-κBp50 DNA binding activity. HIV-1 Tg rat brain also exhibited signs of cell injury, including significantly decreased levels of brain-derived neurotrophic factor (BDNF) and drebrin, a marker of post-synaptic excitatory dendritic spines. Expression of Ca2+-independent iPLA2-VIA and COX-1 was unchanged.

Conclusions

HIV-1 Tg rats show elevated brain markers of neuroinflammation and AA metabolism, with a deficit in several synaptic proteins. These changes are associated with viral proteins and may contribute to cognitive impairment. The HIV-1 Tg rat may be a useful model for understanding progression and treatment of cognitive impairment in HIV-1 patients.

Background

Despite improved survival rates for human immunodeficiency virus (HIV-1)-infected patients due to antiretroviral therapy, HIV-1-associated neurocognitive disorders remain a significant public health burden [1, 2]. Among HIV-1-infected patients, cognitive impairment is a serious complication of HIV-1-infection, and occurs in a substantial (15-50%) proportion of patients [2]. Indeed, a pilot study revealed high rates of asymptomatic neurocognitive impairment in perinatally infected HIV-positive young adults (67%) when compared with older subjects (19%) [3]. Another study highlighted that the prevalence of HIV-associated neurocognitive disorders is high even among long-standing aviremic HIV-positive patients [4].

Deficits in spatial learning also have been demonstrated in aged HIV-1 transgenic (Tg) rats [5, 6]. The HIV-Tg rat contains the HIV-1 virus in its genome, but is not infectious because it lacks the gag and pol replication genes of the virus [7]. HIV-1 Tg rats express the functional viral envelope proteins glycoprotein (gp) 120 and trans-activator of transcription (Tat) in brain and circulating white cells [7]. It has been proposed that these rats can be used to examine effects of these envelope proteins in the absence of infection (viral replication), which may mimic the condition in patients given highly active antiretroviral therapy, who have limited (controlled) viral replication but persistent HIV-1 infection [8]. HIV-1 Tg rats demonstrate reduced spatial learning at 5 months of age, and by 7-9 months show neuroinflammation and upregulated brain arachidonic acid (AA) metabolic rates [5, 6, 9].

Synapto-dendritic injury, a likely cause of cognitive impairment in HIV-1 patients [1012], can be exacerbated by a neuroinflammatory microenvironment [13]. During inflammation, AA is released from membrane phospholipids by AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and secretory sPLA2. This process is associated with increased production of cytokines (e.g., tumor necrosis factor alpha (TNFα) and interleukin (IL)-1β and nitric oxide from activated microglia. Released TNFα and IL-1β can continue to activate AA cascade metabolism by activating transcription factor NF-κB [1417]. Further, the released AA can be converted into pro-inflammatory lipid mediators, such as prostaglandin (PG) H2, leukotrienes, and related compounds by the action of cyclooxygenase (COX), lipoxygenase (LOX) and thromboxane synthase (TXS) enzymes. PGH2 is converted to PGE2 by membrane prostaglandin E synthase (mPGES) or cytosolic PGES (cPGES), or by TXS to TXA2. HIV-1 patients show increased concentrations of PGE2, PGF2 and TXB2 in their cerebrospinal fluid [18], consistent with in vivo and in vitro studies [1921].

A relation of AA and its pro-inflammatory metabolites to neuronal apoptosis and synapse loss has been demonstrated in vivo and in vitro [2226]. Furthermore, reduced dendritic spine density and complexity have been associated with deficits in learning, memory, and general cognitive function [12]. Neuronal loss also may result from insufficient trophic factors, including brain-derived neurotrophic factor (BDNF) [27]. The post-synaptic dendritic proteins, drebrin and neurofilament light chain (L), are abundantly expressed in neurons [2831], and changes in their expression have been used to evaluate neuronal damage [32, 33]. Loss of drebrin has been associated with cognitive impairment in Alzheimer disease and mild cognitive impairment patients [32, 3437]. However, an association between synapse loss and upregulation of the AA cascade has not been identified in vivo. In the current study we used 7- to 9-month-old HIV-1 Tg rats to characterize the brain pro-inflammatory microenvironment and synaptic integrity (determined by levels of drebrin and neurofilament-L). We now show upregulated levels of AA cascade markers and of IL-1β and TNFα in the brain of these HIV-1 Tg rats, in association with lower levels of BDNF, drebrin and neurofilament-L.

Methods

Animals

This protocol was approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23). Seven- to 9-month-old male, specific pathogen-free, Fischer 344/Hsd HIV-1 Tg rats (n = 6) and age-matched parental wild-type inbred Fischer 344/Hsd non-Tg control rats (n = 6) were purchased from Harlan Laboratories (Indianapolis, IN) and housed in an animal facility with controlled temperature, humidity, and 12-h light/dark cycle. Food (Teklad global 18% protein diet, 2018S (sterilized) for controls and 2918 (irradiated) for HIV-1 Tg rats (Harlan) [9] and water were provided ad libitum. After three days of acclimation, rats were anaesthetized with an overdose of CO2 and decapitated. Their brain was rapidly excised, sagittally cut into four sections from the left and right hemispheres, frozen in 2-methylbutane at -50°C, and stored at -80°C until studied. One section from the left hemisphere from each rat was used to isolate the cytosolic fraction, a corresponding section from the right hemisphere was used for total RNA extraction, and remaining sections from both hemispheres were used to prepare nuclear extracts.

Preparation of cytosolic fractions

Cytosolic brain fractions were prepared as reported [38]. One section from each brain was homogenized in a buffer containing 20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 5 mM EDTA, 1.5 mM pepstatin, 2 mM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 U/ml aprotinin, and 2 mM dithiothreitol, using a Polytron homogenizer. The homogenate was centrifuged at 100,000 g for 60 min at 4°C, and the resulting supernatant (cytosolic fraction) collected. Protein concentrations were determined using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA).

Total RNA isolation and real time RT-PCR

Brain tissue was homogenized in Qiagen® lysis solution and total RNA was isolated by phenol-chloroform extraction using a RNeasy® lipid tissue mini kit (Qiagen, Valencia, CA). Complementary DNA was prepared from total RNA using a high-capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). mRNA levels (IL-1β, TNFα, GFAP, CD11b, cPLA2-IVA, sPLA2-IIA, iPLA2-VIA, COX-1, COX-2, mPGES, cPGES, 5-, 12-, 15-LOX, TXS, cytochrome p450 epoxygenase, drebrin and neurofilament-L) were measured by quantitative RT-PCR, using an ABI PRISM 7000 sequence detection system (Applied Biosystems). Specific primers and probes for cPLA2-IVA B, sPLA2-IIA B, iPLA2-VIA, COX-1, COX-2, mPGES, cPGES, 5-, 12-, 15-LOX, TXS, cytochrome p450 epoxygenase, drebrin and neurofilament-L were purchased from TaqManR gene expression assays (Applied Biosystems), and consisted of a 20× mix of unlabeled PCR primers and Taqman minor groove binder (MGB) probe (FAM dye-labeled). The fold-change in gene expression was determined by the ΔΔCT method [39]. Data are expressed as the relative level of the target gene (IL-1β, TNFα, GFAP, CD11b, cPLA2-IVA B, sPLA2-IIA, iPLA2-VIA, COX-1, COX-2, mPGES, cPGES, 5-, 12-, and 15-LOX, TXS, cytochrome p450 epoxygenase, drebrin or neurofilament-L) in the brain of the HIV-1 Tg rat normalized to the endogenous control (β-globulin) and relative to the control (calibrator). All experiments were carried out in triplicate from each control and HIV-1 Tg rat brain (n = 6).

Western blot for protein levels

Proteins from the cytosolic fraction (65 μg) were separated on 4-20% SDS-polyacrylamide gels (PAGE) (Bio-Rad), and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). Cytosolic protein blots were incubated overnight in Tris-buffered-saline containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies for proinflammatory cytokines: IL-1β (1:500), TNFα (1:500); astrocytes: glial fibrillary acidic protein (GFAP) (1:1000); CD11b (1:1000); AA cascade proteins: cPLA2-IVA, sPLA2-IIA, iPLA2-VIA, COX-1 (1:1000), COX-2 (1:1000), cytochrome p450 epoxygenase, TXS, 5-, 12-, 15-LOX, mPGES, cPGES (1:1000); gp120 (1:100); tat (1:100); drebrin (1:1000), BDNF (1:1000) (Santa Cruz, Santa Cruz, CA);); neurofilament-L (1:500) (Cell Signaling Technology, Danvers, MA) and β-actin (1:10,000) (Sigma Aldrich, St. Louis, MO). The cytosolic blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad), and were visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA) and were normalized to β-actin. All experiments were conducted on 6 independent samples. Values are expressed as percent of control.

Transcription factor NF-κBp50 and NF-κBp65 activities

Nuclear extracts were prepared as reported [40, 41] and protein concentrations were determined using Bio-Rad Protein Reagent (Bio-Rad). NF-κBp50 and NF-κBp65 activities were measured according to the manufacturer's instructions (Panomics, Freemont, CA), using nuclear extracts obtained from the control and HIV-1 Tg rats. Briefly, 10 μg of nuclear extract from each sample was preincubated with biotin-labeled NF-κBp50 or p65 oligonucleotides in a separate vial for 60 min. The labeled oligonucleotide-nuclear protein complexes were immobilized on a streptavidin-coated 96-well plate. The bound oligonucleotide nuclear protein complex was detected by adding NF-κBp50 or p65 antibody to the respective NF-κBp50 or p65 complex, followed by addition of secondary antibody conjugated to HRP. Color was developed with tetramethylbenzidine substrate and optical densities were measured at 450 nm. Values are expressed as percent of control. All experiments were conducted on 6 independent samples.

Measurement of active caspase-3 protein

Active caspase-3 protein was measured according to the manufacturer's instructions (Cell Signaling, Danvers, MA), using cytosolic brain fractions from the control and HIV-1 Tg rats Briefly, 100 μl (100 μg) of cytosolic fraction was incubated with pre-coated capture antibody in a microwell plate overnight at 4°C. After incubation, the target protein was captured by coated antibody. Following extensive washing, an HRP-linked secondary antibody was added to recognize the bound antibody complex. Color was developed with tetramethylbenzidine substrate and optical densities were measured at 450 nm. Values are expressed as percent of control. All experiments were conducted on 6 independent samples.

Immunohistochemistry

In a separate cohort of animals, astrocyte and microglia morphology was analyzed by immunohistochemistry. Following CO2 anesthesia, the brain (n = 4) was rapidly excised, cut in the midsagittal plane, and the individual hemispheres immersion-fixed in 4% paraformaldehyde/phosphate buffer (pH 7.2) for 18 h, followed by cryoprotection. Fifty- μm free-floating coronal serial cryosections of the forebrain were stored in solution (FD Neurotechnologies, Baltimore, MD) at -20°C. Sections (between +1.0 and 0.4 mm from bregma) were washed with phosphate buffered saline (PBS), equilibrated to room temperature (RT), transferred to 10 mM citrate buffer containing 0.05% Tween-20 and incubated 30 min at 80°C. Sections were then rinsed in PBS and incubated 2 h in blocking solution (2% goat serum, 1% bovine serum albumin, 0.1% Triton X-100 in automation buffer (Biomedia, Foster City, CA). Sections were incubated with anti-GFAP or ionized calcium binding adopter molecule 1 (Iba-1, 1:500, Dako, Glostrup, Denmark) in blocking solution for 18 h at 4°C, re-equilibrated to RT, washed with PBS, and incubated with Alexa Fluor antibody conjugates (1:250, Invitrogen, Carlsbad, CA) in blocking solution without Triton X-100 for 2 h at RT. Digital images of immunostaining in the somatosensory cortex and the dentate gyrus of the hippocampus were collected using a LSM 410 inverted confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) equipped with argon, HeNe, and iFlex 2000 PSU lasers. Image stacks were collected at 1.5 mm steps (20×) or 1.0 mm steps (63×) and displayed as a single image using 3D maximum projection.

Statistics

Data are expressed as mean ± SEM. We used t-tests for independent samples for group comparisons. We further tested significance using the false discovery rate (FDR) to correct for multiple comparisons. We set alpha = 0.01 to reduce type one error risk. An alpha = 0.01 and an n of 15 markers per mRNA and protein assays would give a 14% chance of at least one false positive for each mRNA and protein assay using the following formula 1-(1-.01)e15. A p value less than 0.01 and 0.001 is represented by ** and *** respectively.

Results

Gp120 and tat proteins and neuroinflammatory markers in HIV-1 Tg rats

Gp120 and tat protein levels were detectable in cytosolic brain fractions of HIV-1 Tg but not of control rats (Figure 1A). Brain mRNA and protein levels for the astrocyte structural protein, GFAP, were not significantly altered in the HIV-1 Tg rats compared to controls (Figure 1B-C). As a molecular marker for activated microglia and macrophages [42], mRNA level of the CD11b was elevated significantly by 7.1-fold in the HIV-1 Tg compared with control brain (Figure 1D), corresponding to a significant 190% elevation in CD11b protein (Figure 1E) (p < 0.001).
Figure 1

(A) Representative immunoblot of gp120 and tat protein in HIV-1 Tg rat brain (A), detected as described in Methods. mRNA levels of brain GFAP (B) and CD11b (D) in control and HIV-1 Tg rat brain, measured using real time RT-PCR, normalized to β-globulin and relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (C) GFAP and (E) CD11b protein in control and HIV-1 Tg rat brain. Bar graphs are ratios of optical densities of individual protein bands to β-actin, expressed as percent of control. Data represent mean ± SEM, statistical significance: **p < 0.01, ***p < 0.001 as determined by unpaired t-test.

To characterize regional specificity of the changes, we measured GFAP immunoreactivity and microglial markers in the hippocampus and somatosensory cortex. In contrast to the initial report on the HIV-1 Tg rat [7], histological examination of the somatosensory cortex (Figures 2 A-B) and of the hippocampus (Figures 2 C-D) did not indicate increased GFAP immunoreactivity in the HIV-1 Tg rats, as their cells maintained a normal thin process-bearing morphology and there was no evidence of astrocyte hypertrophy (Figures 2 E-H). When we examined the morphological phenotype of microglia within various brain regions using Iba-1+ to label diverse phenotypes, minimal differences from control were noted in the HIV-1 Tg rats (Figures 3 A-H). In the somatosensory cortex, microglia maintained a normal appearance with fine ramified processes and had no prominent evidence of activation or of a phagocytic phenotype (Figures 3 A-B). When these immunopositive cells were examined at higher magnification (Figures 3 E-F), the Iba-1+ cells displayed decreased arbor complexity. Given previous reports of deficits in a hippocampal-dependent spatial memory task in HIV-1 Tg rats, we further examined the morphological phenotype of microglia within the dentate gyrus of the hippocampus. Overall labeling of Iba-1+ microglia was not significantly different in the HIV-1 Tg compared to control rats, with no evidence of overt microglia activation or amoeboid phenotype (Figures 3 C-D). At higher magnification, Iba-1+ microglia displayed fine processes and complicated arborization in the control brain (Figure 3G). A distinct difference was noted in the Iba-1+ microglia in the HIV-1 Tg rat hippocampus, with the cells displaying diminished arbor complexity and approximately 50% shortened processes (p < 0.05 by t-test) as determined by a modified Sholl analysis (Figure 3H), but with no evidence of amoeboid phagocytic microglia.
Figure 2

Representative immunofluorescence (gray scale) for GFAP+ astrocytes (white) in layers IV-V of somatosensory cortex of control (A) and HIV-1 Tg rats (B) and in the hippocampus dentate gyrus of control (C) and HIV-1 Tg rats (D) at 7 months of age. Scale bar = 50 microns. In 3-5 sections obtained from each of 4 animals per group, there was no evidence of astrocyte hypertrophy as represented in the higher magnification image of the astrocyte morphology in the (E, F) somatosensory cortex or (G, H) hippocampus. Images represent compiled z-stack images collected through a 50 micron section. Scale bar = 4 microns.

Figure 3

Representative immunofluorescence (gray scale) for Iba-1+ microglia (white) in layers IV-V of the somatosensory cortex of control (A, C) and HIV-1 Tg rats (B, D) and within the dentate gyrus of the hippocampus of control (E, G) and HIV-1 Tg (F, H) rats at 7 months of age. Images represent compiled z-stack images collected throughout a 50 micron section. Higher magnification of individual representative cells demonstrates diminished arborization of Iba-1+ microglia primarily within the hippocampus. Microglia within defined regional areas were randomly selected (10/section/animal) and the projection distance of the processes was determined using a modified Sholl analysis. In the control brain, 90% (± 10%) of the processes projected past the 4th Sholl while in the HIV-1 Tg rat this was decreased to only 40% (± 18%). Estimates of complexity of the dendritic branching were generated by counting the number of processes originating at cell body. The number of processes was not statistically different from the number in the HIV-1 Tg rat hippocampus, ranging between 5 and 6 in Tg rats and between 7 and 8 in controls, although complexity and secondary branching appeared lower.

Increased proinflammatory cytokine response in HIV-1Tg brains

HIV-1 Tg rats showed significantly increased mRNA levels of inflammatory cytokines IL-1β (9.6-fold) (p < 0.001) and TNFα (3.5-fold) (p < 0.01) respectively (Figures 4A, B). These elevations corresponded to elevated brain protein levels of IL-1β (59%) and TNFα (45%) as compared to controls (Figures 4C, D) (p < 0.01). There was a 73% increase in NF-κBp50 activity in HIV-1 Tg compared to control rat brain (Figure 4E) (p < 0.01). However, NF-κBp65 activity did not differ significantly between groups (Figure 4F).
Figure 4

mRNA levels of brain IL-1β (A) and TNFα (B) in control and HIV-1 Tg rats, measured using real time RT-PCR. Data are levels of brain IL-1β and TNFα in the HIV-1 Tg rat normalized to β-globulin and represented relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (C) IL-1β and (D) TNFα protein in control and HIV-1 Tg rat brain. Bar graphs are ratios of optical densities of immunoblots to β-actin, expressed as percent of control (mean ± SEM). Representative brain transcription factor binding activities (DNA-protein complex) of NF-κBp50 (E) and NF-κBp65 (F) in control and HIV-1 Tg rats. DNA binding activity was measured in brain nuclear extracts as described in Methods. Data represent mean ± SEM. Statistical significance: **p < 0.01, ***p < 0.001 as determined by unpaired t-test.

Upregulation of arachidonic cascade enzymes in HIV-1 Tg rat brain

Brain protein and mRNA levels of a number of AA cascade markers were elevated significantly in HIV-1 Tg rats relative to controls. Mean mRNA levels of cPLA2-IVA, sPLA2-IIA and COX-2 were increased (p < 0.01) in HIV-1 Tg compared to control rats by 5-fold, 9-fold and 4.5 fold respectively (Figures 5A-C), but the iPLA2-VIA mRNA level did not differ between groups (HIV-1 Tg 0.92 ± 0.12 vs. control 1.00 ± 0.30). Mean mRNA levels of mPGES (Figure 5D), COX-1 (HIV-1 Tg 0.87 ± 0.20 vs. control 1.00 ± 0.20) and cPGES (HIV-1 Tg 0.97 ± 0.20 vs. control 1.00 ± 0.20) were not significantly different between groups.
Figure 5

mRNA levels of brain cPLA 2 -VIA (A), sPLA 2 -II (B), COX-2 (C), and mPGES (D) in control and HIV-1 Tg rats, determined using real time TaqMan RT-PCR. Data are levels of brain cPLA2-VIA, sPLA2-II, COX-2 and mPGES in the HIV-1 Tg rat normalized to the endogenous control (β-globulin) and relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (E) cPLA2-VIA, (F) sPLA2- IIA (G) COX-2, and (H) mPGES protein in control and HIV-1 Tg rats. Bar graphs represent ratios of optical densities of each individual protein band relative to β-actin, expressed as percent of control mean ± SEM. Mean ± SEM. Data were analyzed by individual unpaired t-tests, statistical significance: **p < 0.01, ***p < 0.001.

The mean protein level of cPLA2-VIA was increased by 119% (p < 0.01), whereas sPLA2 IIA protein was not changed significantly, as the increase was only at p < 0.05 (Figures 5E, F). The mean iPLA2--VIA protein level also did not differ significantly between groups (HIV-1 Tg 114 ± 6.8 vs. control 100 ± 15). The mean protein level of COX-2 was increased significantly by 42% (p < 0.01) (Figure 5G), but the mean mPGES protein level was not (Figure 5H). COX-1 and cPGES protein levels did not differ significantly between groups (COX-1, HIV-1 Tg 118 ± 17 vs. control 100 ± 15; cPGES, HIV-1 Tg 101 ± 11.2 vs. control 100 ± 11).

5-LOX, 15-LOX and p450 epoxygenase expression in HIV-1 Tg rat brain

There were statistically significant increases in mean brain mRNA levels of 5-LOX, 15-LOX and cytochrome p450 expoxygenase in HIV-1 Tg relative to control rats by 2.9-fold (Figure 6A) (p < 0.001), 4.6-fold (Figure 6B) (p < 0.01) and 4.4-fold (Figure 6C), respectively. Upregulation of these was unaccompanied by significant elevations in the respective mean protein levels, whose increases in each case were only at p < 0.05 (Figures 6D-F). Further, there was no significant difference in 12-LOX or TXS protein between groups (data not shown).
Figure 6

mRNA levels of brain 5-LOX BB (A), 15-LOX (B) and cytochrome p450 epoxygenase (C) in control and HIV-1 Tg rats, measured using real time TaqMan RT-PCR. Data represent individual transcript levels normalized to β-globulin, in HIV-1 Tg rat brain relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (D) 5-LOX, (E) 15-LOX, and (F) cytochrome p450 epoxygenase protein in control and HIV-1 Tg rats. Bar graphs display ratios of optical densities of individual protein bands to β-actin, expressed as percent of control. Mean ± SEM, statistical significance: **p < 0.01, ***p < 0.001 as determined by an unpaired t-test.

Indications of neuronal damage and loss in HIV-1 Tg rat

The active caspase 3 protein level (Figure 7A), and levels of neurofilament-L mRNA and protein (Figures 7C, D) did not differ significantly between HIV-1 Tg and control rats, as the former mean decreased at p < 0.05 and the values for neurofilament-L increased only at p < 0.05. BDNF protein (Figure 7B) and drebrin mRNA and protein (Figures 7E, F) were significantly less in HIV Tg than control rats (p < 0.01).
Figure 7

(A) Representative brain active caspase-3 level in control and HIV-1 Tg rats. The active caspase-3 level was measured in brain cytosolic fractions as described in Methods. Bar graphs are relative to control and were compared using an unpaired t-test, mean ± SEM. Representative immunoblots of BDNF (B), neurofilament-L (D) and drebrin (F) protein levels in control and HIV-1 Tg rats. Bar graphs display mean ± SEM optical densities of individual protein bands relative to β-actin, expressed as percent of control. mRNA levels of neurofilament-L (C) and drebrin (E) in control and HIV-1 Tg rat brain, measured using real time TaqMan RT-PCR. Data represent mean ± SEM mRNA levels in the HIV-1 Tg rat brain normalized to β-globulin, relative to control level (calibrator) using the ΔΔCT method. statistical significance: **p < 0.01, ***p < 0.001 as determined by an unpaired t-test.

Discussion

Direct effects of viral gp120 and tat proteins or secondary effects due to neuroinflammatory factors have been associated with HIV-1 infection and HIV-1 related cognitive impairment. HIV-1 Tg rats aged 7-9 months showed gp120 and tat protein in brain, accompanied by significantly elevated AA cascade markers. These differences were accompanied by significant (p < 0.01) elevations in mRNA levels of neuroinflammatory cytokines TNFα and IL-1β, and of the microglial marker CD11b, and reductions in mRNA and protein levels for the synaptic marker, drebrin. These changes occurred in the absence of significantly increased expression of GFAP protein, a marker of astrogliosis.

Elevations in the neuroinflammatory and the AA signaling cascade in HIV-1 Tg rats

We have reported increased cPLA2-IV and sPLA2-IIA activities in the brain of 7- to 9-month-old HIV-1 Tg rats [9]. Consistent with these findings, HIV-1 Tg rat brain in the present study showed elevated protein and mRNA levels (p < 0.01) of cPLA2-IVA and an elevated mRNA level of sPLA2-IIA, without a significant change in iPLA2-VIA or sPLA2-IIA protein levels. COX-2 mRNA and protein levels were significantly higher in HIV-1 Tg rats than controls, whereas COX-1, cPGES or TXS did not differ significantly, consistent with our report of an increased brain concentration of PGE2 but not of TXB2 in HIV-1 Tg rat brain [9]. mPGES protein and mRNA levels were also were not increased in HIV-1 Tg rats.

Our earlier study also showed elevated levels of leukotriene B4, a product of 5-LOX and leukotriene A4 hydrolase, in the brain of HIV-1 Tg rats [9]. Consistent with that report, HIV-1 Tg brain in the present study showed significantly increased 5-LOX mRNA without a significant change in 12-LOX expression. This change was accompanied by increased mRNA levels of cytochrome p450 expoxygenase and 5-LOX. Given that epoxyeicosatrienoic acid produced by cytochrome p450 expoxygenase can be neuroprotective [43, 44], the elevated brain mRNA level of cytochrome p450 epoxygenase in HIV-1 Tg may reflect a compensatory neuroprotective process. While elevations in protein levels of 5-LOX, 15-LOX and cytochrome p450 expoxygenase did not reach significance because of our requirement for multiple comparisons, in each case changes were in the same direction as elevations of the respective mRNA at p < 0.05.

The changes in the AA cascade markers noted in HIV-1 Tg rat brain may be related to microglial activation, with release of proinflammatory cytokines and activation of the NF-κB transcription factor. NF-κB binding sites are present on the promoter region of the gene transcripts of the AA cascade markers, cPLA2-IVA, sPLA2-IIA and COX-2 [4547]. Cell culture studies have shown that IL-1β or TNFα can induce transcription of cPLA2, sPLA2 and COX-2 genes in an NF-κB-dependent manner [1417, 48]. NF-κBp50 is known to regulate transcription of pro-inflammatory genes [49, 50] and can influence HIV-1 gene expression [51]. Elevated DNA binding activity of NF-κBp50 in the HIV-1 Tg rat suggests that the elevated AA cascade markers in the current study may be related to increased levels of IL-1β and TNFα and increased NF-κBp50 DNA binding activity, but are independent of NF-κBp65.

In the absence of HIV-1 replication, the presence of gp120 and tat proteins in HIV-1 Tg rat brain likely account for microglial activation and the increased level of CD11b. In vitro, gp120 directly stimulates microglia and increases expression of CD11b [52]. However, microglia did show retraction of their processes and diminished complexity of arborization, which suggests an early reactive response. As we did not examine animals younger than 7 months, we cannot conclude that these changes were age-related. The altered microglial morphology in the hippocampus is of interest, given the role of the hippocampus in spatial learning tasks and the proposed involvement of microglia during synapse stripping and remodeling [53]. CD11b and Iba-1 cannot be used to distinguish between resident microglia and infiltrating blood borne monocytes. Thus, while we did not observe amoeboid brain macrophages, we cannot rule out a contribution of monocytes from the circulation, especially since gp120 can compromise the blood-brain barrier [54].

In vitro, gp120 can stimulate AA release and PGE2 formation in glial cells [1921] and elevate levels of IL-1β in co-cultures of primary hippocampal neurons and astrocytes [19]. In the initial characterization of the HIV-1 Tg rat, a response of astrocytes was suggested by an increase in GFAP immunoreactivity [7]. In the current study, we did not find an astrocytic response. Consistent with no change in astroglial morphology, protein and mRNA levels of astroglial marker GFAP were not significantly altered. Further studies are needed to understand the role of astrocytes in HIV-1 infection. Similar to gp120, tat protein is also known to stimulate AA release and COX-2 expression in rat brain [5557]. The changes observed with neuroinflammatory and AA cascade markers in HIV-1Tg rats could be due to the presence of tat protein in the HIV-1Tg brain. Altogether, viral proteins can induce neuroinflammatory and AA cascade markers in brain. Despite altered protein levels of the AA cascade enzymes 5-LOX, 12-LOX and p450 epoxygenase a p < 0.05 in HIV-1 Tg brain, these changes did not reach statistical significance at p < 0.01. This may be due to the small sample size; further studies are required to understand changes in HIV-1 Tg brain.

HIV-1 Tg rats show subtle changes in synaptic marker

Neuropathological features of human HIV-1 infection include cortical atrophy, altered dendritic arborization of neurons, and decreased synaptic density [1012, 58]. Neurons are vulnerable to both gp120 and the HIV-1 virus protein, tat [59, 60]. Gp120 and tat are reported to induce apoptosis of neurons in vitro and in vivo [6163] by activating caspases, particularly caspase-3 [59]. The current study did not show a statistically significant increase in the protein level of active caspase-3 in HIV-1 Tg rats. Damage and apoptosis of neurons would be manifest as a loss of neuronal and related markers. Within this framework, we now report a significantly lower mRNA and protein levels of the post-synaptic dendritic marker drebrin, and a reduced protein level of BDNF (p < 0.01). Protein and mRNA levels of neurofilament-L were reduced only at p < 0.05 in the HIV-1 Tg rats. Reduced BDNF is consistent with a report that gp120 reduces BDNF in rat brain in association with neuronal death [59]. A lifelong presence of gp120 in brain may impair neuronal development by reducing neurofilament and microtubule expression [32]. A significantly reduced level of drebrin suggests that altered synaptic structure contributes to cognitive-behavioral defects reported in the HIV-1 Tg rat [5, 6].

In brain, microglia are the primary source of TNFα [64, 65], and its release is implicated in neurotoxicity [66]. In HIV-1 Tg rats, elevated levels of IL-1β and TNFα and increased expression of AA cascade enzymes, have been implicated in neuronal damage [67] and cognitive-behavioral impairment [6873]. A recent study indicates that similar changes could contribute to cognitive impairment in HIV-1 infected patients despite antiretroviral therapy [74]. An association of increased expression of AA cascade enzymes with neurocognitive/neurodegeneration also has been suggested for Alzheimer disease and vascular dementia [75, 76]. In this regard, cPLA2 inhibition or deletion improved learning and memory performance in a transgenic mouse model of Alzheimer disease [72]. Treatment with lithium or sodium valproate also was beneficial in HIV-1 associated dementia patients [77, 78], possibly by attenuating neuroinflammation and an upregulated brain AA cascade [79, 80]. Collectively, these observations suggest that neuroinflammation associated with increased AA metabolism can contribute to cognitive impairment, and that attenuation of AA release by inhibiting cPLA2 may be beneficial.

The significant changes observed with AA cascade and neuroinflammation markers in HIV-1 Tg rats must be interpreted with caution because potential contamination of brain tissue by peripheral cells during cytosolic or total RNA isolation would give higher background levels for measured proteins, except for the neuron-specific marker drebrin. However, such changes are unlikely because we areported increased global brain AA incorporation from plasma in awake HIV-1 Tg rats [9]. Further examination is required to study the extent of activation of neuroinflammation and AA cascade markers in peripheral cells of HIV-1 Tg rats.

While differences between HIV-1 Tg and controls rates in several brain measures at the p < 0.05 level were not considered statistically significant because of the constraint of multiple comparisons (see Methods), they should be given some weight for several reasons, and might be reconsidered in the future with larger samples. This study was exploratory, and was focused on generating hypotheses that could be tested more discretely in the future. Importantly, many of the p < 0.05 changes in a protein occurred with a significant change at p < 0.01 in the respective mRNA, and vice versa, making the p < 0.05 change more credible.

Conclusion

Multiple markers of neuroinflammation and the AA cascade are upregulated, and levels of the postsynaptic markers drebrin and BDNF are reduced, in brain of 7- to 9-month-old HIV-1 Tg rats compared with control rats. These changes may contribute to cognitive impairment in these rats, and likely are related to the presence of viral proteins that trigger activation of several pathways. Our study provides additional critical characterization of neuropathological changes in the mature HIV-1 Tg rat, further establishing this rat as a potentially useful animal model to examine disease progression and effects of therapeutic intervention that can impact treatment and understanding of cognitive and behavioral changes in HIV-1 infected patients.

Notes

Abbreviations

AA: 

arachidonic acid

cPGES: 

cytosolic prostaglandin E synthase

cPLA2

calcium-dependent cytosolic phospholipase A2

COX: 

cyclooxygenase

GFAP: 

glial fibrillary acidic protein

gp120: 

glycoprotein 120

HIV: 

human immunodeficiency virus

IL-1β: 

interleukin-1β

mPGES: 

membrane prostaglandin E synthase

LOX: 

lipoxygenase

NF-κB: 

nuclear factor-kappa B

PG: 

prostaglandin

TNFα: 

tumor necrosis factor α

Tg: 

transgenic

TX: 

thromboxane

TXS: 

thromboxane synthase.

Declarations

Acknowledgements

This research was entirely supported by the Intramural Research Programs of the National Institute on Aging and the National Institute of Mental Health National Institutes of Health, Bethesda, MD, and the National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC. We thank the NIH Fellows Editorial Board for editing the manuscript.

Authors’ Affiliations

(1)
Brain Physiology and Metabolism Section, National Institute on Aging
(2)
National Institute of Mental Health, National Institutes of Health
(3)
Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park

References

  1. Foley J, Ettenhofer M, Wright M, Hinkin CH: Emerging issues in the neuropsychology of HIV infection. Curr HIV/AIDS Rep. 2008, 5: 204-211. 10.1007/s11904-008-0029-x.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Schouten J, Cinque P, Gisslen M, Reiss P, Portegies P: HIV-1 infection and cognitive impairment in the cART era: a review. AIDS. 2011, 25: 561-575. 10.1097/QAD.0b013e3283437f9a.View ArticlePubMedGoogle Scholar
  3. Paramesparan Y, Garvey LJ, Ashby J, Foster CJ, Fidler S, Winston A: High rates of asymptomatic neurocognitive impairment in vertically acquired HIV-1-infected adolescents surviving to adulthood. J Acquir Immune Defic Syndr. 2010, 55: 134-136. 10.1097/QAI.0b013e3181d90e8c.View ArticlePubMedGoogle Scholar
  4. Simioni S, Cavassini M, Annoni JM, Rimbault Abraham A, Bourquin I, Schiffer V, Calmy A, Chave JP, Giacobini E, Hirschel B, Du Pasquier RA: Cognitive dysfunction in HIV patients despite long-standing suppression of viremia. AIDS. 2010, 24: 1243-1250.PubMedGoogle Scholar
  5. Lashomb AL, Vigorito M, Chang SL: Further characterization of the spatial learning deficit in the human immunodeficiency virus-1 transgenic rat. J Neurovirol. 2009, 15: 14-24. 10.1080/13550280802232996.View ArticlePubMedGoogle Scholar
  6. Vigorito M, LaShomb AL, Chang SL: Spatial learning and memory in HIV-1 transgenic rats. J Neuroimmune Pharmacol. 2007, 2: 319-328. 10.1007/s11481-007-9078-y.View ArticlePubMedGoogle Scholar
  7. Reid W, Sadowska M, Denaro F, Rao S, Foulke J, Hayes N, Jones O, Doodnauth D, Davis H, Sill A, et al: An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction. Proc Natl Acad Sci USA. 2001, 98: 9271-9276. 10.1073/pnas.161290298.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Chang SL, Beltran JA, Swarup S: Expression of the mu opioid receptor in the human immunodeficiency virus type 1 transgenic rat model. J Virol. 2007, 81: 8406-8411. 10.1128/JVI.00155-07.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Basselin M, Ramadan E, Igarashi M, Chang L, Chen M, Kraft AD, Harry GJ, Rapoport SI: Imaging upregulated brain arachidonic acid metabolism in HIV-1 transgenic rats. J Cereb Blood Flow Metab. 31: 486-493.Google Scholar
  10. Gray F, Haug H, Chimelli L, Geny C, Gaston A, Scaravilli F, Budka H: Prominent cortical atrophy with neuronal loss as correlate of human immunodeficiency virus encephalopathy. Acta Neuropathol. 1991, 82: 229-233. 10.1007/BF00294450.View ArticlePubMedGoogle Scholar
  11. Masliah E, Ge N, Morey M, DeTeresa R, Terry RD, Wiley CA: Cortical dendritic pathology in human immunodeficiency virus encephalitis. Lab Invest. 1992, 66: 285-291.PubMedGoogle Scholar
  12. Masliah E, Heaton RK, Marcotte TD, Ellis RJ, Wiley CA, Mallory M, Achim CL, McCutchan JA, Nelson JA, Atkinson JH, Grant I: Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders. HNRC Group. The HIV Neurobehavioral Research Center. Ann Neurol. 1997, 42: 963-972. 10.1002/ana.410420618.View ArticlePubMedGoogle Scholar
  13. Rothwell NJ: Annual review prize lecture cytokines-killers in the brain?. J Physiol. 1999, 514 (Pt 1): 3-17.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Moolwaney AS, Igwe OJ: Regulation of the cyclooxygenase-2 system by interleukin-1beta through mitogen-activated protein kinase signaling pathways: a comparative study of human neuroglioma and neuroblastoma cells. Brain Res Mol Brain Res. 2005, 137: 202-212.View ArticlePubMedGoogle Scholar
  15. Laflamme N, Lacroix S, Rivest S: An essential role of interleukin-1beta in mediating NF-kappaB activity and COX-2 transcription in cells of the blood-brain barrier in response to a systemic and localized inflammation but not during endotoxemia. J Neurosci. 1999, 19: 10923-10930.PubMedGoogle Scholar
  16. Blais V, Rivest S: Inhibitory action of nitric oxide on circulating tumor necrosis factor-induced NF-kappaB activity and COX-2 transcription in the endothelium of the brain capillaries. J Neuropathol Exp Neurol. 2001, 60: 893-905.View ArticlePubMedGoogle Scholar
  17. Hernandez M, Bayon Y, Sanchez Crespo M, Nieto ML: Signaling mechanisms involved in the activation of arachidonic acid metabolism in human astrocytoma cells by tumor necrosis factor-alpha: phosphorylation of cytosolic phospholipase A2 and transactivation of cyclooxygenase-2. J Neurochem. 1999, 73: 1641-1649.View ArticlePubMedGoogle Scholar
  18. Griffin DE, Wesselingh SL, McArthur JC: Elevated central nervous system prostaglandins in human immunodeficiency virus-associated dementia. Ann Neurol. 1994, 35: 592-597. 10.1002/ana.410350513.View ArticlePubMedGoogle Scholar
  19. Viviani B, Corsini E, Binaglia M, Galli CL, Marinovich M: Reactive oxygen species generated by glia are responsible for neuron death induced by human immunodeficiency virus-glycoprotein 120 in vitro. Neuroscience. 2001, 107: 51-58. 10.1016/S0306-4522(01)00332-3.View ArticlePubMedGoogle Scholar
  20. Mollace V, Colasanti M, Rodino P, Lauro GM, Nistico G: HIV coating gp 120 glycoprotein-dependent prostaglandin E2 release by human cultured astrocytoma cells is regulated by nitric oxide formation. Biochem Biophys Res Commun. 1994, 203: 87-92. 10.1006/bbrc.1994.2152.View ArticlePubMedGoogle Scholar
  21. Maccarrone M, Navarra M, Corasaniti MT, Nistico G, Finazzi Agro A: Cytotoxic effect of HIV-1 coat glycoprotein gp120 on human neuroblastoma CHP100 cells involves activation of the arachidonate cascade. Biochem J. 1998, 333 (Pt 1): 45-49.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Williams JR, Leaver HA, Ironside JW, Miller EP, Whittle IR, Gregor A: Apoptosis in human primary brain tumours: actions of arachidonic acid. Prostaglandins Leukot Essent Fatty Acids. 1998, 58: 193-200. 10.1016/S0952-3278(98)90113-2.View ArticlePubMedGoogle Scholar
  23. Yagami T, Ueda K, Asakura K, Hata S, Kuroda T, Sakaeda T, Takasu N, Tanaka K, Gemba T, Hori Y: Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis. Mol Pharmacol. 2002, 61: 114-126. 10.1124/mol.61.1.114.View ArticlePubMedGoogle Scholar
  24. Fang KM, Chang WL, Wang SM, Su MJ, Wu ML: Arachidonic acid induces both Na+ and Ca2+ entry resulting in apoptosis. J Neurochem. 2008, 104: 1177-1189. 10.1111/j.1471-4159.2007.05022.x.View ArticlePubMedGoogle Scholar
  25. Okuda S, Saito H, Katsuki H: Arachidonic acid: toxic and trophic effects on cultured hippocampal neurons. Neuroscience. 1994, 63: 691-699. 10.1016/0306-4522(94)90515-0.View ArticlePubMedGoogle Scholar
  26. Farooqui AA, Yi Ong W, Lu XR, Halliwell B, Horrocks LA: Neurochemical consequences of kainate-induced toxicity in brain: involvement of arachidonic acid release and prevention of toxicity by phospholipase A(2) inhibitors. Brain Res Brain Res Rev. 2001, 38: 61-78.View ArticlePubMedGoogle Scholar
  27. Mattson MP, Maudsley S, Martin B: BDNF and 5-HT: a dynamic duo in age-related neuronal plasticity and neurodegenerative disorders. Trends Neurosci. 2004, 27: 589-594. 10.1016/j.tins.2004.08.001.View ArticlePubMedGoogle Scholar
  28. Kojima N, Kato Y, Shirao T, Obata K: Nucleotide sequences of two embryonic drebrins, developmentally regulated brain proteins, and developmental change in their mRNAs. Brain Res. 1988, 464: 207-215.View ArticlePubMedGoogle Scholar
  29. Huang SK, Klein DC, Korf HW: Immunocytochemical demonstration of rod-opsin, S-antigen, and neuron-specific proteins in the human pineal gland. Cell Tissue Res. 1992, 267: 493-498. 10.1007/BF00319371.View ArticlePubMedGoogle Scholar
  30. Aoki C, Sekino Y, Hanamura K, Fujisawa S, Mahadomrongkul V, Ren Y, Shirao T: Drebrin A is a postsynaptic protein that localizes in vivo to the submembranous surface of dendritic sites forming excitatory synapses. J Comp Neurol. 2005, 483: 383-402. 10.1002/cne.20449.View ArticlePubMedGoogle Scholar
  31. Terry-Lorenzo RT, Inoue M, Connor JH, Haystead TA, Armbruster BN, Gupta RP, Oliver CJ, Shenolikar S: Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density. J Biol Chem. 2000, 275: 2439-2446. 10.1074/jbc.275.4.2439.View ArticlePubMedGoogle Scholar
  32. McCarthy M, Vidaurre I, Geffin R: Maturing neurons are selectively sensitive to human immunodeficiency virus type 1 exposure in differentiating human neuroepithelial progenitor cell cultures. J Neurovirol. 2006, 12: 333-348. 10.1080/13550280600915347.View ArticlePubMedGoogle Scholar
  33. Harigaya Y, Shoji M, Shirao T, Hirai S: Disappearance of actin-binding protein, drebrin, from hippocampal synapses in Alzheimer's disease. J Neurosci Res. 1996, 43: 87-92. 10.1002/jnr.490430111.View ArticlePubMedGoogle Scholar
  34. Julien C, Tremblay C, Bendjelloul F, Phivilay A, Coulombe MA, Emond V, Calon F: Decreased drebrin mRNA expression in Alzheimer disease: correlation with tau pathology. J Neurosci Res. 2008, 86: 2292-2302. 10.1002/jnr.21667.View ArticlePubMedGoogle Scholar
  35. Chuang DM, Chen RW, Chalecka-Franaszek E, Ren M, Hashimoto R, Senatorov V, Kanai H, Hough C, Hiroi T, Leeds P: Neuroprotective effects of lithium in cultured cells and animal models of diseases. Bipolar Disord. 2002, 4: 129-136. 10.1034/j.1399-5618.2002.01179.x.View ArticlePubMedGoogle Scholar
  36. Hatanpaa K, Isaacs KR, Shirao T, Brady DR, Rapoport SI: Loss of proteins regulating synaptic plasticity in normal aging of the human brain and in Alzheimer disease. J Neuropathol Exp Neurol. 1999, 58: 637-643. 10.1097/00005072-199906000-00008.View ArticlePubMedGoogle Scholar
  37. Counts SE, Nadeem M, Lad SP, Wuu J, Mufson EJ: Differential expression of synaptic proteins in the frontal and temporal cortex of elderly subjects with mild cognitive impairment. J Neuropathol Exp Neurol. 2006, 65: 592-601. 10.1097/00005072-200606000-00007.View ArticlePubMedGoogle Scholar
  38. Dwivedi Y, Rizavi HS, Rao JS, Pandey GN: Modifications in the phosphoinositide signaling pathway by adrenal glucocorticoids in rat brain: focus on phosphoinositide-specific phospholipase C and inositol 1,4,5-trisphosphate. J Pharmacol Exp Ther. 2000, 295: 244-254.PubMedGoogle Scholar
  39. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
  40. Lahiri DK: An region upstream of the gene promoter for the beta-amyloid precursor protein interacts with proteins from nuclear extracts of the human brain and PC12 cells. Brain Res Mol Brain Res. 1998, 58: 112-122.View ArticlePubMedGoogle Scholar
  41. Rao JS, Ertley RN, Rapoport SI, Bazinet RP, Lee HJ: Chronic NMDA administration to rats up-regulates frontal cortex cytosolic phospholipase A2 and its transcription factor, activator protein-2. J Neurochem. 2007, 102: 1918-1927. 10.1111/j.1471-4159.2007.04648.x.View ArticlePubMedGoogle Scholar
  42. Ford AL, Goodsall AL, Hickey WF, Sedgwick JD: Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol. 1995, 154: 4309-4321.PubMedGoogle Scholar
  43. Iliff JJ, Jia J, Nelson J, Goyagi T, Klaus J, Alkayed NJ: Epoxyeicosanoid signaling in CNS function and disease. Prostaglandins Other Lipid Mediat. 2010, 91: 68-84. 10.1016/j.prostaglandins.2009.06.004.View ArticlePubMedGoogle Scholar
  44. Caro AA, Cederbaum AI: Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity. Free Radic Biol Med. 2006, 40: 364-375. 10.1016/j.freeradbiomed.2005.10.044.View ArticlePubMedGoogle Scholar
  45. Morri H, Ozaki M, Watanabe Y: 5'-flanking region surrounding a human cytosolic phospholipase A2 gene. Biochem Biophys Res Commun. 1994, 205: 6-11. 10.1006/bbrc.1994.2621.View ArticlePubMedGoogle Scholar
  46. Antonio V, Brouillet A, Janvier B, Monne C, Bereziat G, Andreani M, Raymondjean M: Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1beta in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-kappaB and Ets transcription factors. Biochem J. 2002, 368: 415-424. 10.1042/BJ20020658.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Tanabe T, Tohnai N: Cyclooxygenase isozymes and their gene structures and expression. Prostaglandins Other Lipid Mediat. 2002, 68-69: 95-114.View ArticlePubMedGoogle Scholar
  48. Hernandez M, Fuentes L, Fernandez Aviles FJ, Crespo MS, Nieto ML: Secretory phospholipase A(2) elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis. Circ Res. 2002, 90: 38-45. 10.1161/hh0102.102978.View ArticlePubMedGoogle Scholar
  49. Pereira SG, Oakley F: Nuclear factor-kappaB1: regulation and function. Int J Biochem Cell Biol. 2008, 40: 1425-1430. 10.1016/j.biocel.2007.05.004.View ArticlePubMedGoogle Scholar
  50. Niederberger E, Schmidtko A, Gao W, Kuhlein H, Ehnert C, Geisslinger G: Impaired acute and inflammatory nociception in mice lacking the p50 subunit of NF-kappaB. Eur J Pharmacol. 2007, 559: 55-60. 10.1016/j.ejphar.2006.11.074.View ArticlePubMedGoogle Scholar
  51. Dandekar DH, Ganesh KN, Mitra D: HIV-1 Tat directly binds to NFkappaB enhancer sequence: role in viral and cellular gene expression. Nucleic Acids Res. 2004, 32: 1270-1278. 10.1093/nar/gkh289.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Roy A, Fung YK, Liu X, Pahan K: Up-regulation of microglial CD11b expression by nitric oxide. J Biol Chem. 2006, 281: 14971-14980. 10.1074/jbc.M600236200.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Perry VH, O'Connor V: The role of microglia in synaptic stripping and synaptic degeneration: a revised perspective. ASN Neuro. 2010, 2.Google Scholar
  54. Kanmogne GD, Schall K, Leibhart J, Knipe B, Gendelman HE, Persidsky Y: HIV-1 gp120 compromises blood-brain barrier integrity and enhances monocyte migration across blood-brain barrier: implication for viral neuropathogenesis. J Cereb Blood Flow Metab. 2007, 27: 123-134. 10.1038/sj.jcbfm.9600330.View ArticlePubMedGoogle Scholar
  55. Flora G, Pu H, Hennig B, Toborek M: Cyclooxygenase-2 is involved in HIV-1 Tat-induced inflammatory responses in the brain. Neuromolecular Med. 2006, 8: 337-352. 10.1385/NMM:8:3:337.View ArticlePubMedGoogle Scholar
  56. Flora G, Lee YW, Nath A, Hennig B, Maragos W, Toborek M: Methamphetamine potentiates HIV-1 Tat protein-mediated activation of redox-sensitive pathways in discrete regions of the brain. Exp Neurol. 2003, 179: 60-70. 10.1006/exnr.2002.8048.View ArticlePubMedGoogle Scholar
  57. Xiong H, Zeng YC, Lewis T, Zheng J, Persidsky Y, Gendelman HE: HIV-1 infected mononuclear phagocyte secretory products affect neuronal physiology leading to cellular demise: relevance for HIV-1-associated dementia. J Neurovirol. 2000, 6 (Suppl 1): S14-23.PubMedGoogle Scholar
  58. Kobayashi R, Sekino Y, Shirao T, Tanaka S, Ogura T, Inada K, Saji M: Antisense knockdown of drebrin A, a dendritic spine protein, causes stronger preference, impaired pre-pulse inhibition, and an increased sensitivity to psychostimulant. Neurosci Res. 2004, 49: 205-217. 10.1016/j.neures.2004.02.014.View ArticlePubMedGoogle Scholar
  59. Acquas E, Bachis A, Nosheny RL, Cernak I, Mocchetti I: Human immunodeficiency virus type 1 protein gp120 causes neuronal cell death in the rat brain by activating caspases. Neurotox Res. 2004, 5: 605-615.View ArticlePubMedGoogle Scholar
  60. Haughey NJ, Mattson MP: Calcium dysregulation and neuronal apoptosis by the HIV-1 proteins Tat and gp120. J Acquir Immune Defic Syndr. 2002, 31 (Suppl 2): S55-61.View ArticlePubMedGoogle Scholar
  61. Alirezaei M, Watry DD, Flynn CF, Kiosses WB, Masliah E, Williams BR, Kaul M, Lipton SA, Fox HS: Human immunodeficiency virus-1/surface glycoprotein 120 induces apoptosis through RNA-activated protein kinase signaling in neurons. J Neurosci. 2007, 27: 11047-11055. 10.1523/JNEUROSCI.2733-07.2007.View ArticlePubMedGoogle Scholar
  62. New DR, Maggirwar SB, Epstein LG, Dewhurst S, Gelbard HA: HIV-1 Tat induces neuronal death via tumor necrosis factor-alpha and activation of non-N-methyl-D-aspartate receptors by a NFkappaB-independent mechanism. J Biol Chem. 1998, 273: 17852-17858. 10.1074/jbc.273.28.17852.View ArticlePubMedGoogle Scholar
  63. Bonavia R, Bajetto A, Barbero S, Albini A, Noonan DM, Schettini G: HIV-1 Tat causes apoptotic death and calcium homeostasis alterations in rat neurons. Biochem Biophys Res Commun. 2001, 288: 301-308. 10.1006/bbrc.2001.5743.View ArticlePubMedGoogle Scholar
  64. Gregersen R, Lambertsen K, Finsen B: Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab. 2000, 20: 53-65. 10.1097/00004647-200001000-00009.View ArticlePubMedGoogle Scholar
  65. Hanisch UK: Microglia as a source and target of cytokines. Glia. 2002, 40: 140-155. 10.1002/glia.10161.View ArticlePubMedGoogle Scholar
  66. Taylor DL, Jones F, Kubota ES, Pocock JM: Stimulation of microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis factor alpha-induced neurotoxicity in concert with microglial-derived Fas ligand. J Neurosci. 2005, 25: 2952-2964. 10.1523/JNEUROSCI.4456-04.2005.View ArticlePubMedGoogle Scholar
  67. Beghi E, Citterio A, Cornelio F, Filippini G, Grilli R, Liberati A: Practice guidelines: a more rational approach to diagnosis and treatment and a more effective use of health care resources. Ital J Neurol Sci. 1998, 19: 120-123. 10.1007/BF02427570.View ArticlePubMedGoogle Scholar
  68. Fiore M, Angelucci F, Alleva E, Branchi I, Probert L, Aloe L: Learning performances, brain NGF distribution and NPY levels in transgenic mice expressing TNF-alpha. Behav Brain Res. 2000, 112: 165-175. 10.1016/S0166-4328(00)00180-7.View ArticlePubMedGoogle Scholar
  69. Fiore M, Probert L, Kollias G, Akassoglou K, Alleva E, Aloe L: Neurobehavioral alterations in developing transgenic mice expressing TNF-alpha in the brain. Brain Behav Immun. 1996, 10: 126-138. 10.1006/brbi.1996.0013.View ArticlePubMedGoogle Scholar
  70. Gemma C, Bickford PC: Interleukin-1beta and caspase-1: players in the regulation of age-related cognitive dysfunction. Rev Neurosci. 2007, 18: 137-148. 10.1515/REVNEURO.2007.18.2.137.View ArticlePubMedGoogle Scholar
  71. Hauss-Wegrzyniak B, Dobrzanski P, Stoehr JD, Wenk GL: Chronic neuroinflammation in rats reproduces components of the neurobiology of Alzheimer's disease. Brain Res. 1998, 780: 294-303. 10.1016/S0006-8993(97)01215-8.View ArticlePubMedGoogle Scholar
  72. Sanchez-Mejia RO, Newman JW, Toh S, Yu GQ, Zhou Y, Halabisky B, Cisse M, Scearce-Levie K, Cheng IH, Gan L, et al: Phospholipase A2 reduction ameliorates cognitive deficits in a mouse model of Alzheimer's disease. Nat Neurosci. 2008, 11: 1311-1318. 10.1038/nn.2213.View ArticlePubMedPubMed CentralGoogle Scholar
  73. Lukiw WJ, Bazan NG: Neuroinflammatory signaling upregulation in Alzheimer's disease. Neurochem Res. 2000, 25: 1173-1184. 10.1023/A:1007627725251.View ArticlePubMedGoogle Scholar
  74. Harezlak J, Buchthal S, Taylor M, Schifitto G, Zhong J, Daar E, Alger J, Singer E, Campbell T, Yiannoutsos C, et al: Persistence of HIV-associated cognitive impairment, inflammation, and neuronal injury in era of highly active antiretroviral treatment. AIDS Epub Feb 3. 2011Google Scholar
  75. Yagami T: Cerebral arachidonate cascade in dementia: Alzheimer's disease and vascular dementia. Curr Neuropharmacol. 2006, 4: 87-100. 10.2174/157015906775203011.View ArticlePubMedPubMed CentralGoogle Scholar
  76. Sun GY, Xu J, Jensen MD, Simonyi A: Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases. J Lipid Res. 2004, 45: 205-213.View ArticlePubMedGoogle Scholar
  77. Schifitto G, Peterson DR, Zhong J, Ni H, Cruttenden K, Gaugh M, Gendelman HE, Boska M, Gelbard H: Valproic acid adjunctive therapy for HIV-associated cognitive impairment: a first report. Neurology. 2006, 66: 919-921. 10.1212/01.wnl.0000204294.28189.03.View ArticlePubMedGoogle Scholar
  78. Letendre SL, Woods SP, Ellis RJ, Atkinson JH, Masliah E, van den Brande G, Durelle J, Grant I, Everall I: Lithium improves HIV-associated neurocognitive impairment. AIDS. 2006, 20: 1885-1888. 10.1097/01.aids.0000244208.49123.1b.View ArticlePubMedGoogle Scholar
  79. Basselin M, Ramadan E, Chen M, Rapoport SI: Anti-inflammatory effects of chronic aspirin on brain arachidonic acid metabolites. Neurochem Res. 2011, 36: 139-145. 10.1007/s11064-010-0282-4.View ArticlePubMedGoogle Scholar
  80. Basselin M, Villacreses NE, Lee HJ, Bell JM, Rapoport SI: Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation. J Neurochem. 2007, 102: 761-772. 10.1111/j.1471-4159.2007.04593.x.View ArticlePubMedGoogle Scholar

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